Modulation of proteasome subunit selectivity of syringolins.

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure-activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure-activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding ß5 and ß2 subunits.

Synthesis and biological evaluation of echinomycin analogues as potential colon cancer agent.

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.

Photoinduced dual bond rotation of a nitrogen-containing system realized by chalcogen substitution.

Photoinduced concerted multiple-bond rotation has been proposed in some biological systems. However, the observation of such phenomena in synthetic systems, in other words, the synthesis of molecules that undergo photoinduced multiple-bond rotation upon photoirradiation, has been a challenge in the photochemistry field. Here we describe a chalcogen-substituted benzamide system that exhibits photoinduced dual bond rotation in heteroatom-containing bonds. Introduction of the chalcogen substituent into a sterically hindered benzamide system provides sufficient kinetic stability and photosensitivity to enable the photoinduced concerted rotation. The presence of two different substituents on the phenyl ring in the thioamide derivative enables the generation of a pair of enantiomers and E/Z isomers. Using these four stereoisomers as indicators of which bonds are rotated, we monitor the photoinduced C-N/C-C concerted bond rotation in the thioamide derivative depending on external stimuli such as temperature and photoirradiation. Theoretical calculations provide insight on the mechanism of this selective photoinduced C-N/C-C concerted rotation.

Determining the structure of protein-bound ceramides, essential lipids for skin barrier function.

Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the ß-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides.

Solid-Phase Synthesis of Nannocystin Ax and Its Analogues.

Solid-phase total synthesis of nannocystin Ax (1) was disclosed. A coupling reaction between a peptide and a polyketide moiety was conducted on a solid support, and macrocyclization was achieved by Mitsunobu cyclization. The established synthetic route was efficient to prepare its analogues, which contain different types of peptide moieties.

Solid-Phase Total Synthesis of Sandramycin and Its Analogues.

Solid-phase total synthesis of sandramycin (1), which is a C2-symmetric cyclic decadepsipeptide natural product, and its analogues is described. On-resin ester formation and [5+5] peptide coupling allowed the preparation of a range of desymmetrized analogues. An amino acid residue that would not hamper the biological activity of 1 was successfully identified, and probe molecules and dimeric analogues were prepared on the basis of the result of the structure-activity relationship study.

Discovery of Biologically Optimized Polymyxin Derivatives Facilitated by Peptide Scanning and In Situ Screening Chemistry.

Peptides can be converted to highly active compounds by introducing appropriate substituents on the suitable amino acid residue. Although modifiable residues in peptides can be systematically identified by peptide scanning methodologies, there is no practical method for optimization at the "scanned" position. With the purpose of using derivatives not only for scanning but also as a starting point for further chemical functionalization, we herein report the "scanning and direct derivatization" strategy through chemoselective acylation of embedded threonine residues by a serine/threonine ligation (STL) with the help of in situ screening chemistry. We have applied this strategy to the optimization of the polymyxin antibiotics, which were selected as a model system to highlight the power of the rapid derivatization of active scanning derivatives. Using this approach, we explored the structure-activity relationships of the polymyxins and successfully prepared derivatives with activity against polymyxin-resistant bacteria and those with Pseudomonas aeruginosa selective antibacterial activity. This strategy opens up efficient structural exploration and further optimization of peptide sequences.

Synthesis of macrocyclic nucleoside antibacterials and their interactions with MraY.

The development of new antibacterial drugs with different mechanisms of action is urgently needed to address antimicrobial resistance. MraY is an essential membrane enzyme required for bacterial cell wall synthesis. Sphaerimicins are naturally occurring macrocyclic nucleoside inhibitors of MraY and are considered a promising target in antibacterial discovery. However, developing sphaerimicins as antibacterials has been challenging due to their complex macrocyclic structures. In this study, we construct their characteristic macrocyclic skeleton via two key reactions. Having then determined the structure of a sphaerimicin analogue bound to MraY, we use a structure-guided approach to design simplified sphaerimicin analogues. These analogues retain potency against MraY and exhibit potent antibacterial activity against Gram-positive bacteria, including clinically isolated drug resistant strains of S. aureus and E. faecium. Our study combines synthetic chemistry, structural biology, and microbiology to provide a platform for the development of MraY inhibitors as antibacterials against drug-resistant bacteria.

Solid-Phase Total Synthesis of Plusbacin A3.

The total synthesis of the depsipeptide natural product plusbacin A3 (1) utilizing solid-phase peptide synthesis (SPPS) was disclosed. A 3-hydroxy-proline derivative compatible with Fmoc SPPS was prepared by a diastereoselective Joullié-Ugi three-component reaction (JU-3CR)/hydrolysis sequence. After peptide elongation on the solid support, cleavage of the peptide from the resin, followed by macrolactamization and global deprotection, gave plusbacin A3 (1).

Structure, solubility, and permeability relationships in a diverse middle molecule library.

To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.

Elucidating the Structural Requirement of Uridylpeptide Antibiotics for Antibacterial Activity.

The synthesis and biological evaluation of analogues of uridylpeptide antibiotics were described, and the molecular interaction between the 3'-hydroxy analogue of mureidomycin A (3'-hydroxymureidomycin A) and its target enzyme, phospho-MurNAc-pentapeptide transferase (MraY), was analyzed in detail. The structure-activity relationship (SAR) involving MraY inhibition suggests that the side chain at the urea-dipeptide moiety does not affect the MraY inhibition. However, the anti-Pseudomonas aeruginosa activity is in great contrast and the urea-dipeptide motif is a key contributor. It is also suggested that the nucleoside peptide permease NppA1A2BCD is responsible for the transport of 3'-hydroxymureidomycin A into the cytoplasm. A systematic SAR analysis of the urea-dipeptide moiety of 3'-hydroxymureidomycin A was further conducted and the antibacterial activity was determined. This study provides a guide for the rational design of analogues based on uridylpeptide antibiotics.

