Expression and Characterization of a Novel Cold-Adapted and Stable β-Agarase Gene agaW1540 from the Deep-Sea Bacterium Shewanella sp. WPAGA9

The neoagaro-oligosaccharides, degraded from agarose by agarases, are important natural substances with many bioactivities. In this study, a novel agarase gene, agaW1540, from the genome of a deep-sea bacterium Shewanella sp. WPAGA9, was expressed, and the recombinant AgaW1540 (rAgaW1540) displayed the maximum activity under the optimal pH and temperature of 7.0 and 35 °C, respectively. rAgaW1540 retained 85.4% of its maximum activity at 0 °C and retained more than 92% of its maximum activity at the temperature range of 20–40 °C and the pH range of 4.0–9.0, respectively, indicating its extensive working temperature and pH values. The activity of rAgaW1540 was dramatically suppressed by Cu²⁺ and Zn²⁺, whereas Fe²⁺ displayed an intensification of enzymatic activity. The K_m and V_max of rAgaW1540 for agarose degradation were 15.7 mg/mL and 23.4 U/mg, respectively. rAgaW1540 retained 94.7%, 97.9%, and 42.4% of its maximum activity after incubation at 20 °C, 25 °C, and 30 °C for 60 min, respectively. Thin-layer chromatography and ion chromatography analyses verified that rAgaW1540 is an endo-acting β-agarase that degrades agarose into neoagarotetraose and neoagarohexaose as the main products. The wide variety of working conditions and stable activity at room temperatures make rAgaW1540an appropriate bio-tool for further industrial production of neoagaro-oligosaccharides.     

The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidans

Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α(1,3)-fucoidanase encoding gene, Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca²⁺ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per μM enzyme: U_f/μM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10⁻³ U_f/μM and 3.6 × 10⁻³ U_f/μM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α(1,3)-linked fucosyl residues with unique and novel α(1,4)-linked fucosyl branches, not previously identified in fucoidans from S. latissima.     

Secondary Metabolites with α-Glucosidase Inhibitory Activity from Mangrove Endophytic Fungus Talaromyces sp. CY-3

Eight new compounds, including two sambutoxin derivatives (1–2), two highly oxygenated cyclopentenones (7–8), four highly oxygenated cyclohexenones (9–12), together with four known sambutoxin derivatives (3–6), were isolated from semimangrove endophytic fungus Talaromyces sp. CY-3, under the guidance of molecular networking. The structures of new isolates were elucidated by analysis of detailed spectroscopic data, ECD spectra, chemical hydrolysis, ¹³C NMR calculation, and DP4+ analysis. In bioassays, compounds 1–5 displayed better α-glucosidase inhibitory activity than the positive control 1-deoxynojirimycin (IC₅₀ = 80.8 ± 0.3 μM), and the IC₅₀ value was in the range of 12.6 ± 0.9 to 57.3 ± 1.3 μM.     

A Novel Agarase,Gaa16B,Isolated from the Marine Bacterium Gilvimarinus agarilyticus JEA5, and the Moisturizing Effect of Its Partial Hydrolysis Products

We recently identified a β-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of β-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 °C and pH 6–7, respectively, and the protein was highly stable at 55 °C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl₂. The K_m and V_max of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.     

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

Genome sequencing of Catenovulum agarivorans YM01^T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01^T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a K_m of 3.78 mg mL⁻¹ for agarose and a V_max of 1.14 × 10⁴ U mg⁻¹. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.     

