Photochemical properties of a bacteriorhodopsin analog with 13-demethyl-13-trifluoromethyl retinal

It was shown that the substitution of the CF₃ group in the structure of retinal for the methyl group at C13 causes not only a decrease in the affinity of the proton for the nitrogen in the Schiff base (pK ～ 8.4) but also considerably changes the photochemical properties of the bacteriorhodopsin analog. At pH > 6.5, the rate of the Schiff base reprotonation during M decay depends on the proton concentration in the medium. In the photocycle of the yellow M-like form with the deprotonated Schiff base, a long-wavelength product absorbing at 625 nm is formed, which has a similar pH dependence of decay kinetics. The two processes also have similar activation energies (about 15 ± 1 kcal/mol). It is concluded that both cases involve proton transfer from an aqueous medium through the donor part of the channel to the Schiff base and Asp96, respectively. In the analog, however, the structure of water molecules necessary for the stabilization of the proton on the Schiff base is broken. As a result, dehydration of the preparation gives rise to a fraction of M-like form of bacteriorhodopsin with the deprotonated Schiff base.     

Electrogenic transport properties of bacteriorhodopsin containing chemically modified retinal analogues

The electrical activity of bacteriorhodopsin (BR) containing the 13-substituted retinal analogues 13-demethyl and 13-methoxy as well as the naturally occurring retinal carrying a methyl group at C13 is compared. White membrane patches reconstituted with the different retinals are attached to a black lipid membrane, and the dependency of the photocurrent on light intensity is measured. This allows a comparison of the overall photocycle time and the number of protons transported per cycle for the various preparations. From previous work (Gärtner, W., Towner, P., Hopf, H. and Oesterhelt, D. (1983) Biochem. 22, 2637–2644, see also Gärtner, W. and Oesterhelt, D., unpublished data) the equilibrium isomeric distribution (all-trans and 13-cis) of the different retinals in the binding site is known. Taking into account that only all-trans retinal BR contributes to the pumping activity (Fahr, A. and Bamberg, E. (1982) FEBS Lett. 140, 251–253), it is shown, that the cycle time for the modified BRs is moderately changed, whereas the number of protons transported per cycle and transporting all-trans BR molecule is not affected by the substituent. It is concluded, that substituting the methyl group at position 13 of the retinal molecule by a hydrogen atom or a methoxy group only slightly affects the pumping activity of the trans-photocycle, but rather controls the biological function of BR via the equilibrium isomeric distribution of the retinal molecule in the binding site.     

Optical picosecond studies of bacteriorhodopsin containing a sterically fixed retinal

The photochemical behaviour of an analogous bacteriorhodopsin (9,12-Ph-BR) which contains the sterically fixed 9,12-phenylretinal has been investigated with picosecond spectroscopy. The following results have been obtained. No ground-state intermediate photoproduct is found in agreement with the previous observation that 9,12-Ph-BR does not exhibit proton pumping under illumination. The excited singlet state has a lifetime of τ_S = 10 ± 2 ps. This lifetime agrees favourably with the value calculated from the radiative lifetime τ_rad = 6.2 ns and the fluorescence quantum efficiency of 1.2·10⁻³. Excited-state absorption occurs which results in fluorescence in the ultraviolet region. These various observations differ drastically from the corresponding findings on bacteriorhodopsin. Most important for an understanding of the differences is the fact that 9,12-phenylretinal does not isomerize in the protein's binding site in contrast to retinal. Our data therefore suggest that the formation of the intermediate K observed in bacteriorhodopsin is accompanied by the all-trans to 13-cis isomerization.     

