Kohei Torikai

Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides

We report here the ribosomal synthesis of methyllanthionine-containing cyclic peptides involving a site-specific incorporation of vinylglycine under the reprogrammed genetic code, followed by the isomerization of the vinylglycine to dehydrobutyrine, and the subsequent intramolecular Michael addition of a cysteine residue placed at a downstream position of the vinylglycine.

Surface plasmon resonance-based detection of ladder-shaped polyethers by inhibition detection method

Ladder-shaped polyether (LSP) compounds represented by brevetoxins and ciguatoxins were largely discovered in association with seafood poisoning. Thus, a quick quantification method for LSPs is potentially important. We examined a surface plasmon resonance method using desulfated-yessotoxin (dsYTX) immobilized on a sensor chip and phosphodiesterase PDEII in a inhibition detection mode. Yessotoxin, brevetoxin B and synthetic LSP derivatives showed clear inhibition against PDEII binding to the immobilized dsYTX, by which their half inhibitory concentrations were successfully estimated. This inhibition method appeared to be superior in specificity to direct binding assays where binding proteins to LSP was immobilized on a sensor chip.

Interaction of ladder-shaped polyethers with transmembrane α-helix of glycophorin A as evidenced by saturation transfer difference NMR and surface plasmon resonance

Ladder-shaped polyether (LSP) compounds are thought to interact with transmembrane alpha-helices, but direct evidence has scarcely obtained for these interactions. We adopted a transmembrane alpha-helix of glycophorin A, and quantitatively evaluated its interaction with LSPs such as yessotoxin (YTX), desulfated YTX and artificial LSPs, using surface plasmon resonance and saturation transfer difference NMR. As a result, dissociation constants (K(D)) of YTX and desulfated YTX to a transmembrane domain peptide of glycophorin A were determined to be in the submillimolar range. Furthermore, in saturation transfer difference NMR, the signals at the polyene side chain and the angular methyl groups of YTX were significantly attenuated, which probably comprised an interacting interface of LSPs with a transmembrane alpha-helix. These results suggest that hydrophobic interaction plays an important role in molecular recognition of the alpha-helix peptide by LSPs.

Synthesis and DNA cleaving activity of water-soluble non-conjugated thienyl tetraynes.

Water-soluble non-conjugated thienyl tetraynes (3-6) were synthesized and their DNA cleaving activity was evaluated using electrophoresis, atomic force microscopy (AFM) and Escherichia coli (E. coli) transformation techniques. The amino-functionalized compound 4 was shown to possess an activity to cleave plasmid DNA by both electrophoresis and E. coli transformation techniques. AFM also showed a cleavage of the circular DNA into a linear form with a formation of burst-star-shaped architectures, which were envisaged to be cross-linked DNA oligomers.

Ribosomal synthesis of an amphotericin-B inspired macrocycle.

Here we report in vitro ribosomal synthesis of a natural product-like macrocyclic peptide, inspired by the structure of amphotericin B (AmB), an amphiphilic and membrane-interacting antifungal natural product. This AmB-inspired macrocyclic peptide (AmP), one side of which is composed of hydrophobic terpene, and the other side comprises a peptidic chain, was synthesized utilizing flexizyme-assisted in vitro translation via an unusual but successful initiation with a D-cysteine derivative. The established method for the synthesis of AmPs is applicable to the generation of a diverse AmP library coupled with an in vitro display format, with the potential to lead to the discovery of artificial bioactive amphiphilic macrocycles.

Artificial ladder-shaped polyethers that inhibit maitotoxin-induced Ca2+ influx in rat glioma C6 cells

Maitotoxin (MTX) is a ladder-shaped polyether produced by the epiphytic dinoflagellate Gambierdiscus toxicus. It is known to elicit potent toxicity against mammals and induce influx of Ca(2+) into cells. An artificial ladder-shaped polyether possessing a 6/7/6/6/7/6/6 heptacyclic ring system, which was designed for elucidating interactions with transmembrane proteins, was found to be the most potent inhibitor against MTX-induced Ca(2+) influx that has ever been reported.

Synthesis of Artificial Ladder-Shaped Polyethers Containing a 6/7 cis-Fused Ring System

Ladder-shaped tetracyclic and heptacyclic ethers containing a 6/7 cis-fused ring system have been synthesized. The synthesis features convergent coupling of monocyclic building blocks through esterification, ring-closing reaction using a low-valent titanium complex, and hydroxy dithioacetal cyclization. The double reaction strategy enabled expeditious synthesis of the heptacyclic ether in only thirteen steps from the building blocks.

Guest responsivity of a two-dimensional coordination polymer incorporating a cholesterol-based co-ligand.

To implement specific guest responsivity, a hydrophobic cholesterol-based co-ligand, cholest-5-en-3-yl-4-isonicotinate (Cholpy), was incorporated into a two-dimensional Hofmann-type Co(II)Ni(II) coordination polymer. The chemically programmed structure successfully demonstrated the unique guest response with remarkable chromatic changes.