Interactions of Globular and Ribbon [γ4E]GID with α4β2 Neuronal Nicotinic Acetylcholine Receptor

The α4β2 nAChR is implicated in a range of diseases and disorders including nicotine addiction, epilepsy and Parkinson’s and Alzheimer’s diseases. Designing α4β2 nAChR selective inhibitors could help define the role of the α4β2 nAChR in such disease states. In this study, we aimed to modify globular and ribbon α-conotoxin GID to selectively target the α4β2 nAChR through competitive inhibition of the α4(+)β2(−) or α4(+)α4(−) interfaces. The binding modes of the globular α-conotoxin [γ4E]GID with rat α3β2, α4β2 and α7 nAChRs were deduced using computational methods and were validated using published experimental data. The binding mode of globular [γ4E]GID at α4β2 nAChR can explain the experimental mutagenesis data, suggesting that it could be used to design GID variants. The predicted mutational energy results showed that globular [γ4E]GID is optimal for binding to α4β2 nAChR and its activity could not likely be further improved through amino-acid substitutions. The binding mode of ribbon GID with the (α4)₃(β2)₂ nAChR was deduced using the information from the cryo-electron structure of (α4)₃(β2)₂ nAChR and the binding mode of ribbon AuIB. The program FoldX predicted the mutational energies of ribbon [γ4E]GID at the α4(+)α4(−) interface, and several ribbon[γ4E]GID mutants were suggested to have desirable properties to inhibit (α4)₃(β2)₂ nAChR.     

A Novel α4/7-Conotoxin QuIA Selectively Inhibits α3β2 and α6/α3β4 Nicotinic Acetylcholine Receptor Subtypes with High Efficacy

α6β4 nAChR is expressed in the peripheral and central nervous systems and is associated with pain, addiction, and movement disorders. Natural α-conotoxins (α-CTxs) can effectively block different nAChR subtypes with higher efficacy and selectivity. However, the research on α6β4 nAChR is relatively poor, partly because of the lack of available target-specific α-CTxs. In this study, we synthesized a novel α-4/7 conotoxin QuIA that was found from Conus quercinus. We investigated the efficacy of this peptide to different nAChR subtypes using a two-electrode voltage-clamp technique. Remarkably, we found α-QuIA inhibited the neuronal α3β2 and α6/α3β4 nAChR subtypes with significantly high affinity (IC₅₀ was 55.7 nM and 90.68 nM, respectively), and did not block other nAChR subtypes even at a high concentration of 10 μM. In contrast, most α-CTxs have been determined so far to effectively block the α6/α3β4 nAChR subtype while also maintaining a similar higher efficacy against the closely related α6β2β3 and/or α3β4 subtypes, which are different from QuIA. In conclusion, α-QuIA is a novel α4/7-CTx, which has the potential to develop as an effective neuropharmacology tool to detect the function of α6β4 nAChR.     

Anti-Inflammatory Components of the Starfish Astropecten polyacanthus

Inflammation is important in biomedical research, because it plays a key role in inflammatory diseases including rheumatoid arthritis and other forms of arthritis, diabetes, heart disease, irritable bowel syndrome, Alzheimer’s disease, Parkinson’s disease, allergies, asthma, and even cancer. In the present study, we describe the inhibitory effect of crude extracts and steroids isolated from the starfish Astropecten polyacanthus on pro-inflammatory cytokine (Interleukin-12 (IL-12) p40, interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α)) production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). Among those tested, compounds 5 and 7 showed potent inhibitory effects on the production of all three pro-inflammatory cytokines with IC₅₀ values ranging from 1.82 ± 0.11 to 7.00 ± 0.16 μM. Potent inhibitory activities were also observed for compound 1 on the production of IL-12 p40 and IL-6 with values of 3.96 ± 0.12 and 4.07 ± 0.13 μM, respectively, and for compounds 3 and 4 on the production of IL-12 p40 with values of 6.55 ± 0.18 and 5.06 ± 0.16 μM, respectively. Moreover, compounds 2 (IC₅₀ = 34.86 ± 0.31 μM) and 6 (IC₅₀ = 79.05 ± 2.05 μM) exhibited moderate inhibitory effects on the production of IL-12 p40, whereas compounds 3 (IC₅₀ = 22.80 ± 0.21 μM) and 4 (IC₅₀ = 16.73 ± 0.25 μM) moderately inhibited the production of TNF-α and IL-6, respectively.     

