Improved Electrochemical Peptide Synthesis Enabled by Electron-Rich Triaryl Phosphines.

While remarkable progress has been made in the development of peptide medicines, many problems related to peptide synthesis remain unresolved. Previously, we reported electrochemical peptide synthesis using a phosphine as a potentially recyclable coupling reagent. However, there was room for improvement from the point of view of reaction efficiency, especially in the carboxylic acid activation step and the peptide bond formation step. To overcome these challenges, we searched for the optimal phosphine. Among phosphines with various electronic properties, we found that electron-rich triaryl phosphines improved the reaction efficiency. Consequently, we successfully performed electrochemical peptide synthesis on sterically hindered and valuable amino acids. We also synthesized oligopeptides that were challenging with our previous method. Finally, we examined the effect of substituents on the phosphine cations, and gained some insights into reactivity, which will aid researchers designing reactions involving phosphine cations.

Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types.

BACKGROUND: The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system. RESULTS: The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut. CONCLUSIONS: These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Characterization and alteration of product specificity of Beijerinckia indica subsp. indica ß-fructosyltransferase.

The trisaccharide 1-kestose, a major constituent of fructooligosaccharide, has strong prebiotic effects. We used high-performance liquid chromatography and 1H nuclear magnetic resonance spectroscopy to show that BiBftA, a ß-fructosyltransferase belonging to glycoside hydrolase family 68, from Beijerinckia indica subsp. indica catalyzes transfructosylation of sucrose to produce mostly 1-kestose and levan polysaccharides. We substituted His395 and Phe473 in BiBftA with Arg and Tyr, respectively, and analyzed the reactions of the mutant enzymes with 180 g/L sucrose. The ratio of the molar concentrations of glucose and 1-kestose in the reaction mixture with wild-type BiBftA was 100:8.1, whereas that in the reaction mixture with the variant H395R/F473Y was 100:45.5, indicating that H395R/F473Y predominantly accumulated 1-kestose from sucrose. The X-ray crystal structure of H395R/F473Y suggests that its catalytic pocket is unfavorable for binding of sucrose while favorable for transfructosylation.

Identification of aminobenzoic acids as selective inhibitors of the N-terminal phosphatase of soluble epoxide hydrolase.

Soluble epoxide hydrolase (EC 3.3.2.10) is a key enzyme in the regulation of inflammation and metabolism, whereas, the role of its N-terminal phosphatase activity (N-phos) has been poorly understood because of a lack of selective inhibitors. Here we report 4-aminobenzoic (Ki 15.3 µm) and 3-amino-4-hydroxy benzoic acid (Ki 11.7 µm) as selective competitive inhibitors of N-phos.

Anti-Barnacle Activities of Isothiocyanates Derived from ß-Citronellol and Their Structure-Activity Relationships.

Antifouling agents with low toxicity are in high demand for sustaining marine industries and the environment. This study aimed to synthesize 15 isothiocyanates derived from ß-citronellol and evaluate their antifouling activities and toxicities against cypris larvae of the barnacle Amphibalanus amphitrite. The synthesized isothiocyanates exhibited effective antifouling activities (EC50 =0.10-3.33 µg mL-1 ) with high therapeutic ratios (LC50 /EC50 >30). Four isothiocyanates with an amide or isocyano group showed great potential as effective antifouling agents (EC50 =0.10-0.32 µg mL-1 , LC50 /EC50 =104-833). The enantiomers of the isothiocyanates only slightly differed in their antifouling activities. These results may serve as a basis for further research and development of ß-citronellol-derived isothiocyanates as effective low-toxic antifouling agents. To the best of our knowledge, this study is the first to report the antifouling activities of isothiocyanates derived from accessible natural products.

Oxidation Potential Gap (ΔEox ): The Hidden Parameter in Redox Chemistry.

