Shinichiro Fuse

Efficient amide bond formation through a rapid and strong activation of carboxylic acids in a microflow reactor.

The development of highly efficient amide bond forming methods which are devoid of side reactions, including epimerization, is important, and such a method is described herein and is based on the concept of rapid and strong activation of carboxylic acids. Various carboxylic acids are rapidly (0.5 s) converted into highly active species, derived from the inexpensive and less-toxic solid triphosgene, and then rapidly (4.3 s) reacted with various amines to afford the desired peptides in high yields (74 %–quant.) without significant epimerization (≤3 %). Our process can be carried out at ambient temperature, and only CO2 and HCl salts of diisopropylethyl amine are generated. In the long history of peptide synthesis, a significant number of active coupling reagents have been abandoned because the highly active electrophilic species generated are usually susceptible to side reactions such as epimerization. The concept presented herein should renew interest in the use of these reagents.

Structural Analysis of the Contacts Anchoring Moenomycin to Peptidoglycan Glycosyltransferases and Implications for Antibiotic Design

Peptidoglycan glycosyltransferases (PGTs), enzymes that catalyze the formation of the glycan chains of the bacterial cell wall, have tremendous potential as antibiotic targets. The moenomycins, a potent family of natural product antibiotics, are the only known active site inhibitors of the PGTs and serve as blueprints for the structure-based design of new antibacterials. A 2.8 A structure of a Staphylococcus aureus PGT with moenomycin A bound in the active site appeared recently, potentially providing insight into substrate binding; however, the protein-ligand contacts were not analyzed in detail and the implications of the structure for inhibitor design were not addressed. We report here the 2.3 A structure of a complex of neryl-moenomycin A bound to the PGT domain of Aquifex aeolicus PBP1A. The structure allows us to examine protein-ligand contacts in detail and implies that six conserved active site residues contact the centrally located F-ring phosphoglycerate portion of neryl-moenomycin A. A mutational analysis shows that all six residues play important roles in enzymatic activity. We suggest that small scaffolds that maintain these key contacts will serve as effective PGT inhibitors. To test this hypothesis, we have prepared, via heterologous expression of a subset of moenomycin biosynthetic genes, a novel moenomycin intermediate that maintains these six contacts but does not contain the putative minimal pharmacophore. This compound has comparable biological activity to the previously proposed minimal pharmacophore. The results reported here may facilitate the design of antibiotics targeted against peptidoglycan glycosyltransferases.

Complete characterization of the seventeen step moenomycin biosynthetic pathway

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

Elucidating the Structure–Property Relationships of Donor–π-Acceptor Dyes for Dye-Sensitized Solar Cells (DSSCs) through Rapid Library Synthesis by a One-Pot Procedure

The creation of organic dyes with excellent high power conversion efficiency (PCE) is important for the further improvement of dye-sensitized solar cells. We wish to describe the rapid synthesis of a 112-membered donor-π-acceptor dye library by a one-pot procedure, evaluation of PCEs, and elucidation of structure-property relationships. No obvious correlations between ε, and the η were observed, whereas the HOMO and LUMO levels of the dyes were critical for η. The dyes with a more positive E(HOMO), and with an E(LUMO)<-0.80 V, exerted higher PCEs. The proper driving forces were crucial for a high J(sc), and it was the most important parameter for a high η. The above criteria of E(HOMO) and E(LUMO) should be useful for creating high PCE dyes; nevertheless, that was not sufficient for identifying the best combination of donor, π, and acceptor blocks. Combinatorial synthesis and evaluation was important for identifying the best dye.

