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A simplified analog (3) of aplysiatoxin was synthesized. Compound 3 has only one tetrahydropyran ring at positions 3-7, the A-ring of the spiroketal moiety, which is the conformation-controlling unit for the macrolactone ring. Nuclear magnetic resonance (NMR) analysis and density functional theory (DFT) calculations indicated that 3 existed as an equilibrium mixture of two conformers arising from inversion of the chair conformation of the 2,6-trans-tetrahydropyran ring. The des-B-ring analog 3 binds protein kinase C isozymes and exhibits antiproliferative activity toward human cancer cell lines, comparable to 18-deoxy-aplog-1 with a spiroketal moiety.

The teleocidin B family members are terpene indole compounds isolated from Streptomyces bacteria, and they strongly activate protein kinase C (PKC). Their unique structures have attracted many researchers in the natural product chemistry and pharmacology fields, and numerous isolation and bioactivity studies have been conducted. The accumulated information has facilitated the identification of the enzymatic reactions in teleocidin biosynthesis, and new developments in structural biology have strongly aided efforts to clarify the finer points of these reactions. This review describes the recent biochemical and structural biological studies to reveal their reaction mechanisms, with a primary focus on the terpene cyclization triggered by the C-N bond formation by P450 oxygenase (TleB), the prenyltransferase (TleC), and the methyltransferase (TleD). This new knowledge will benefit future engineering studies to create unnatural PKC activators.

Toxic seaweed dermatitis is caused by toxins of the marine cyanobacterium Lyngbya majuscula after sea bathing in some tropical area. This rarely described irritant dermatitis results in papular and bullous lesions, mainly in the genital area but severe ocular lesions can occur.

Simplified analogs of aplysiatoxin (ATX) such as 10-Me-aplog-1 exhibit potent antiproliferative activity toward human cancer cell lines by activating protein kinase C (PKC). However, the synthesis of 10-Me-aplog-1 involved a 23-step longest linear sequence (LLS). Therefore, we have been working toward the development of a more synthetically accessible analog of ATX. In this study, we designed a new analog of ATX wherein a cyclic ketal moiety derived from (R)-(−)-carvone replaced the spiroketal moiety in 18-deoxy-aplog-1. The new analog's synthesis proceeded in an eight-step LLS. Although the configuration at position 3 of the cyclic ketal in the (R)-(−)-carvone-based analog was opposite to those of ATX and 18-deoxy-aplog-1, the antiproliferative activity toward human cancer cell lines of the carvone-based analog was comparable to that of 18-deoxy-aplog-1. The obtained results indicate the potential of the carvone-based analog as a basis for discovering PKC-targeting molecules requiring a decreased number of synthetic steps.

C-S bond formation reactions are widely distributed in the biosynthesis of biologically active molecules, and thus have received much attention over the past decades. Herein, we report intramolecular C-S bond formation by a P450 monooxygenase, TleB, which normally catalyzes a C-N bond formation in teleocidin biosynthesis. Based on the proposed reaction mechanism of TleB, a thiol-substituted substrate analogue was synthesized and tested in the enzyme reaction, which afforded the unprecedented sulfur-containing thio-indolactam V, in addition to an unusual indole-fused 6/5/8-tricyclic product whose structure was determined by the crystalline sponge method. Interestingly, conformational analysis revealed that the SOFA conformation is stable in thio-indolactam V, in sharp contrast to the major TWIST form in indolactam V, resulting in differences in their biological activities.

Covering: up to July 2019 Terpene synthases (TSs) are responsible for generating much of the structural diversity found in the superfamily of terpenoid natural products. These elegant enzymes mediate complex carbocation-based cyclization and rearrangement cascades with a variety of electron-rich linear and cyclic substrates. For decades, two main classes of TSs, divided by how they generate the reaction-triggering initial carbocation, have dominated the field of terpene enzymology. Recently, several novel and unconventional TSs that perform TS-like reactions but do not resemble canonical TSs in sequence or structure have been discovered. In this review, we identify 12 families of non-canonical TSs and examine their sequences, structures, functions, and proposed mechanisms. Nature provides a wide diversity of enzymes, including prenyltransferases, methyltransferases, P450s, and NAD+-dependent dehydrogenases, as well as completely new enzymes, that utilize distinctive reaction mechanisms for TS chemistry. These unique non-canonical TSs provide immense opportunities to understand how nature evolved different tools for terpene biosynthesis by structural and mechanistic characterization while affording new probes for the discovery of novel terpenoid natural products and gene clusters via genome mining. With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand.

