A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Photoactivation of the orange carotenoid protein requires two light-driven reactions mediated by a metastable monomeric intermediate.

The orange carotenoid protein (OCP) functions as a sensor of the ambient light intensity and as a quencher of bilin excitons when it binds to the core of the cyanobacterial phycobilisome. We show herein that the photoactivation mechanism that converts the resting, orange-colored state, OCPO, to the active red-colored state, OCPR, requires a sequence of two reactions, each requiring absorption of a single photon by an intrinsic ketocarotenoid chromophore. Global analysis of absorption spectra recorded during continuous illumination of OCPO preparations from Synechocystis sp. PCC 6803 detects the reversible formation of a metastable intermediate, OCPI, in which the ketocarotenoid canthaxanthin exhibits an absorption spectrum with a partial red shift and a broadened vibronic structure compared to that of the OCPO state. While the dark recovery from OCPR to OCPI is a first-order, unimolecular reaction, the subsequent conversion of OCPI to the resting OCPO state is bimolecular, involving association of two OCPO monomers to form the dark-stable OCPO dimer aggregate. These results indicate that photodissociation of the OCPO dimer to form the monomeric OCPO intermediate is the first step in the photoactivation mechanism. Formation of the OCPO monomer from the dimer increases the mean value and broadens the distribution of the solvent-accessible surface area of the canthaxanthin chromophore measured in molecular dynamics trajectories at 300 K. The second step in the photoactivation mechanism is initiated by absorption of a second photon, by canthaxanthin in the OCPO monomer, which obtains the fully red-shifted and broadened absorption spectrum detected in the OCPR product state owing to displacement of the C-terminal domain and the translocation of canthaxanthin more than 12 Å into the N-terminal domain. Both steps in the photoactivation reaction of OCP are likely to involve changes in the structure of the C-terminal domain elicited by excited-state conformational motions of the ketocarotenoid.

Re-examining Cannabidiol: Conversion to Tetrahydrocannabinol Using Only Heat.

Introduction: In the last decade, the market for Cannabidiol (CBD) has grown to become a near $2 billion dollar industry in the United States alone. This growth can be attributed to a growing social acceptance of marijuana, a more detailed understanding of many health benefits attributed to cannabinoids, and the low cost and wide availibility of hemp-derived cannabinoids. Due to the complex legal histories of marijuana and cannabinoids, the stability and safety of CBD is still an area of interest as research has been restricted globally. Conversion of CBD to its psychoactive isomers, most notably delta-9-Tetrahydrocannabinol (Δ9-THC), presents a significant safety issue for consumers and producers of CBD products. Methods: Previous studies investigating the stability of CBD have focused mainly on replicating conditions experienced during long-term storage at room temperature or lower. Here, we report the thermal stability of CBD at 175°C. Dynamic 1H-NMR experiments and computational electronic structure calculations were used to characterize possible reaction paths from CBD to THC. Results: After 30 minutes of heating, Δ9-THC was produced in detectable amounts in aerobic and anaerobic conditions without an acid catalyst. Conclusions: Our findings support an energetically feasible reaction route that is favorable due to both an increase in phenol acidity at high temperatures and the presence of intramolecular OH-π hydrogen bonding.

Syntheses and Aromaticity Parameters of Hexahydroxypyrrocorphin, Porphotrilactones, and Their Oxidation State Intermediates.

Triple OsO4-mediated dihydroxylation of meso-tetrakis(pentafluorophenyl)porphyrin formed a non-aromatic hexahydroxypyrrocorphin as a single stereo-isomer. A one-step oxidative conversion of all three diol functionalities to lactone moieties generated three out of the four possible porphotrilactone regioisomers that were spectroscopically and structurally characterized. This conversion recovered most of the porphyrinic macrocycle aromatic ring current, as seen in their 1H NMR spectra and modeled using DFT computations. Stepwise OsO4-mediated dihydroxylations of porpho-mono- and -di-lactones generated intermediate oxidation state compounds between the pyrrole-three pyrroline macrocycle of the pyrrocorphin and the pyrrole-three oxazolone chromophore of the trilactones. The aromaticity of these chromophores was reduced with increasing number of oxazolone to pyrroline replacements, showing the importance for the presence of three lactone moieties for the retention of the macrocycle aromaticity in the tris-ß,ß'-modified macrocycles. This work first describes hexahydoxypyrrocorphins, porphotrislactones, and the oxidation state intermediates between them; furthers the understanding of the roles of ß-lactone moieties in the expression of porphyrinic macrocycle aromaticity; and generally broadens access to chemically stable pyrrocorphins and pyrrocorphin analogues.