Total Synthesis of Acaulide and Acaulone A.

Acaulide and acaulone A, which contain 14-membered macrodiolides, were isolated from a culture of Acaulium sp. H-JQSF. The antiosteoporosis activity of acaulide is expected to contribute to drug discovery research for an aging society. We herein report the first total synthesis of acaulide, acaulone A, and 10-keto-acaudiol A. Acaulide and acaulone A were synthesized via the late stage Michael addition to the 14-membered macrodiolide, which was inspired by plausible biosynthetic pathways. This approach succeeded in the construction of the acaulide skeleton, which revealed the specific conformation of the 14-membered macrodiolide for late stage functionalization.

Total Synthesis of Echinomycin and Its Analogues.

The first total synthesis of echinomycin (1) was accomplished by featuring the late-stage construction of the thioacetal moiety via Pummerer rearrangement and simultaneous cyclization, as well as two-directional elongation of the peptide chains to construct a C2-symmetrical bicyclic octadecadepsipeptide bridged with a sulfide linkage. This strategy can be applicable to a variety of echinomycin analogues.

A Synthesis Strategy for the Production of a Macrolactone of Gulmirecin A via a Ni(0)-Mediated Reductive Cyclization Reaction.

A synthesis strategy for the production of a key synthetic intermediate of gulmirecin A was described. The key reaction in the preparation of the 12-membered macrolactone is the Ni(0)-mediated reductive cyclization reaction of ynal using an N-heterocyclic carbene ligand and silane reductant. In addition, the α-selective glycosylation reaction of the macrolactone was performed to demonstrate the synthesis of gulmirecin and disciformycin precursors.

Synthesis and biological evaluation of a MraY selective analogue of tunicamycins.

Tunicamycins, which are nucleoside natural products, inhibit both bacterial phospho-N-acetylmuraminic acid (MurNAc)-pentapeptide translocase (MraY) and human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). The improved synthesis and detailed biological evaluation of an MraY-selective inhibitor, 2, where the GlcNAc moiety was modified to a MurNAc amide, has been described.

Development of cyclic peptide derivatives from the N-terminal region of LANA for targeting the nucleosome acidic patch.

Kaposi's sarcoma-associated herpesvirus (KSHV) is known to be a carcinogenic agent that causes AIDS-associated Kaposi's sarcoma (KS). When KSHV infects host's cells, one of the virus's proteins, latency-associated nuclear antigen 1 (LANA), binds to the host's nucleosomes to retain episomes and create latency circumstances. Although the infectious mechanism of KSHV is partly elucidated, the development of drug candidates for targeting KS is ongoing. In this study, we developed cyclic peptides corresponding to an N-terminal LANA sequence that disrupt the LANA-nucleosome interaction. The cyclic peptides showed a different secondary structure compared to their corresponding linear peptide derivatives, which suggests that our cyclization strategy imitates the N-terminal LANA binding conformation on nucleosomes.

Chemical logic of MraY inhibition by antibacterial nucleoside natural products.

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.

Structural requirement of tunicamycin V for MraY inhibition.

Elucidating a structure-activity relationship study by evaluating a series of truncated analogues is a simple but important and effective tactic in medicinal chemistry based on natural products with a large and complex chemical structure. In this study, a series of truncated analogues of tunicamycin V were designed and synthesized and their MraY inhibitory activity was investigated in order to gain insight into the effect of these moieties on MraY inhibition.

Synthesis and Medicinal Chemistry of Muraymycins, Nucleoside Antibiotics.

Muraymycins, isolated from a culture broth of Streptomyces sp., are members of a class of naturally occurring nucleoside antibiotics. They are strong inhibitors of the phospho-MurNAc-pentapeptide translocase (MraY), which is responsible for the peptidoglycan biosynthesis. Since MraY is an essential enzyme among bacteria, muraymycins are expected to be a novel antibacterial agent. In this review, our efforts to synthesize muraymycin D2, simplify the chemical structure, improve antibacterial spectrum, and solve the X-ray crystal structure of the muraymycin D2/MraY complex are described.

Total Synthesis of Plusbacin A3 and Its Dideoxy Derivative Using a Solvent-Dependent Diastereodivergent Joullié-Ugi Three-Component Reaction.

Full details of our synthetic studies toward plusbacin A3 (1), which is a depsipeptide with antibacterial activity, and its dideoxy derivative are described. To establish an efficient synthetic route of 1, a solvent-dependent diastereodivergent Joullié-Ugi three-component reaction (JU-3CR) was used to construct trans-Pro(3-OH) in a small number of steps. Two strategies were investigated toward the total synthesis. In the first synthetic strategy, the key steps were the trans-selective JU-3CR and a macrolactonization at the final stage of the synthesis. The JU-3CR using alkyl isocyanides in 1,1,1,3,3,3-hexafluoroisopropanol provided the trans products, and the coupling of the fragments to prepare the macrocyclization precursor proceeded smoothly. However, attempts toward the macrolactonization did not provide the desired product. Then, the second strategy that included esterification in an initial stage was investigated. Methods for constructing trans-Pro(3-OH) were examined using a convertible isocyanide, which could be converted to a carboxylic acid required for the following amidation. Ester bond formation was achieved through an intermolecular coupling using a hydroxyl-Asp derivative and the corresponding alcohol, and the amidation afforded a linear depsipeptide. The macrolactamization of the linear peptide gave the cyclic depsipeptide, and then the global deprotection accomplished the total synthesis of 1 and its dideoxy derivative.