PHOTOISOMERIZATION OF SIXTEEN ISOMERS OF RETINAL. INITIAL PRODUCT DISTRIBUTION IN DIRECT AND SENSITIZED IRRADIATION. PHOTOCHEMISTRY OF POLYENES 31*

Abstract— Initial product distributions of all 16 geometric isomers of retinal in hexane solutions have been determined. With direct irradiation, the product ratios are characterized by a preference for isomerization at the 13,14-bond. In particular, all isomers containing the 13-cis geometry give the corresponding 13-trans isomer as the major product. Preference for one-photon-one-bond isomerization was also noted, although a substantial amount of the all-trans isomer was detected for all poly-cis, 13-trans isomers. In sensitized irradiation, the initial mixture shows extensive one-photon-two(or multiple)-bond isomerization to the corresponding unhindered isomers. In cases of hindered isomers, multiple-bond isomerized products are dominant. The different results are accountable by the different shapes of the excited state potential curves for singlet and triplet states.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.     

TIME-RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185→E: ASP-96 REPROTONATES DURING O FORMATION; ASP-85 AND ASP-212 DEPROTONATE DURING O DECAY

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.     

Recherches dans la série des cyclitols XLII. Synthèses de cyclitols tout-cis dérivés du cyclopentane; préparation des cyclopentane-tétrol et - pentol tout-cis

All-cis cyclitols derived from cyclopentane have been prepared in two ways: (i) by LiAlH₄ reduction of all-cis epoxypolyos; (ii) by dimethyl sulfoxide treatment, in the presence of sodium hydrogencarbonate, of brominated derivatives containing a neighbouring benzoyloxy group; under these conditions, the halogen is replaced by a cis-oriented hydroxyl group. Thus all-cis cyclopentane-tetrol and -pentol have been prepared; their configuration has been confirmed by NMR. spectroscopy.     

Seven new hindered isomeric rhodopsins: A reexamination of the stereospecificity of the binding site of bovine opsin

Results of interaction of seven new geometric isomers of retinal (7-cis, 7,9-dicis; 7,11-dicis, 7,13-dicis; 9,11-dicis 7,9,11-tricis 7,9,13-tricis) with bovine opsin are reported. All of them form pigments with absorption maxima varying between 450 and 480 nm. The rates of pigment formation were generally considerably lower than those of 11-cis-retinal and the yields were less than quantitative. Implications of these results for the stereospecificity of the binding site of opsin are discussed.     

EFFECT OF PHOTOSELECTION UPON SATURATION AND THE DICHROIC RATIO IN FLASH EXPERIMENTS UPON EFFECTIVELY IMMOBILIZED SYSTEMS

Abstract— Flash spectroscopy of photochemical or photobiological systems, such as bacteriorhodopsin in purple membrane, for which the chromophore transition dipole moment does not undergo complete reorientation during the time of the flash, is considered as a function of light intensity. Due to photoselection, saturation proceeds very slowly with increasing flash intensity and the linear dichroic ratio decays rapidly from a maximum of three at zero flash intensity. Simple formulae are derived to describe these effects under stringent assumptions. Calculations are also performed which relax the assumptions by taking into account (i) non-zero optical density, (ii) thermal decay of the photoproduct during the flash and (iii) non-zero angle y between the initial chromophore and its photoproduct. Agreement with experiments on bacteriorhodopsin in purple membranes is excellent.     

Cis/trans ISOMERIZATION OF CAROTENOIDS BY THE TRIPLET CARBONYL SOURCE 3-HYDROXYMETHYL-3,4,4-TRIMETHYL-1,2-DIOXETANE*

Abstract— The interaction of biological carotenoids with 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), a thermodissociable source of electronically excited ketones, was investigated using reversed-phase high-performance liquid chromatography. Incubation of the all-trans isomers of β-carotene, lycopene and canthaxanthin with HTMD led to significant trans-to-cis isomerization, with cis isomers accounting for 20–50% of products formed (the balance assigned as oxidation products). The isomers forming from all-trans-β-carotene were identified as 9-cis-, 13-cis- and 15-cis-β-carotene by cochromatography of cis isomer standards and by on-line diode array absorbance spectroscopy. An HTMD-dependent cis-to-trans isomerization was observed in incubations started with 15-cis-β-carotene, and it occurred more rapidly and to a greater extent than the isomerization of all-trans-β-carotene. The isomer patterns generated from lycopene and β-carotene are generally similar to those reported recently for various human tissues (Stahl et al, 1992, Arch. Biochem. Biophys. 294 , 173–177).     

