Engineering P450 Peroxygenase to Catalyze Highly Enantioselective Epoxidation of cis‐β‐Methylstyrenes

P450 119 peroxygenase and its site‐directed mutants are discovered to catalyze the enantioselective epoxidation of methyl‐substituted styrenes. Two new site‐directed P450 119 mutants, namely T213Y and T213M, which were designed to improve the enantioselectivity and activity for the epoxidation of styrene and its methyl substituted derivatives, were studied. The T213M mutant is found to be the first engineered P450 peroxygenase that shows highly enantioselective epoxidation of cis‐β‐methylstyrenes, with up to 91 % ee. Molecular modeling studies provide insights into the different catalytic activity of the T213M mutant and the T213Y mutant in the epoxidation of cis‐β‐methylstyrene. The results of the calculations also contribute to a better understanding of the substrate specificity and configuration control for the regio‐ and stereoselective peroxygenation catalyzed by the T213M mutant.     

Wavepacket Splitting and Two‐Pathway Deactivation in the Photoexcited Visual Pigment Isorhodopsin

Isorhodopsin is the visual pigment analogue of rhodopsin. It shares the same opsin environment but it embeds 9‐cis retinal instead of 11‐cis. Its photoisomerization is three times slower and less effective. The mechanistic rationale behind this observation is revealed by combining high‐level quantum‐mechanical/molecular‐mechanical simulations with ultrafast optical spectroscopy with sub‐20 fs time resolution and spectral coverage extended to the near‐infrared. Whereas in rhodopsin the photoexcited wavepacket has ballistic motion through a single conical intersection seam region between the ground and excited states, in isorhodopsin it branches into two competitive deactivation pathways involving distinct conical intersection funnels. One is rapidly accessed but unreactive. The other is slower, as it features extended steric interactions with the environment, but it is productive as it follows forward bicycle pedal motion.     

Quantum yield of CHAPSO-solubilized rhodopsin and 3-hydroxy retinal containing bovine opsin.

The quantum yields of bleaching for two artificial pigments, bovine opsin combined with (3R)-3-hydroxy retinal or (3R,S)-3-methoxy retinal, were determined in comparison to the value for regenerated bovine rhodopsin. Regeneration of the visual pigments was performed by incubation of 3-[(3-Cholamidopropyl)-dimethylammonio]-2-hydroxy-1- propanesulfonate (CHAPSO)-solubilized opsin with the 11-cis isomers of retinal and the respective retinal derivatives. The extinction coefficients of the pigments in CHAPSO were determined to 35,000 M-1 cm-1 (native rhodopsin), 35,300 M-1 cm-1 (regenerated rhodopsin) and 34,500 M-1 cm-1 (3-OH retinal opsin). With respect to rhodopsin (lambda max: 500 nm), the pigments carrying the substituted chromophores exhibit blue shifted absorbance maxima (3-hydroxy and 3-methoxy retinal opsin: 488 nm). In parallel experiments under absolutely identical conditions we find related to the value of CHAPSO solubilized rhodopsin (identical to 1) a quantum efficiency of bleaching for the 3-hydroxy pigment of 1.2.     

Thermal [2+3]‐Cycloadditions of trans‐1‐Methyl‐2,3‐diphenylaziridine with CS and CC Dipolarophiles: An Unexpected Course with Dimethyl Dicyanofumarate

The thermal reaction of trans‐1‐methyl‐2,3‐diphenylaziridine (trans‐ 1a ) with aromatic and cycloaliphatic thioketones 2 in boiling toluene yielded the corresponding cis‐2,4‐diphenyl‐1,3‐thiazolidines cis‐ 4 via conrotatory ring opening of trans‐ 1a and a concerted [2+3]‐cycloaddition of the intermediate (E,E)‐configured azomethine ylide 3a (Scheme 1). The analogous reaction of cis‐ 1a with dimethyl acetylenedicarboxylate ( 5 ) gave dimethyl trans‐2,5‐dihydro‐1‐methyl‐2,5‐diphenylpyrrole‐3,4‐dicarboxylate (trans‐ 6 ) in accord with orbital‐symmetry‐controlled reactions (Scheme 2). On the other hand, the reactions of cis‐ 1a and trans‐ 1a with dimethyl dicyanofumarate ( 7a ), as well as that of cis‐ 1a and dimethyl dicyanomaleate ( 7b ), led to mixtures of the same two stereoisomeric dimethyl 3,4‐dicyano‐1‐methyl‐2,5‐diphenylpyrrolidine‐3,4‐dicarboxylates 8a and 8b (Scheme 3). This result has to be explained via a stepwise reaction mechanism, in which the intermediate zwitterions 11a and 11b equilibrate (Scheme 6). In contrast, cis‐1,2,3‐triphenylaziridine (cis‐ 1b ) and 7a gave only one stereoisomeric pyrrolidine‐3,4‐dicarboxylate 10 , with the configuration expected on the basis of orbital‐symmetry control, i.e., via concerted reaction steps (Scheme 10). The configuration of 8a and 10 , as well as that of a derivative of 8b , were established by X‐ray crystallography.     

Electron density deformations provide new insights into the spectral shift of rhodopsins

Spectral shifts of rhodopsin, which are related to variations of the electron distribution in 11‐cis‐retinal, are investigated here using the method of deformed atoms in molecules. We found that systems carrying the M207R and S186W mutations display large perturbations of the π‐conjugated system with respect to wild‐type rhodopsins. These changes agree with the predicted behavior of the bond length alternation (BLA) and the blue shifts of vertical excitation energies of these systems. The effect of the planarity of the central and Schiff‐base regions of retinal chain on the electronic structure of the chromophore is also investigated. By establishing nonlinear polynomial relations between BLA, chain distortions, and vertical excitation energies, we are also able to provide a semiquantitative approach for the understanding of the mechanisms regulating spectral shifts in rhodopsin and its mutants. © 2013 Wiley Periodicals, Inc.     

cis‐trans Peptide‐Bond Isomerization in α‐Methylproline Derivatives

α‐Methyl‐L ‐proline is an α‐substituted analog of proline that has been previously employed to constrain prolyl peptide bonds in a trans conformation. Here, we revisit the cis‐trans prolyl peptide bond equilibrium in derivatives of α‐methyl‐L ‐proline, such as N‐Boc‐protected α‐methyl‐L ‐proline and the hexapeptide H‐Ala‐Tyr‐αMePro‐Tyr‐Asp‐Val‐OH. In Boc‐α‐methyl‐L ‐proline, we found that both cis and trans conformers were populated, whereas, in the short peptide, only the trans conformer was detected. The energy barrier for the cis‐trans isomerization in Boc‐α‐methyl‐L ‐proline was determined by line‐shape analysis of NMR spectra obtained at different temperatures and found to be 1.24 kcal/mol (at 298 K) higher than the corresponding value for Boc‐L ‐proline. These findings further illuminate the conformationally constraining properties of α‐methyl‐L ‐proline.     

Synthesis and Cytotoxicity Studies of New (Dimethylamino)‐Functionalised and 7‐Azaindole‐Substituted ‘Titanocene’ Anticancer Agents (7‐Azaindole=1H‐Pyrrolo[2,3‐b]pyridine)

From the carbolithiation of 1‐(cyclopenta‐2,4‐dien‐1‐ylidene)‐N,N‐dimethylmethanamine (=6‐(dimethylamino)fulvene; 3 ) and different lithiated azaindoles 2 (1‐methyl‐7‐azaindol‐2‐yl, 1‐[(diethylamino)methyl]‐7‐azaindol‐2‐yl, and 1‐(methoxymethyl)‐7‐azaindol‐2‐yl), the corresponding lithium cyclopentadienide intermediates 4a – 4c were formed (7‐azaindole=1H‐pyrrolo[2,3‐b]pyridine). The latter underwent a transmetallation reaction with TiCl₄ resulting in the (dimethylamino)‐functionalised ‘titanocenes’ 5a – 5c . When the ‘titanocenes’ 5a – 5c were tested against LLC‐PK cells, the IC₅₀ values obtained were of 8.8, 12, and 87 μM , respectively. The most cytotoxic ‘titanocene’, 5a , with an IC₅₀ value of 8.8 μM is nearly as cytotoxic as cis‐platin, which showed an IC₅₀ value of 3.3 μM when tested on the epithelial pig kidney LLC‐PK cell line, and ca. 200 times better than ‘titanocene dichloride’ itself.     