Oxidative biaryl coupling of aryls with different electronic features generally fails. However, this has not been systematically studied via theoretical analysis, and thus, the crucial factor governing coupling efficiency remains unclear. Herein, we propose that the "oxidation potential gap (ΔEox )" is a key parameter in predicting the efficiency of an intramolecular oxidative coupling reaction, with ΔEox defined as a difference in the oxidation potentials of the relevant aromatic rings. Our experimental and computational analyses revealed that the efficiency of an aromatic intramolecular coupling reaction correlates with the activation energy (ΔE≠ ) of C-C bond formation of the radical cation intermediates. Furthermore, ΔE≠ correlates with ΔEox . Therefore, we demonstrate the tuning of ΔEox by attaching cleavable extra electron-donating/-withdrawing groups, enabling the rational synthesis of a phenanthridone skeleton using aromatic rings with an electronic gap.

Click Chemistry of Melamine Dendrimers: Comparison of "Click-and-Grow" and "Grow-Then-Click" Strategies Using a Divergent Route to Diversity.

Dendrimers are attractive macromolecules for a broad range of applications owing to their well-defined shapes and dimensions, highly branched and globular architectures, and opportunities for exploiting multivalency. Triazine dendrimers in particular offer advantages such as ease of synthesis, stability, well-defined spherical structure, multivalency, potential to achieve acceptable drug loadings, and low polydispersity. In this study, the potential utility of alkyne-azide "click" cycloadditions of first-, second-, and third-generation triazine dendrimers containing three or six alkynyl groups with benzyl azide was examined using copper catalysts. "Click-and-grow" and "grow-then-click" strategies were employed. For the first- and second- generation dendrimers, the desired triazole derivatives were obtained in high yields and purified by simple reprecipitation without column chromatography; however, some difficulties were observed in the preparation of third-generation dendrimers. The desired reaction proceeded under microwave irradiation as well as with simple heating. This click chemistry can be utilized for various melamine dendrimers that are fabricated with other amine linkers.

Biphasic electrochemical peptide synthesis.

The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint. To overcome this issue, we report an electrochemical peptide synthesis in a biphasic system. Anodic oxidation of triphenylphosphine (Ph3P) generates a phosphine radical cation, which serves as the coupling reagent to activate carboxylic acids, and produces triphenylphosphine oxide (Ph3P[double bond, length as m-dash]O) as a stoichiometric byproduct. In combination with a soluble tag-assisted liquid-phase peptide synthesis, the selective recovery of desired peptides and Ph3P[double bond, length as m-dash]O was achieved. Given that methods to reduce Ph3P[double bond, length as m-dash]O to Ph3P have been reported, Ph3P[double bond, length as m-dash]O could be a recyclable byproduct unlike byproducts from typical coupling reagents. Moreover, a commercial peptide active pharmaceutical ingredient (API), leuprorelin, was successfully synthesized without the use of traditional coupling reagents.

Hydrosilane-Mediated Electrochemical Reduction of Amides.

Electrochemical reduction of amides was achieved by using a hydrosilane without any toxic or expensive metals. The key reactive ketyl radical intermediate was generated by cathodic reduction. Continuous reaction with anodically generated silyl radicals or zinc bromide resulted in chemoselective deoxygenation to give the corresponding amines.

Construction of TUATinsecta database that integrated plant and insect database for screening phytophagous insect metabolic products with medicinal potential.

Phytophagous insect larvae feed on plants containing secondary metabolic products with biological activity against other predatory organisms. Phytophagous insects can use their specialised metabolic systems to covert these secondary metabolic products into compounds with therapeutic properties useful to mankind. Some Asians drink tea decoctions made from phytophagous insect frass which is believed to be effective against inflammatory diseases. However, insects that can convert plant-derived secondary metabolic products into useful human therapeutic agents remain poorly studied. Here, we constructed the TUATinsecta database by integrating publicly plant/insect datasets for the purpose of selecting insect species. Using TUAT-insecta we selected the Asian swallowtail butterfly, Papilio xuthus larvae fed on several species of Rutaceous plants and examined whether the plant-derived secondary metabolites, especially those present in frass, were chemically altered or not. We extracted metabolic products from frass using three organic solvents with different polarities, and evaluated solvent fractions for their cytotoxic effects against several human cell lines. We found that chloroform frass extracts from P. xuthus larvae fed on Poncirus trifoliata leaves contained significant cytotoxic activity. Our findings demonstrate that screening of insect species using the 'TUATinsecta' database provides an important pipeline for discovering novel therapeutic agents that might be useful for mankind.