TIME-RESOLVED FLUOROMETRY OF BACTERIORHODOPSIN

Abstract A mode-locked Nd:YAG laser was used to excite the aromatic amino acid residues of bacteriorhodopsin in the purple membrane and the tryptophan (Trp) fluorescence decay analyzed with a streak camera (λ> 380 nm). The decay kinetics are resolvable into two first-order half-times (1.5 and 0.17 ns, respectively), while for retinylidene-free bacterioopsin, only the longer-lived Trp emission was observed. The shorter-lived species reappeared upon regeneration of bacteriorhodopsin by addition of retinal to bacterioopsin but not on treatment of the latter with an equivalent of retinol. It is proposed that these results are consistent with a structural model in which the 7-8 Trp's distributed among sections A, C, E and F of the seven helical segments A-G of native bacteriorhodopsin are distinguishable by their distances from the chromophore. Assuming a Förster mechanism for energy transfer with R_o= 25 and 32 Å, respectively, for retinylidene chromophore and retinol the Trp's may be divided into two groups: (i) those completely quenched by retinol and partly quenched by retinal (τ= 0.17) with R ≃ 18 Å and (ii) those (τ= 1.5 ns) which are quenched neither by chromophore nor retinol with R > ca. 30 Å. These results are consistent with and support some of the best models of Engelman et al. (1980) for the protein conformation in the purple membrane.     

PHOTOCHROMIC SYNERGISM OF BACTERIORHODOPSIN- and HALORHODOPSIN-MEDIATED PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantitative action spectroscopy was performed in Halobacterium halobium. using four suited pigment mutants, namely the bacteriorhodopsin and halorhodopsin positive mutant strain M-l (BR⁺, HR⁺), the bacteriorhodopsin positive but halorhodopsin negative strain M-18 (BR⁺, HR^-), the bacteriorhodopsin negative but halorhodopsin positive strain L-33 (BR^-, HR⁺), and the bacteriorhodopsin and halorhodopsin negative strain L-07 (BR^-, HR⁺). The approached questions were: First, photoenergetic synergism of halorhodopsin and bacteriorhodopsin in intact cells; second, photochromism and cellular function of the blue light-absorbing intermediates, i.e. M-412 and HR-410 in bacteriorhodopsin and in halorhodopsin, respectively. Dark-adapted cells of mutant strain M-l show wavelength-dependency of quantum yield of photo-phosphorylation, φ_ATP. An 1.4-fold enhancement was found at 575 nm wavelength where the long wavelength absorbance bands of bacteriorhodopsin and halorhodopsin intersect. The enhancement vanished after a 30 min pulse of orange light (600 Wm^-2 bandpass from 495 to 750 nm), but was restored after a 30 min pulse of blue light (100 Wm^-2 bandpass from 325 to 480 nm). Photoreversibility of this enhancement probably reflects phototransformation of halorhodopsin from its ground state into its inactive intermediate, HR-410, and vice versa. The halorhodopsin-mediated enhancement with maximum quantum yield of photophosphorylation, φ_ATP= 0.06, i.e. a quantum requirement of = 17 photons/ATP, is partly substituted by a rise in phosphate potential and explained in terms of a voltage-regulated gating effect on the H⁺-driven ATP-synthase, superimposed on the chemiosmotic mechanism of energy coupling. The blue-absorbing photochromic intermediate, M-412 of bacteriorhodopsin, dissipates light energy upon photoexcitation that is reflected by a spectral decline in quantum yield of photophosphorylation to a minimum value of = 0.01 at 415 nm, i.e. a quantum requirement of = 100 photons/ATP.     

