Potential of Polar Lipids Isolated from the Marine Sponge Haliclona (Halichoclona) vansoesti against Melanoma.

Marine sponges represent a good source of natural metabolites for biotechnological applications in the pharmacological, cosmeceutical, and nutraceutical fields. In the present work, we analyzed the biotechnological potential of the alien species Haliclona (Halichoclona) vansoesti de Weerdt, de Kluijver & Gomez, 1999, previously collected in the Mediterranean Sea (Faro Lake, Sicily). The bioactivity and chemical content of this species has never been investigated, and information in the literature on its Caribbean counterpart is scarce. We show that an enriched extract of H. vansoesti induced cell death in human melanoma cells with an IC50 value of 36.36 µg mL-1, by (i) triggering a pro-inflammatory response, (ii) activating extrinsic apoptosis mediated by tumor necrosis factor receptors triggering the mitochondrial apoptosis via the involvement of Bcl-2 proteins and caspase 9, and (iii) inducing a significant reduction in several proteins promoting human angiogenesis. Through orthogonal SPE fractionations, we identified two active sphingoid-based lipid classes, also characterized by nuclear magnetic resonance and mass spectrometry, as the main components of two active fractions. Overall, our findings provide the first evaluation of the anti-cancer potential of polar lipids isolated from the marine sponge H. (Halichoclona) vansoesti, which may lead to new lead compounds with biotechnological applications in the pharmaceutical field.

New lectin isolated from the tropical sponge Haliclona (Reniera) implexiformis (Hechtel, 1965) shows antibiofilm effect.

A lectin from the marine sponge Haliclona (Reniera) implexiformis (HiL) was isolated by affinity chromatography on Sepharose™ matrix. HiL showed specificity for galactose and its derivatives. The glycoproteins porcine stomach mucin (PSM) and bovine stomach mucin (BSM) were potent inhibitors. Hemagglutinating activity of the lectin was maximal between pH 5.0 and 9.0. The lectin remained active until 60°C. The presence of CaCl2 and EDTA did not affect the hemagglutinating activity. In SDS-PAGE, HiL showed a single band of 20 kDa under reduced conditions, whereas in the non-reducing conditions, it showed a band of 20 kDa and one additional band of 36 kDa. The average molecular mass determined by Electrospray Ionization Mass Spectrometry (ESI-MS) was 35.874 ± 2 Da in native and non-reducing conditions, whereas carboxyamidomethylated-lectin showed 18,111 Da. These data indicated that HiL consists in a dimer formed by identical subunits linked by disulfide bonds. Partial amino acid sequence of HiL was determined by mass spectrometry, and revealed that it is a new type of lectin, which showed no similarity with any protein. Secondary structure consisted of 6% α-helice, 31% ß-sheet, 18% ß-turn and 45% random coil. HiL showed significant reduction in the number of viable cells of Staphylococcus biofilms.

Isoquinolinequinone Derivatives from a Marine Sponge (Haliclona sp.) Regulate Inflammation in In Vitro System of Intestine.

Using bio-guided fractionation and based on the inhibitory activities of nitric oxide (NO) and prostaglandin E2 (PGE2), eight isoquinolinequinone derivatives (1-8) were isolated from the marine sponge Haliclona sp. Among these, methyl O-demethylrenierate (1) is a noble ester, whereas compounds 2 and 3 are new O-demethyl derivatives of known isoquinolinequinones. Compound 8 was assigned as a new 21-dehydroxyrenieramycin F. Anti-inflammatory activities of the isolated compounds were tested in a co-culture system of human epithelial Caco-2 and THP-1 macrophages. The isolated derivatives showed variable activities. O-demethyl renierone (5) showed the highest activity, while 3 and 7 showed moderate activities. These bioactive isoquinolinequinones inhibited lipopolysaccharide and interferon gamma-induced production of NO and PGE2. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and the phosphorylation of MAPKs were down-regulated in response to the inhibition of NF-κB nuclear translocation. In addition, nuclear translocation was markedly promoted with a subsequent increase in the expression of HO-1. Structure-activity relationship studies showed that the hydroxyl group in 3 and 5, and the N-formyl group in 7 may be key functional groups responsible for their anti-inflammatory activities. These findings suggest the potential use of Haliclona sp. and its metabolites as pharmaceuticals treating inflammation-related diseases including inflammatory bowel disease.

