Stabilization of sp-Hybridized Nitrogen Cation by Lewis Acid-Base Complex Formation with Intramolecular Iodine.

Here we show that the sp-hybridized nitrogen cation is strongly stabilized by a peri-iodine substituent in the tetralone system. The cation is captured by anionic species such as CF3 CO2 - , affording hypervalent iodine(III) compounds with a short nitrogen-iodine (N-I) bond, in which the cation serves as a Lewis acid. Notably, the O-I bond of the O-trifluoroacetate or O-acetate is intrinsically weaker than the N-I bond due to its more ionic character and is further weakened by protonation in trifluoroacetic acid. As a result, the oxygen ligand can dissociate in the presence of a Brønsted acid, affording a I+ cation intermediate that retains the N-I bond. We isolated the cation as the tetrafluoroborate, and characterized it experimentally by 1 H NMR spectroscopy and X-ray structure analysis, and theoretically by means of DFT calculation. The results suggest that the N-I bonded cation is intrinsically stable, and is weakly coordinated with water and the BF4 counter anion or trifluoroacetate anion. This cation can be employed as a reagent for α-oxidation of ketones.

Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides.

In this study, we explored the potential of the photoremovable o-nitrobenzyl (oNB) group as a tool to manipulate the membrane permeability and regulate the conformation of linear peptides by means of experimental and computational studies. We found that the introduction of one or more oNB groups markedly increased the permeability and altered the conformation, as compared to the corresponding unmodified peptides. We thoroughly investigated the impact of peptide length, number of oNB group, oNB insertion position, and introduction of N- and C-terminal protecting groups on the passive membrane permeability by means of parallel artificial membrane permeability assay (PAMPA). Photoreaction of peptides containing one or two oNB groups proceeded cleanly in moderate to high yields, releasing the unprotected parent linear peptide. The oNB-modified peptides showed a cis/trans conformational equilibrium, while after photolysis, the unprotected linear peptides showed only the trans-amide conformation. Furthermore, a comprehensive comparison of oNB-modified peptides and N-methylated peptides was conducted, encompassing conformational analysis and physicochemical properties. N-Substituted peptides favored a folded-like structure, which may contribute to the improvement in permeability.

Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6.

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

Isosteric Replacement of Ester Linkage of Lysophospholipids with Heteroaromatic Rings Retains Potency and Subtype Selectivity.

Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.

Friedel-Crafts Acylation of Aminocarboxylic Acids in Strong Brønsted Acid Promoted by Lewis Base P4O10.

The amino group in aminocarboxylic acids is sufficiently basic to be protonated in strong acids, and consequently, ionization of the carboxylic acid to an acylium ion is blocked due to charge-charge repulsion. Thus, acylation of aromatic compounds is significantly retarded in Friedel-Craft type reactions. We found that Friedel-Crafts acylation with aminocarboxylic acids can proceed smoothly even in a strong Brønsted acid (triflic acid, TfOH) if the Lewis base P4O10 is added. Here we describe the Friedel-Crafts acylation reactions of anthranilic acid and α- to δ-aminocarboxylic acids with benzene derivatives in the presence of P4O10. Non-amino-containing carboxylic acids as well as N-containing heteroaromatic carboxylic acids are available, and α-amino acids can be directly utilized without any protective group. Most substrates afford acylation products in high yields, although some epimerization/racemization may occur. Density functional theory (DFT) calculations suggested that P4O10 neutralizes the protonated amine, converting the N-H covalent bond to a N-hydrogen bond and allowing the carboxylic acid OH functionality to serve as a good leaving group.

Neighboring Group Participation of Nitrogen Cation to Form an Unusual Six-Membered Ring Intermediate During Oxime Rearrangement.

Neighboring group participation involving a 6-membered ring structure is rare, despite the privilege of 6-membered ring transition structures in organic chemistry. We examined the putative role of a 6-membered cyclic intermediate with neighboring group participation of nitrogen cation in syn-migration of peri-ester indanone oximes. Direct observation of a peri-methyl ester-iminylium intermediate in solution by means of 1H NMR supported the existence of the 6-membered cation intermediate. Density functional theory (DFT) calculations also supported the intervention of this intermediate in the rearrangement and indicated that it has a planar structure stabilized by electron delocalization.

Contribution of Solvents to Geometrical Preference in the Z/E Equilibrium of N-Phenylthioacetamide.

We studied the Z/E preference of N-phenylthioacetamide (thioacetanilide) derivatives in various solvents by means of 1H NMR spectroscopy, as well as molecular dynamics (MD) and other computational analyses. Our experimental results indicate that the Z/E isomer preference of secondary (NH)thioamides of N-phenylthioacetamides shows substantial solvent dependency, whereas the corresponding amides do not show solvent dependency of the Z/E isomer ratios. Detailed study of the solvent effects based on molecular dynamics simulations revealed that there are two main modes of hydrogen (H)-bond formation between solvent and (NH)thioacetamide, which influence the Z/E isomer preference of (NH)thioamides. DFT calculations of NH-thioamide in the presence of one or two explicit solvent molecules in the continuum solvent model can effectively mimic the solvation by multiple solvent molecules surrounding the thioamide in MD simulations and shed light on the precise nature of the interactions between thioamide and solvent. Orbital interaction analysis showed that, counterintuitively, the Z/E preference of NH-thioacetamides is mainly determined by steric repulsion, while that of sterically congested N-methylthioacetamides is mainly determined by thioamide conjugation.

Elaboration of Non-naturally Occurring Helical Tripeptides as p53-MDM2/MDMX Interaction Inhibitors.

Protein-protein interactions (PPIs) are often mediated by helical, strand and/or coil secondary structures at the interface regions. We previously showed that non-naturally occurring, stable helical trimers of bicyclic ß-amino acids (Abh) with all-trans amide bonds can block the p53-MDM2/MDMX α-helix-helix interaction, which plays a role in regulating p53 function. Here, we conducted docking and molecular dynamics calculations to guide the structural optimization of our reported compounds, focusing on modifications of the C-terminal/N-terminal residues. We confirmed that the modified peptides directly bind to MDM2 by means of thermal shift assay, isothermal titration calorimetry, and enzyme-linked immunosorbent assay (ELISA) experiments. Biological activity assay in human osteosarcoma cell line SJSA-1, which has wild-type p53 and amplification of the Mdm2 gene, indicated that these peptides are membrane-permeable p53-MDM2/MDMX interaction antagonists that can rescue p53 function in the cells.

Membrane Phospholipid Analogues as Molecular Rulers to Probe the Position of the Hydrophobic Contact Point of Lysophospholipid Ligands on the Surface of G-Protein-Coupled Receptor during Membrane Approach.

When lipid mediators bind to G-protein-coupled receptors (GPCRs), the ligand first enters the lipid bilayer, then diffuses laterally in the cell membrane to make hydrophobic contact with the receptor protein, and finally enters the receptor's binding pocket. In this process, the location of the hydrophobic contact point on the surface of the receptor has been little discussed even in cases in which the crystal structure has been determined, because the ligand binding pocket is buried inside the transmembrane (TM) domains. Here, we coupled an activator ligand to a series of membrane phospholipid surrogates, which constrain the depth of entry of the ligand into the lipid bilayer. Consequently, via measurement of the receptor-activating activity as a function of the depth of entry into the membrane, these surrogates can be used as molecular rulers to estimate the location of the hydrophobic contact point on the surface of GPCR. We focused on lysophosphatidylserine (LysoPS) receptor GPR34 and prepared a series of simplified membrane-lipid-surrogate-conjugated lysophospholipid analogues by attaching alkoxy amine chains of varying lengths to the hydrophobic tail of a potent GPR34 agonist. As expected, the activity of these lipid-conjugated LysoPS analogues was dependent on chain length. The predicted contact position matches the position of the terminal benzene ring of a nonlipidic ligand that protrudes between TMs 4 and 5 of the receptor. We further found that the nature of the terminal hydrophilic functional group of the conjugated membrane lipid surrogate strongly influences the activity, suggesting that lateral hydrophilic contact of LysoPS analogues with the receptor's surface is also crucial for ligand-GPCR binding.

Conformational preference of bicyclic ß-amino acid dipeptides.

Bridged bicyclic amino acids have high potential applicability as self-organized, conformationally constrained synthetic building blocks that do not require assistance from hydrogen bond formation. We systematically investigated the intrinsic conformational propensities of dipeptides of bridged bicyclic ß-amino acids by means of accelerated molecular dynamics simulation and density functional theory (DFT) calculations in methanol, chloroform, and water. While the main-chain conformation, represented by φ and θ values, is fixed by the nature of the bicyclic ring structure, rotation of the C-terminal carbonyl group (ψ) is also restricted, converging to one or two minima. In endo-type dipeptides, in which the two N- and C-terminal amides are spatially close to each other, the C-terminal amide plane is placed horizontally. In exo-type dipeptides, in which the two amides are on opposite sides of the ring plane, the C-terminal carbonyl group can take two types of positions: either parallel/antiparallel with the N-terminal carbonyl or beneath the bicyclic ring, forcing the amide NHMe moiety to lie outside of the ring. We also examined the cis-trans preference of model bicyclic amides. Although the parent amides exhibit cis-trans equilibrium without any preference, addition of a methyl group on one of the bridgehead positions tips the equilibrium towards trans.

Non-naturally Occurring Helical Molecules Can Interfere with p53-MDM2 and p53-MDMX Protein-Protein Interactions.

We have discovered that ß-amino acid homooligomers with cis- or trans-amide conformation can fold themselves into highly ordered helices. Moreover, unlike α-amino acid peptides, which are significantly stabilized by intramolecular hydrogen bonding, these helical structures are autogenous conformations that are stable without the aid of hydrogen bonding and irrespective of solvent (protic/aprotic/halogenated) or temperature. A structural overlap comparison of helical cis/trans bicyclic ß-proline homooligomers with typical α-helix structure of α-amino acid peptides reveals clear differences of pitch and diameter per turn. Bridgehead substituents of the present homooligomers point outwards from the helical surface. We were interested to know whether such non-naturally occurring divergent helical molecules could mimic α-helix structures. In this study, we show that bicyclic ß-proline oligomer derivatives inhibit p53-MDM2 and p53-MDMX protein-protein interactions, exhibiting MDM2-antagonistic and MDMX-antagonistic activities.

Contrasting C- and O-Atom Reactivities of Neutral Ketone and Enolate Forms of 3-Sulfonyloxyimino-2-methyl-1-phenyl-1-butanones.

The mechanisms of intramolecular cyclization of 3-sulfonyloxyimino-2-methyl-1-phenyl-1-butanones (1) under basic (DABCO and t-BuOK) and acidic (AcOH and TFA) conditions were investigated by means of experimental and computational methods. The ketone, enol, and enolate forms of 1 can afford different intramolecular cyclization products (2, 3, 4), depending on the conditions. The results of the reaction of 1 under basic conditions suggest intermediacy of neutral enol (DABCO) and anionic enolate (t-BuOK), while the results under acidic conditions (AcOH and TFA) indicate involvement of neutral ketones, which exhibit reactivities arising from both the oxygen lone-pair electrons (O atom reactivity) and carbon σ-electrons (C atom reactivity). The neutral enol in DABCO afforded 2H-azirine 4. On the other hand, the products (isoxazole 2 and oxazole 3) generated from the ketone form and from the enolate form are the same, but the reaction mechanisms are apparently different. The results demonstrate ambident-like reactivity of neutral ketone in the 3-sulfonyloxyimino-2-methyl-1-phenyl-1-butanone system.

Application of C-Terminal 7-Azabicyclo[2.2.1]heptane to Stabilize ß-Strand-like Extended Conformation of a Neighboring α-Amino Acid.

ß-Strands are formed by extended linear peptide chains that are usually paired to form ß-sheet structure through interstrand hydrogen bonding. Linking a structured organic molecule with α-amino acid(s) can enforce or stabilize ß-strand-like extended structures of the jointed amino acids. Spectroscopic and simulation studies indicated that the presence of a C-terminal 7-azabicyclo[2.2.1]heptane amine (Abh) favors a ß-strand-like extended conformation of the adjacent α-amino acid on the N side. The bridgehead substitution of the Abh unit biases the amide cis-trans equilibrium of the adjacent α-amino acid residue to cis conformation. The proximity, specified by the presence of bond paths (such as H-H bond path) between the bridgehead proton of Abh and the α-proton of the α-amino acid provides a driving force favoring the extended conformation, which is independent of solvents. These results provide a basis for de novo design of ß-strand-mimicking extended peptides by using ß-strand enforcer/stabilizer even in the absence of the interstrand hydrogen bonding.

