Differential Metabolic Stability of 4α,25- and 4ß,25-Dihydroxyvitamin D3 and Identification of Their Metabolites.

Vitamin D3 (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D3 (6a) and 4ß,25-dihydroxyvitamin D3 (6b) are both produced from 25-hydroxyvitamin D3 (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4ß,24R,25-trihydroxyvitamin D3 (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the ß-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.

Regulation of thrombin activity by ligand-induced topological alteration in a thrombin-binding aptamer.

Thrombin-binding aptamer (TBA), which forms a G-quadruplex (G4) structure with anti-parallel topology, interacts with thrombin to inhibit its enzymatic activity. Here we show that the G4-topology-altering ligand L2H2-2M2EA-6LCO (6LCO) changes the anti-parallel topology of TBA G4 to the parallel topology, thereby abrogating the thrombin-inhibitory activity of TBA. This finding suggests that G4 ligands that alter topology may be promising drug candidates for diseases involving G4-binding proteins.

Mechanism of rate controllability of water-soluble bifunctional cyclooctadiynes through cation-anion interactions.

Click reactions are used for chemoselective functionalization in many research fields. Despite the utility of small, bioinert azide groups as a counterpart, applications of strain-promoted alkyne-azide cycloaddition (SPAAC) reactions for this purpose are still limited by slow reaction kinetics. Here, we report ion-pair-guided reaction rate enhancement by the use of water-soluble cyclooctadiynes (WS-CODYs) composed of bifunctional strained alkynes and polar side chains. Arrhenius plot analysis revealed that the rate enhancement by WS-CODYs is due to a kinetic salt effect between the polar substituents and the target azide. We demonstrate the utility of these compounds for rapid protein labelling and isoelectric point-dependent labelling.

Ion-Pair-Enhanced Double-Click Driven Cell Adhesion and Altered Expression of Related Genes.

Bio-orthogonal ligations that crosslink living cells with a substrate or other cells require high stability and rapid kinetics to maintain the nature of target cells. In this study, we report water-soluble cyclooctadiyne (WS-CODY) derivatives that undergo an ion-pair enhanced double-click reaction. The cationic side chain of WS-CODY accelerated the kinetics on the azide-modified cell surface due to proximity effect. Cationic WS-CODY was able to crosslink azide-modified, poorly adherent human lung cancer PC-9 cells not only to azide-grafted glass substrates but also to other cells within 5-30 min. We discovered that cell-substrate crosslinking induced the ITGA5 gene expression, whereas cell-cell crosslinking induced the CTNNA1 gene, according to the adhesion partner. Ion-pair-enhanced WS-CODY can be applied to a wide range of cells with established azide modifications and is expected to provide a powerful tool to regulate cell-substrate and cell-cell interactions.

Synthesis of Deuterium-Labeled Vitamin D Metabolites as Internal Standards for LC-MS Analysis.

Blood levels of the vitamin D3 (D3) metabolites 25-hydroxyvitamin D3 (25(OH)D3), 24R,25-dihydroxyvitamin D3, and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) are recognized indicators for the diagnosis of bone metabolism-related diseases, D3 deficiency-related diseases, and hypercalcemia, and are generally measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using an isotope dilution method. However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum.

Synthesis of C2-Alkoxy-Substituted 19-Nor Vitamin D3 Derivatives: Stereoselectivity and Biological Activity.

The active form of vitamin D3 (D3), 1a,25-dihydroxyvitamn D3 (1,25D3), plays a central role in calcium and bone metabolism. Many structure-activity relationship (SAR) studies of D3 have been conducted, with the aim of separating the biological activities of 1,25D3 or reducing its side effects, such as hypercalcemia, and SAR studies have shown that the hypercalcemic activity of C2-substituted derivatives and 19-nor type derivatives is significantly suppressed. In the present paper, we describe the synthesis of 19-nor type 1,25D3 derivatives with alkoxy groups at C2, by means of the Julia-Kocienski type coupling reaction between a C2 symmetrical A ring ketone and a CD ring synthon. The effect of C2 substituents on the stereoselectivity of the coupling reaction was evaluated. The biological activities of the synthesized derivatives were evaluated in an HL-60 cell-based assay. The a-methoxy-substituted C2α-7a was found to show potent cell-differentiating activity, with an ED50 value of 0.38 nM, being 26-fold more potent than 1,25D3.

Vinylnaphthalene-bearing hexaoxazole as a fluorescence turn-on type G-quadruplex ligand.

Oxazole-type fluorophores show an increase of fluorescence intensity upon interaction with nucleic acids, and therefore can be used as tools for nucleic acid-sensing and fluorescence imaging. Here, we developed a novel stilbene-type fluorophore, MO-VN (1), consisting of a mono oxazole bearing a vinyl naphthalene moiety. This compound (1) was embedded in a trioxazole 2 and a cyclic hexaoxazole 3a. The fluorescence properties of 1, 2, and 3a were evaluated in the presence of various nucleic acid sequences. Compound 3 showed significant fluorescent enhancement upon interacting with G-quadruplex (G4) structure, which plays critical roles in various biological phenomena. Further structural development focusing on the vinyl naphthalene moiety of 3a afforded a turn-on type G4 ligand 3e that shows G4-specific fluorescence. Measurement of the fluorescence of 3e during titration of a telomeric DNA, telo24, with its C-rich complementary sequence, which unwinds the G4 structure, allowed us to monitor the dynamics of G4.

