Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

FTIR Spectroscopy and DFT Calculations to Probe the Kinetics of ß-Carotene Thermal Degradation.

The thermal degradation of ß-carotene in air was investigated. The sample was heated at different temperatures (90, 100, 115, and 130 °C) for periods of up to 8 h to perform a complete kinetic study, the product analysis having been carried out via infrared spectroscopy in attenuated total reflectance mode coupled to density functional theory (DFT) calculations. The kinetics of this thermal degradation process was found to follow a first-order scheme, with rate coefficients varying from k90 °C = (2.0 ± 0.3) × 10-3 to k130 °C = (11.0 ± 0.7) × 10-3 min-1, the experimental activation energy having been calculated as (52 ± 1) kJ mol-1. This Ea value is close to the DFT energies corresponding to a C15-15' or a C13-14 cis-trans isomerization, followed by the formation of a carotene-oxygen diradical, which was characterized for the first time. Comparison between the experimental and calculated infrared data confirmed the C15-15'- cis rupture as the predominant reaction pathway and retinal as the major degradation product.

Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins.

Three all-trans retinals containing multiple 13 C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsin and channelrhodopsin. The synthetic approaches allowed specific introduction of 13 C labels in ring substituents and at different positions in the polyene chain to probe structural features such as ring orientation and interaction of the chromophore with the protein in the ground state and in photointermediates. [10-18-13 C9 ]-All-trans-retinal (1b), [12,15-13 C2 ]-all-trans-retinal (1c), and [14,15-13 C2 ]-all-trans-retinal (1d) were synthesized in in 12, 8, and 7 linear steps from ethyl 2-oxocyclohexanecarboxylate (5) or ß-ionone (4), respectively.

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore.

By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been optimized for mammalian cell imaging. By targeting both the protein and its chromophore, we overcome inherent challenges associated with engineering bright NIR fluorescence into Archaerhodopsin. This work demonstrates an efficient strategy for engineering non-natural, tailored properties into microbial opsins, properties relevant for imaging and interrogating biological systems.

Retinal-conjugated pH-sensitive micelles induce tumor senescence for boosting breast cancer chemotherapy.

Evoking tumor cellular senescence, an irreversible status of cell growth quiescence, has been recently proposed as a potential strategy to improve the efficacy of cancer treatment. In the current study, all-trans retinal, the precursor of all-trans retinoic acid, was conjugated to dextran via hydrazone bond to generate amphiphilic dextran-retinal (DR) conjugates, which self-assembled into pH-sensitive DR micelles. Our results showed that DR micelles moderately inhibited MCF-7 breast cancer cell growth through inducing p21-associated cellular senescence, which relied on retinoic acid receptors (RARs) and was accompanied by significant G0/G1 cell cycle arrest. Moreover, DR micelles were capable of encapsulating doxorubicin (DOX) to generate DOX-loaded DD micelles, facilitating the uptake and release of DOX in cancer cells. Compared with free DOX, DD micelles more effectively suppressed tumor growth and prolonged survival time of mouse xenograft model through inducing tumor apoptosis and cellular senescence. However, blocking cellular senescence diminished DD-caused apoptosis in MCF-7 cells by 40-50%. Therefore, pH-sensitive DR micelles not only served as a potent platform for DOX delivery, but also enhanced the anti-tumor effect of DOX by inducing tumor cellular senescence. These data reveal a great potential of evoking tumor senescence with retinal-conjugated micelles for boosting breast cancer chemotherapy.

Increased bioavailability of rifampicin from stimuli-responsive smart nano carrier.

Stimuli responsive polymeric nanocarrier (RCOP-2) functionalized with frontline antituberculosis drug (Rifampicin) is demonstrated for sustained release. Bioavailability of Rifampicin is taken care of by conjugating this drug through a acylhydrazine linker to the polymeric backbone. The poly(ethylene glycol) structural motif is introduced in the copolymer architecture for water solubility. Releasing retinal along with Rifampicin is hypothesized to reduce the risk of side effects due to Rifampicin. The self-assembly of RCOP-2, due to the amphiphilicity present in the copolymer, is explored in detail. The pH responsiveness of RCOP-2 is demonstrated in mild acidic environment as well as in cell lines. The 4T cell line, due to its acidic nature, shows time-dependent cellular internalization. On the basis of the results, our unique design is expected to provide an increased bioavalaibility of Rifampicin with reduced side effects. From the flow cytometry results on A549 cell lines, it is clear that the newly designed copolymer RCOP-2 can internalize efficiently and serve as an effective Rifampicin delivery system.

Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach.