Facile synthesis of 2,3-benzodiazepines using one-pot two-step phosphate-assisted acylation-hydrazine cyclization reactions.

Here, we report new methodology for synthesizing 2,3-benzodiazepines and their analogues by means of phosphate-assisted acylation reaction of 1-arylpropan-2-ones with a carboxylic acid followed by hydrazine cyclization in a one-pot two-step manner. An unprotected amino group is tolerated in this reaction. This method provides a direct access to 2,3-benzodiazepines containing aromatic 7,8-dimethoxy and 1-p-aminophenyl groups, which are generally considered important for bioactivity. The presence of 3,4-dimethoxy or 3-methoxy substitution on the benzene ring of the 1-arylpropan-2-one is important for high regioselectivity in the acylation reaction.

Unexpected Resistance to Base-Catalyzed Hydrolysis of Nitrogen Pyramidal Amides Based on the 7-Azabicyclic[2.2.1]heptane Scaffold.

Non-planar amides are usually transitional structures, that are involved in amide bond rotation and inversion of the nitrogen atom, but some ground-minimum non-planar amides have been reported. Non-planar amides are generally sensitive to water or other nucleophiles, so that the amide bond is readily cleaved. In this article, we examine the reactivity profile of the base-catalyzed hydrolysis of 7-azabicyclo[2.2.1]heptane amides, which show pyramidalization of the amide nitrogen atom, and we compare the kinetics of the base-catalyzed hydrolysis of the benzamides of 7-azabicyclo[2.2.1]heptane and related monocyclic compounds. Unexpectedly, non-planar amides based on the 7-azabicyclo[2.2.1]heptane scaffold were found to be resistant to base-catalyzed hydrolysis. The calculated Gibbs free energies were consistent with this experimental finding. The contribution of thermal corrections (entropy term, ⁻TΔS‡) was large; the entropy term (ΔS‡) took a large negative value, indicating significant order in the transition structure, which includes solvating water molecules.

A simple and effective preparation of quercetin pentamethyl ether from quercetin.

Among the five hydroxy (OH) groups of quercetin (3,5,7,3',4'-pentahydroxyflavone), the OH group at 5 position is the most resistant to methylation due to its strong intramolecular hydrogen bonding with the carbonyl group at 4 position. Thus, it is generally difficult to synthesize the pentamethyl ether efficiently by conventional methylation. Here, we describe a simple and effective per-O-methylation of quercetin with dimethyl sulfate in potassium (or sodium) hydroxide/dimethyl sulfoxide at room temperature for about 2 hours, affording quercetin pentamethyl ether (QPE) quantitatively as a single product. When methyl iodide was used in place of dimethyl sulfate, the C-methylation product 6-methylquercetin pentamethyl ether was also formed. A computational study provided a rationale for the experimental results.

Lysophosphatidylserine suppresses IL-2 production in CD4 T cells through LPS3/GPR174.

Lysophosphatidylserine (LysoPS) has been shown to have lipid mediator-like actions to induce mast cell degranulation and suppress T lymphocyte proliferation. Recently, three G protein-coupled receptors (GPCRs), LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, were found to react specifically with LysoPS, raising the possibility that LysoPS exerts its roles through these receptors. In this study, we show that LPS3 is expressed in various T cell subtypes and is involved in suppression of Interleukin-2 (IL-2) production in CD4 T cells. We found that LysoPS suppressed the IL-2 production from activated T cells at the mRNA and protein levels. In addition, LysoPS did not have such an effect on the splenocytes and CD4 T cells isolated from LPS3-deficient mice. In LPS3-deficient splenocytes and CD4 T cells, anti-CD3/anti-CD28-triggered IL-2 production is somewhat increased. Interestingly, LysoPS with various fatty acids was up-regulated upon T cell activation. The present study raised the possibility that LysoPS exerts its immunosuppressive roles by down-regulating IL-2 production through a LysoPS-LPS3 axis in T cells.