Melanopsin and the Non‐visual Photochemistry in the Inner Retina of Vertebrates

Melanopsin (Opn4), a member of the G‐protein‐coupled receptor family, is a vitamin A‐based opsin in the vertebrate retina that has been shown to be involved in the synchronization of circadian rhythms, pupillary light reflexes, melatonin suppression and other light‐regulated tasks. In nonmammalian vertebrates there are two Opn4 genes, Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. Opn4x is only expressed in nonmammalian vertebrates including reptiles, fish and birds, while Opn4m is found in a subset of retinal ganglion cells (RGCs), the intrinsically photosensitive (ip) RGCs of the inner retina of both mammals and nonmammalian vertebrates. All opsins described utilize retinaldehyde as chromophore, photoisomerized from 11‐cis‐ to all‐trans‐retinal upon light exposure. Visual retinal photoreceptor cones and rods, responsible for day and night vision respectively, recycle retinoids through a process called the visual cycle that involves the retinal pigment epithelium or glial Müller cells. Although Opn4 has been characterized as a bistable photopigment, little is known about the mechanism/s involved in its chromophore regeneration. In this review, we will attempt to shed light on the visual cycle taking place in the inner retina and discuss the state of the art in the nonvisual photochemistry of vertebrates.     

One Pot Synthesis of Medicinally Important cis‐2‐Methyl‐4‐amino substituted‐1,2,3,4‐tetrahydroquinolines

A simple synthesis of medicinally important cis‐2‐methyl‐4‐azapan‐2‐one‐1,2,3,4‐tetrahydroquinolines/cis‐9‐(2‐methyl‐1,2,3,4‐tetrahydroquinolin‐4‐yl)‐9H‐carbazole was reported. Multicomponent one pot synthesis with anilines and N‐vinylcaprolactam/N‐vinyl carbazole via imino Diels‐Alder reaction by using antimony trichloride as catalyst and acetonitrile as solvent was employed. NMR technique (2D) was used to study the regio‐ and stereo‐chemistry of newly synthesized compounds. The cis diastereo‐selectivity of the products was predicted by COSY and NOESY studies.     

Synthesis and 1H and 13C NMR structural analysis of cis‐ and trans‐2‐imino‐1,3‐ and ‐3,1‐perhydrobenzoxazines and their 3‐ and 1‐N‐methyl derivatives

cis‐ and trans‐2‐imino‐1,3‐ and ‐3,1‐perhydrobenzoxazines and the N‐methyl derivatives of the latter were synthesized from the corresponding cyclic 1,3‐amino alcohol with cyanogen bromide. The configurations of the studied compounds were confirmed by ¹H and ¹³C NMR spectra. All trans‐fused compounds exist in biased chair–chair conformations as expected, whereas the cis‐fused 1,3‐benzoxazines attain exclusively the O‐in conformations. The cis‐fused 3,1‐benzoxazines, especially the 1‐methyl‐substituted derivatives, tend to favor the N‐out form, obviously owing to the favorable axial orientation of this N‐methyl. Copyright © 2003 John Wiley & Sons, Ltd.     

Comparative Analysis of GPCR Crystal Structures†

The phototransduction cascade is perhaps the best understood model system for G protein‐coupled receptor (GPCR) signaling. Phototransduction links the absorption of a single photon of light to a decrease in cytosolic cGMP. Depletion of the cGMP pool induces closure of cGMP‐gated cation channels resulting in the hyperpolarization of photoreceptor cells and consequently a neuronal response. Many biochemical and both low‐ and high‐resolution structural approaches have been utilized to increase our understanding of rhodopsin, the key molecule of this signaling cascade. Rhodopsin, a member of the GPCR or seven‐transmembrane spanning receptor superfamily, is composed of a chromophore, 11‐cis‐retinal that is covalently bound by a protonated Schiff base linkage to the apo‐protein opsin at Lys²⁹⁶ (in bovine opsin). Upon absorption of a photon, isomerization of the chromophore to an all‐trans‐retinylidene conformation induces changes in the rhodopsin structure, ultimately converting it from an inactive to an activated state. This state allows it to activate the heterotrimeric G protein, transducin, by triggering nucleotide exchange. To fully understand the structural and functional aspects of rhodopsin it is necessary to critically examine crystal structures of its different photointermediates. In this review we summarize recent progress on the structure and activation of rhodopsin in the context of other GPCR structures.     