Eutrophicated waters frequently support bloom-forming cyanobacteria, many of which produce potent cyanobacterial toxins (cyanotoxins). Cyanotoxins can cause adverse health effects in a wide range of organisms where the toxins may target the liver, other internal organs, mucous surfaces and the skin and nervous system. This review surveyed more than 100 studies concerning the cardiovascular toxicity of cyanotoxins and related topics. Over 60 studies have described various negative effects on the cardiovascular system by seven major types of cyanotoxins, i.e. the microcystin (MC), nodularin (NOD), cylindrospermopsin (CYN), anatoxin (ATX), guanitoxin (GNTX), saxitoxin (STX) and lyngbyatoxin (LTX) groups. Much of the research was done on rodents and fish using high, acutely toxin concentrations and unnatural exposure routes (such as intraperitoneal injection), and it is thus concluded that the emphasis in future studies should be on oral, chronic exposure of mammalian species at environmentally relevant concentrations. It is also suggested that future in vivo studies are conducted in parallel with studies on cells and tissues. In the light of the presented evidence, it is likely that cyanotoxins do not constitute a major risk to cardiovascular health under ordinary conditions met in everyday life. The risk of illnesses in other organs, in particular the liver, is higher under the same exposure conditions. However, adverse cardiovascular effects can be expected due to indirect effects arising from damage in other organs. In addition to risks related to extraordinary concentrations of the cyanotoxins and atypical exposure routes, chronic exposure together with co-existing diseases could make some of the cyanotoxins more dangerous to cardiovascular health. © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a β-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains.

Fast Detection Strategy for Cyanobacterial blooms and associated Cyanotoxins (FDSCC) is a multidisciplinary strategy that allows early detection, in 24 man-hours, of cyanobacteria and related cyanotoxins in water and bivalve samples. This approach combines the advantages of remote/proximal sensing with those of analytical/bioinformatics analyses, namely, LC-HRMS-based molecular networking. The detection of Lyngbyatoxin A, a lipophilic cyanotoxin, in all analyzed water samples and in bivalves, commonly used as food, was the proof of the reliability of the new method.

Neo-debromoaplysiatoxin C (1), a new member of the aplysiatoxin family, was isolated from the marine cyanobacterium Lyngbya sp. The structure of 1 was elucidated based on spectroscopic data, and its stereochemistry was determined from NOESY spectrum and biosynthetic considerations. This new compound presents an intriguing 10-membered lactone ring skeleton derived from debromoaplysiatoxin by structural rearrangement, which is the first example in the aplysiatoxin family. Its biological properties were evaluated for cytotoxicity, PKCδ activation and inhibitory effects on potassium channel.

Debromoaplysiatoxin (DAT) is a potent protein kinase C (PKC) activator with tumor-promoting and pro-inflammatory activities. Irie and colleagues have found that 10-methyl-aplog-1 (1), a simplified analog of DAT, has strong anti-proliferative activity against several cancer cell lines with few adverse effects. Therefore, 1 is a potential lead compound for cancer therapy. We synthesized a new derivative 2 which has a naphthalene ring at the side chain terminal position instead of a benzene ring, to increase CH/π interactions with Pro-241 of the PKCδ-C1B domain. Based on the synthetic route of 1, 2 was convergently synthesized in 26 linear steps from 6-hydroxy-1-naphthoic acid with an overall yield of 0.18%. Although the anti-proliferative activity of 2 was more potent than that of 1, the binding potency of 2 to the PKCδ-C1B domain did not exceed that of 1. Molecular dynamics simulation indicated the capability of 2 to simultaneously form hydrogen bonds and CH/π interactions with the PKCδ-C1B domain. Focusing on the hydrogen bonds, their geometry in the binding modes involving the CH/π interactions seemed to be sub-optimal, which may explain the slightly lower affinity of 2 compared to 1. This study could be of help in optimizing such interactions and synthesizing a promising lead cancer compound.

Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid–liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.

Teleocidins are potent protein kinase C activators, and possess a unique indole-fused nine-membered lactam structure. Teleocidin biosynthesis starts from the formation of a dipeptide by non-ribosomal peptide synthetase (NRPS), followed by oxidative C-N bond formation by a cytochrome P450 oxidase, reverse-prenylation by a prenyltransferase, and methylation-initiated terpene cyclization by a C-methyltransferase. This minireview focuses on recent research progress toward the elucidation of the molecular basis for the remarkable P450-catalyzed intramolecular C-N bond-forming reaction, which is challenging in synthetic chemistry, to generate the indolactam scaffold. In addition, precursor-directed biosynthesis with the promiscuous P450 enzymes led to the formation of a series of unnatural and novel molecular scaffolds, including a sulfur-substituted indolactam with a different conformation from that of indolactam V.

10-Methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes and potent antiproliferative activity against several cancer cells with few adverse effects. A recent study has suggested that its phenol group in the side chain is involved in hydrogen bonding and CH/π interactions with the binding cleft-forming loops in the PKCδ-C1B domain. To clarify the effects of the side chain length on these interactions, four analogs of 1 with various lengths of side chains (2-5) were prepared. The maximal PKC binding affinity and antiproliferative activity were observed in 1. Remarkably, the introduction of a bromine atom into the phenol group of 2 increased not only these activities but also proinflammatory activity. These results indicated that 1 has the optimal side chain length as an anticancer seed. This conclusion was supported by docking simulations of 1-5 to the PKCδ-C1B domain.

Pine wilt disease (PWD) is a plant disease that causes serious damage to pine trees. PWD occurs when the host plant is infected with pinewood nematode (PWN), Bursaphelenchus xylophilus. In this study, a compound with nematicidal activity was isolated from actinomycetes and its efficacy was investigated in vitro.We screened and selected Streptomyces sp. 680560, which had nematicidal activity against B. xylophilus. Based on 16S rRNA sequence analysis, it showed 99.93% similarity with Streptomyces blastmyceticus NRRB-5480This study was carried out to isolate actinomycete metabolites from pine tree endophytes from various natural environments for control of PWD. A compound with nematicidal activity was isolated from a selected strain and its structure was identified as teleocidin B4. The nematicidal effect of the isolated active substance, teleocidin B4, was confirmed. This is the first report of the effect of teleocidin B4 on B. xylophilus, suggesting its possibility as a PWD control agent. © 2020 Society of Chemical Industry.

A new aplysiatoxin derivative, neo-aplysiatoxin A (1), along with seven known compounds, neo-debromoaplysiatoxin A (2), dolastatin 3 (3), lyngbic acid (4), malyngamide M (5), hermitamide A (6), (−)-loliolide (7), and (+)-epiloliolide (8), was isolated from the Okinawan cyanobacterium Moorea producens. Their structures were elucidated on the basis of spectroscopic data, including high-resolution mass spectrometry and nuclear magnetic resonance. The compounds were evaluated for cytotoxic and diatom growth inhibition activities.

Human proteinase-activated receptor 2 (PAR2), a transmembrane G-protein-coupled receptor (GPCR), is an attractive target for a novel anticancer therapy, as it plays a critical role in cell migration and invasion. Selective PAR2 inhibitors therefore have potential as anti-metastatic drugs. Knowing that the natural product teleocidin A2 is able to inhibit PAR2 in tumor cells, the goal of the present study was to elaborate structure-activity relationships and to identify potent PAR2 inhibitors with lower activity against the adverse target, protein kinase C (PKC). For this purpose, an efficient gram-scale total synthesis of indolactam V (i.e., the parent structure of all teleocidins) was developed, and a library of derivatives was prepared. Some compounds were indeed found to exhibit high potency as PAR2 inhibitors at low nanomolar concentrations with improved selectivity (relative to teleocidin A2). The pseudopeptidic fragment bridging the C3 and C4 positions of the indole core proved to be essential for target binding, whereas activity and target selectivity depends on the substituents at N1 or C7. This study revealed novel derivatives that show high efficacy in PAR2 antagonism combined with increased selectivity.