Base-Induced Transformation of 2-Acyl-3-alkyl-2H-azirines to Oxazoles: Involvement of Deprotonation-Initiated Pathways.

An experimental study of base-induced transformation reaction of 2-acyl-3-alkyl-2H-azirines to oxazoles indicated that a deprotonation-initiated mechanism is involved, in addition to nucleophilic addition to the imine functionality. Calculations suggested the participation of a ketenimine (ethenimine) intermediate generated by azirine ring opening of the carbanion intermediate formed by α-deprotonation of 2H-azirine. The ketenimine intermediate possessing methyl substituents at C(3) appears to be more stable than the tautomeric nitrile ylide which was proposed to be involved in photoinduced and pyrolysis reactions of 2-acyl-3-alkyl-2H-azirines to afford oxazoles. Thus, intermediacy of ketenimine is consistent with both experimental and computational results, at least under strongly basic reaction conditions.

Elucidation of the E-Amide Preference of N-Acyl Azoles.

The conformational properties of N-acyl azoles (imidazole, pyrazole, and triazole) were examined. The N-2',4',6'-trichlorobenzoyl azoles were stable in methanol at room temperature, and no hydrolyzed products were observed over 7 days in the presence of 5% trifluoroacetic acid or 5% triethylamine in CDCl3. The high stability may be explained by the double-bond amide character caused by the steric hindrance due to the ortho-substituents in the benzoyl group. While specific E-amide preferences were observed in N-acyl pyrazoles/triazoles, the amides of the imidazoles gave a mixture of E and Z. One of the conceivable ideas to rationalize this conformational preference may be repulsive interaction between two sets of lone-pair electrons on the pyrazole 2-nitrogen (nN) and the carbonyl oxygen atoms (nO) in the Z-conformation of N-acyl pyrazoles/triazoles. However, analysis of orbital interactions suggested that in the case of the E-conformation of N-acyl pyrazoles, such electron repulsion is small because of distance. The interbond energy calculations suggested that the Z-conformer is involved in strong vicinal σ-σ repulsion along the amide linkage between the σN1N2 and σC1C2 orbitals in the anti-periplanar arrangement and between the σN1C5 and σC1C2 orbitals in the syn-periplanar arrangement, which lead to the overwhelming E-preference in N-acyl pyrazoles/triazoles. In the case of N-acyl imidazoles, similar vicinal σ-σ repulsions were counterbalanced, leading to a weak preference for the E-conformer over the Z-conformer. The chemically stable and E-preferring N-acyl azoles may be utilized as scaffolds in future drug design.

Electrophilic activation of aminocarboxylic acid by phosphate ester promotes Friedel-Crafts acylation by overcoming charge-charge repulsion.

Friedel-Crafts acylation of aromatic compounds with aminocarboxylic acids proceeds efficiently in the presence of a tailored phosphate ester and a strong Brønsted acid, despite the strong charge-charge repulsion associated with acylium ion formation. Here, we investigate the mechanism of this electrophilic aromatic acylation reaction, focusing on how the aminocarboxylic acid is activated by the phosphate ester and how the charge-charge repulsion is overcome. In the first step of the reaction, an acyl phosphate is generated from the aminocarboxylic acid through the intervention of the phosphate ester, which possesses three methyl salicylate ester linkages. The o-methyl salicylates enhance the reactivity of the phosphate ester via a protonation-induced conformational change, thereby overwhelming the charge-charge repulsion associated with the acylium ion formation. Weakening of the resonance interaction in the C(O)-O(P) bond by the lone-pair electrons of the ether oxygen atom of the carboxylic acid functionality contributes to the rapid formation of the acylium ion. Thus, our results show that the formation of aromatic ketones from various carboxylic acids proceeds because the strong leaving ability of the acyl phosphate overwhelms the charge-charge repulsion associated with the formation of the acylium ion. This information will be helpful to improve the design of tailored phosphate reagents.