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.

Diazaquinomycin Biosynthetic Gene Clusters from Marine and Freshwater Actinomycetes.

Tuberculosis is an infectious disease of global concern. Members of the diazaquinomycin (DAQ) class of natural products have shown potent and selective activity against drug-resistant Mycobacterium tuberculosis. However, poor solubility has prevented further development of this compound class. Understanding DAQ biosynthesis may provide a viable route for the generation of derivatives with improved properties. We have sequenced the genomes of two actinomycete bacteria that produce distinct DAQ derivatives. While software tools for automated biosynthetic gene cluster (BGC) prediction failed to detect DAQ BGCs, comparative genomics using MAUVE alignment led to the identification of putative BGCs in the marine Streptomyces sp. F001 and in the freshwater Micromonospora sp. B006. Deletion of the identified daq BGC in strain B006 using CRISPR-Cas9 genome editing abolished DAQ production, providing experimental evidence for BGC assignment. A complete model for DAQ biosynthesis is proposed based on the genes identified. Insufficient knowledge of natural product biosynthesis is one of the major challenges of productive genome mining approaches. The results reported here fill a gap in knowledge regarding the genetic basis for the biosynthesis of DAQ antibiotics. Moreover, identification of the daq BGC shall enable future generations of improved derivatives using biosynthetic methods.

Two novel quinomycins discovered by UPLC-MS from Stretomyces sp. HCCB11876.

Two novel quinomycins I (1) and J (3) were discovered by UPLC-MS, then the two novel compounds and five known quinomycins A(2), B(4), E(5), C(6) and monosulfoxide quinomycin (7) were isolated from the culture broth of Streptomyces sp. HCCB11876. The structures of these compounds were elucidated through MS and NMR spectroscopic analysis. Compounds 1-7 showed significant antibacterial and cytotoxic activities. The structure-activity relationship indicated that sulfoxide group in N-methylcysteine of quinomycins (1, 3 and 7) would significantly decrease the antibacterial and cytotoxic activities. Moreover, the antibacterial and cytotoxic activities were decreased with the increase of carbon chain in amino-acid residues.

Quinomycins H1 and H2, new cytotoxic antibiotics from Streptomyces sp. RAL404.

Two new cytotoxic antibiotics designated quinomycins H1 (2) and H2 (3) were isolated from the culture broth of Streptomyces sp. RAL404. The molecular formula of both compounds was established as C52H65N11O13S2 by electrospray ionization mass spectrometry (ESI-MS). Their structures were determined as echinomycin (1) derivatives containing a 3-hydoxyquinaldic acid molecule in place of one of the two quinoxaline-2-carboxylic acid chromophores. Quinomycins H1 (2) and H2 (3) showed selective cytotoxicity against RG-E1-4 cells bearing the adenovirus oncogenes with IC50s of 11 nM and 12 nM, respectively.

A New Analogue of Echinomycin and a New Cyclic Dipeptide from a Marine-Derived Streptomyces sp. LS298.

Quinomycin G (1), a new analogue of echinomycin, together with a new cyclic dipeptide, cyclo-(l-Pro-4-OH-l-Leu) (2), as well as three known antibiotic compounds tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated from Streptomyces sp. LS298 obtained from a marine sponge Gelliodes carnosa. The planar and absolute configurations of compounds 1 and 2 were established by MS, NMR spectral data analysis and Marfey's method. Furthermore, the differences in NMR data of keto-enol tautomers in tirandamycins were discussed for the first time. Antibacterial and anti-tumor activities of compound 1 were measured against 15 drug-sensitive/resistant strains and 12 tumor cell lines. Compound 1 exhibited moderate antibacterial activities against Staphylococcuse pidermidis, S. aureus, Enterococcus faecium, and E. faecalis with the minimum inhibitory concentration (MIC) values ranged from 16 to 64 µg/mL. Moreover, it displayed remarkable anti-tumor activities; the highest activity was observed against the Jurkat cell line (human T-cell leukemia) with an IC50 value of 0.414 µM.

Synthesis and biological evaluation of quinaldopeptin.