Microsolvation Effects in the Spectral Tuning of Heliorhodopsin.

Heliorhodopsins (HeR) are a new category of heptahelical transmembrane photoactive proteins with a covalently linked all-trans retinal. The protonated Schiff base (PSB) nitrogen in the retinal is stabilized by a negatively charged counterion. It is well-known that stronger or weaker electrostatic interactions with the counterion cause a significant spectral blue- or red-shift, respectively, in both microbial and animal rhodopsins. In HeR, however, while Glu107 acts as the counterion, mutations of this residue are not directly correlated with a spectral shift. A molecular dynamics analysis revealed that a water cluster pocket produces a microsolvation effect on the Schiff base, compensating to various extents the replacement of the native counterion. Using a combination of molecular dynamics and quantum mechanical/molecular mechanics (QM/MM), we study this microsolvation effect on the electronic absorption of the retinylidene Schiff base chromophore of HeR.

Electronic energy levels of porphyrins are influenced by the local chemical environment.

Self-assembled islands of 5,10,15,20-tetrakis(pentafluoro-phenyl)porphyrin (2HTFPP) on Au(111) contain two bistable molecular species that differ by shifted electronic energy levels. Interactions with the underlying gold herringbone reconstruction and neighboring 2HTFPP molecules cause approximately 60% of molecules to have shifted electronic energy levels. We observed the packing density decrease from 0.64 ± 0.04 molecules per nm2 to 0.38 ± 0.03 molecules per nm2 after annealing to 200 °C. The molecules with shifted electronic energy levels show longer-range hexagonal packing or are adjacent to molecular vacancies, indicating that molecule-molecule and molecule-substrate interactions contribute to the shifted energies. Multilayers of porphyrins do not exhibit the same shifting of electronic energy levels which strongly suggests that molecule-substrate interactions play a critical role in stabilization of two electronic species of 2HTFPP on Au(111).

A favorable path to domain separation in the orange carotenoid protein.

The orange carotenoid protein (OCP) is responsible for nonphotochemical quenching (NPQ) in cyanobacteria, a defense mechanism against potentially damaging effects of excess light conditions. This soluble two-domain protein undergoes profound conformational changes upon photoactivation, involving translocation of the ketocarotenoid inside the cavity followed by domain separation. Domain separation is a critical step in the photocycle of OCP because it exposes the N-terminal domain (NTD) to perform quenching of the phycobilisomes. Many details regarding the mechanism and energetics of OCP domain separation remain unknown. In this work, we apply metadynamics to elucidate the protein rearrangements that lead to the active, domain-separated, form of OCP. We find that translocation of the ketocarotenoid canthaxanthin has a profound effect on the energetic landscape and that domain separation only becomes favorable following translocation. We further explore, characterize, and validate the free energy surface (FES) using equilibrium simulations initiated from different states on the FES. Through pathway optimization methods, we characterize the most probable path to domain separation and reveal the barriers along that pathway. We find that the free energy barriers are relatively small (<5 kcal/mol), but the overall estimated kinetic rate is consistent with experimental measurements (>1 ms). Overall, our results provide detailed information on the requirement for canthaxanthin translocation to precede domain separation and an energetically feasible pathway to dissociation.

Easily accessible non-aromatic heterocycles with handles: 4-bromo-2,3-dihydrofurans from 1,2-dibromohomoallylic alcohols.

The first general preparation of 4-bromo-2,3-dihydrofurans is reported. These non-aromatic heterocycles containing a useful coupling handle are accessed via Cu-catalyzed intramolecular cyclization of 1,2-dibromohomoallylic alcohols, which are themselves available in just two steps from aromatic and aliphatic aldehydes and ketones. Molecular dynamics simulations using the simple substrates and key geometric parameters provide a rationale for the selectivities observed. The synthetic utility of the 4-bromodihydrofurans is also demonstrated.

High-Throughput Non-targeted Chemical Structure Identification Using Gas-Phase Infrared Spectra.

The high-throughput identification of unknown metabolites in biological samples remains challenging. Most current non-targeted metabolomics studies rely on mass spectrometry, followed by computational methods that rank thousands of candidate structures based on how closely their predicted mass spectra match the experimental mass spectrum of an unknown. We reasoned that the infrared (IR) spectra could be used in an analogous manner and could add orthologous structure discrimination; however, this has never been evaluated on large data sets. Here, we present results of a high-throughput computational method for predicting IR spectra of candidate compounds obtained from the PubChem database. Predicted spectra were ranked based on their similarity to gas-phase experimental IR spectra of test compounds obtained from the NIST. Our computational workflow (IRdentify) consists of a fast semiempirical quantum mechanical method for initial IR spectra prediction, ranking, and triaging, followed by a final IR spectra prediction and ranking using density functional theory. This approach resulted in the correct identification of 47% of 258 test compounds. On average, there were 2152 candidate structures evaluated for each test compound, giving a total of approximately 555,200 candidate structures evaluated. We discuss several variables that influenced the identification accuracy and then demonstrate the potential application of this approach in three areas: (1) combining IR and mass spectra rankings into a single composite rank score, (2) identifying the precursor and fragment ions using cryogenic ion vibrational spectroscopy, and (3) the incorporation of a trimethylsilyl derivatization step to extend the method compatibility to less-volatile compounds. Overall, our results suggest that matching computational with experimental IR spectra is a potentially powerful orthogonal option for adding significant high-throughput chemical structure discrimination when used with other non-targeted chemical structure identification methods.

Spectral Features of Canthaxanthin in HCP2. A QM/MM Approach.

The increased interest in sequencing cyanobacterial genomes has allowed the identification of new homologs to both the N-terminal domain (NTD) and C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP). The N-terminal domain homologs are known as Helical Carotenoid Proteins (HCPs). Although some of these paralogs have been reported to act as singlet oxygen quenchers, their distinct functional roles remain unclear. One of these paralogs (HCP2) exclusively binds canthaxanthin (CAN) and its crystal structure has been recently characterized. Its absorption spectrum is significantly red-shifted, in comparison to the protein in solution, due to a dimerization where the two carotenoids are closely placed, favoring an electronic coupling interaction. Both the crystal and solution spectra are red-shifted by more than 50 nm when compared to canthaxanthin in solution. Using molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies of HCP2, we aim to simulate these shifts as well as obtain insight into the environmental and coupling effects of carotenoid-protein interactions.

Spectral Signatures of Canthaxanthin Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is involved in the photoprotective processes in cyanobacteria via nonphotochemical quenching. Triggered by blue-green light absorption, the carotenoid chromophore undergoes translocation, displacing around 12 Å from the C-terminal domain (CTD) to the N-terminal domain (NTD). The detailed molecular rearrangements that occur within the carotenoid and the protein during this process remain largely elusive. By using a combination of molecular dynamics, well-tempered metadynamics, and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations, we were able to mimic the translocation of the carotenoid from the inactive OCPO and obtain metastable red-shifted states in the photoactivation mechanism, replicating the λmax values of reference experimental spectra. In addition, our simulations give insight into the structure of the red-shifted form of the inactive state of OCP.

Amide-Linked C4″-Saccharide Modification of KRN7000 Provides Potent Stimulation of Human Invariant NKT Cells and Anti-Tumor Immunity in a Humanized Mouse Model.

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Fluorescence Anisotropy Detection of Barrier Crossing and Ultrafast Conformational Dynamics in the S2 State of ß-Carotene.

