Biosynthesis of dillapiole/apiole in dill (Anethum graveolens): characterization of regioselective phenylpropene O-methyltransferase.

The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.

Formation of chalcogen-bonding interactions and their role in the trans-trans conformation of thiourea.

The chalcogen-bonding interactions formed at both sides of the thiocarbonyl group in thiourea were investigated. In particular, the role of these chalcogen-bonding interactions in the trans-trans conformation of thiourea was evaluated via single-crystal X-ray diffraction analysis and DFT calculations. The obtained results indicate that the Se⋯S⋯Se dual chalcogen-bonding interactions play a stronger role in controlling the planar structure than the S⋯S⋯S interactions.

Palladium-catalyzed C-C bond cleavage of N-cyclopropyl acylhydrazones.

Despite their utility as directing groups, the C-C bond cleavage of cyclopropanes utilizing hydrazones has not been explored. Herein, Pd-catalyzed C-C bond cleavage reaction of N-cyclopropyl acylhydrazones, followed by cycloisomerization to yield pyrazoles, has been developed. The protocol enables the synthesis of various α-pyrazole carbonyl compounds, which have a potential of biological activity. Control experiments and DFT calculations suggest that ß-carbon elimination of a stable 6-membered chelate palladium complex occurs, generating a conjugated azine as a reaction intermediate for the following cycloisomerization.

Dual Chalcogen-Bonding Interactions for the Conformational Control of Urea.

Dual chalcogen-bonding interactions is proposed as a novel means for the conformational control of urea derivatives. The formation of a chalcogen-bonding interaction at both sides of the urea carbonyl group was unambiguously confirmed by X-ray diffraction as well as computational studies including non-covalent interaction (NCI) plot index analysis, quantum theory of atoms in molecules (QTAIM) analysis, and natural bond orbital (NBO) analysis via DFT calculations. By virtue of this dual interaction, urea derivatives that bear chalcogen atoms (X=S and Se) adopt a planar structure via the carbonyl oxygen (O) with an X⋅⋅⋅O⋅⋅⋅X arrangement on the same side of the molecule. The rigidity of the conformational lock was evaluated using the molecular arrangement in the crystal and the rotational barrier of benzochalcogenophene ring, which indicated a stronger conformational lock in benzoselenophene than in benzothiophene urea derivatives. Furthermore, the acidity of the urea derivatives increases according to the Lewis-acidic properties of the chalcogen-bonding interactions, whereby benzoselenophene urea is more acidic than benzothiophene urea. Tweezer-shaped urea derivatives were prepared, and their stereostructure proved the viability of the conformational control for defining the location of the substituents on the urea framework.

Oxidative Deprotection of Benzyl Protecting Groups for Alcohols by an Electronically Tuned Nitroxyl-Radical Catalyst.

The oxidative deprotection of benzyl (Bn) groups using nitroxyl-radical catalyst 1 and co-oxidant phenyl iodonium bis(trifluoroacetate) (PIFA) is reported. This catalyst is highly active for the oxidation of benzylic ethers because of the electronic tuning on account of the electron-withdrawing ester groups next to the catalytically active center. This catalytic system promotes deprotections at ambient temperature and has a broad substrate scope, including substrates possessing hydrogenation-sensitive functional groups, while the deprotection hardly proceeds when using well-known nitroxyl-radical catalysts such as 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO). The 1/PIFA system also promotes the deprotection of several benzylic protecting groups, including 2-naphthylmethyl (NAP) and 4-methylbenzyl (MBn) groups. Catalyst 1 was also effective for the direct synthesis of ketones and aldehydes from Bn ethers via deprotected alcohols using an excess of the co-oxidant PIFA.

One-Pot Preparation of (NH)-Phenanthridinones and Amide-Functionalized [7]Helicene-like Molecules from Biaryl Dicarboxylic Acids.

A one-pot transformation of biaryl dicarboxylic acids to (NH)-phenanthridinone derivatives based on a Curtius rearrangement and subsequent basic hydrolysis was developed. This method is also applicable for the preparation of optically active amide-functionalized [7]helicene-like molecules. Furthermore, aza[5]helicene derivatives with a phosphate moiety were isolated as a product of the Curtius rearrangement step in the case of substrates that bear chalcogen atoms. The stereostructures of these products, revealed by X-ray diffraction analysis, suggested that chalcogen-bonding and pnictogen-bonding interactions might contribute to their stabilization. The configurational stability of the helicene-like molecules and their chiroptical properties were further investigated.

Syntheses, and Structural and Physical Properties of Axially Chiral Biaryl Dicarboxylic Acids Bearing Chalcogen Atoms.