Characterization of Extracellular Protease from the Haloarcheon Halococcus sp. Strain GUGFAWS-3 (MF425611).

Halococcus agarilyticus GUGFAWS-3 (MF425611) was isolated from a marine white sponge of Haliclona sp., inhabiting the rocks in the intertidal region of Anjuna, Goa, India. Uniquely, the microbe simultaneously produces two halo-extremozymes in 25% NaCl, namely protease and lipase at 49.5 ± 0.4 and 3.67 ± 0.02 (U mL-1), respectively. The protease is constitutively produced in starch mineral salts medium with consistent 4 ± 1.0 mm zone of enzyme production, regardless of the non-availability of protein as substrate. The ethanol precipitated enzyme on dialysis and Sephadex G-200 gel filtration chromatography was partially purified to 12.26-fold and was active between 20 and 80 °C, 0-5 M NaCl, and pH 3-13. Optimum activity, however, was at 70 °C, 3 M NaCl, and pH 7. The enzyme was thermo stable at 70 °C with 50.26 ± 2.40% of relative enzyme activity at 75 min. Furthermore, it was stable in the presence of polar and non-polar organic solvents, detergents, and hydrocarbons. Several metal cations enhanced its activity in the order of Ca2+ > Ni2+ > Fe3+ > Co2+ > Mg2+ > Cu2+ > Mn2+. Dependence of enzyme on cysteine; serine, and metal ions was confirmed by ß-mercaptoethanol; PMSF and EDTA, respectively which induced its partial inhibition. Additionally, protease inhibited in vitro biofilm formation in Staphylococcus aureus. Conclusively, the production of a neutral halo-thermophilic protease is reported for the first time in the genus Halococcus.

Osirisynes G-I, New Long-Chain Highly Oxygenated Polyacetylenes from the Mayotte Marine Sponge Haliclona sp.

Chemical study of the CH2Cl2-MeOH (1:1) extract from the sponge Haliclona sp. collected in Mayotte highlighted three new long-chain highly oxygenated polyacetylenes, osirisynes G-I (1-3) together with the known osirisynes A (4), B (5), and E (6). Their structures were elucidated by 1D and 2D NMR spectra and HRESIMS and MS/MS data. All compounds were evaluated on catalase and sirtuin 1 activation and on CDK7, proteasome, Fyn kinase, tyrosinase, and elastase inhibition. Five compounds (1; 3-6) inhibited proteasome kinase and two compounds (5-6) inhibited CDK7 and Fyn kinase. Osirisyne B (5) was the most active compound with IC50 on FYNB kinase, CDK7 kinase, and proteasome inhibition of 18.44 µM, 9.13 µM, and 0.26 µM, respectively.

Gracilosulfates A-G, Monosulfated Polyoxygenated Steroids from the Marine Sponge Haliclona gracilis.

Seven new polyoxygenated steroids belonging to a new structural group of sponge steroids, gracilosulfates A-G (1-7), possessing 3ß-O-sulfonato, 5ß,6ß epoxy (or 5(6)-dehydro), and 4ß,23-dihydroxy substitution patterns as a common structural motif, were isolated from the marine sponge Haliclona gracilis. Their structures were determined by NMR and MS methods. The compounds 1, 2, 4, 6, and 7 inhibited the expression of prostate-specific antigen (PSA) in 22Rv1 tumor cells.

Silicatein expression in Haliclona indistincta (Phylum Porifera, Order Haplosclerida) at different developmental stages.

Silicatein is the main protein responsible for the formation of spicules, tiny structures that constitute the silica skeleton of marine demosponges (Phylum Porifera). A unique innovation in Porifera that evolved from the cathepsin L family of proteins, it has been reported that two amino acids (S and H) are necessary to form the catalytic triad (SHN) to enable silica condensation. However, a diversity of silicatein sequence variants has since been reported with a variable pattern of presence/absence across sponge groups. Variants containing CHN or C/SQN at the active site appear more common in sponges from the Haplosclerida. Here, we report the expression levels of five silicatein variants through different developmental stages in the haplosclerid Haliclona indistincta. All five silicatein variants were expressed at low levels in the free-swimming larvae, which lack spicules and expression significantly increased at the two developmental phases in which spicules were visible. At these two phases, silicateins of CHN and C/SQN types were much more highly expressed than the SHN type indicating a possible ability of active sites with these alternative amino acids to condense silica and a more complex evolutionary story for spicule formation in marine demosponges than previously understood.

Stable and Enriched Cenarchaeum symbiosum and Uncultured Betaproteobacteria HF1 in the Microbiome of the Mediterranean Sponge Haliclona fulva (Demospongiae: Haplosclerida).

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

Bioactive Nitrogenous Secondary Metabolites from the Marine Sponge Genus Haliclona.

Marine sponge genus Haliclona, one of the most prolific sources of natural products, contains over 600 species but only a small part of them had been classified and chemically investigated. On the basis of extensive literature search, this review firstly summarizes 112 nitrogenous secondary metabolites from classified and unclassified Haliclona sponges as well as from their symbiotic microorganisms. Most of these substances have only been found in Haliclona sponges, and display diverse bioactive properties with potential applications in new drug discovery.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Metabolome variability for two Mediterranean sponge species of the genus Haliclona: specificity, time, and space.

INTRODUCTION: The study of natural variation of metabolites brings valuable information on the physiological state of the organisms as well as their phenotypic traits. In marine organisms, metabolome variability has mostly been addressed through targeted studies on metabolites of ecological or pharmaceutical interest. However, comparative metabolomics has demonstrated its potential to address the overall and complex metabolic variability of organisms. OBJECTIVES: In this study, the intraspecific (temporal and spatial) variability of two Mediterranean Haliclona sponges (H. fulva and H. mucosa) was investigated through an untargeted and then targeted metabolomics approach and further compared to their interspecific variability. METHODS: Samples of both species were collected monthly during 1 year in the coralligenous habitat of the Northwestern Mediterranean sae at Marseille and Nice. Their metabolomic profiles were obtained by UHPLC-QqToF analyses. RESULTS: Marked variations were noticed in April and May for both species including a decrease in Shannon's diversity and concentration in specialized metabolites together with an increase in fatty acids and lyso-PAF like molecules. Spatial variations across different sampling sites could also be observed for both species, however in a lesser extent. CONCLUSIONS: Synchronous metabolic changes possibly triggered by physiological factors like reproduction and/or environmental factors like an increase in the water temperature were highlighted for both Mediterranean Haliclona species inhabiting close habitats but displaying different biosynthetic pathways. Despite significative intraspecific variations, metabolomic variability remains minor when compared to interspecific variations for these congenerous species, therefore suggesting the predominance of genetic information of the holobiont in the observed metabolome.

Proposal of Litorimonas haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A bright-orange-pigmented, Gram-stain-negative, motile, and rod-shaped bacterium, strain MAA42T, was isolated from a marine sponge of the genus Haliclona, which is in long-time culture in a marine aquarium system at the Justus Liebig University Giessen, Germany. The strain grew at 4-34 °C (optimum 28 °C), in the presence of 0.5-9.5 % (w/v) NaCl (optimum 3.5 %) and at pH 4.5-10.0 (optimum pH 7.5). Strain MAA42T shared the highest 16S rRNA gene sequence similarity (98.1 %) with the type strain of Litorimonas taeanensis. Sequence similarities to all other closely related type strains were below 97 %. DNA-DNA hybridization of strain MAA42T with L. taeanensis DSM 22008T resulted in values of 4.7 % (reciprocal 17.7 %). Major cellular fatty acids of strain MAA42T were C18 : 1ω7c (66.2 %), C18 : 1 2-OH (17.4 %), and C18 : 0 (14.1 %). Spermidine was predominant in the polyamine pattern, and ubiquinone Q-10 was the major respiratory quinone. The polar lipid profile contained the major compounds phosphatidylglycerol, monoglycosyldiglyceride, three unidentified phospholipids, and one unidentified glycolipid. Glucuronopyranosyldiglyceride was present as a minor compound. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The genomic DNA G+C content was 52.8 mol%. Based on the genotypic, chemotaxonomic, and phenotypic analyses, strain MAA42T represents a novel species of the genus Litorimonas, for which the name Litorimonas haliclonae is proposed. The type strain is MAA42T (=CCM 8709T=CIP 111178T=LMG 29765T).

Can Stereoclusters Separated by Two Methylene Groups Be Related by DFT Studies? The Case of the Cytotoxic Meroditerpenes Halioxepines.

QM/NMR-DFT (quantum mechanics combined with nuclear magnetic resonance parameters calculated by density functional theory approximations) studies allowed us to link two stereoclusters separated by two methylene groups present in the new meroditerpenes halioxepine B (2) and halioxepine C (3) and the known halioxepine (1), isolated from two Indonesian sponges of the genus Haliclona (Reniera). DP4 and DP4+ probabilities were used to discriminate the two diastereotopic arrangements of the two stereoclusters, whose unconnected relative configurations were determined by ROESY and J-based configurational analysis. To confirm the DFT studies, the full relative configuration of 1 was deduced using a mixture of benzene-d6 and pyridine-d5 as the NMR solvent. ROESY measurements connected the two stereoclusters and demonstrated that DFT calculations accurately predict the configuration when two methylenes separate the two stereoclusters. The different arrangements of the distant stereoclusters C-1/C-2/C-7 and C-10/C-15 for compounds 2 and 3 were deduced by DFT calculations and explained the opposite optical rotations observed for the two compounds. Halioxepines B (2) and C (3) display moderate cytotoxicity against different human cancer cell lines.

Skin Delivery of Hydrophilic Biomacromolecules Using Marine Sponge Spicules.

We report the development of sponge Haliclona sp. spicules, referred to as SHS, and its topical application in skin delivery of hydrophilic biomacromolecules, a series of fluorescein isothiocyanate-dextrans (FDs). SHS are silicious oxeas which are sharp-edged and rod-shaped (∼120 µm in length and ∼7 µm in diameter). SHS can physically disrupt skin in a dose-dependent manner and retain within the skin over at least 72 h, which allows sustained skin penetration of hydrophilic biomacromolecules. The magnitude of enhancement of FD delivery into skin induced by SHS treatment was dependent on its molecular weight. Specifically, SHS topical application enhanced FD-10 (MW: 10 kDa) penetration into porcine skin in vitro by 33.09 ± 7.16-fold compared to control group (p < 0.01). SHS dramatically increased the accumulation of FD-10 into and across the dermis by 62.32 ± 13.48-fold compared to the control group (p < 0.01). In vivo experiments performed using BALB/c mice also confirmed the effectiveness of SHS topical application; the skin absorption of FD-10 with SHS topical application was 72.14 ± 48.75-fold (p < 0.05) and 15.39 ± 9.91-fold (p < 0.05) higher than those from the PBS and Dermaroller microneedling, respectively. Further, skin irritation study and transepidermal water loss (TEWL) measurement using guinea pig skin in vivo indicated that skin disruption induced by SHS treatment is self-limited and can be recovered with time and efficiently. SHS can offer a safe, effective, and sustained skin delivery of hydrophilic biomacromolecules and presents a promising platform technology for a wide range of cosmetic and medical applications.

Winogradskyella haliclonae sp. nov., isolated from a marine sponge of the genus Haliclona.

A yellow-pigmented, Gram-stain-negative, motile and rod-shaped bacterium, strain M1A16T, was isolated from the internal tissue of a sponge of the genus Haliclona, which was long-term cultured in the CEMarin aquaria system at Justus Liebig University of Giessen. The strain grew well at 20-32 °C (optimum 25 °C), in the presence of 0-6 % NaCl (optimum 3 %), and at pH 5.5-9.0 (optimum pH 7.0-8.0). Phylogenetic analysis based on its 16S rRNA gene sequence placed the strain within the monophyletic cluster of the genus Winogradskyella with highest sequence similarity to Winogradskyella jejuensis CP32T (98.3 % 16S rRNA gene sequence similarity). Sequence similarities to all other type strains were 98.0 % or less. DNA-DNA hybridization of strain M1A16T with W. jejuensis CP32T resulted in hybridization values of 44.1 % (reciprocal 68.1 %). Major cellular fatty acids of strain M1A16T were iso-C15 : 1 G (18.1 %), iso-C15 : 0 (13.7 %), C16 : 1ω7c (12.9 %), iso-C17 : 0 3-OH (10.6 %) and iso-C16 : 0 3-OH (10.2 %). The overall polyamine content was very low with major components being cadaverine, spermidine and sym-homospermidine. The major quinone was menaquinone MK-6. The polar lipid profile contained predominantly phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids devoid of a detectable functional group. The genomic DNA G+C content was 32.7 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic analyses, strain M1A16T represents a novel species of the genus Winogradskyella, for which the name Winogradskyella haliclonae sp. nov. is proposed. The type strain is M1A16T (=DSM 103138T=CCM 8681T=LMG 29588T=CIP 111091T).

Anti-mycobacterial alkaloids, cyclic 3-alkyl pyridinium dimers, from the Indonesian marine sponge Haliclona sp.

Three new dimeric 3-alkyl pyridinium alkaloids, named haliclocyclamines A-C (1-3), were isolated together with five known congeners, cyclostellettamines A (4), B (5), C (6), E (7), and F (8), from the Indonesian marine sponge Haliclona sp. The structures of 1-3 were assigned based on their spectroscopic data (1D and 2D NMR, HRFABMS, ESIMS/MS, UV, and IR). Compounds 1-8 exhibited antimicrobial activities against Mycobacterium smegmatis with inhibition zones of 17, 10, 13, 14, 8, 8, 12, and 12mm, respectively, at 10µg/disc. Compounds 3 and 8 also modestly inhibited the activity of vaccinia H-1-related phosphatase (VHR), a dual-specificity phosphatase, at 17-18µM.

Metabolic Profiling as a Screening Tool for Cytotoxic Compounds: Identification of 3-Alkyl Pyridine Alkaloids from Sponges Collected at a Shallow Water Hydrothermal Vent Site North of Iceland.

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as Haliclona (Rhizoniera) rosea (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. Haliclona rosea metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.

Antibiotic activity and microbial community of the temperate sponge, Haliclona sp.

AIMS: Sessile marine invertebrates engage in a diverse array of beneficial interactions with bacterial symbionts. One feature of some of these relationships is the presence of bioactive natural products that can defend the holobiont from predation, competition or disease. In this study, we investigated the antimicrobial activity and microbial community of a common temperate sponge from coastal North Carolina. METHODS AND RESULTS: The sponge was identified as a member of the genus Haliclona, a prolific source of bioactive natural products, based on its 18S rRNA gene sequence. The crude chemical extract and methanol partition had broad activity against the assayed Gram-negative and Gram-positive pathogenic bacteria. Further fractionation resulted in two groups of compounds with differing antimicrobial activity, primarily against Gram-positive test organisms. There was, however, notable activity against the Gram-negative marine pathogen, Vibrio parahaemolyticus. Microbial community analysis of the sponge and surrounding sea water via denaturing gradient gel electrophoresis (DGGE) indicates that it harbours a distinct group of bacterial associates. CONCLUSIONS: The common temperate sponge, Haliclona sp., is a source of multiple antimicrobial compounds and has some consistent microbial community members that may play a role in secondary metabolite production. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that common temperate sponges can be a source of bioactive chemical and microbial diversity. Further studies may reveal the importance of the microbial associates to the sponge and natural product biosynthesis.

Antibacterial activity and mutagenesis of sponge-associated Pseudomonas fluorescens H41.

Marine sponges (phylum Porifera) are well known to harbour a complex and diverse bacterial community. Some of these sponge-associated bacteria have been shown to be the real producers of secondary metabolites with a wide range of activities from antimicrobials to anticancer agents. Previously, we revealed that the strain Pseudomonas fluorescens H41 isolated from the sponge Haliclona sp. (collected at the coast of Rio de Janeiro, Brazil) showed a strong antimicrobial activity against clinical and marine bacteria. Thus, in this study the genes involved in the antimicrobial activity of P. fluorescens H41 were identified. To this end, a library of mutants was generated via miniTnphoA3 transposon mutagenesis and the resulting clones were characterized for their antimicrobial activity. It was demonstrated that genes involved in the biosynthesis of the pyoverdine siderophore are related to the inhibitory activity of P. fluorescens H41. Therefore, this strain might play an important role in the biocontrol of the host sponge.

Chemistry of Renieramycins. Part 14: Total Synthesis of Renieramycin I and Practical Synthesis of Cribrostatin 4 (Renieramycin H).

The first total synthesis of (±)-renieramycin I, which was isolated from the Indian bright blue sponge Haliclona cribricutis, is described. The key step is the selenium oxide oxidation of pentacyclic bis-p-quinone derivative (3) stereo- and regioselectively. We also report a large-scale synthesis of cribrostatin 4 (renieramycin H) via the C3-C4 double bond formation in an early stage based on the Avendaño's protocol, from readily available 1-acetyl-3-(3-methyl-2,4,5-trimethylphenyl)methyl-piperazine-2,5-dione (8) in 18 steps (8.3% overall yield). The synthesis provides unambiguous evidence supporting the original structure of renieramycin I.