CHEMOTAXONOMY IN MARINE ALGAE: CHEMICAL SEPARATION OF SOME LAURENCIA SPECIES (RHODOPHYTA) FROM THE GULF OF CALIFORNIA1

Species separation in the genus Laurencia (Rhodomelaceae, Rhodophyta) is complicated by the high degree of morphological variation within the species. Chemical investigations on a worldwide basis of over 15 species indicate that 1 or more of the halogenated natural products synthesized by Laurencia are unique to each species. Our chemical investigations of Laurencia pacifica, as presently understood from the Gulf of California, indicate that more than 1 species had been included under this name. Thin layer chromatographic (TLC) comparisons of the halogenated components of 3 recognizable forms of “L. pacifica” were completed. The results revealed 3 distinct forms, with halogenated products unique to each form. In each form the observed chemical characters had been previously isolated and identified and could now be positively assigned to their algal source. Comparisons were also conducted with L. pacifica Kylin (1941) from the type locality of the species, La Jolla, California, and revealed that it contained another halogenated product different from those isolated from the Gulf species. We conclude that 3 species of Laurencia have been elucidated in the Gulf of California and these are separate from L. pacifica Kylin. Each species can be distinguished by its characteristic array of halogenated compounds. Comparative thin layer chromatography of the lipid components of morphologically similar Laurencia species should prove to be a useful new taxonomic aid.     

Antibacterial activity of halogenated sesquiterpenes from Malaysian Laurencia spp

During our studies on Malaysian Laurencia species, brominated metabolites, tiomanene, acetylmajapolene B, and acetylmajapolene A were isolated from an unrecorded species collected at Pulau Tioman, Pahang along with known majapolene B and majapolene A. Acetylmajapolene A was a mixture of diastereomers as in the case of majapolene A. Tiomanene may be a plausible precursor for acetylmajapolenes B and A. In addition, three known halogenated sesquiterpenes and two known halogenated C₁₅ acetogenins were found from other two unrecorded species collected at Pulau Karah, Terengganu and Pulau Nyireh, Terengganu, respectively. Some of these halogenated metabolites showed moderate antibacterial activity against some marine bacteria.     

Chemical relationship between red algae genus Laurencia and sea hare (Aplysia dactylomela Rang) in the North Borneo Island

Red algae genus Laurencia is an interesting alga with the ability to produce halogenated secondary metabolites that exhibits ecological and pharmaceutical potential. In nature, Laurencia is selectively grazed by sea hares (Aplysia dactylomela). In this study, Laurencia populations in three islands (Mantanani, Sulug, and Dinawan Islands) in the coastal waters of North Borneo were investigated and their chemical relationship with sea hare determined. Four species of Laurencia were found to grow abundantly in these waters, Laurencia snackeyi, Laurencia majuscula, Laurencia nangii, and Laurencia similis. Sea hares, Aplysia dactylomela, found grazing on Laurencia were collected and their chemical composition determined. A total of 20 halogenated metabolites were isolated and identified via spectroscopic data. Isolated compounds could be grouped into syndrean (5), chamigrane (6), non-chamigrane sesquiterpene (3), cuparane (1), bromoindole (2), and C15 acetogenin (acetylene type) (3). Sea hares from Mantanani, Sulug, and Dinawan Islands contained a total of 9, 10, and 10 compounds, respectively. In addition, 12-acetoxypalisadin B (1), which was isolated from sea hares of Sulug Island is a first record of its existence in nature.     

Brominated metabolites from an Okinawan Laurencia intricata

Two halogenated C₁₅ acetogenins, itomanallenes A and B, with a terminal bromoallene moiety along with a halogenated sesquiterpene, itomanol, have been isolated from the red alga Laurencia intricata collected in Okinawan waters. Their structures were deduced from 1D and 2D NMR experiments including ¹H–¹H COSY, HSQC, HMBC, and NOESY methods. The alcohol corresponding to itomanallene B seems to be a plausible precursor of itomanallene A, which has an unusual 2,10-dioxabicyclo[7.3.0]dodecene skeleton. Itomanol was found to be a selinane-type bromosesquiterpenoid, and is the first example of a selinane to be isolated from Japanese Laurencia species.     