The preparation, optical resolution, and structural investigations of a series of axially chiral biaryl dicarboxylic acids bearing oxygen, sulfur, and selenium atoms were carried out. The crystal structures of sulfur- and selenium-containing derivatives revealed that the carboxy groups of these compounds are located in a co-planar geometry with the fused aromatic rings including the chalcogen atoms. These conformational controls were found to be achieved by chalcogen-bonding interactions between chalcogen atoms in the aromatic rings and oxygen atoms in the carboxy groups. Even in the case of a binaphthofuran derivative, in which the formation of chalcogen-bonding interactions was expected to be negligible, the carboxy groups were also found to be located in a co-planar geometry toward its fused cyclic rings. Natural bond orbital (NBO) analyses of these dicarboxylic acids indicated the formation not only for the chalcogen-bonding interactions for S and Se derivatives, but also the tetrel-bonding interactions between the oxygen atoms in the carboxy groups and the carbon atoms in the fused cyclic rings for all biaryl dicarboxylic acids. These tetrel-bonding interactions were thought to contribute to conformational control in the binaphthofuran derivative. Physical and chiroptical properties such as the racemization barriers and circular dichroism (CD) spectra of these biaryl dicarboxylic acids were also revealed.

Nitro-Substituted Benzaldehydes in the Generation of Azomethine Ylides and Retro-1,3-Dipolar Cycloadditions.

1,3-Dipolar cycloaddition of 2- and 3-nitrobenzaldehydes with 2-aminomethylpyridine and ethyl (2E)-2-cyano-3-(4-nitrophenyl)prop-2-enoate yielded endo-cycloadducts as the sole products under various reaction conditions. Fortuitously, 4-nitrobenzaldehyde behaved differently in three- and four-component cascades to produce a mixture of endo- and exo'-cycloadducts. This reaction is solvent- and temperature-dependent, and consequently, both the endo- and exo'-cycloadducts were synthesized in an excellent regio-, stereo-, and chemoselective fashion. Retro-1,3-dipolar cycloadditions of the endo-cycloadducts were conducted under mild reaction conditions, and the generated syn-dipoles were stereomutated into anti-dipoles which recycloadded with the dipolarophiles to provide the exo'-cycloadducts. Mechanistic studies were carried out to support the proposed mechanisms. Unprecedentedly, particular arylidene scaffolds participated as aldehyde or activated methylene precursors. Density functional theory calculations were performed to shed light on the importance of AcOH in the generation and isomerization of dipoles and to explain the high selectivity and the possibility of retro-cycloaddition.

A bench-stable N-trifluoroacetyl nitrene equivalent for a simple synthesis of 2-trifluoromethyl oxazoles.

ortho-Nitro-substituted N-trifluoroacetyl imino-λ3-iodane is a bench-stable trifluoroacetyl nitrene precursor, in which intra- and intermolecular halogen bonding (XB) plays an important role. Potential synthetic applications of this novel precursor were explored.

Axial chirality in biaryl N,N-dialkylaminopyridine derivatives bearing an internal carboxy group.

Axial chirality in N,N-dimethylaminopyridines as well as N,N-dipropylaminopyridines bearing an internal carboxy group were evaluated based on their racemization barriers and circular dichroism spectra. The half-life of racemization of N,N-dipropylaminopyridine derivative 2 was estimated to be 19.7 days at 20°C. Its enantiomers isolated as optically active forms showed positive-negative and negative-positive Cotton effects for (+)-2 and (-)-2, respectively, from 310 to 210 nm. Furthermore, (-)-2 was applied as a chiral nucleophilic catalyst and exhibited asymmetric induction in acylative kinetic resolution of 1-(1-naphthyl)ethane-1-ol.

Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells.

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.

The RING finger- and PDZ domain-containing protein PDZRN3 controls localization of the Mg2+ regulator claudin-16 in renal tube epithelial cells.

Ion exchange in the renal tubules is fundamental to the maintenance of physiological ion levels. Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle's loop in the kidney, with dephosphorylation of CLDN16 increasing its intracellular distribution and decreasing paracellular Mg2+ permeability. CLDN16 is located in the tight junctions, but the mechanism regulating its localization is unclear. Using yeast two-hybrid systems, we found that CLDN16 binds to PDZRN3, a protein containing both RING-finger and PDZ domains. We also observed that the carboxyl terminus of the cytoplasmic CLDN16 region was required for PDZRN3 binding. PZDRN3 was mainly distributed in the cytosol of rat kidney cells and upon cell treatment with the protein kinase A inhibitor H-89, colocalized with CLDN16. H-89 also increased mono-ubiquitination and the association of CLDN16 with PDZRN3. Mono-ubiquitination levels of a K275A mutant were lower, and its association with PDZRN3 was reduced compared with wild-type (WT) CLDN16 and a K261A mutant, indicating that Lys-275 is the major ubiquitination site. An S217A mutant, a dephosphorylated form of CLDN16, localized to the cytosol along with PDZRN3 and the endosomal marker Rab7. PDZRN3 siRNA increased cell-surface localization of WT CLDN16 in H-89-treated cells or containing the S217A mutant and also suppressed CLDN16 endocytosis. Of note, H-89 decreased paracellular Mg2+ flux in WT CLDN16 cells, and PDZRN3 siRNA increased Mg2+ flux in the H-89-treated WT CLDN16 and S217A mutant cells. These results suggest that PDZRN3 mediates endocytosis of dephosphorylated CLDN16 and represents an important component of the CLDN16-trafficking machinery in the kidney.