10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.

Potassium channel Kv1.5 has been considered a key target for new treatments of atrial tachyarrhythmias, with few side effects. Four new debromoaplysiatoxin analogues with a 6/6/12 fused ring system were isolated from marine cyanobacterium Lyngbya sp. Their planar structures were elucidated by HRESIMS, 1D and 2D NMR. The absolute configuration of oscillatoxin J (1) was determined by single-crystal X-ray diffraction, and the absolute configurations of oscillatoxin K (2), oscillatoxin L (3) and oscillatoxin M (4) were confirmed on the basis of GIAO NMR shift calculation followed by DP4 analysis. The current study confirmed the absolute configuration of the pivotal chiral positions (7S, 9S, 10S, 11R, 12S, 15S, 29R and 30R) at traditional ATXs with 6/12/6 tricyclic ring system. Compound 1, 2 and 4 exhibited blocking activities against Kv1.5 with IC50 values of 2.61 ± 0.91 µM, 3.86 ± 1.03 µM and 3.79 ± 1.01 µM, respectively. However, compound 3 exhibited a minimum effect on Kv1.5 at 10 µM. Furthermore, all of these new debromoaplysiatoxin analogs displayed no apparent activity in a brine shrimp toxicity assay.

A total synthesis of the proposed structure of nhatrangin A is described. This strategy relies on two aldol reactions to install the chiral centers at C3/C4 and C3'/C4', a lithium-mediated coupling between an advanced intermediate alkyne and a Weinreb amide to complete the C1-C13 alkyl scaffold, and a Yamaguchi esterification to set the side chain. Discrepancies in the spectroscopic data between synthetic and natural nhatrangins led us to synthesize six more diastereoisomers of the proposed structure of nhatrangin A.

Cyanobacteria are prolific producers of natural products and genome mining has shown that many orphan biosynthetic gene clusters can be found in sequenced cyanobacterial genomes. New tools and methodologies are required to investigate these biosynthetic gene clusters and here we present the use of Anabaena sp. strain PCC 7120 as a host for combinatorial biosynthesis of natural products using the indolactam natural products (lyngbyatoxin A, pendolmycin, and teleocidin B-4) as a test case. We were able to successfully produce all three compounds using codon optimized genes from Actinobacteria. We also introduce a new plasmid backbone based on the native Anabaena 7120 plasmid pCC7120ζ and show that production of teleocidin B-4 can be accomplished using a two-plasmid system, which can be introduced by co-conjugation.

A pair of stereoisomers possessing novel structures with 6/6/5 fused-ring systems, neo-debromoaplysiatoxin E (1) and neo-debromoaplysiatoxin F (2), were isolated from the marine cyanobacterium Lyngbya sp. Their structures were elucidated using various spectroscopic techniques including high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR). The absolute stereochemistry was determined by calculated electronic circular dichroism (ECD) and gauge-independent atomic orbital (GIAO) NMR shift calculation followed by DP4+ analysis. Significantly, this is the first report on aplysiatoxin derivatives with different absolute configurations at C9&ndash, C12 (1: 9S, 10R, 11S, 12S, 2: 9R, 10S, 11R, 12R). Compounds 1 and 2 exhibited potent blocking activities against Kv1.5 with IC50 values of 1.22 &plusmn, 0.22 &mu, M and 2.85 &plusmn, 0.29 &mu, M, respectively.

Mass spectrometric methods for the quantitative and qualitative analyses of algal biotoxins are often complicated by co-eluting compounds that present analytically as interferences. This issue is particularly critical for organic polyamines, where co-eluting materials can suppress the formation of cations during electrospray ionization. Here we present an extraction procedure designed specifically to overcome matrix-derived ion suppression of algal toxins in samples of Lyngbya wollei, a filamentous benthic algae known to produce several saxitoxin analogues. Lyngbya wollei samples were collected from a large, persistent harmful algal bloom in Lake Wateree, SC. Six known Lyngbya wollei-specific toxins (LWT1–6) were successfully resolved and quantified against saxitoxin using hydrophilic interaction liquid chromatography coupled with triple quadrupole and quadrupole time-of-flight mass spectrometry. The parent ions [M(2+) – H(+)](+) were observed for LWTs 1–6 and the [M](2+) ion was observed for LWT5. High resolution mass spectra and unique fragmentation ions were obtained for LWTs 1–6. A dilution factor of 50 resulted in a linear calibration of saxitoxin in the algae matrix. Ion suppression was resolved by sample dilution, which led to linear, positive correlations between peak area and mass of the extracted sample (R(2) > 0.96). Optimized sample extraction method and instrument parameters are presented.

A unified and modular approach to the teleocidin B family of natural products is presented that proceeds in 11 steps and features an array of interesting strategies and methods. Indolactam V, the known biosynthetic precursor to this family, was accessed through electrochemical amination, Cu-mediated aziridine opening, and a remarkable base-induced macrolactamization. Guided by a desire to minimize concession steps, the tactical combination of C–H borylation and a Sigman–Heck transform enabled the convergent, stereo-controlled synthesis of the teleocidins.

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin-lyngbyatoxin A-and its biosynthetic intermediates by heterologous expression of the Streptomyces-derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.

A mass spectrometry (MS)-guided isolation has led to the purification of a new cyanobactin, wewakazole B (1), along with the known compound curacin D from a Red Sea Moorea producens. The planar structure of 1 was elucidated using a combination of NMR and MS techniques. After ozonolysis and acid hydrolysis, the absolute configurations of the amino acid components of 1 were determined by chiral-phase LC-MS and HPLC analyses. Notably, compound 1 exhibited cytotoxic activity toward human MCF7 breast cancer cells (IC50 = 0.58 μM) and human H460 lung cancer cells (IC50 = 1.0 μM) and was also found to be inactive in a siderophore assay.

Aplog-1 is a simplified analog of debromoaplysiatoxin (DAT) with potent tumor-promoting and proinflammatory activities. Aplog-1 and DAT exhibited anti-proliferative activities against several human cancer cell lines, whereas aplog-1 did not have tumor-promoting nor proinflammatory activities. We have recently found 10-methyl-aplog-1 (1) to have strong anti-proliferative activity compared with aplog-1. To further investigate the structural factors involved in the tumor-promoting, proinflammatory, and anti-proliferative activities, two dimethyl derivatives of aplog-1 (2, 3) were synthesized, where two methyl groups were installed at positions 4 and 10 or 10 and 12. 10,12-Dimethyl-aplog-1 (2) had stronger inhibitory effects on the growth of several human cancer cell lines than 1 and DAT, but exhibited no tumor-promoting and proinflammatory activities. In contrast, 4,10-dimethyl-aplog-1 (3) displayed weak tumor-promoting and proinflammatory activities along with anti-proliferative activity similar to that of 1 and DAT. Compound 2 would be the optimized seed for anticancer drugs among the simplified analogs of DAT.

Certain strains of cyanobacteria produce a wide array of cyanotoxins, such as microcystins, lyngbyatoxins and aplysiatoxins, that are associated with public health issues. In this pilot study, an approach combining LC-MS/MS and molecular networking was employed as a rapid analytical method to detect aplysiatoxins present in four environmental marine cyanobacterial samples collected from intertidal areas in Singapore. Based on 16S-ITS rRNA gene sequences, these filamentous cyanobacterial samples collected from Pulau Hantu were determined as Trichodesmium erythraeum, Oscillatoria sp. PAB-2 and Okeania sp. PNG05-4. Organic extracts were prepared and analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) for the presence of aplysiatoxin-related molecules. From the molecular networking, six known compounds, debromoaplysiatoxin (1), anhydrodebromoaplysiatoxin (2), 3-methoxydebromoaplysiatoxin (3), aplysiatoxin (4), oscillatoxin A (5) and 31-noroscillatoxin B (6), as well as potential new analogues, were detected in these samples. In addition, differences and similarities in molecular networking clusters related to the aplysiatoxin molecular family were observed in extracts of Trichodesmium erythraeum collected from two different locations and from different cyanobacterial species found at Pulau Hantu, respectively.

Cyanobacterial natural products offer new possibilities for drugs and lead compounds but many factors can inhibit the production of sufficient yields for pharmaceutical processes. While Escherichia coli and Streptomyces sp. have been used as heterologous expression hosts to produce cyanobacterial natural products, they have not met with resounding success largely due to their inability to recognize cyanobacterial promoter regions. Recent work has shown that the filamentous freshwater cyanobacterium Anabaena sp. strain PCC 7120 recognizes various cyanobacterial promoter regions and can produce lyngbyatoxin A from the native promoter. Introduction of Anabaena sigma factors into E. coli might allow the native transcriptional machinery to recognize cyanobacterial promoters. Here, all 12 Anabaena sigma factors were expressed in E. coli and subsets were found to initiate transcription from several cyanobacterial promoters based on transcriptional fusions to the chloramphenicol acetyltransferase (CAT) reporter. Expression of individual Anabaena sigma factors in E. coli did not result in lyngbyatoxin A production from its native cyanobacterial gene cluster, possibly hindered by deficiencies in recognition of cyanobacterial ribosomal binding sites by native E. coli translational machinery. This represents an important step toward engineering E. coli into a general heterologous expression host for cyanobacterial biosynthetic gene cluster expression.

Teleocidin B is a terpenoid indole alkaloid with unique structures including indolactam and cyclic terpenoid, and is a strong protein kinase C activator. Its unique structure and bioactivity have drawn vast interest from chemists and biologists, and numerous isolation and bioactivity studies on this molecule have been performed. Recently, its biosynthetic enzymes were identified, and some of their crystal structures were reported. In this review, we describe the isolation of teleocidin derivatives, biosynthetic studies, and detailed analyses of biosynthetic enzymes, to clarify their biosynthetic reactions toward the enzymatic synthesis of bioactive teleocidin compounds.

Teleocidin B, with its unique indolactam-terpenoid scaffold, is a potent activator of protein kinase C. This short review summarizes our recent research progress on the biosynthesis of teleocidins in Streptomyces blastmyceticus NBRC 12747. We first identified the biosynthetic genes for teleocidin B, which include genes encoding a non-ribosomal peptide synthetase (tleA), a cytochrome P450 monooxygenase (tleB), an indol prenyltransferase (tleC), and a C-methyltransferase (tleD). Notably, the tleD gene is located outside the tleABC cluster. Our in vivo and in vitro analyses revealed that TleD not only catalyzes the C-methylation of the prenyl chain but also produces the indole-fused cyclic terpene structure. This is the first report of terpene cyclization initiated by the C-methylation of the prenyl double bond. In contrast, TleC catalyzes the geranylation of the C-7 position of the indole ring, in the reverse fashion. Our X-ray crystallographic analyses provided the structural basis for the reverse prenylation reactions, and structure-based mutagenesis successfully resulted in the production of unnatural, novel prenylated indolactams.

Lyngbyabellin M is a non-ribosomal peptide synthetase/polyketide synthase derived metabolite isolated from the cyanobacterium M. bouillonii displaying thiazole rings and a distinct chlorinated octanoic acid chain. Its absolute configuration was proposed based on the comparison of its spectroscopic data with those of other representatives of this family of marine natural products, as well as degradation and derivatization studies. Here the first total synthesis of (+)-lyngbyabellin M is described based on the coupling of three key intermediates: two chiral thiazole moieties and an anti hydroxycarboxylic acid prepared stereoselectively via a boron enolate mediated aldol reaction directed by Masamune’s chiral auxiliary.

Itralamides A and B were isolated from the lipophilic extract of Lyngbya majuscula collected from the eastern Caribbean. Itralamide B (1) showed cytotoxic activity towards human embryonic kidney cells (HEK293, IC50 = 6 μM). Preliminary studies disapproved the proposed stereochemistry of itralamide. In this paper, we will provide a full account of the total synthesis of four stereoisomers of itralamide B and the results derived from biological tests of these structural congeners.

An acetylene-containing lipopeptide, jahanyne, was isolated from the marine cyanobacterium Lyngbya sp. Its gross structure was established by spectroscopic analyses, and the absolute configuration was clarified based on a combination of chiral HPLC analyses, spectroscopic analyses, and derivatization reactions. Jahanyne significantly inhibited the growth of human cancer cells and induced apoptosis in HeLa cells.

Teleocidin B is an indole terpenoid isolated from Streptomyces. Due to its unique chemical structure and ability to activate protein kinase C, it has attracted interest in the areas of organic chemistry and cell biology. Here, we report the identification of genes encoding enzymes for teleocidin B biosynthesis, including nonribosomal peptide synthetase (tleA), P-450 monooxygenase (tleB), prenyltransferase (tleC), and methyltransferase (tleD). The tleD gene, which is located outside of the tleABC cluster on the chromosome, was identified by transcriptional analysis and heterologous expression. Remarkably, TleD not only installs a methyl group on the geranyl moiety of the precursor but also facilitates the nucleophilic attack from the electron-rich indole to the resultant cation, to form the indole-fused six-membered ring. This is the first demonstration of a cation, generated from methylation, triggering successive terpenoid ring closure.

Lyngbyatoxin A from the marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) is known as the causative agent of “swimmer’s itch” with its highly inflammatory effect. A new toxic compound was isolated along with lyngbyatoxin A from an ethyl acetate extract of M. producens collected from Hawaii. Analyses of HR-ESI-MS and NMR spectroscopies revealed the isolated compound had the same planar structure with that of lyngbyatoxin A. The results of optical rotation and CD spectra indicated that the compound was a new lyngbyatoxin A derivative, 12-epi-lyngbyatoxin A (1). While 12-epi-lyngbyatoxin A showed comparable toxicities with lyngbyatoxin A in cytotoxicity and crustacean lethality tests, it showed more than 100 times lower affinity for protein kinase Cδ (PKCδ) using the PKCδ-C1B peptide when compared to lyngbyatoxin A.

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC50 values of 1.3 μM and 2.7 μM, respectively, and selectivity indices of 10.9 and 9.2, respectively.

Cyanobacteria have been shown to produce a number of bioactive compounds, including toxins. Some bioactive compounds obtained from a marine cyanobacterium Moorea producens (formerly Lyngbya majuscula) have been recognized as drug leads; one of these compounds is aplysiatoxin. We have isolated various aplysiatoxin derivatives from a M. producens sample obtained from the Okinawan coastal area. The frozen sample was extracted with organic solvents. The ethyl acetate layer was obtained from the crude extracts via liquid−liquid partitioning, then separated by HPLC using a reversed-phase column. Finally, 1.1 mg of the compound was isolated. The chemical structure of the isolated compound was elucidated with spectroscopic methods, using HR-MS and 1D and 2D NMR techniques, and was revealed to be oscillatoxin I, a new member of the aplysiatoxin family. Oscillatoxin I showed cytotoxicity against the L1210 mouse lymphoma cell line and diatom growth-inhibition activity against the marine diatom Nitzschia amabilis.

Cultivation of the marine cyanobacterium Moorea producens, collected from the Nabq Mangroves in the Gulf of Aqaba (Red Sea), led to the isolation of new apratoxin analogues, apratoxin H (1) and apratoxin A sulfoxide (2), together with the known apratoxins A-C, lyngbyabellin B and hectochlorin. The absolute configuration of these new potent cytotoxins was determined by chemical degradation, MS, NMR, and CD spectroscopy. Apratoxin H (1) contains pipecolic acid in place of the proline residue present in apratoxin A, expanding the known suite of naturally occurring analogues that display amino acid substitutions within the final module of the apratoxin biosynthetic pathway. The oxidation site of apratoxin A sulfoxide (2) was deduced from MS fragmentation patterns and IR data, and 2 could not be generated experimentally by oxidation of apratoxin A. The cytotoxicity of 1 and 2 to human NCI-H460 lung cancer cells (IC50 = 3.4 and 89.9 nM, respectively) provides further insight into the structure–activity relationships in the apratoxin series. Phylogenetic analysis of the apratoxin-producing cyanobacterial strains belonging to the genus Moorea, coupled with the recently annotated apratoxin biosynthetic pathway, supports the notion that apratoxin production and structural diversity may be specific to their geographical niche.