Ribosomal Synthesis of Backbone-Macrocyclic Peptides Containing γ-Amino Acids

Pepstatin, discovered in 1970 as an extraordinarily potent inhibitor of pepsin, is a classical example of a g-amino acid-containing natural product. It has been proven that the statine residue, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, mimics the tetrahedral transition-state structure of peptidebond hydrolysis. This knowledge led to the development of specific inhibitors of clinically relevant aspartic proteases, including renin and HIV proteases. More recently, a new class of g-amino acid-containing natural products has been discovered that is active against a non-protease enzyme. Spiruchostatin A, which was isolated from Pseudomonas sp., is a representative example; it has a macrocyclic structure containing a dvaline-derived (3S,4R)-statine and acts as a histone deacetylase inhibitor. Clearly, the statine residue in this molecule does not act as a transition-state analogue but rather serves as a critical component of the macrocyclic scaffold. Moreover, didemnins B, isolated from the genus Trididemnum as an agent against kidney and epithelial ovarian cancer, has a g-amino acid, isostatine, and also has a macrocyclic moiety. These examples imply that statine and probably other g-amino acids in a macrocyclic scaffold can be versatile structural elements for constructing bioactive peptides. Despite the attractiveness of g-amino acid-containing peptides, their availability still relies on the traditional methodologies, that is, the serendipitous discovery of bioactive g-amino acid-containing peptides from secondary-metabolite sources or by chemical synthesis from small libraries. We here report a new methodology involving genetic code reprogramming to ribosomally express backbone-cyclized peptides containing g-amino acids (Figure 1 A) that potentially gives us more diverse libraries of g-amino acid-containing peptides with a greater ease. Since the translation apparatus allows for the facile construction of a peptide library with vast diversity, ribosomal synthesis of drug-like peptides can be a powerful tool for discovering novel bioactive compounds. However, despite a number of reports describing the incorporation of various nonstandard amino acids into nascent peptide chains by using a conventional strategy such as nonsense or frame-shift suppression, to the best of our knowledge, a successful demonstration of ribosomal incorporation of g-amino acids has never appeared in literature. There are two reasons for this failure: 1) g-Aminoacyl-tRNA is not stable under near-neutral aqueous conditions as the g-amino group tends to intramolecularly attack the acyl group on the 3’ terminus of tRNA; this results in self-deacylation. 2) Even if some fractions of g-aminoacyl-tRNA can be brought into the ribosome A site, the ribosome very likely fails to promote the peptidyl-transfer reaction as its catalytic environment is incompatible with bond formation between the gamino group and the acyl group on the P site tRNA. As a result, no successful example of expressing any peptide containing g-amino acids has been reported. We have recently developed two new methods of generating unique peptides by using a custom-made, reconstituted, cell-free translation system integrated with flexizymes (flexible tRNA aminoacylation ribozymes) and referred to as FIT (flexible in-vitro translation) system. Using this system, we reprogrammed an initiation event in translation in which a FIT system lacking methionine was supplemented with a tRNA CAU charged with short exotic peptides by flexizyme; thereby, peptides containing unusual auxiliary residues at the N terminus were expressed. The second method was ribosomal synthesis of backbone-cyclic peptides in a FIT system in which a codon was assigned to glycolic acid (G). Expression of linear peptides bearing cystidyl-prolidyl-glycolate (C-P-G) resulted in self-rearrangement into peptides with a C-terminal diketopiperadine (dkp) thioester upon cleavage of the ester bond between P and G. The presence of two recombinant enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP), in the FIT system generates an N-terminal free amino group that spontaneously reacts with the intramolecular dkpthioester to afford the backbone-cyclized peptides. Although direct acylation of g-amino acid onto tRNA would suffer from self-deacylation through intramolecular cyclization, acylation of a dipeptide consisting of gand a-amino acids onto tRNA could avoid such an undesired side reaction. To test whether this approach would yield stable peptidyl-tRNAs, we [a] Dr. Y. Goto, Prof. Dr. H. Suga Department of Chemistry, Graduate School of Science The University of Tokyo 7-3-1, Hongo, Bunkyo, Tokyo 113-0033 (Japan) Fax: (+ 81) 3-5841-8372 E-mail : hsuga@chem.s.u-tokyo.ac.jp [b] Y. Ohshiro, Prof. Dr. H. Suga Department of Advanced Interdisciplinary Studies Graduate School of Engineering, The University of Tokyo 4-6-1, Komaba, Meguro, Tokyo 153-8904 (Japan) [c] Dr. E. Nakajima, Prof. Dr. H. Suga Department of Chemistry and Biotechnology Graduate School of Engineering, The University of Tokyo 7-3-1, Hongo, Bunkyo, Tokyo 113-8656 (Japan) [d] Dr. S. Fuse, Prof. Dr. T. Takahashi Department of Applied Chemistry, Tokyo Institute of Technology 2-12-1, Ookayama, Meguro, Tokyo 152-8552 (Japan) [e] Prof. Dr. T. Doi Graduate School of Pharmaceutical Sciences, Tohoku University 6-3, Aza-aoba, Aramaki, Aoba, Sendai, Miyagi 980-8578 (Japan) [f] Prof. Dr. H. Suga WCU Department of Molecular Medicine and Biopharmaceutical Sciences Seoul National University, Seoul 151-742 (Korea) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100104.

Functional and Structural Analysis of a Key Region of the Cell Wall Inhibitor Moenomycin

Moenomycin A (MmA) belongs to a family of natural products that inhibit peptidoglycan biosynthesis by binding to the peptidoglycan glycosyltransferases, the enzymes that make the glycan chains of peptidoglycan. MmA is remarkably potent, but its clinical utility has been hampered by poor physicochemical properties. Moenomycin contains three structurally distinct regions: a pentasaccharide, a phosphoglycerate, and a C25 isoprenyl (moenocinyl) lipid tail that gives the molecule its name. The phosphoglycerate moiety links the pentasaccharide to the moenocinyl chain. This moiety contains two negatively charged groups, a phosphoryl group and a carboxylate. Both the phosphoryl group and the carboxylate have previously been implicated in target binding but the role of the carboxylate has not been explored in detail. Here we report the synthesis of six MmA analogues designed to probe the importance of the phosphoglycerate. These analogues were evaluated for antibacterial and enzyme inhibitory activity; the specific contacts between the phosphoglycerate and the protein target were assessed by X-ray crystallography in conjunction with molecular modeling. Both the phosphoryl group and the carboxylate of the phosphoglycerate chain play roles in target binding. The negative charge of the carboxylate, and not its specific structure, appears to be the critical feature in binding since replacing it with a negatively charged acylsulfonamide group produces a more active compound than replacing it with the isosteric amide. Analysis of the ligand-protein contacts suggests that the carboxylate makes a critical contact with an invariant lysine in the active site. The reported work provides information and validated computational methods critical for the design of analogues based on moenomycin scaffolds.

Rapid assembly of resorcylic acid lactone frameworks through sequential palladium-catalyzed coupling reactions.

kinase inhibitors amongst the RALs have revived interest in this family of natural products. Interestingly, target proteins of RALs vary according to rather small differences in their functional groups. Therefore, development of a shortstep, diversity-oriented synthetic route that would allow the construction of a variety of RAL analogues is important. The key point in RAL analogue synthesis is the construction of a 6-substituted-2,4-dihydroxybenzoate structure (shown by the dashed box in Figure 1). In 2006, Winssinger and coworkers reported the successful synthesis of the aigialomycin D (1) analogues library. They constructed the key structure via Mitsunobu esterification and an alkylation of benzylthioether at the 1’ position. Reductive or oxidative removal of the phenylthio group afforded either an alkane or an alkene at the 1’and 2’-positions. Interestingly, they reported that the result of the Mitsunobu reaction was greatly influenced by the 2-OH group. In the late 1970s, we reported a zearalenone (2) analogue synthesis through a palladium-catalyzed carbonylative esterification and an alkylation of benzylthioether at the 1’ position, followed by the reductive, or oxidative, removal of the phenylthio group to form an alkane or alkene at the 1’and 2’positions. Although carbonylative esterification required the activation of the sterically hindered ortho-disubstituted aryl iodide, the reaction proceeded well to give the desired benzoate in good yield. We also reported the syntheses of a

JBIR-56 and JBIR-57, 2(1H)-Pyrazinones from a Marine Sponge-Derived Streptomyces sp. SpD081030SC-03

Strain SpD081030SC-03, representing a novel species of Streptomyces, was isolated from a marine sponge. Two 3,5,6-trisubstituted 2(1H)-pyrazinones, JBIR-56 (1) and JBIR-57 (2), were isolated from a culture of SpD081030SC-03. The planar structures of 1 and 2 were assigned on the basis of extensive NMR and MS analyses. In addition, analyses of the methylated derivative of 1 confirmed a 3,5,6-trisubstituted 2(1H)-pyrazinone moiety. The absolute configurations of the amino acid residues were determined by application of Marfeys method. Because 1 did not appear to comprise the normal connection of amino acid units, we confirmed its structure by the total synthesis of 1. Biosynthetically, 1 consists of a unique skeleton connected to the peptide chain at C-5 of the pyrazinone ring.

Rapid Synthesis of Thiophene‐Based, Organic Dyes for Dye‐Sensitized Solar Cells (DSSCs) by a One‐Pot, Four‐Component Coupling Approach

This one-pot, four-component coupling approach (Suzuki-Miyaura coupling/C-H direct arylation/Knoevenagel condensation) was developed for the rapid synthesis of thiophene-based organic dyes for dye-sensitized solar cells (DSSCs). Seven thiophene-based, organic dyes of various donor structures with/without the use of a 3,4-ethylenedioxythiophene (EDOT) moiety were successfully synthesized in good yields based on a readily available thiophene boronic acid pinacol ester scaffold (one-pot, 3-step, 35-61%). Evaluation of the photovoltaic properties of the solar cells that were prepared using the synthesized dyes revealed that the introduction of an EDOT structure beside a cyanoacrylic acid moiety improved the short-circuit current (Jsc) while decreasing the fill factor (FF). The donor structure significantly influenced the open-circuit voltage (Voc), the FF, and the power conversion efficiency (PCE). The use of a n-hexyloxyphenyl amine donor, and our originally developed, rigid, and nonplanar donor, both promoted good cell performance (η=5.2-5.6%).

Total synthesis and stereochemistry revision of mannopeptimycin aglycone.

Development of efficient methods for preparation of bioactive nonribosomal peptides, containing densely functionalized nonproteinogenic amino acids, is an important task in organic synthesis. We have employed a concise synthesis for such amino acids by asymmetric aldol addition coupled with an isomeric resolution via diastereoselective cyclization. This approach is successfully applied to the first total synthesis of the cyclic hexapeptide aglycone of the mannopeptimycins, a group of glycopeptides known for potent activity against drug-resistant bacteria. The facile preparation of the key amino acids and the synthesis of the aglycone pave the way for further studies on this class of antibiotics and the development of new lead compounds with therapeutic potential. In addition, our studies have led to the revision of the stereochemistry of the β-methylphenylalanine residue in the mannopeptimycin aglycone.