C(13)-substituted bacteriorhodopsin analogs

13-Ethyl-, 13-isopropyl-, 13-tert-butyl-, 13-phenyl-, 13-alpha-naphthyl-, and 13-demethyl-retinals were synthesized and incubated with bacterioopsin (bO) to give the corresponding bacteriorhodopsin (bR) analogs. The capability of the 13-tert-butyl- and 13-alpha-naphthyl-bRs to exist and to photocycle shows that apparently around C(13) of the chromophore there lies a large enough cavity. A study of the light-induced conversions of the artificial pigments prepared has shown that the introduction at position 13 of the chromophore of the hydrocarbon substituents bulkier than that of the natural bR diminished the amplitudes of the electric photoresponses. Bulky C(13)-substituents or absence of substitution at that position decelerated the relaxation of the M-intermediates and disturbed the 13-cis-in equilibrium all-trans-isomerization.     

Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups.

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro- and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.     

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.     

Synthesis and biological properties of new chimeric galanin analogue GAL(1-13)-[Ala10,11]ET-1(6-21)-NH2.

Several chimeric peptides consisting of the N-terminal fragment of galanin (GAL) and C-terminal fragments of other bioactive peptides (e.g. substance P, bradykinin, neuropeptide Y, mastoparan) have been synthesized and reported as high-affinity galanin receptor antagonists. Recently we have synthesized a new chimeric peptide, GAL(1-13)-[Ala(10,11)]ET-1(6-21)-NH(2), consisting of the N-terminal fragment of GAL and the C-terminal fragment of endothelin-1 (ET-1) analogue. This chimera was previously shown to be a moderate-affinity ligand to hypothalamic galanin receptors with a K(D) value of 205 nM. However, its biological action has been unknown so far. In our studies we characterized the biological properties of this new chimeric analogue, investigating its action on rat isolated gastric smooth muscles and influence on insulin secretion from rat isolated islets of Langerhans. Data acquired in the course of our studies suggest that analogue GAL(1-13)-[Ala(10,11)]ET-1(6-21)-NH(2) does not seem to be a potent galanin receptor antagonist in the gastrointestinal tract.     

Effect of variation of retinal polyene side-chain length on formation and function of bacteriorhodopsin analogue pigments

The effect of the length of the retinal polyene side chain on bacterioopsin pigment formation and function has been investigated with two series of synthetic retinal analogues. Cyclohexyl derivatives with polyene chains one carbon longer and one or more carbons shorter than retinal and linear polyenes with no ring have been synthesized and characterized. Compounds of six carbons or less in the polyene chain form pigments very poorly or not at all with bacterioopsin. Compounds containing at least seven carbons in the chain are found to form reasonably stable bacterioopsin pigments that show a small shift in absorbance on irradiation. However, photocycling and proton photorelease are not detected. The analogue with nine carbons in the polyene chain (one less than retinal) forms a stable pigment with an M-type intermediate but demonstrates reduced amounts of photocycling and light-activated proton release. The analogue with a polyene chain identical with that of retinal, but containing no ring, forms a pigment that shows both an efficient light-activated proton photocycle and release. The pigment containing the chromophore with the polyene chain one carbon longer than retinal is likewise fully active. We thus conclude that the length of the polyene chain must be at least 9 carbons for the formation of a stable pigment that photocycles and must be 10 carbons for both the photocycle and light-activated proton release to have a high quantum efficiency.     

Conformation and backbone dynamics of bacteriorhodopsin revealed by (13)C-NMR

It is demonstrated here how the secondary structure and dynamics of transmembrane helices, as well as surface residues, such as interhelical loops and N- or C-terminus of bacteriorhodopsin (bR) in purple membrane, can be determined at ambient temperature based on very simple (13)C-NMR measurements, together with a brief experimental background. In contrast to the static picture of bR, currently available from X-ray diffraction or cryo-electron microscopy, the structure consists of dynamically heterogeneous domains which undergo various types of local fluctuations with a frequency range of 10(2)--10 (8) Hz. The significance of this picture is discussed in relation to the biological function of this protein.     

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.     

Synthesis of nucleotide derivatives containing 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol for photoaffinity modification of proteins and nucleic acid

1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4'- dimethoxytrityl)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)15 were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8-9 degrees C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA-protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Discovery of a series of aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamides as TRPA1 antagonists

We describe the discovery and advancement of a novel series of TRPA1 antagonist having an aryl-N-(3-(alkylamino)-5-(trifluoromethyl)phenyl)benzamide scaffold. The physical and in vitro DMPK profiles are discussed.     

Synthesis and pharmacological evaluation of trifluoromethyl containing 4-(2-pyrimidinylamino)benzamides as Hedgehog signaling pathway inhibitors

In present study, a series of novel containing trifluoromethyl 4-(2-pyrimidinylamino)benzamide derivatives were designed by the fluorine scan strategy. Their Hh signaling inhibitory activities were evaluated by Gli-luciferase reporter method. The comprehensive SAR was discussed and several derivatives were found to display more potent Hh signaling inhibitory activity than positive drug vismodegib. Compound 13d was the most potent compound with IC₅₀ of 1.44 nM against Hh signaling pathway and also exhibited optimal PK properties in the in vivo PK properties study, deserved as an ideal lead compound for further study in future.     

Semisynthesis and properties of a fluorescent phosphatidyl-inositol analogue containing a cis-parinaroyl moiety

A method for the preparation of a fluorescent phosphatidylinositol analogue, 1-acl,-2-prinaroyl-sn-glycero-3-phospho-sn-1-myo-inositol has been developed. This method makes use of yeast phosphatidylinositol as the starting material and includes the following steps: (1) acetylation of the free hydroxyl groups of the inositol moeity; (2) removal of the fatty acid from the sn-2-position of the glycerol moiety by phospholipase A₂; (3) reacylation with parimaroyl anhydride; (4) removal of the protecting acetyl groups by alkaline solvolysis. The identity of the product was established by thin-layer chromatography (TLC), UV absorption spectroscopy, enzymatic degradation and by a transfer assay using the phosphatidylinositol transfer protein from bovine brain.Some properties of the fluorescent phosphatidylinositol analogue are reported.     

A (13)C NMR study on [3-(13)C]-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

We have recorded (13)C NMR spectra of selectively [3-(13)C]Ala-, [1-(13)C]Ala-, or [1-(13)C]Val-labeled synthetic transmembrane peptides of bacteriorhodopsin (bR) and enzymatically cleaved C-2 fragment in the solid and dimyristoylphosphatidylcholine bilayer. It turned out that these transmembrane peptides either in hexafluoroisopropanol or cast from it take an ordinary alpha-helix (alpha(I)-helix) irrespective of their amino acid sequences with reference to the conformation-dependent (13)C chemical shifts of (Ala)(n) taking the alpha-helix form. These transmembrane peptides are not always static in the lipid bilayer as in the solid state but undergo rigid-body motions with various frequencies as estimated from suppressed peaks either by fast isotropic or large-amplitude motions (>10(8) Hz) or intermediate frequencies (10(5) or 10(3) Hz). Further, (13)C chemical shifts of the [3-(13)C]Ala-labeled peptides in the bilayer were displaced downfield by 0.3-1.1 ppm depending upon amino acid sequence with respect to those in the solid state, which were explained in terms of local conformational fluctuation (10(2) Hz) deviated from the torsion angles (alpha(II)-helix) from those of standard alpha-helix, under anisotropic environment in lipid bilayer, in addition to the above-mentioned rigid-body motions. The carbonyl (13)C peaks, on the other hand, are not sensitively displaced by such local anisotropic fluctuations, because they are more sensitive to the manner of hydrogen-bond interactions. The amino acid sequences of these peptides inserted within the bilayer were not always the same as those of intact bR, causing disposition of the transmembrane alpha-helical segment from that of intact bR. Finally, we confirmed that the (13)C NMR peak positions of the random coil form are located at the boundary between the alpha-helix and a turned structure in loop regions.     

Chemoenzymatic synthesis and properties of Schiff bases containing (R)-1-(9-anthryl)ethylamine

Racemic 1-(9-anthryl)ethylamine (10), obtained in 70% overall yield from commercial 9-cyanoanthracene, was kinetically resolved by the Candida antarctica A lipase-catalyzed acetylation with isopropyl acetate as acyl donor, affording (R)-(+)-10 with 95.8% enantiomeric excess (e.e.) (E-value 43.5), which afforded Schiff bases (R)-4 and(R)-8. (1)H-NMR, CD, and MM2 calculations offer a consistent picture of the conformational properties of these potential ligands and an explanation for the limited enhancement of enantioselectivity in cyclopropanation of styrene by their Cu(I) complexes, as compared with previously studied ligands in this series.     

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.     

The spectral properties and photosensitivities of analogue photopigments regenerated with 10- and 14-substituted retinal analogues

Analogues of 11-cis- and 9-cis-retinal with substitutions at positions 10 and 14 were used to regenerate analogue photopigments with two opsins: that of the transmuted (cone-like) 521-pigment of Gekko gekko and that of the rhodopsin of Porichthys notatus. The spectral absorbances and photosensitivities of the regenerated photopigments were determined and compared, first, between the two systems of analogue photopigments, and second, in the responses to the two opsins. Unlike the 10-fluoropigments, the comparable 14-compounds were significantly red-shifted by 19-30 nm and their sensitivity to light was similar to that of the parent 11-cis- and 9-cis-pigments. These were the results for both analogue pigments. In contrast, the 10-pigments were spectrally located close to the wavelengths of the parent compounds and the photosensitivity was significantly reduced, especially in the case of the 9-cis-analogues. Evidence was obtained for a steric hindrance effect at position 14, for no regeneration was obtained when methyl or ethyl groups were at this carbon. In the 10-substituted retinals, steric hindrance was noted only for the gecko; only the fluorosubstituted, but not the chloro-, the methyl- or the ethyl-substituted, retinals reacted. With the fish opsin, pigments were regenerated with all but the ethyl-substituted retinal. The gecko opsin appears to have a more restricted binding site. Another feature of the gecko was related to the chloride bathochromic and hyperchromic effects, in which the 521-pigment prepared in a chloride-deficient state has a blue-shifted spectrum compared with the spectrum obtained after the addition of chloride, and its extinction is raised by the addition of chloride to give a mean ratio of 1.23 for the two extinctions, one with, the other without, added chloride. The 11-cis-10-F-analogue pigment gave both chloride effects and the hyperchromic ratio was the same as that recorded for the native visual pigment. In contrast, the pigment formed with 11-cis-14-F-retinal gave a hyperchromic ratio significantly greater than 1.23. A similar contrast in the responses to chloride was obtained with the analogue photopigments regenerated with the 9-cis-10-F- and 9-cis-14-F-chromophores. This difference between the two systems is interpreted as the result of a specific configurational feature of the gecko opsin when in the chloride-deficient state that is relevant to the binding of the retinal analogue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of 2-(3-(3,5-bis(trifluoromethyl)phenyl)thioureido)-3-((dimethylamino)methyl)camphor organocatalysts

In a stereo-divergent synthesis, three novel camphor-derived bifunctional thiourea organocatalysts 7-9 have been prepared in five steps starting from (+)-camphor. In addition, borneol-derived bifunctional thiourea organocatalysts 19/19' have been prepared in three steps from (1S)-(+)-camphorquinone. Novel organocatalysts 7-9, 19/19' have been evaluated in a model reaction of Michael addition of dimethyl malonate to trans-β-nitrostyrene with low to moderate enantioselectivities (20%-60% ee). Configuration of all novel compounds has been meticulously determined using nuclear magnetic resonance (NMR) techniques.     

Synthesis and antiparasitic activity of 2-(trifluoromethyl)-benzimidazole derivatives

2-(Trifluoromethyl)benzimidazole derivatives substituted at the 1-, 5-, and 6-positions have been synthesized and in vitro tested against the protozoa Giardia lamblia, Entamnoeha histolytica. and the helminth Trichinella spiralis. Results indicate that all the compounds tested are more active as antiprotozoal agents than Albendazole and Metronidazole. One compound (20) was as active as Albendazole against T. spiralis. These compounds were also tested for their effect on tubulin polymerization and none inhibited tubulin polymerization.