Confirmation of the absolute configuration of Stachybotrin C using single-crystal X-ray diffraction analysis of its 4-bromobenzyl ether derivative.

The absolute configuration of Stachybotrin C was confirmed in this study. After synthesizing the dimethyl ethers of Stachybotrin C, the C-8 epimer was analyzed by 1D NOESY. However, the stereochemistry determination was difficult through the NOE correlations. Instead, the di(4-bromobenzyl) ether of Stachybotrin C was derived and used for X-ray diffraction analysis, because its single crystal was easier to obtain than that of the original Stachybotrin C. The stereochemistry of Stachybotrin C was determined to be (8S, 9R). This derivatization approach may also be used to prepare single crystals of the analogues.

Isocyanides Derived from α,α-Disubstituted Amino Acids: Synthesis and Antifouling Activity Assessment.

Herein, we contribute to the development of environmentally friendly antifoulants by synthesizing eighteen isocyanides derived from α,α-disubstituted amino acids and evaluating their antifouling activity/toxicity against the cypris larvae of the Balanus amphitrite barnacle. Almost all isocyanides showed good antifouling activity without significant toxicity and exhibited EC50 values of 0.07 - 7.30 µg/mL after 120-h exposure. The lowest EC50 values were observed for valine-, methionine-, and phenylalanine-derived isocyanides, which achieved > 95% cypris larvae settlement inhibition at concentrations of less than 30 µg/mL without exhibiting significant toxicity. Thus, the prepared isocyanides should be useful for further research focused on the development of environmentally friendly antifouling agents.

Anti-barnacle Activity of Isocyanides Derived from Amino Acids.

Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid-isocyanides exhibited potent anti-barnacle activity with EC50 values of 0.07 - 10.34 µg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 µg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid-isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants.

Isoprene Side-chain of SMTP is Essential for Soluble Epoxide Hydrolase Inhibition and Cellular Localization.

SMTPs, a family of natural small molecules that effectively treat ischemic stroke, are subject to clinical development. SMTPs enhance plasminogen activation and inhibit soluble epoxide hydrolase (sEH), leading to promotion of endogenous thrombolysis and anti-inflammation. The SMTP molecule consists of atricyclic γ-lactam moiety, an isoprene side-chain, and an N-linked side-chain. Here, we investigate the yet-to-be-characterized function of the isoprene side- chain of SMTPs in sEH inhibition and cellular distribution. The results demonstrated that oxidative modification as well as truncation of the side-chain abolished epoxide hydrolase inhibition. The introduction of a terminal hydroxy group exceptionally unaffected epoxide hydrolase, but led to impaired cellular localization, resulting in diminution of cellular epoxide hydrolase inhibition. Thus, the isoprene side-chain of SMTP is an important pharmacophore for epoxide hydrolase inhibition and cellular localization.

Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.

Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with ß-D-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-D-glucopyranosyl-α-D-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK.

Electrochemical synthesis of azanucleoside derivatives using a lithium perchlorate-nitromethane system.

We have developed a highly efficient synthetic method for azanucleosides using a lithium perchlorate-nitromethane reaction medium, allowing direct and exclusive installation of various nucleophiles, including protected nucleobases into prolinol derivatives at the preferred 5-position.

The mode of action of isocyanide in three aquatic organisms, Balanus amphitrite, Bugula neritina and Danio rerio.

Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml(-1)), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the "entry enzyme" of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused "wavy" notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish.