All‐Trans Retinal Mediates Light‐Induced Oxidation in Single Living Rod Photoreceptors†

All‐trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all‐trans retinal is reduced within the outer segment to all‐trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all‐trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light‐induced oxidation was measured with fluorescence imaging using an oxidation‐sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light‐induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all‐trans retinal. Furthermore, light‐induced oxidation increased with the concentration of exogenously added all‐trans retinal. The results show that the all‐trans retinal generated during the detection of light can mediate light‐induced oxidation. Its removal through reduction to all‐trans retinol protects photoreceptor outer segments against light‐induced oxidative damage.     

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.     

Cytotoxicity of All‐Trans‐Retinal Increases Upon Photodegradation†

All‐trans‐retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time‐resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ～330 nm. Toxicity of dAtRal was concentration‐dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.     

Influence of Arrestin on the Photodecay of Bovine Rhodopsin

Continued activation of the photocycle of the dim‐light receptor rhodopsin leads to the accumulation of all‐trans‐retinal in the rod outer segments (ROS). This accumulation can damage the photoreceptor cell. For retinal homeostasis, deactivation processes are initiated in which the release of retinal is delayed. One of these processes involves the binding of arrestin to rhodopsin. Here, the interaction of pre‐activated truncated bovine visual arrestin (Arr^Tr) with rhodopsin in 1,2‐diheptanoyl‐sn‐glycero‐3‐phosphocholine (DHPC) micelles is investigated by solution NMR techniques and flash photolysis spectroscopy. Our results show that formation of the rhodopsin–arrestin complex markedly influences partitioning in the decay kinetics of rhodopsin, which involves the simultaneous formation of a meta II and a meta III state from the meta I state. Binding of Arr^Tr leads to an increase in the population of the meta III state and consequently to an approximately twofold slower release of all‐trans‐retinal from rhodopsin.     

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.     

Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.     

A flash photolysis study of the formation of retinal Schiff bases in a native photoreceptor cell

It is believed that the accumulation of all-trans-retinal (ATR) in retinal cells is responsible for photoinduced damage to the retina. However, ATR is formed within a photoreceptor cell disk in the process of photolysis and transferred to the cell cytoplasm, where it is converted in a high yield into the nonphototoxic compound retinol. Within the disk where ATR can be accumulated, retinol can form Schiff bases, which are also nonphototoxic compounds, with the amino groups of proteins and lipids. Thus, it is unclear in which concentrations free ATR can be accumulated inside a cell. In this study, it has been proposed to evaluate the concentration of free ATR in the cell from the yield of an excited triplet state because neither Schiff bases nor retinol can be transformed into an excited triplet state. It has been found that 70% ATR forms Schiff bases in the native cell in the equilibrium state, thereby, a considerably decreasing the probability that ATR is the main inducer of photodamage.     

High Reactivity of Strained Seven‐Membered‐Ring trans‐Alkenes

trans‐Oxasilacycloheptenes are highly reactive strained alkenes. Competition reactions showed that these seven‐membered ring trans‐alkenes underwent [4+2] cycloaddition reactions faster than a trans‐cyclooctene. They also reacted with quinones and dimethyl acetylenedicarboxylate to form adducts with high diastereoselectivity. Kinetic studies showed that ring strain increases nucleophilicity by approximately 10⁹.     

Microwave‐induced Stereoselectivity in Synthesis of trans‐4‐Aryl‐3‐methyl‐6‐oxo‐4,5,6,7‐tetrahydro‐2H‐pyrazolo[3,4‐b]pyridine‐5‐carbonitriles

A facile synthesis of trans isomers of 4‐aryl‐3‐methyl‐6‐oxo‐4,5,6,7‐tetrahydro ‐ 2H ‐ pyrazolo[3,4‐b]pyridine‐5‐carbonitriles via three‐component condensation reaction of an aldehyde, 3‐amino‐5‐methylpyrazole and ethyl cyanoacetate in acetonitrile has been developed under microwave irradiation. This one‐pot reaction proceeds without any catalyst in short times and gives the product in high selectivities and high yields.     

New Synthetic Analogs of Retinoids: Synthesis of Aromatic Analogs of 9‐Methylidene‐ and 13‐Demethyl‐9‐methylidene‐retinol, ‐retinal,and Ethyl 13‐Demethyl‐9‐methylideneretinoate

Aromatic analogs of 9‐methylidene and 13‐demethyl‐9‐methylideneretinol, ‐retinal, and ethyl 13‐demethyl‐9‐methylideneretinoate were synthesized via a new β‐methylidene‐aldehyde synthon.     