Synthesis of η6‐(4a‐Methyl‐1,2,3,4‐tetrahydro‐4aH‐carbazole)‐tricarbonylchromium complex and stereoelectronic behaviour with organolithium reagents: an apparent frontal attack to the (CO)3Cr group

The synthesis of η⁶‐(4a‐methyl‐1,2,3,4‐tetrahydro‐4aH‐carbazole)tricarbonylchromium ( 3 ) is described, and its reactivity with organolithium reagents have been analysed. The addition of RLi (R= H, Me, n‐Bu, tert‐Bu) to 3 affords the corresponding endo/exo tricarbonylchromium complexes of cis‐4a‐methyl‐9a‐substituted‐1,2,3,4‐tetrahydro‐4aH‐carbazole, which permit the consideration of the stereoelectronic behaviour of the tricarbonylchromium group on 4a‐methyl and the 9a substituent or on the methylenes of the cyclohexene moiety in the complexes.     

Synthesis and transformations of hydrazine substituted 6‐fluoro derivatives of 1,3,4‐thiadiazolo[3,2‐a]pyrimidine

2‐Alkyl and 2‐arylhydrazine derivatives of 5H‐2‐R‐6‐fluoro‐7‐methyl‐1,3,4‐thiadiazolo[3,2‐a]pyrimidin‐5‐one were prepared by reaction of 5H‐2‐bromo‐6‐fluoro‐7‐methyl‐1,3,4‐thiadiazolo[3,2‐a]pyrimidin‐5‐one with hydrazine derivatives. A convenient procedure was developed for the preparation of new hydrazine derivatives of 5H‐2‐R‐6‐fluoro‐7‐methyl‐1,3,4‐thiadiazolo[3,2‐a]pyrimidin‐5‐one. J. Heterocyclic Chem., (2011).     

Synthesis of the Chiral Pair of a Novel Thromboxane Antagonist, 3‐[2‐(3‐Benzenesulfonylamino‐7‐oxabicyclo[2.2.1]hept‐2‐yl‐methyl)phenyl] Propionic Acid

Preparation of the enantiomeric pair of 3‐[2‐(3‐benzenesulfonylamino‐7‐oxabicyclo[2.2.1]hept‐2‐yl‐methyl)phenyl] propionic acid, a novel thromboxane antagonist is reported. They are synthesized from either enantiomers of known (1R,2R,3R,4S)‐3‐[2‐(3‐carboxy‐7‐oxabicyclo[2,2,1]hept‐2‐yl‐methyl)phenyl]‐propionic acid methyl ester via epimerization, modified Curtius' rearrangement and sulfonylamino formation. Other derivatives may be prepared similarly.     

Decomposition of model alkoxyamines in simple and polymerizing systems. II. Diastereomeric N‐(2‐methylpropyl)‐N‐(1‐diethyl‐phosphono‐2,2‐dimethyl‐propyl)‐aminoxyl‐based compounds

Thermal reactions of the alkoxyamine diastereomers DEPN‐R′ [DEPN: N‐(2‐methylpropyl)‐N‐(1‐diethylphosphophono‐2,2‐dimethyl‐propyl)‐aminoxyl; R′: methoxy‐carbonylethyl and phenylethyl] with (R,R) + (S,S) and (R,S) + (S,R) configurations have been investigated by ¹H NMR at 100 °C. During the overall decay the diastereomers interconvert, and an analytical treatment of the combined processes is presented. Rate constants are obtained for the cleavage and reformation of DEPN‐R′ from NMR, electron spin resonance, and chemically induced dynamic nuclear polarization experiments also using 2,2,6,6‐tetramethylpiperidinyl‐1‐oxyl (TEMPO) as a radical scavenger. The rate constants depend on the diastereomer configuration and the residues R′. Simulations of the kinetics observed with styrene and methyl methacrylate containing solutions yielded rate constants for unimeric and polymeric alkoxyamines DEPN‐(M)_n‐R′. The results were compatible with the known DEPN mediation of living styrene and acrylate polymerizations. For methyl methacrylate the equilibrium constant of the reversible cleavage of the dormant chains DEPN‐(M)_n‐R′ is very large and renders successful living polymerizations unlikely. Mechanistic and kinetic differences of DEPN‐ and TEMPO‐mediated polymerizations are discussed. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3264–3283, 2002     