Synthesis and Structural Properties of Axially Chiral Binaphthothiophene Dicarboxylic Acid.

Axially chiral binaphthothiophene dicarboxylic acid was prepared as a novel functionalized chiral dicarboxylic acid. The crystal structures of both the racemic form and its salt with chiral diamine revealed the intramolecular S···O interactions (chalcogen bonds) between the sulfur in the naphthothiophene rings and the oxygen of the carboxy groups. The negative-positive and the positive-negative Cotton effects from longer to shorter wavelengths were observed for (R)- and (S)-enantiomers, respectively, in the circular dichroism (CD) spectra.

Total Synthesis of Ellagitannins via Sequential Site-Selective Functionalization of Unprotected D-Glucose.

A short-step total synthesis of the natural glycosides pterocarinin C and tellimagrandin II (eugeniin) has been performed by sequential and site-selective functionalization of free hydroxy groups of unprotected D-glucose. The key reactions are ß-selective glycosidation of a gallic acid derivative using unprotected D-glucose as a glycosyl donor and catalyst-controlled site-selective introduction of a galloyl group into the inherently less reactive hydroxy group of the glucoside.

Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase.

MAIN CONCLUSION: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

A Concise Access to C2-Symmetric Chiral 4-Pyrrolidinopyridine Catalysts with Dual Functional Side Chains.

A practical method was developed for the preparation of a diastereomeric library of C2-symmetric chiral 4-pyrrolidinopyridine catalysts with dual amide side chains. Use of a racemic precursor is the key to the concise production of catalysts with diverse stereochemisty.

Asymmetric Synthesis of Multisubstituted Dihydrobenzofurans by Intramolecular Conjugate Addition of Short-Lived C-O Axially Chiral Enolates.

Enantioselective intramolecular conjugate addition reactions of short-lived C-O axially chiral enolates have been developed. The reactions proceeded with inversion of the configuration and provided dihydrobenzofurans with contiguous tetra- and trisubstituted carbon centers in up to 96% enantiomeric excess (ee).

Organocatalytic Site-Selective Acylation of Avermectin B2a, a Unique Endectocidal Drug.

The organocatalytic site-selective monoacylation of avermectin B2a, an insecticidal and anti-parasitic drug, was accomplished. Although an acetylation of avermectin B2a using a 4-dimethylaminopyridine (DMAP) as a catalyst gave poor site-selectivity, use of our organocatalyst increased site-selectivity of the acylation at the C-5-OH as well as the yield of monoacetate. This catalyst was also effective in other acylations. Interestingly, trihaloacetylation under same conditions gave poor site-selectivity. However, the use of an enantiomer of our organocatalyst provided the C-4″-O-trihaloacetyl avermectin B2a with excellent site-selectivity. These results indicate that the site-selective acylation of avermectin B2a can be controlled by the combination of a suitable organocatalyst and an acid anhydride.

Organocatalytic Site-Selective Acylation of 10-Deacetylbaccatin III.

Organocatalytic site-selective diversification of 10-deacetylbaccatin III, a key natural product for the semisynthesis of taxol, has been achieved. Various acyl groups were selectively introduced into the C(10)-OH of 10-deacetylbaccatin III. The C(10)-OH selective acylation was also applied to acylative site-selective dimerization of 10-deacetylbaccatin III to provide the structurally defined dimer.

Origin of high E-selectivity in 4-pyrrolidinopyridine-catalyzed tetrasubstituted α,α'-alkenediol: a computational and experimental study.

We have developed 4-pyrrolidinopyridine catalysts for the geometry-selective (E-selective) acylation of tetrasubstituted α,α'-alkenediols. To elucidate the major factors of the high geometry selectivity, experimental and computational studies were carried out. The control experiments with respect to the substituent of the substrate indicated the fundamental hydrogen bonding of the acidic hydrogen of NHNs and the Z-OH in the substrate. Comparison between C2- and C1-symmetric catalysts exhibited the necessity of the C2-symmetric catalyst structure. The computationally proposed transition state (TS) model well explained the experimental results. Whereas the fundamental NH/amide-CO and the two-point free-OH/acetate anion hydrogen bonds stabilize the transition state (TS), affording the E-product, the steric repulsion between the N-protecting group and the amide side chain destabilizes TS, affording the Z-product. The role of the two amide side chains of the catalyst in a C2-symmetric fashion is the enhancement of the molecular recognition ability through the additional hydrogen bond in a cooperative manner.