含2-苯基-1,2,3-三唑基单环β-内酰胺类化合物的合成

寻找能口服、广谱的单环β-内酰胺类抗生素已经发展成为一个重要研究领域, 烯酮-亚胺环加成是最有效的合成方法之一. 在三乙胺与含2-苯基-1,2,3-三唑基的Schiff碱的苯溶液中逐滴加入邻苯二甲酰亚氨基乙酰氯或丙酰氯的苯溶液, 加热回流, 通过环加成反应得到含有2-苯基-1,2,3-三唑基和邻苯二甲酰亚胺基的单环β-内酰胺类化合物. 产物均为反式构型, 其结构由元素分析, IR, ¹H NMR, MS以及NOESY谱得到确证.     

Molecular Dynamics Simulation of the Excited-State Dynamics of Bacteriorhodopsin

Abstract— The excited-state dynamics of bacteriorhodopsin was studied by molecular dynamics simulation. For the purpose of suppressing large displacement of amino acid residues on the surface of bacteriorhodopsin, positional restraints were imposed on these residues. A new method was developed to investigate the movement of amino acid residues upon photoexcitation and their role on the ultrafast photoisomerization of the chromophore. The structural change of bacteriorhodopsin was then traced up to 200 fs, i.e. until the formation of the intermediate I. We found that when all the conjugated bonds of the chromophore were allowed to twist freely in the excited state, many bonds including the C13=C14 bond twist in large scale within 100 fs. When only the C13=C14 bond and the single bonds were allowed to twist freely, the twisting took place at most 20° within 200 fs. From these results, it is claimed that a special potential surface is provided for the C13=C14 bond twisting by the protein environment in the course of the isomerization reaction, giving rise to the specific, ultrafast photoisomerization of bacteriorhodopsin. As a trace of such a mechanism, we observed that several functionally important residues incuding Asp85, Asp212 and Tyr185 responded quickly to the photoexcitation of the chromophore.     

SPECTROSCOPIC STUDIES ON THE STRUCTURES OF RETINOCHROME and METARETINOCHROME*

Abstract– Spectroscopic measurements of retinochrome and the related photoproduct, metaretino-chrome, were carried out to determine the conformation of the retinal and the protein. Absorption spectra with fourth derivatives indicate that the tryptophan residues are located in a hydrophobic core and that the environment around these residues does not change after light irradiation. Circular dichroism measurements indicate that retinochrome has a high helical content which is not altered by the conversion of retinochrome to metaretinochrome. Fourier transform infrared difference spectra demonstrate that retinochrome has the all-trans retinal and metaretinochrome has the 11-CM retinal. Retinochrome has an absorption due to amino acid residue(s) which changes in metaretinochrome. This work demonstrates that conformational changes are not induced during the conversion but the electrical charge(s) of the protein are changed by irradiation.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

PHOTOSELECTION AND TRANSIENT LINEAR DICHROISM WITH ORIENTED IMMOBILIZED PURPLE MEMBRANES: EVIDENCE FOR MOTION OF THE C(20)-METHYL GROUP OF THE CHROMOPHORE TOWARDS THE CYTOPLASMIC SIDE OF THE MEMBRANE*

Abstract— We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time-resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time-resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the M-intermediate, the transition dipole moment tilts out of the plane of the membrane by about 3°. On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11° tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 Å and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M-intermediate.     

A new triterpenoid saponin from Gleditsia sinensis and its antiproliferative activity

Chemical investigation of the anomalous fruits of Gleditsia sinensis led to the isolation and identification of a new triterpenoid saponin, 3-O-β-D-xylopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyrano--syl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 3)-β-D-glucopyranosyl ester (1), along with other nine known compounds (2–10). All the isolates from this species were reported for the first time. The structure of Compound 1 was determined by a detailed analysis using various analytical techniques, including 1D and 2D NMR. In vitro antiproliferative activities of Compound 1 on MCF-7 and Hep-G2 tumor cell lines were evaluated. IC₅₀ values against the two cell lines were 9.5 and 11.6 μM, respectively.     