Structural diversity and geographical distribution of halogenated secondary metabolites in red algae,Laurencia nangii Masuda (Rhodomelaceae,Ceramiales), in the coastal waters of North Borneo Island

The red algae genus Laurencia (Rhodomelaceae, Ceramiales) is known as a prolific producer of halogenated secondary metabolites with a high level of species diversity and geographical distribution. In North Borneo Island, Malaysia, there are four main Laurencia species: Laurencia snackeyi, Laurencia majuscula, Laurencia similis and L. nangii. Although the chemistry of Laurencia is well studied, the diversity of compounds in L. nangii has not been thoroughly investigated. Therefore, we studied the chemical constituents of seven populations of L. nangii from Tunku Abdul Rahman Marine Park (two populations), Dinawan Island (one population), Tun Mustapha Marine Park (two populations) and Tun Sakaran Marine Park (two populations). Halogenated compounds were isolated and the structures determined via spectroscopic methods. A total of 20 metabolites belonging to the classes of sesquiterpenes, acetylenes, bromoallenes, diterpenes and triterpenes were identified. Populations from Tunku Abdul Rahman Marine Park and Dinawan Island contained non-chamigrane-type sesquiterpenes, acetylenes and diterpenes. Populations from Tun Mustapha Marine Park contained chamigrane-type sesquiterpenes, acetylenes and diterpenes. However, the chemical compositions of populations from Tun Sakaran Marine Park were found to differ significantly, containing chamigrane-type and non-chamigrane-type sesquiterpenes, bromoallenes and triterpenes. This investigation has revealed the presence of interesting chemotaxonomical markers in populations of L. nangii and the existence of chemical races in this species.     

An experimental approach to study the biosynthesis of brominated metabolites by the red algal genus Laurencia

The production of labeled brominated metabolites with radioactive ⁸²Br in Laurencia species was investigated as part of a study of the biosynthesis of halogenated metabolites from species belonging to the red algal genus Laurencia (Rhodomelaceae, Ceramiales). Radiobromide [⁸²Br], thin-layer chromatography (TLC), and TLC–autoradioluminography (ARLG) were used. When cultured in artificial seawater medium (ASP₁₂NTA including Na⁸²Br) under 16:8 h light:dark (LD) illumination cycles for 24 h, each of the strains of Laurencia, Laurencia japonensis Abe et Masuda, Laurencia nipponica Yamada (laurencin-producing race and laureatin-producing race), and Laurencia okamurae Yamada, produced species- (or race-) specific ⁸²Br-containing metabolites. In the case of the laurencin-producing race of L. nipponica, laurencin and deacetyllaurencin were found to be produced in approximately 1:1 ratio, though laurencin is the major metabolite in the wild sample. Furthermore, when cultured in the dark, the production rates of brominated metabolites in Laurencia spp. were found to be diminished. The present study strongly indicates that the use of radiobromine [⁸²Br] in combination with the TLC–ARLG method is an effective approach for investigating the biosynthesis of brominated metabolites in Laurencia.     

Teanol,a new brominated sesquiterpene from the Thailand Laurencia mariannensis

We examined the constitution of Laurencia mariannensis collected from the east coast of Tean Island, Thailand. The structure of a new brominated rearranged cyclolaurane-type sesquiterpene, named teanol (1), is reported. This is the first report on biology and chemistry of the Thailand red algal genus Laurencia (Rhodomelaceae, Ceramiales).     

Chemical composition of Laurencia spp. collected from the Seto Inland Sea of Japan

The chemical composition of three Laurencia spp., Laurencia sp., L. okamurae and L. saitoi, which were collected from the Seto Inland Sea of Japan, has been examined. Laurencia sp. collected from the coast of Matoba Park, Takehara, Hiroshima Prefecture, contained a brominated chamigrane-type sesquiterpene (1), named matobol, as the main metabolite. The structure of matobol was determined as (+)-(2R,3R,6R,10S)-2,10-dibromochamigr-7(14)-en-3-ol (1). This is the first time that the optically active 1 has been isolated from Laurencia. On the other hand, L. okamurae from the coast of Ikunoshima Island, Hiroshima Prefecture, produced laurinterol (2) that is a known cyclolaurane-type sesquiterpene characteristic to this species in Japan. L. saitoi from the coast of Matoba Park contained a known bromoallenic C₁₅-acetogenin, neolaurallene (3).     

Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by Nitrosomonas europaea

Ammonia monooxygenase (AMO) from Nitrosomonas europaea catalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of N. europaea. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with N. europaea cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by N. europaea. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH₄⁺ in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH₃ to concentrations (8 to 80 μM) similar to the K_s value of AMO for NH₃ (~29 μM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.     

Chloromethane, a Novel Methyl Donor for Biosynthesis of Esters and Anisoles in Phellinus pomaceus

Chloromethane (CH₃Cl), a gaseous natural product released as a secondary metabolite by many woodrotting fungi of the family Hymenochaetaceae, has been shown to act as a methyl donor for biosynthesis of methyl esters of benzoic and furoic acid in the primary metabolism of Phellinus pomaceus. The broad-specificity methylating system could esterify a wide range of aromatic and aliphatic acids. In addition to CH₃Cl, both bromo- and iodomethanes acted as methyl donors. Methylation did not appear to proceed via methanol or a coenzyme A intermediate. The initial growth-related accumulation of methyl benzoate during culture of P. pomaceus was paralleled by an increase in activity of the methylating system in the mycelium. Changes in percent incorporation of C²H₃ from exogenous C²H₃Cl during growth indicated that although utilization of CH₃Cl was initially closely coupled to biosynthesis of the compound, the system became less tightly channeled later in growth. This phase coincided with release of gaseous CH₃Cl by the fungus. A biochemically distinct CH₃Cl-utilizing system capable of methylating phenols and thiophenol was also identified in the fungus, but in contrast with the carboxylic acid-methylating system, it attained maximum activity in the idiophase. Preliminary investigations of a non-CH₃Cl-releasing fungus, Fomitopsis pinicola, have shown the presence of a CH₃Cl-utilizing system capable of methylating benzoic acid, suggesting that CH₃Cl biosynthesis may occur in non-hymenochaetaceous fungi. Halogenated compounds hitherto found in nature are mainly stable end products of metabolism. The participation of CH₃Cl in primary fungal metabolism demonstrates that some halometabolites may have a previously unrecognized role as intermediates in the biosynthesis of nonhalogenated natural products.     

Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules: BIOGENESIS OF THE BROAD SPECTRUM ANTIFUNGAL AND ANTI-OOMYCETE COMPOUND,OOCYDIN A

Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties.     

A rearranged chamigrene derivative and its potential biogenetic precursor from a new species of the marine red algal genus Laurencia (rhodomelaceae)

The structures of a novel rearranged sesquiterpenoid and a biogenetically-related chamigrene derivative have been determined by combined spectral and chemical methods. These sesquiterpenoids were components of an undescribed Laurencia species, and each was toxic toward the damselfish Pomacentrus coeruleus.     

Additional analysis of chemical diversity of the red algal genus Laurencia (Rhodomelaceae) from Japan

The halogenated secondary metabolite constitution of four species of the red algal genus Laurencia (Rhodomelaceae) from southern Japan is reported. Laurencia composita Yamada from Tanegashima Island (Kagoshima Prefecture) bears five sesquiterpenoids (2,10‐dibromo‐3‐chloro‐α‐chamigrene and 2,10‐dibromo‐3‐chloro‐9‐hydroxy‐α‐chamigrene, in addition to pre‐pacifenol epoxide, johnstonol and pacifenol, which are known in other populations of this species). Laurencia intricata Lamouroux from Chinzei (Saga Prefecture) and Oomura Bay (Nagasaki Prefecture) bear a C₁₅ aceto‐genin, okamurallene. Laurencia majuscula (Harvey) Lucas from Tanegashima Island produces three sesquiterpenoids, (Z)‐10,15‐dibromo‐9‐hydroxy‐chamigra‐1, 3(15),7(14)‐triene, 10‐bromo‐7‐hydroxylaurene and 10,11‐dibromo‐7‐hydroxylaurene, corresponding to those of one of its chemical races. Laurencia venusta Yamada from Tanegashima Island produces two sesquiterpenoids, cupalaurenol and cyclolaurenol, which were known only from a sea hare, Aplysia dactylomela Rang. This strongly suggests that Aplysia consumes L. venusta and concentrates these halogenated compounds.     

Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface

The pregnancy-related serine protease HtrA3 plays an important role in human placental development and has recently been recognized as a potential therapeutic target in the treatment of cancer. Previously, a C-terminal pentapeptide FGRWV^–COOH was identified to bind at the PDZ domain of HtrA3 with a moderate affinity. Here, based on the high-resolution complex crystal structure of HtrA3 PDZ domain with the pentapeptide ligand we successfully introduced a rationally designed halogen bond to the complex interface by substituting R₄-hydrogen atom of the indole moiety of peptide Trp_-1 residue with a halogen atom. High-level theoretical calculations suggested that bromine is the best choice that can render strong interaction energy for the halogen bond and can confer high affinity to the PDZ–peptide complex. Fluorescence spectroscopy characterizations revealed that the resulting R₄-brominated peptide (K _d = 0.15 ± 0.03 μM) exhibited 12-fold affinity improvement relative to its nonhalogenated counterpart (K _d = 1.8 ± 0.4 μM). In contrast, the PDZ-binding affinity of R₆-brominated peptide (K _d = 1.2 ± 0.1 μM), a negative control that was unable to form the halogen bond according to theoretical investigations, did not change substantially as compared to the nonhalogenated peptide.     

Antiprotozoal activities of marine polyether triterpenoids

Chagas disease and leishmaniasis are tropical neglected diseases caused by kinetoplastids protozoan parasites of Trypanosoma and Leishmania genera, and a public health burden with high morbidity and mortality rates in developing countries. Among difficulties with their epidemiological control, a major problem is their limited and toxic treatments to attend the affected populations; therefore, new therapies are needed in order to find new active molecules. In this work, sixteen Laurencia oxasqualenoid metabolites, natural compounds 1–11 and semisynthetic derivatives 12–16, were tested against Leishmania amazonensis, Leishmania donovani and Trypanosoma cruzi. The results obtained point out that eight substances possess potent activities, with IC₅₀ values in the range of 5.40–46.45 µM. The antikinetoplastid action mode of the main metabolite dehydrothyrsiferol (1) was developed, also supported by AFM images.The semi-synthetic active compound 28-iodosaiyacenol B (15) showed an IC₅₀ 5.40 µM against Leishmania amazonensis, turned to be non-toxic against the murine macrophage cell line J774A.1 (CC₅₀ > 100). These values are comparable with the reference compound miltefosine IC₅₀ 6.48 ± 0.24 and CC₅₀ 72.19 ± 3.06 μM, suggesting that this substance could be scaffold for development of new antikinetoplastid drugs.     

Identification of novel inhibitors of phospho-MurNAc-pentapeptide translocase MraY from library screening: Isoquinoline alkaloid michellamine B and xanthene dye phloxine B

The National Cancer Institute (NCI) Diversity Set was screened for potential inhibitors of phospho-MurNAc-pentapeptide translocase MraY from Escherichia coli using a primary fluorescence enhancement assay, followed by a secondary radiochemical assay. One new MraY inhibitor was identified from this screen, a naphthylisoquinoline alkaloid michellamine B, which inhibited E. coli MraY (IC₅₀ 456 μM) and Bacillus subtilis MraY (IC₅₀ 386 μM), and which showed antimicrobial activity against B. subtilis (MIC 16 μg/mL). Following an earlier report of halogenated fluoresceins identified from a combined MraY/MurG screen, three halogenated fluoresceins were tested as inhibitors of E. coli MraY and E. coli MurG, and phloxine B was identified as an inhibitor of E. coli MraY (IC₅₀ 32 μM). Molecular docking of inhibitor structures against the structure of Aquifex aeolicus MraY indicates that phloxine B appears to bind to the Mg²⁺ cofactor in the enzyme active site, while michellamine B binds to a hydrophobic groove formed between transmembrane helices 5 and 9.     

Chemical variation in a new bromochamigrene derivative from the red seaweed Laurencia pacifica

The secondary metabolite chemistry of the red seaweed Laurencia pacifica Kylin varies geographically. The La Jolla, Calif. population (type locality) contains the sesquiterpene prepacifenol, while L. pacifica collected south of Ensenada, Mexico contains a new bromochamigrene derivative, the structure of which is described herein. Since chamigrene synthesis in Laurencia species has been demonstrated to be reasonably species-specific, L. pacifica may be a mixture of morphologically similar forms.