Catalyst‐Directed Diastereo‐ and Site‐Selectivity in Successive Nucleophilic and Electrophilic Allylations of Chiral 1,3‐Diols: Protecting‐Group‐Free Synthesis of Substituted Pyrans

The iridium‐catalyzed, protecting group‐free synthesis of 4‐hydroxy‐2,6‐cis‐ or trans‐pyrans through successive nucleophilic and electrophilic allylations of chiral 1,3‐diols occurs with complete levels of catalyst‐directed diastereoselectivity in the absence of protecting groups, premetallated reagents, or discrete alcohol‐to‐aldehyde redox reactions.     

Synthesis of a Photostable Near‐Infrared‐Absorbing Photosensitizer for Selective Photodamage to Cancer Cells

A new class of near‐infrared (NIR)‐absorptive (>900 nm) photosensitizer based on a phenothiazinium scaffold is reported. The stable solid compound, o‐DAP, the oxidative form of 3,7‐bis(4‐methylaminophenyl)‐10H‐phenothiazine, can generate reactive oxygen species (ROS, singlet oxygen and superoxide) under appropriate irradiation conditions. After biologically evaluating the intracellular uptake, localization, and phototoxicity of this compound, it was concluded that o‐DAP is photostable and a potential selective photodynamic therapy (PDT) agent under either NIR or white light irradiation because its photodamage is more efficient in cancer cells than in normal cells and is without significant dark toxicity. This is very rare for photosensitizers in PDT applications.     

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 .     

Retinal Photodamage by Endogenous and Xenobiotic Agents†

The human eye is constantly exposed to sunlight and artificial lighting. Light transmission through the eye is fundamental to its unique biological functions of directing vision and circadian rhythm and therefore light absorbed by the eye must be benign. However, exposure to the very intense ambient radiation can pose a hazard particularly if the recipient is over 40 years of age. There are age‐related changes in the endogenous (natural) chromophores (lipofuscin, A2E and all‐trans‐retinal derivatives) in the human retina that makes it more susceptible to visible light damage. Intense visible light sources that do not filter short blue visible light (400–440 nm) used for phototherapy of circadian imbalance (i.e. seasonal affective disorder) increase the risk for age‐related light damage to the retina. Moreover, many drugs, dietary supplements, nanoparticles and diagnostic dyes (xenobiotics) absorb ocular light and have the potential to induce photodamage to the retina, leading to transient or permanent blinding disorders. This article will review the underlying reasons why visible light in general and short blue visible light in particular dramatically raises the risk of photodamage to the human retina.     

New Syntheses of Retinal and Its Acyclic Analog γ‐Retinal by an Extended Aldol Reaction with a C6 Building Block That Incorporates a C5 Unit after Decarboxylation. A Formal Route to Lycopene and β‐Carotene

Since the C₁₅ β‐end‐group aldehyde 10 ((β‐ionylidene)acetaldehyde), an excellent intermediate in the syntheses of retinoids, can be synthesized in many ways from β‐ionone, and since the corresponding acyclic C₁₅ ψ‐end‐group aldehyde 5 can easily be synthesized from citral ( 1 ) (Scheme 3), we applied the C₁₅+C₅ route to the syntheses of γ‐retinal ((all‐E)‐ 8 ) (Scheme 3) and retinal ((all‐E)‐ 13 ) (Scheme 4), and therefore, by coupling (2×C₂₀→C₄₀), to the preparation of lycopene ( 14 ) and β‐carotene ( 15 ) (Scheme 5). Our new syntheses of retinal ((all‐E)‐ 13 ) and γ‐retinal ((all‐E)‐ 8 use an extended aldol reaction with a C₆ building block that incorporates a C₅ unit after decarboxylation.     

Retinal Conformation and Dynamics in Activation of Rhodopsin Illuminated by Solid‐state 2H NMR Spectroscopy†

Solid‐state NMR spectroscopy gives a powerful avenue for investigating G protein‐coupled receptors and other integral membrane proteins in a native‐like environment. This article reviews the use of solid‐state ²H NMR to study the retinal cofactor of rhodopsin in the dark state as well as the meta I and meta II photointermediates. Site‐specific ²H NMR labels have been introduced into three regions (methyl groups) of retinal that are crucially important for the photochemical function of rhodopsin. Despite its phenomenal stability ²H NMR spectroscopy indicates retinal undergoes rapid fluctuations within the protein binding cavity. The spectral lineshapes reveal the methyl groups spin rapidly about their three‐fold (C₃) axes with an order parameter for the off‐axial motion of For the dark state, the ²H NMR structure of 11‐cis‐retinal manifests torsional twisting of both the polyene chain and the β‐ionone ring due to steric interactions of the ligand and the protein. Retinal is accommodated within the rhodopsin binding pocket with a negative pretwist about the C11=C12 double bond. Conformational distortion explains its rapid photochemistry and reveals the trajectory of the 11‐cis to trans isomerization. In addition, ²H NMR has been applied to study the retinylidene dynamics in the dark and light‐activated states. Upon isomerization there are drastic changes in the mobility of all three methyl groups. The relaxation data support an activation mechanism whereby the β‐ionone ring of retinal stays in nearly the same environment, without a large displacement of the ligand. Interactions of the β‐ionone ring and the retinylidene Schiff base with the protein transmit the force of the retinal isomerization. Solid‐state ²H NMR thus provides information about the flow of energy that triggers changes in hydrogen‐bonding networks and helix movements in the activation mechanism of the photoreceptor.     