A new acylated flavonoid tetraglycoside with anti-inflammatory activity from Tipuana tipu leaves

A new acylated kaempferol glycoside, kaempferol 3-O-α-l-rhamnopyranosyl-(1 → 6)-O-[β-d-glucopyranosyl-(1 → 2)-4-O-acetyl-α-l-rhamnopyranosyl-(1 → 2)]-β-d-galactopyranoside, has been isolated from the leaves of Tipuana tipu (Benth.) Lillo growing in Egypt, along with three known flavonol glycosides, kaempferol 3-O-rutinoside, quercetin 3-O-rutinoside (rutin) and kaempferol 3-O-[α-l-rhamnopyranosyl-(1 → 6)]-[α-l-rhamnopyranosyl-(1 → 2]-β-d-glucopyranoside. Structure elucidation was achieved through different spectroscopic methods. Structure relationship with anti-inflammatory activity using carrageenin-induced rat paw oedema model is discussed.     

The Importance of Charge Transfer between the Retinal Chromophore and the Protein Environment in Bacteriorhodopsin: A Theoretical Analysis on Reduced and Oxidized Chromophores

Abstract— The importance of charge transfer(CT) between the retinal chromophore and the protein environment in the ground state of bacteriorhodopsin(BR) has been verified by using ab initio and semiempirical molecular orbital methods. We hypothesize that the chromophore is stabilized in BR by highest occupied molecular orbital-lowest unoccupied molecular orbital(HOMO-LUMO) interaction with the protein environment. If sufficient charge is transferred between two sites due to the strong HOMO-LUMO interaction, the chromophore might be treated as a one-electron reduced species(when it behaves as an electron acceptor), or as a one-electron oxidized one (when it acts as an electron donor).In both optimized geometries, the -conjugated systems exhibit a drastic decrease in bond alternation. To estimate the rotational barrier for thermal isomerization between the al-trans and the 13,15-dicis form, the potential energy curve around these two bonds was computed. The first -_* transition energy was also calculated for an inspection of the opsin shift. The barrier height and the transition energy became much lower as a result of the chromophore reduction. The site selectivity in photo- and thermal isomerization and the opsin shift in BR can be well explained by considering CT from the protein environment to the chromophore.     

FURTHER CHARACTERIZATION OF ANESTHETIC-TREATED PURPLE MEMBRANES

Abstract— The absorption maximum of bacteriorhodopsin is shifted from 568 nm to 480 nm when halogenated volatile anesthetics (enflurane; halothane) are added to purple membranes. Analysis of the rate of formation of this new species upon addition of the anesthetic and of the back-formation of native bacteriorhodopsin upon its removal indicate that in purple membranes, the dark-adapted chromophore is much less reactive than its light-adapted counterpart. Lipid-soluble molecules thus have a lower accessibility to the dark-adapted chromophore.
In addition, activity of the 480 nm bacteriorhodopsin was investigated. Flash and steady-state photolysis experiments reveal that this blue shifted chromophore has full photochemical activity. It has a meta-intermediate absorbing maximally at 380 nm. The photocycle ofBR–480 is mainly characterized by a slow decay of the "O" intermediate, enabling the direct observation of the branching reaction between the "M" intermediate and the parentBR–480 pigment.     

Photocatalyzed E→Z Contra-thermodynamic Isomerization of Vinyl Boronates with Binaphthol

The photocatalytic contra-thermodynamic E→Z isomerization of vinyl boronates by using a binaphthol catalyst is disclosed. The reaction, based on the transient formation of a suitable chromophore with a BINOL derivative as the catalyst, allowed geometrical isomerization in good-to-excellent Z/E ratio and excellent-to-quantitative yields. The mechanism of this E→Z contra-thermodynamic isomerization was studied, and the formation of a transient chromophore species is suggested.