Carotenoids are usually only weakly fluorescent despite being very strong absorbers in the mid-visible region because their first two excited singlet states, S1 and S2, have very short lifetimes. To probe the structural mechanisms that promote the nonradiative decay of the S2 state to the S1 state, we have carried out a series of fluorescence lineshape and anisotropy measurements with a prototype carotenoid, ß-carotene, in four aprotic solvents. The anisotropy values observed in the fluorescence emission bands originating from the S2 and S1 states reveal that the large internal rotations of the emission transition dipole moment, as much as 50° relative to that of the absorption transition dipole moment, are initiated during ultrafast evolution on the S2 state potential energy surface and persist upon nonradiative decay to the S1 state. Electronic structure calculations of the orientation of the transition dipole moment account for the anisotropy results in terms of torsional and pyramidal distortions near the center of the isoprenoid backbone. The excitation wavelength dependence of the fluorescence anisotropy indicates that these out-of-plane conformational motions are initiated by passage over a low-activation energy barrier from the Franck-Condon S2 structure. This conclusion is consistent with detection over the 80-200 K range of a broad, red-shifted fluorescence band from a dynamic intermediate evolving on a steep gradient of the S2 state potential energy surface after crossing the activation barrier. The temperature dependence of the oscillator strength and anisotropy indicate that nonadiabatic passage from S2 through a conical intersection seam to S1 is promoted by the out-of-plane motions of the isoprenoid backbone with strong hindrance by solvent friction.

Aluminosilicate Mineralogy and the Sorption of Organic Cations: Interplay between Electrostatic Barriers and Compound Structural Features.

Current predictive models of organic cation sorption assume that sorbates interact with all sites on aluminosilicate minerals in the same manner. To examine whether differences in aluminosilicate structure and the resultant changes in electrostatic potential influence the sorption of organic cations, seven smectites were chosen with different proportions of isomorphic substitutions (origin of clay charge) located in octahedral versus tetrahedral layers and with the presence or absence of aluminosilicate interlayers. Sorption coefficients for 14 benzylamine derivatives with systematic differences in compound structures were collected to understand the possible influence of aluminosilicate mineralogy. Benzylamine compounds with methyl group substitution on the charged amine or with electron-donating or -withdrawing ring substituents displayed decreases in cation exchange-normalized sorption coefficients (KCEC), by up to one order of magnitude, between hectorite (100% isomorphic substitution in the octahedral layer) and nontronite (100% isomorphic substitution in the tetrahedral layer). To understand this difference across aluminosilicates, stochastic molecular models of the various aluminosilicate minerals with interlayers were performed. These models showed that negative charge density associated with tetrahedral sites results in high positive electrostatic energy barriers within the interlayer, creating a penalty for compounds with positive charge spread over a larger compound surface area as occurs from primary to quaternary amines. Conversely, clays with charge originating from octahedral sites produce low electrostatic potential barriers within the interlayer, decreasing the penalty for quaternary amine sorption. Trends for nine cationic pharmaceutical compounds, which varied in size, group alkylation, and/or polar substituents, demonstrated similar decreases in KCEC values to aluminosilicate minerals with high electrostatic energy barriers. Overall, aluminosilicate mineralogy was found to exert a large influence (0.5-1 order of magnitude in sorption coefficients) on organic cation sorption. The application of atomistic electrostatic potential mapping of both sorbent and sorbate structures provided insights to explain trends in sorption coefficients that could not be described by the basic electrostatic potential theory or by assuming that sorbate structure moieties yielded additive sorption contributions.

Origins of the Electronic Modulations of Bacterio- and Isobacteriodilactone Regioisomers.