The second-generation total synthesis of quinaldopeptin (1) was established via a Staudinger/aza-Wittig/diastereoselective Ugi three-component reaction sequence and a racemization-free [5 + 5] coupling and macrolactamization. A single-crystal X-ray structure of the chromophore analogue 26 confirmed the structural and stereochemical assignments of the macrocycle. Synthetic 1 successfully unwound supercoiled DNA to form a relaxed DNA in a dose-dependent manner, the binding affinity of 1 to four dsODNs was within a similar range (K(b) = 1.45-2.53 × 10(7) M(-1)), and the sequence selectivity was subtle. It was suggested that 1 possesses biological behaviors similar to those of sandramycin (2) in terms of cytotoxic activity against human cancer cell lines (IC50 = 3.2-12 nM) and HIF-1 inhibitory activity.

RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening.

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Total synthesis of quinaldopeptin and its analogues.

The first total synthesis of quinaldopeptin (1) was accomplished. Our approach to the synthesis of 1 includes the solid-phase peptide synthesis of the linear decapeptide 4 followed by macrocyclization and introduction of the quinoline chromophores 2 at a late stage of the synthesis. As for the preparation of 4, a fragment coupling approach was applied considering the C2 symmetrical structure of 1. Chromophore analogues 22 and 23 and desmethyl analogue 27 were also prepared in a manner similar to the synthesis of 1. Synthetic 1 exhibits a strong cytotoxicity with the IC50 value of 3.2 nM. On the other hand, the activity of 23 and 27 was largely reduced.

Non-covalent duplex to duplex crosslinking of DNA in solution revealed by single molecule force spectroscopy.

Small molecules that interact with DNA, disrupting the binding of transcription factors or crosslinking DNA into larger structures, have significant potential as cancer therapies and in nanotechnology. Bisintercalators, including natural products such as echinomycin and rationally designed molecules such as the bis-9-aminoacridine-4-carboxamides, are key examples. There is little knowledge of the propensity of these molecules to crosslink duplex DNA. Here we use single molecule force spectroscopy to assay the crosslinking capabilities of bisintercalators. We show that bis-9-aminoacridine-4-carboxamides with both rigid and flexible linkers are able to crosslink duplex strands of DNA, and estimate the equilibrium free energy of a 9-aminoacridine-4-carboxamide bisintercalator from DNA at 5.03 kJ mol(-1). Unexpectedly, we find that echinomycin and its synthetic analogue TANDEM are capable of sequence-specific crosslinking of the terminal base pairs of two duplex DNA strands. In the crowded environment of the nucleosome, small molecules that crosslink neighbouring DNA strands may be expected to have significant effects on transcription, while a small molecule that facilitates sequence-specific blunt-end ligation of DNA may find applications in the developing field of DNA nanotechnology.

Involvement of common intermediate 3-hydroxy-L-kynurenine in chromophore biosynthesis of quinomycin family antibiotics.

Quinomycin antibiotics, represented by echinomycin, are an important class of antitumor antibiotics. We have recently succeeded in identification of biosynthetic gene clusters of echinomycin and SW-163D, and have achieved heterologous production of echinomycin in Escherichia coli. In addition, we have engineered echinomycin non-ribosomal peptide synthetase to generate echinomycin derivatives. However, the biosynthetic pathways of intercalative chromophores quinoxaline-2-carboxylic acid (QXC) and 3-hydroxyquinaldic acid (HQA), which are important for biological activity, were not fully elucidated. Here, we report experiments involving incorporation of a putative advanced precursor, (2S, 3R)-[6'-(2)H]-3-hydroxy-L-kynurenine, and functional analysis of the enzymes Swb1 and Swb2 responsible for late-stage biosynthesis of HQA. On the basis of these experimental results, we propose biosynthetic pathways for both QXC and HQA through the common intermediate 3-hydroxy-L-kynurenine.

Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product.

Natural products display impressive activities against a wide range of targets, including viruses, microbes, and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily scalable production of valuable yet hard-to-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, triostin A, and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and triostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we report an identification of a new gene cluster responsible for the biosynthesis of SW-163s that involves previously unknown biosynthesis of (+)-(1S, 2S)-norcoronamic acid and generation of aliphatic side chains of various sizes via iterative methylation of an unactivated carbon center. Substituting an echinomycin biosynthetic gene with a gene from the newly identified SW-163 biosynthetic gene cluster, we were able to rationally re-engineer the plasmid-based echinomycin biosynthetic pathway for the production of a novel bioactive compound in E. coli.

Improved production of triostin A in engineered Escherichia coli with furnished quinoxaline chromophore by design of experiments in small-scale culture.

Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de novo production of triostin A by means of high cell density fed-batch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with small-scale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.

Relative and absolute configuration of antitumor agent SW-163D.

Our interest on engineering non-ribosomal synthetase responsible for SW-163 biosynthesis prompted us to determine the relative and absolute configuration of antitumor cyclic depsipeptide SW-163s. We first isolated and identified SW-163 homologs D, F and G as known compounds UK-63598, UK-65662 and UK-63052, respectively. Both enantiomers of the unusual constitutive amino acid, N-methylnorcoromic acid, were synthesized in chiral forms starting from (R)- and (S)-1,2-propanediol. The hydrolyzate of SW-163D, a major constituent of this family, was converted with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA), and the resulting mixture of amino acid derivatives was subjected to an LC/MS analysis. Compared with authentic samples, the analytical data unambiguously show that SW-163D consisted of L-Ala, D-Ser and (1S, 2S)-N-methylnorcoronamic acid. The remaining stereochemistry of the N-methylcysteine moieties was determined from NOE data.

Role of stacking interactions in the binding sequence preferences of DNA bis-intercalators: insight from thermodynamic integration free energy simulations.

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities.

In vitro antibacterial activity of echinomycin and a novel analogue, YK2000, against vancomycin-resistant enterococci.

The in vitro inhibitory and bactericidal activity of echinomycin and its the novel synthetic analogues of echinomycin,YK2000 and YK2005, were evaluated using 93 clinical isolates of vancomycin-resistant enterococci (VRE). In agar dilution tests, the MIC(90) of echinomycin and YK2000 were 0.125 and 8 mg/l, respectively, using Mueller-Hinton II agar, while that of YK2005 was 32 mg/l. Bactericidal activity of echinomycin and YK2000 were two to four times higher than the MIC in time-kill assay experiments. These results suggest that echinomycin and its analogues might be useful as anti-VRE drugs.

Echinomycin and a novel analogue induce apoptosis of HT-29 cells via the activation of MAP kinases pathway.

Echinomycin, in typical DNA minor groove binder, had comparable efficacy compared to 5-FU in the phase II trail of colon cancer treatment. To improve echinomycin's drawback (hydrophobicity, toxicity), we synthesized the YK-2000 series (echinomycin analogues). Among these, YK-2000 had the best in vitro cytotoxicity on six different human solid cancer cell lines. Echinomycin and YK-2000 were enabled to induce the apoptosis on the HT-29 colorectal cancer cell line. The hypothesis that apoptosis in the HT-29 cell was triggered by echinomycin and YK-2000 were supported through DNA laddering, poly-(ADP-ribose) polymerase (PARP) cleavage, and flow cytometric analysis. In order to explore the signaling pathway of echinomycin and YK-2000, we examined the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAP kinase. However, what the mechanism of cancer cell death would be induced by echinomycin and YK-2000 is unknown. Here, we present some evidence that one of the major apoptotic signaling pathways induced by echinomycin and YK-2000 is possibly the MAP kinases pathway in HT-29 human colon cancer cells.

Synthesis and biological activity of new quinoxaline antibiotics of echinomycin analogues.

Novel quinoxaline antibiotics having the methylenedithioether bridge as an analogue of echinomycin have been synthesized by insertion of methylene moiety between -S-S- bond. The compound 1a shows remarkable cytotoxicities against human tumor various cell lines, and is active VRE (vancomycin-resistant enterococci) within MIC range 0.5-8 microg/mL. According to the eukaryotic or prokaryotic data, 1a might be a first analogue to replace echinomycin.

SW-163C and E, novel antitumor depsipeptides produced by Streptomyces sp. II. Structure elucidation.

SW-163C and E are novel antitumor antibiotics, which belong to quinomycin family, isolated from the culture broth of Streptomyces sp. SNA15896. These compounds were determined to be cyclic depsipeptides having 3-hydroxyquinaldic acid as a chromophore and a sulfur-containing intramolecular cross linkage through various spectroscopic analyses.