Characterization of an α 4/7-Conotoxin LvIF from Conus lividus That Selectively Blocks α3β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptor (nAChR), a member of pentameric ligand-gated ion channel transmembrane protein composed of five subunits, is widely distributed in the central and peripheral nervous system. The nAChRs are associated with various neurological diseases, including schizophrenia, Alzheimer’s disease, Parkinson’s disease, epilepsy and neuralgia. Receptors containing the α3 subunit are associated with analgesia, generating our interest in their role in pharmacological studies. In this study, α-conotoxin (α-CTx) LvIF was identified as a 16 amino acid peptide using a genomic DNA clone of Conus lividus (C. lividus). The mature LvIF with natural structure was synthesized by a two-step oxidation method. The blocking potency of α-CTx lvIF on nAChR was detected by a two-electrode voltage clamp. Our results showed that α-CTx LvIF was highly potent against rα3β2 and rα6/α3β2β3 nAChR subtypes, The half-maximal inhibitory concentration (IC₅₀) values of α-CTx LvIF against rα3β2 and rα6/α3β2β3 nAChRs expressed in Xenopus oocytes were 8.9 nM and 14.4 nM, respectively. Furthermore, α-CTx LvIF exhibited no obvious inhibition on other nAChR subtypes. Meanwhile, we also conducted a competitive binding experiment between α-CTxs MII and LvIF, which showed that α-CTxs LvIF and MII bind with rα3β2 nAChR at the partial overlapping domain. These results indicate that the α-CTx LvIF has high potential as a new candidate tool for the studying of rα3β2 nAChR related neurophysiology and pharmacology.     

Genome Mining of α-Pyrone Natural Products from Ascidian-Derived Fungus Amphichorda felina SYSU-MS7908

Culturing ascidian-derived fungus Amphichorda felina SYSU-MS7908 under standard laboratory conditions mainly yielded meroterpenoid, and nonribosomal peptide-type natural products. We sequenced the genome of Amphichorda felina SYSU-MS7908 and found 56 biosynthetic gene clusters (BGCs) after bioinformatics analysis, suggesting that the majority of those BGCSs are silent. Here we report our genome mining effort on one cryptic BGC by heterologous expression in Aspergillus oryzae NSAR1, and the identification of two new α-pyrone derivatives, amphichopyrone A (1) and B (2), along with a known compound, udagawanone A (3). Anti-inflammatory activities were performed, and amphichopyrone A (1) and B (2) displayed potent anti-inflammatory activity by inhibiting nitric oxide (NO) production in RAW264.7 cells with IC₅₀ values 18.09 ± 4.83 and 7.18 ± 0.93 μM, respectively.     

Microwave-Assisted Extraction of Phlorotannins from Fucus vesiculosus

Microwave-assisted extraction (MAE) was carried out to maximize the extraction of phlorotannins from Fucus vesiculosus using a hydroethanolic mixture as a solvent, as an alternative to the conventional method with a hydroacetonic mixture. Optimal MAE conditions were set as ethanol concentration of 57% (v/v), temperature of 75 °C, and time of 5 min, which allowed a similar recovery of phlorotannins from the macroalgae compared to the conventional extraction. While the phlorotannins richness of the conventional extract was slightly superior to that of MAE (11.1 ± 1.3 vs. 9.8 ± 1.8 mg PGE/g DW_extract), both extracts presented identical phlorotannins constituents, which included, among others, tetrafucol, pentafucol, hexafucol, and heptafucol structures. In addition, MAE showed a moderate capacity to scavenge ABTS^•+ (IC₅₀ of 96.0 ± 3.4 µg/mL) and to inhibit the activity of xanthine oxidase (IC₅₀ of 23.1 ± 3.4 µg/mL) and a superior ability to control the activity of the key metabolic enzyme α-glucosidase compared to the pharmaceutical drug acarbose.     