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

Synthesis and use of stable isotope enriched retinals in the field of vitamin A.

The role of vitamin A and its metabolites in the life processes starting with the historical background and its up to date information is discussed in the introduction. Also the role of 11Z-retinal in vision and retinoic acid in the biological processes is elucidated. The essential role of isotopically enriched systems in the progress of vision research, nutrition research etc. is discussed. In part B industrial commercial syntheses of vitamin A by the two leading companies Hoffmann-La Roche (now DSM) and BASF are discussed. The knowledge obtained via these pioneering syntheses has been essential for the further synthetic efforts in vitamin A field by other scientific groups. The rest of the paper is devoted to the synthetic efforts of the Leiden group that gives an access to the preparation of site directed high level isotope enrichment in retinals. First the synthesis of the retinals with deuterium incorporation in the conjugated side chain is reviewed. Then, 13C-labeled retinals are discussed. This is followed by the discussion of a convergent synthetic scheme that allows a rational access to prepare any isotopomer of retinals. The schemes that provide access to prepare any possible isotope enriched chemically modified systems are discussed. Finally, nor-retinals and bridged retinals that give access to a whole (as yet incomplete) library of possible isotopomers are reviewed.

[Synthesis of spyropyran derivatives of retinal and study of their interaction with bacterioopsin from Halobacterium salinarum].

The synthesis of retinal analogue series that contain a spyropyran moiety instead of a trimethylcyclohexene ring was proposed. The process of the retinal analogue interaction with bacterioopsin from apomembranes of Halobacterium salinarum and the spectral properties of the newly formed pigments were studied. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

A convenient and stereoselective synthesis of 11Z-3,4-didehydroretinal by Horner-Emmons reaction using diphenyl phosphonate.

A convenient synthesis of 3,4-didehydroretinal was developed. The Horner-Emmons reaction between 3,4-didehydro-beta-ionone and diphenyl phosphonate in the presence of crown ether gave the retinonitrile as an isomeric mixture, in which the newly produced double bond was predominantly 11Z-form. After separation of the 11Z-form retinonitrile, it was converted into the corresponding retinal in good yield without isomerization of the double bonds.

Novel retinylidene iminium salts for defining opsin shifts: synthesis and intrinsic chromophoric properties.

Retinal Schiff bases serve as chromophores in many photoactive proteins that carry out functions such as signalling and light-induced ion translocation. The retinal Schiff base can be found as neutral or protonated, as all-trans, 11-cis or 13-cis isomers and can adopt different conformations in the protein binding pocket. Here we present the synthesis and characterisation of isomeric retinylidene iminium salts as mimics blocked towards isomerisation at the C11 position and conformationally restrained. The intrinsic chromophoric properties are elucidated by gas phase absorption studies. These studies reveal a small blue-shift in the S0-->S1 absorption for the 11-locked derivative as compared to the unlocked one. The gas phase absorption spectra of all the cationic mimics so far investigated show almost no absorption in the blue region. This observation stresses the importance of protein interactions for colour tuning, which allows the human eye to perceive blue light.

Synthesis of enantiopure C3- and C4-hydroxyretinals and their enzymatic reduction by ADH8 from Xenopus laevis.

(R)-all-trans-3-hydroxyretinal 1, (S)-all-trans-4-hydroxyretinal and (R)-all-trans-4-hydroxyretinal have been synthesized stereoselectively by Horner-Wadsworth-Emmons and Stille cross-coupling as bond-forming reactions. The CBS method of ketone reduction was used in the enantioface-differentiation step to provide the precursors for the synthesis of the 4-hydroxyretinal enantiomers. The kinetic constants of Xenopus laevis ADH8 with these retinoids have been determined.

Synthesis of N-heteroaryl retinals and their artificial bacteriorhodopsins.

N-Heteroaryl retinals derived from indole, 1-indolizine and 3-indolizine (10 a-c) have been synthesized after their UV/Vis red-shifted absorption properties had been predicted by time-dependent density functional theory (TD-DFT) computations. The three new analogues form artificial pigments upon recombination with bacterioopsin: indolyl retinal 10 a undergoes fast and efficient reconstitution to form a species with a UV/Vis absorbance maximum similar to that of wild-type bacteriorhodopsin, whilst the indolizinyl retinals 10 b and 10 c also reconstitute in significant proportion to give noticeably red-shifted, although unstable, pigments. Significant changes in the pK(a) values of these artificial bacteriorhodopsins are interpreted as arising from nonoptimal binding-site occupancy by the chromophore due to steric constraints.