Stabilization of telomeric G-quadruplex by ligand binding increases susceptibility to S1 nuclease.

The extent of thermodynamic stabilization of telomeric G-quadruplex (G4) by isomers of G4 ligand L2H2-6OTD, a telomestatin analog, is inversely correlated with susceptibility to S1 nuclease. L2H2-6OTD facilitated the S1 nuclease activities through the base flipping in G4, unlike the conventional role of G4 ligands which inhibit the protein binding to DNA/RNA upon ligand interactions.

Target identification of a macrocyclic hexaoxazole G-quadruplex ligand using post-target-binding visualization.

Macrocyclic hexaoxazoles (6OTDs) are G-quadruplex (G4) ligands, and some derivatives, such as L2H2-6OTD (1a) bearing two aminobutyl side chains, show cytotoxicity towards cancer cells. To identify the cellular target of 1a, we employed a post-target-binding strategy utilizing click reaction (Huisgen cyclization) between the azide-conjugated ligand L2H2-6OTD-Az (1b) and the cell-permeable dye CO-1 bearing a strained alkyne moiety and the BODIPY fluorophore under Cu-free conditions. We confirmed that introduction of the small azide group did not alter the physical or biological properties, including anti-cancer activity, of 1a, and we also demonstrated bias-free localization of CO-1. The post-binding visualization strategy suggested that L2H2-6OTD (1a) colocalized with RNA G4 in living cells.

Cross-linking cellular nucleic acids via a target-directing double click reagent.

Bioorthogonal ligation reactions are powerful tools for characterizing DNA metabolism, DNA-protein binding interactions, and they even provide new leads for therapeutic strategies. Nucleoside analogs can deliver bioorthogonal functional groups into chromatin via cellular metabolic pathways, however, insufficient phosphorylation by endogenous kinases often limits the efficiency of their incorporation. Even when successfully metabolized into biopolymers, steric hindrance of the modified nucleotide by chromatin can inhibit subsequent click reactions. In this chapter, we describe methods that overcome these limitations. Nucleotide monophosphate triesterers can bypass the need for cellular nucleoside kinase activity and thereby enable efficient incorporation of azide groups into cellular DNA. Steric access to and modification of the azide groups within natively folded chromatin can then be accomplished using a bioorthogonal "intercalating reagent" comprised of a cationic Sondheimer diyne that reversibly intercalates into duplexes where it undergoes tandem, strain-promoted cross-linking of two azides to give DNA-DNA interstrand crosslinks or DNA-fluorophore conjugation, depending on the relative number and spatial orientation of the azide groups in the DNA.

Author Correction: Identification of a binding protein for sesamin and characterization of its roles in plant growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Identification of a binding protein for sesamin and characterization of its roles in plant growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Linear consecutive hexaoxazoles as G4 ligands inducing chair-type anti-parallel topology of a telomeric G-quadruplex.

G-quadruplex structures (G4s) in guanine-rich regions of DNA play critical roles in various biological phenomena, including replication, translation, and gene expression. There are three types of G4 topology, i.e., parallel, anti-parallel, and hybrid, and ligands that selectively interact with or stabilize a specific topology have been extensively explored to enable studies of topology-related functions. Here, we describe the synthesis of a new series of G4 ligands based on 6LCOs (6-linear consecutive oxazoles), i.e., L2H2-2M2EA-6LCO (2), L2A2-2M2EAc-6LCO (3), and L2G2-2M2EG-6LCO (4), which bear four aminoalkyl, acetamidealkyl, and guanidinylalkyl side chains, respectively. Among them, ligand 2 stabilized telomeric G4 and induced anti-parallel topology independently of the presence of cations. The anti-parallel topology induced by 2 was identified as chair-type by means of 19F NMR spectroscopy and fluorescence experiments with 2-aminopurine-labeled DNA.

Three-Component Bioorthogonal Reactions on Cellular DNA and RNA.

Metabolic incorporation of bioorthogonal functional groups into chromatin, followed by copper-free conjugation reactions, often gives low yields due to steric hindrance. Here we report that a cationic Sondheimer diyne derivative "DiMOC" rapidly reacts with azide groups in duplex DNA that are otherwise unreactive toward aliphatic cyclooctynes such as bicyclo[6.1.0]nonyne (BCN). DiMOC reversibly intercalates into duplex DNA and undergoes tandem strain-promoted cross-linking of two different azide groups to give DNA-DiMOC-"X" cross-links, where "X" theoretically represents a fluorescent probe, affinity tag, RNA, protein, carbohydrate, and so forth. As a proof of principle, the metabolic incorporation of azide-modified nucleosides into cellular DNA or RNA, followed by treatment with DiMOC and a fluorescent azide enabled visualization of newly synthesized nucleic acids in whole cells.