Low‐Concentration 1,2‐trans β‐Selective Glycosylation Strategy and Its Applications in Oligosaccharide Synthesis

This study develops an operationally easy, efficient, and general 1,2‐trans β‐selective glycosylation reaction that proceeds in the absence of a C2 acyl function. This process employs chemically stable thioglycosyl donors and low substrate concentrations to achieve excellent β‐selectivities in glycosylation reactions. This method is widely applicable to a range of glycosyl substrates irrespective of their structures and hydroxyl‐protecting functions. This low‐concentration 1,2‐trans β‐selective glycosylation in carbohydrate chemistry removes the restriction of using highly reactive thioglycosides to construct 1,2‐trans β‐glycosidic bonds. This is beneficial to the design of new strategies for oligosaccharide synthesis, as illustrated in the preparation of the biologically relevant β‐(1→6)‐glucan trisaccharide, β‐linked Gb₃ and isoGb₃ derivatives.     

1,3‐Thiazolidine‐dicarboxylates from Thioketones and Thermally Generated Azomethine Ylides

The reaction of 9H‐fluorene‐9‐thione ( 1 ) with the cis‐ and trans‐isomers of dimethyl 1‐(4‐methoxyphenyl)aziridine‐2,3‐dicarboxylate (cis‐ and trans‐ 2 , resp.) in xylene at 110° yielded exclusively the spirocyclic cycloadduct with trans‐ and cis‐configurations, respectively (trans‐ and cis‐ 3 , resp.; Scheme 1). Analogously, less‐reactive thioketones, e.g., thiobenzophenone ( 5 ), and cis‐ 2 reacted stereoselectively to give the corresponding trans‐1,3‐thiazolidine‐2,4‐dicarboxylate (e.g., trans‐ 8 ; Scheme 2). On the other hand, the reaction of 5 and trans‐ 2 proceeded in a nonstereoselective course to provide a mixture of trans‐ and cis‐substituted cycloadducts. This result can be explained by an isomerization of the intermediate azomethine ylide. Dimethyl 1,3‐thiazolidine‐2,2‐dicarboxylates 14 and 15 were formed in the thermal reaction of dimethyl aziridine‐2,2‐dicarboxylate 11 with aromatic thioketones (Scheme 3). On treatment of 14 and 15 with Raney‐Ni in refluxing EtOH, a desulfurization and ring‐contraction led to the formation of azetidine‐2,2‐dicarboxylates 17 and 18 , respectively (Scheme 4).     

Module structure of interphotoreceptor retinoid-binding protein (IRBP) may provide bases for its complex role in the visual cycle – <Emphasis Type="Italic">structure/function study of Xenopus IRBP</Emphasis>

Background  

Interphotoreceptor retinoid-binding protein's (IRBP) remarkable module structure may be critical to its role in mediating the transport of all-trans and 11-cis retinol, and 11-cis retinal between rods, cones, RPE and Müller cells during the visual cycle. We isolated cDNAs for Xenopus IRBP, and expressed and purified its individual modules, module combinations, and the full-length polypeptide. Binding of all-trans retinol, 11-cis retinal and 9-(9-anthroyloxy) stearic acid were characterized by fluorescence spectroscopy monitoring ligand-fluorescence enhancement, quenching of endogenous protein fluorescence, and energy transfer. Finally, the X-ray crystal structure of module-2 was used to predict the location of the ligand-binding sites, and compare their structures among modules using homology modeling.     

o‐ and m‐Benzenedicarbaldehyde

o‐Benzene­dicarb­aldehyde (systematic name: benzene‐1,2‐dicarb­aldehyde), C₈H₆O₂, exhibits a weak intramolecular hydrogen bond between an aldehyde H atom and the O atom of the adjacent aldehyde group, with a C?O distance of 2.852 (2) Å. m‐Benzene­dicarb­aldehyde (systematic name: benzene‐1,3‐dicarb­aldehyde), C₈H₆O₂, occurs as two different isomorphs. In all three crystals, there are intermolecular C—­H?O contacts involving both aldehyde and ring H atoms.