Advances in the utilization of porphyrinoids for photomedicine, catalysis, and artificial photosynthesis require a fundamental understanding of the relationships between their molecular connectivity and resulting electronic structures. Herein, we analyze how the replacement of two pyrrolic Cß═Cß bonds of a porphyrin by two lactone (O═C-O) moieties modulates the ground-state thermodynamic stability and electronic structure of the resulting five possible pyrrole-modified porphyrin isomers. We made these determinations based on density functional theory (DFT) and time-dependent DFT computations of the optical spectra of all regioisomers. We also analyzed the computed magnetically induced currents of their aromatic π-systems. All regioisomers adopt the tautomeric state that maximizes aromaticity, whether or not transannular steric strains are incurred. In all isomers, the O═Cß-Oß bonds were found to support a macrocycle diatropic ring current. We attributed this to the delocalization of nonbonding electrons from the ring oxa- and oxo-atoms into the macrocycle. As a consequence of this delocalization, the dilactone regioisomers are as-or even more-aromatic than their hydroporphyrin congeners. The electronic structures follow different trends for the bacteriochlorin- and isobacteriochlorin-type isomers. The presence of either oxo- or oxa-oxygens conjugated with the macrocyclic π-system was found to be the minimal structural requirement for the regioisomers to exhibit distinct electronic properties. Our computational methods and mechanistic insights provide a basis for the systematic exploration of the physicochemical properties of porphyrinoids as a function of the number, relative orientation, and degree of macrocycle-π-conjugation of ß-substituents, in general, and for dilactone-based porphyrinic chromophores, in particular.

Bacterio- and Isobacteriodilactones by Stepwise or Direct Oxidations of meso-Tetrakis(pentafluorophenyl)porphyrin.

Porpholactones are porphyrinoids in which one or more ß,ß'-bonds of the parent chromophore were replaced by lactone moieties. Accessible to varying degrees by direct and nonselective oxidations of porphyrins, the rational syntheses of all five dilactone isomers along stepwise, controlled, and high-yielding routes via porphyrin → tetrahydroxyisobacteriochlorin metal complexes → isobacteriochlorindilactone metal complexes or porphyrin → tetrahydroxybacteriochlorin → bacteriochlorindilactone (and related) pathways, respectively, are described. A major benefit of these complementary routes over established methods is the simplicity of the isolation of the dilactones because of the reduced number of side products formed. In an alternative approach we report the direct and selective conversion of free base meso-tetrakis(pentafluorophenyl)porphyrin to all isomers of free base isobacteriodilactones using the oxidant cetyltrimethylN+MnO4-. The solid-state structures of some of the isomers and their precursors are reported, providing data on the conformational modulation induced by the derivatizations. We also rationalize computationally their differing thermodynamic stability and electronic properties. In making new efficient routes toward these dilactone isomers available, we enable the further study of this diverse class of porphyrinoids.

Carotenoid-Chlorophyll Interactions in a Photosynthetic Antenna Protein: A Supramolecular QM/MM Approach.

Multichromophoric interactions control the initial events of energy capture and transfer in the light harvesting peridinin-chlorophyll a protein (PCP) from marine algae dinoflagellates. Due to the van der Waals association of the carotenoid peridinin (Per) with chlorophyll a in a unique 4:1 stoichiometric ratio, supramolecular quantum mechanical/molecular mechanical (QM/MM) calculations are essential to accurately describe structure, spectroscopy, and electronic coupling. We show that, by enabling inter-chromophore electronic coupling, substantial effects arise in the nature of the transition dipole moment and the absorption spectrum. We further hypothesize that inter-protein domain Per-Per interactions are not negligible, and are needed to explain the experimental reconstruction features of the spectrum in wild-type PCP.

The limited extent of the electronic modulation of chlorins and bacteriochlorins through chromene-annulation.

Optical data (UV-vis absorption and fluorescence emission spectra, including fluorescence yields and lifetimes) and electrochemical measurements are used to quantify the modulation of the electronic properties of meso-tetrakis(pentafluorophenyl)-chlorin diol and -bacteriochlorin tetraols upon intramolecular chromene-annulation, including the investigation of regio- and stereoisomers. The small modulations of the frontier orbitals of the porphyrinoids are rationalized using DFT computations and can be traced to small electronic effects due to the co-planarized meso-aryl groups in combination with conformational effects.

Dual Modifications of α-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity.

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.