Practical Synthesis of DOPA Derivative for Biosynthetic Production of Potent Antitumor Natural Products, Saframycins and Ecteinascidin 743

A practical synthetic route of DOPA derivative 2, which should be useful for direct biosynthetic production of potent antitumor natural products, saframycins and ecteinascidin 743 was established. The developed strategy features i) easy-to-handle reactions without special care upon both dryness and inert atmopsphere, and ii) the facile HPLCfree purification of 2 via recrystallization enabling scalable synthesis of 2. Introduction Saframycins (SMs), produced by Streptomyces and various soil bacteria as well as marine vertebrates such as ascidians and sponges, are potent antitumor antibiotics [1]. In particular, a highly potent SM analog, ecteinascidin 743 [2] (ET-743), has recently been in use as an anticancer drug against soft-tissue sarcoma [3]. ETs share the central pentacyclic tetrahydroisoquinoline core with SMs, except for the oxidation state of their terminal rings and the additional ten-membered lactone bridge found in ET-743. Due to the short supply from natural resources, the production of ET-743 should depend on a semi-synthesis including 21 synthetic steps [4]. In order to facilitate the direct biosynthetic production of SMs including ETs, unremitting bioinfomatic analyses were carried out, and it was found that SMs are biosynthesized from L-alanine, glycine and two molecules of 3,4dihydroxyphenylalanine (DOPA) derivative 1 [5] through dual Pictet-Spengler (PS) mechanism [6] (Scheme 1). Briefly, tetrahydroisoquinoline core is constructed by the following three steps; i) Schiff base is formed between DOPA derivative 1 and dipeptidic aldehyde 3, generated from glycine and Lalanine by the aid of non-ribosomal polypeptide synthetases (NRPSs); ii) PS cyclization occurs to give 4; and iii) enzymatic region is reductively eliminated to afford aldehyde 5, which is involved in the same sequence to furnish SMs and ETs through intermediates 6 and 7. Thus, to develop an engineered perpetual SM-producing system, we cloned necessary biosynthetic gene clusters for SMs and expressed them in model creatures, however, it was unsuccessful to detect the production of SMs even by mass spectrometric analysis [7]. We envisaged that one of the reasons would be insufficient amount of endogenous non-natural amino acid 1, and if 2 were fed from outside the system, enough supply of SMs would be realized. Herein, we report practical and scalable synthesis of amino acid 2 to tolerate the feeding experiments. Results and Discussion Synthesis of DOPA derivative 2 commenced with N-t-butoxycabonyl (N-Boc) tyrosine 8 according to the Schmidt’s report [8], one of the most expeditious and easy-to-handle methods to date [9] (Scheme 2). Aldehyde 9 was prepared by Reimer-Tiemann formylation [10a] and the subsequent esterification of N-Boc tyrosine 8 as reported previously [8,10b]. Although we attempted the Scheme 1. Hypothetical Biosynthesis of Saframycin A and Ecteinascidin 743. conversion of 9 into iodobenzene 10 by the action of I2 and H2O2, the reaction could not be reproduced even in refluxing ethanol. Hence, we decided to set a robust and reproducible route for the two-steps-introduction of iodine as follows; (i) NaBH4 reduction of aldehyde 9 into the corresponding alcohol [9a], and (ii) iodination of the resulting alcohol with I2/H2O2 combination. As a result, probably due to the fact that electronwithdrawing formyl group was converted to electron-donating hydroxymethyl group, electrophilic iodination proceeded smoothly to afford 11 in good yield (69% for two steps). Our next task was to oxidize alcohol 11 into Schmidt’s intermediate 10 with MnO2 under argon atmosphere, however, our endeavor was wasteful only to observe the decomposition of substrate 11. We postulated that o-hydroxybenzaldehyde structure of 10 would be unstable even to mild heterogeneous oxidant under inert atmosphere, therefore, methylation of phenolic hydroxy group was first performed. After selective methylation of 11 using methyl iodide and potassium carbonate Practical Synthesis of DOPA Derivative for Biosynthetic Production of Potent Antitumor Natural Products, Saframycins and Ecteinascidin 743

Design and synthesis of benzoacridines as estrogenic and anti-estrogenic agents

Estrogens play undisputedly important physiological roles, but lifetime exposure to estrogens has also been linked to the development of breast cancer. Moreover, imbalanced estrogen levels have been associated with various symptoms such as osteoporosis and menopausal disorders. For the improvement of such estrogen imbalances, estrogenic reagents with regulatory properties have shown promising potential. Herein, we report the construction of a 12-arylbenzoacridine library via a diversity-oriented strategy that furnished non-toxic estrogenic and anti-estrogenic agents. Derivatives with a hydroxy group at the molecular edge exhibit potent binding affinity to the estrogen receptor α (ERα) and ERβ (IC50 < μM), while binding to the estrogen-related receptor γ (ERRγ), i.e., an orphan nuclear receptor on which estrogens often trigger unfavorable events, was not observed. These findings offer valuable insights into 12-arylbenzoacridines as a novel platform for the development of selective estrogen-receptor modulators (SERMs).