Synthesis and properties of some 2,3‐disubstituted 6‐fluoro‐7‐(4‐methyl‐1‐piperazinyl)quinoxalines

The 2,3‐disubstituted 6‐fluoro‐7‐(4‐methyl‐1‐piperazinyl)‐quinoxalines ( 3–11 ) were synthesized for bioassay via reaction of 1.2‐diamino‐4‐fluoro‐5‐(4‐methyl‐1‐piperazinyl)benzene (2) with the appropriate 1,2‐dicarbonyl compounds. However, none of the tested compounds 3–11 showed significant in vitro activ ity against E. coli ATCC11229, S. aureus ATCC6538 and C.albicans SATCC10231.     

Analogues of α‐Campholenal (= (1R)‐2,2,3‐Trimethylcyclopent‐3‐ene‐1‐acetaldehyde) as Building Blocks for (+)‐β‐Necrodol (= (1S,3S)‐2,2,3‐Trimethyl‐4‐methylenecyclopentanemethanol) and Sandalwood‐like Alcohols

To complete our panorama in structure–activity relationships (SARs) of sandalwood‐like alcohols derived from analogues of α‐campholenal (= (1R)‐2,2,3‐trimethylcyclopent‐3‐ene‐1‐acetaldehyde), we isomerized the epoxy‐isopropyl‐apopinene (?)‐ 2d to the corresponding unreported α‐campholenal analogue (+)‐ 4d (Scheme 1). Derived from the known 3‐demethyl‐α‐campholenal (+)‐ 4a , we prepared the saturated analogue (+)‐ 5a by hydrogenation, while the heterocyclic aldehyde (+)‐ 5b was obtained via a Bayer‐Villiger reaction from the known methyl ketone (+)‐ 6 . Oxidative hydroboration of the known α‐campholenal acetal (?)‐ 8b allowed, after subsequent oxidation of alcohol (+)‐ 9b to ketone (+)‐ 10 , and appropriate alkyl Grignard reaction, access to the 3,4‐disubstituted analogues (+)‐ 4f,g following dehydration and deprotection. (Scheme 2). Epoxidation of either (+)‐ 4b or its methyl ketone (+)‐ 4h , afforded stereoselectively the trans‐epoxy derivatives 11a,b , while the minor cis‐stereoisomer (+)‐ 12a was isolated by chromatography (trans/cis of the epoxy moiety relative to the C₂ or C₃ side chain). Alternatively, the corresponding trans‐epoxy alcohol or acetate 13a,b was obtained either by reduction/esterification from trans‐epoxy aldehyde (+)‐ 11a or by stereoselective epoxidation of the α‐campholenol (+)‐ 15a or of its acetate (?)‐ 15b , respectively. Their cis‐analogues were prepared starting from (+)‐ 12a . Either (+)‐ 4h or (?)‐ 11b , was submitted to a Bayer‐Villiger oxidation to afford acetate (?)‐ 16a . Since isomerizations of (?)‐ 16 lead preferentially to β‐campholene isomers, we followed a known procedure for the isomerization of (?)‐epoxyverbenone (?)‐ 2e to the norcampholenal analogue (+)‐ 19a . Reduction and subsequent protection afforded the silyl ether (?)‐ 19c , which was stereoselectively hydroborated under oxidative condition to afford the secondary alcohol (+)‐ 20c . Further oxidation and epimerization furnished the trans‐ketone (?)‐ 17a , a known intermediate of either (+)‐β‐necrodol (= (+)‐(1S,3S)‐2,2,3‐trimethyl‐4‐methylenecyclopentanemethanol; 17c ) or (+)‐(Z)‐lancifolol (= (1S,3R,4Z)‐2,2,3‐trimethyl‐4‐(4‐methylpent‐3‐enylidene)cyclopentanemethanol). Finally, hydrogenation of (+)‐ 4b gave the saturated cis‐aldehyde (+)‐ 21 , readily reduced to its corresponding alcohol (+)‐ 22a . Similarly, hydrogenation of β‐campholenol (= 2,3,3‐trimethylcyclopent‐1‐ene‐1‐ethanol) gave access via the cis‐alcohol rac‐ 23a , to the cis‐aldehyde rac‐ 24 .     

Interphotoreceptor Retinoid‐Binding Protein Protects Retinoids from Photodegradation

Retinol degrades rapidly in light into a variety of photoproducts. It is remarkable that visual cycle retinoids can evade photodegradation as they are exchanged between the photoreceptors, retinal pigment epithelium and Müller glia. Within the interphotoreceptor matrix, all‐trans retinol, 11‐cis retinol and retinal are bound by interphotoreceptor retinoid‐binding protein (IRBP). Apart from its role in retinoid trafficking and targeting, could IRBP have a photoprotective function? HPLC was used to evaluate the ability of IRBP to protect all‐trans and 11‐cis retinols from photodegradation when exposed to incandescent light (0 to 8842 μW cm^?2); time periods of 0–60 min, and bIRBP: retinol molar ratios of 1:1 to 1:5. bIRBP afforded a significant prevention of both all‐trans and 11‐cis retinol to rapid photodegradation. The effect was significant over the entire light intensity range tested, and extended to the bIRBP: retinol ratio 1:5. In view of the continual exposure of the retina to light, and the high oxidative stress in the outer retina, our results suggest IRBP may have an important protective role in the visual cycle by reducing photodegradation of all‐trans and 11‐cis retinols. This role of IRBP is particularly relevant in the high flux conditions of the cone visual cycle.     

Novel Synthesis of 2‐Aryl‐4,5,6,7‐tetrahydro‐1,2‐benzisothiazol‐3(2H)‐ones and Their S‐Oxides

2‐Aryl‐4,5,6,7‐tetrahydro‐1,2‐benzisothiazol‐3(2H)‐ones 1a – e were synthesized by cyclocondensation of 2‐(thiocyanato)cyclohexene‐1‐carboxanilides 9 as a convenient new method. Their S‐oxides 10 were prepared by two routes, either by oxidation of 1 or dehydration of rac‐cis‐3‐hydroperoxysultims 11 . Furthermore, compounds 1 have been identified by HPLC? API‐MS‐MS as intermediates in the oxidation process of the salts 6 . The hydroperoxides 12b and rac‐trans‐ 11b have been unambiguously detected by HPLC? MS investigations and in the reaction of rac‐cis‐ 13b with H₂O₂ to the hydroperoxides rac‐trans‐ 11b and rac‐cis‐ 11b .     

Synthesis of alkyl 6‐methyl‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates possessing a N‐3 nitro substituent to determine calcium channel modulation structure‐activity relationships

The Bigenelli acid catalyzed condensation of 2‐trifluoromethylbenzaldehyde ( 1 ), urea ( 2 ) and an alkyl acetoacetate ( 3 ) afforded the respective alkyl (Me, Et, i‐Pr, i‐Bu) 6‐methyl‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylate ( 4‐7 ). Subsequent N³‐nitration of the alkyl esters ( 4‐7 ) using Cu(NO₃)₂ 3H₂O and Ac₂O furnished the target alkyl 6‐methyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates ( 8‐11 ). The N³‐nitro compounds ( 8‐11 ) were less potent calcium channel antagonists (IC₅₀ values in the 1.9 × 10^?7 to 3.9 × 10^?6 M range) on guinea pig ileal longitudinal smooth muscle than the reference drug nifedipine (Adalat®, IC₅₀ = 1.4 × 10^?8 M). In vitro calcium channel modulation studies on guinea pig left atrium (GPLA) showed that the methyl and ethyl esters ( 8‐9 ) induced a weak‐to‐modest positive inotropic (agonist) effect, and that the inactive isopropyl ( 10 ) and isobutyl ( 11 ) esters did not alter the cardiac contractile force of GPLA.