Identification of a binding protein for sesamin and characterization of its roles in plant growth.

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

Intercalation-enhanced "Click" Crosslinking of DNA.

DNA-DNA cross-linking agents constitute an important family of chemotherapeutics that non-specifically react with endogenous nucleophiles and therefore exhibit undesirable side effects. Here we report a cationic Sondheimer diyne derivative "DiMOC" that exhibits weak, reversible intercalation into duplex DNA (Kd =15 µm) where it undergoes tandem strain-promoted cross-linking of azide-containing DNA to give DNA-DNA interstrand crosslinks (ICLs) with an exceptionally high apparent rate constant kapp =2.1×105 m-1 s-1 . This represents a 21 000-fold rate enhancement as compared the reaction between DIMOC and 5-(azidomethyl)-2'-deoxyuridine (AmdU) nucleoside. As single agents, 5'-bispivaloyloxymethyl (POM)-AmdU and DiMOC exhibited low cytotoxicity, but highly toxic DNA-DNA ICLs were generated by metabolic incorporation of AmdU groups into cellular DNA, followed by treatment of the cells with DiMOC. These results provide the first examples of intercalation-enhanced bioorthogonal chemical reactions on DNA, and furthermore, the first strain-promoted double click (SPDC) reactions inside of living cells.

In Vivo Incorporation of Azide Groups into DNA by Using Membrane-Permeable Nucleotide Triesters.

Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl-2'-deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low-fidelity thymidine kinase, but not by wild-type HeLa cells. Here we report that membrane-permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild-type cells and animals. AmdU monophosphate derivatives bearing either 5'-bispivaloyloxymethyl (POM), 5'-bis-(4-acetoxybenzyl) (AB), or "Protide" protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative "POM-AmdU" exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than "AB-AmdU". Remarkably, the addition of POM-AmdU to the water of zebrafish larvae enabled the biosynthesis of azide-modified DNA throughout the body.

Author Correction: Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame.

The original version of of the Supplementary Information associated with this Article inadvertently omitted oligonucleotide primer sequences from Supplementary Table 3 and Supplementary Methods describing the molecular cloning of CYP92B14, CPR1 and CYP81Q cDNA fragments. The HTML has been updated to include a corrected version of the Supplementary Information.

Oxidative rearrangement of (+)-sesamin by CYP92B14 co-generates twin dietary lignans in sesame.

(+)-Sesamin, (+)-sesamolin, and (+)-sesaminol glucosides are phenylpropanoid-derived specialized metabolites called lignans, and are rich in sesame (Sesamum indicum) seed. Despite their renowned anti-oxidative and health-promoting properties, the biosynthesis of (+)-sesamolin and (+)-sesaminol remained largely elusive. Here we show that (+)-sesamolin deficiency in sesame is genetically associated with the deletion of four C-terminal amino acids (Del4C) in a P450 enzyme CYP92B14 that constitutes a novel clade separate from sesamin synthase CYP81Q1. Recombinant CYP92B14 converts (+)-sesamin to (+)-sesamolin and, unexpectedly, (+)-sesaminol through an oxygenation scheme designated as oxidative rearrangement of α-oxy-substituted aryl groups (ORA). Intriguingly, CYP92B14 also generates (+)-sesaminol through direct oxygenation of the aromatic ring. The activity of CYP92B14 is enhanced when co-expressed with CYP81Q1, implying functional coordination of CYP81Q1 with CYP92B14. The discovery of CYP92B14 not only uncovers the last steps in sesame lignan biosynthesis but highlights the remarkable catalytic plasticity of P450s that contributes to metabolic diversity in nature.

Design and synthesis of a berberine dimer: a fluorescent ligand with high affinity towards G-quadruplexes.

G-quadruplexes (G4) are thought to be important factors for telomerase inhibition and transcriptional/translational modulations. Bioinformatic analyses imply that the human genome and mRNA contain a multitude of G4-forming sequences; however, their analysis requires selective and detectable ligands. Given that two molecules of fluorescent berberine (BBR) coordinate to telomeric G4 in their co-crystals, we designed hydrocarbon-linked BBR-analogue dimers because we expected the alignment of two BBR chromophores would avoid Watson-Crick base pair intercalation, which should result in high selectivity towards G4. An alkene-cis-C2 BBR dimer showed the highest affinity (Kd ≤2.6 nM) and selectivity (ca. 900-fold vs. duplex) towards G4. The intrinsic "light-up" fluorescence properties of this BBR dimer, derived from its conformational switching by G4, allowed a selective visualization of various G4 in the gel without using additional bulky fluorescence dyes, which, combined with the observed lack of conformational change of the ligand, suggested future applications in in vitro detection systems.