Synthesis of ring-oxidized retinoids as substrates of mouse class I alcohol dehydrogenase (ADH1).

Ring-oxidized retinoids have been synthesized stereoselectively using the Stille cross-coupling reaction. Kinetic constants of mouse class I alcohol dehydrogenase (ADH1) with these retinoids were determined.

Constraints of the 9-methyl group binding pocket of the rhodopsin chromophore probed by 9-halogeno substitution.

Sterical constraints of the 9-methyl-binding pocket of the rhodopsin chromophore are probed using retinal analogues carrying substituents of increasing size at the 9 position (H, F, Cl, Br, CH(3), and I). The corresponding 11-Z retinals were employed to investigate formation of photosensitive pigment, and the primary photoproduct was identified by Fourier transform infrared difference spectroscopy. In addition, any effects of cumulative strain were studied by introduction of the 9-Z configuration and/or the alpha-retinal ring structure. Our results show that the 9-F analogue still can escape from the 9-methyl-binding pocket and that its photochemistry behaves very similar to the 9-demethyl analogue. The 9-Cl and 9-Br analogues behave very similar to the native 9-methyl pigments, but the 9-I retinal does not fit very well and shows poor pigment formation. This puts an upper limit on the radial dimension of the 9-methyl pocket at 0.45-0.50 nm. Introduction of the alpha-retinal ring constraint in the 11-Z series results in cumulative strain, because the 9-I and 9-Br derivatives cannot bind to generate a photopigment. The 9-Z configuration can partially compensate for the additional alpha-retinal strain. The corresponding 9-Br analogue does form a photopigment, and the other derivatives give increased photopigment yields compared to the corresponding 11-Z derivatives. In fact, 9-Z-alpha-retinal would be an interesting candidate for retinal supplementation studies. Our data provide direct support for the concept that the 9-methyl group is an important determinant in ligand anchoring and activation of the protein and in general agree with a three-point interaction model involving the ring, 9-methyl group, and aldehyde function.

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models.

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.

Rhodopsin with 11-cis-locked chromophore is capable of forming an active state photoproduct.

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C(11)=C(12) double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C(11)=C(12) double bond is prevented by a 6-member ring structure (Rh(6.10)). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C(11)=C(12) double bond being locked, isomerization around the C(9)=C(10) or the C(13)=C(14) double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.

The preparation of all-trans uniformly (13)C-labeled retinal via a modular total organic synthetic strategy. emerging central contribution of organic synthesis toward the structure and function study with atomic resolution in protein research.

Uniformly [(13)C(20)]-labeled all-trans-retinal (1) has been prepared via a convergent modular total organic strategy with high isotope incorporation (>99%) and without isotope dilution starting from commercially available 99% enriched (13)C-labeled starting materials. For this purpose we have developed a strategy that is based on four different modules: [1,2,3,4,(3-CH(3))-(13)C(5)]-4-(diethylphosphono)-3-methyl-2-butenenitrile (3), [1,2,3,4-(13)C(4)]-ethyl acetoacetate (7), [U-(13)C(5)]-4-bromo-2-methyl-2-butene (13), and [U-(13)C(10)]-2,6,6-trimethylcyclohex-2-ene-1-ylcarbonitrile (16). This scheme permits the synthesis of the full cassette of all isotopomers with (13)C-labels at any position or combination of positions by using different (13)C-labeled starting materials. In addition, modifications of the synthesized modules will give access to a broad range of chemically modified (13)C-labeled retinoids and carotenoids. This modular strategy enables the synthesis of multifold and uniformly stable isotopically labeled (bio)macromolecules that can be used for studying proteins with atomic resolution, providing detailed functional information of the studied biological system.

Synthesis of 11-cis-locked bicyclo[5.1.0]octanyl retinal and an enantioselective binding to bovine opsin.

Both enantiomers of 13-(E) and 13-(Z) isomers of 11-cis-locked bicyclo[5.1.0]octanyl retinal were prepared by an improved synthesis and incubated with bovine opsin. The synthesis also establishes the absolute configuration of the enantiomers. Only one of the enantiomers binds to opsin, thus showing the steric restrictions regarding the middle polyene moiety of the retinoid molecule; this is in sharp contrast to the known leniency of the ring moiety binding site of retinoids. However, although one enantiomer is incorporated into the pigment, the circular dichroic spectrum of the pigment incorporating the bound enantiomer yields only a very weak Cotton effect, showing that, once incorporated, the bicyclo[5.1.0]octanyl chromophore is flattened by the opsin binding site. The titled retinoid was synthesized for study of the absolute conformation of the retinal pigment in rhodopsin.