Bioactive Metabolites from Mangrove Endophytic Fungus Aspergillus sp. 16-5B

Chemical investigation of the endophytic fungus Aspergillus sp. 16-5B cultured on Czapek’s medium led to the isolation of four new metabolites, aspergifuranone (1), isocoumarin derivatives (±) 2 and (±) 3, and (R)-3-demethylpurpurester A (4), together with the known purpurester B (5) and pestaphthalides A (6). Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The absolute configuration of Compound 1 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra, and that of Compound 4 was revealed by comparing its optical rotation data and CD with those of the literature. The structure of Compound 6 was further confirmed by single-crystal X-ray diffraction experiment using CuKα radiation. All isolated compounds were evaluated for their α-glucosidase inhibitory activities, and Compound 1 showed significant inhibitory activity with IC₅₀ value of 9.05 ± 0.60 μM. Kinetic analysis showed that Compound 1 was a noncompetitive inhibitor of α-glucosidase. Compounds 2 and 6 exhibited moderate inhibitory activities.     

Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson’s disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3β2β3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers’ yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH₄HCO₃ or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.     

α-Conotoxin TxID and [S9K]TxID, α3β4 nAChR Antagonists,Attenuate Expression and Reinstatement of Nicotine-Induced Conditioned Place Preference in Mice

Tobacco smoking has become a prominent health problem faced around the world. The α3β4 nicotinic acetylcholine receptor (nAChR) is strongly associated with nicotine reward and withdrawal symptom. α-Conotoxin TxID, cloned from Conus textile, is a strong α3β4 nAChR antagonist, which has weak inhibition activity of α6/α3β4 nAChR. Meanwhile, its analogue [S9K]TxID only inhibits α3β4 nAChR (IC₅₀ = 6.9 nM), and has no inhibitory activity to other nAChRs. The present experiment investigates the effect of α3β4 nAChR antagonists (TxID and [S9K]TxID) on the expression and reinstatement of nicotine-induced conditioned place preference (CPP) and explores the behaviors of acute nicotine in mice. The animal experimental results showed that TxID and [S9K] TxID could inhibit the expression and reinstatement of CPP, respectively. Moreover, both had no effect in acute nicotine experiment and the locomotor activity in mice. Therefore, these findings reveal that the α3β4 nAChR may be a potential target for anti-nicotine addiction treatment. [S9K]TxID, α3β4 nAChR antagonist, exhibit a superior effect for anti-nicotine addiction, which is promising to develop a novel smoking cessation drug.     

Epipolythiodiketopiperazines from the Marine Derived Fungus Dichotomomyces cejpii with NF-κB Inhibitory Potential

The Ascomycota Dichotomomyces cejpii was isolated from the marine sponge Callyspongia cf. C. flammea. A new gliotoxin derivative, 6-acetylmonodethiogliotoxin (1) was obtained from fungal extracts. Compounds 2 and 3, methylthio-gliotoxin derivatives were formerly only known as semi-synthetic compounds and are here described as natural products. Additionally the polyketide heveadride (4) was isolated. Compounds 1, 2 and 4 dose-dependently down-regulated TNFα-induced NF-κB activity in human chronic myeloid leukemia cells with IC_50s of 38.5 ± 1.2 µM, 65.7 ± 2.0 µM and 82.7 ± 11.3 µM, respectively. The molecular mechanism was studied with the most potent compound 1 and results indicate downstream inhibitory effects targeting binding of NF-κB to DNA. Compound 1 thus demonstrates potential of epimonothiodiketopiperazine-derived compounds for the development of NF-κB inhibitors.     

Inhibitory Effect of N,N-Didesmethylgrossularine-1 on Inflammatory Cytokine Production in Lipopolysaccharide-Stimulated RAW 264.7 Cells

N,N-Didesmethylgrossularine-1 (DDMG-1), a compound with a rare α-carboline structure, was isolated from an Indonesian ascidian Polycarpa aurata as responsible for the observed inhibitory activity against TNF-α production in lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells. DDMG-1 inhibited the mRNA level of mTNF-α, IκB-α degradation, and binding of NF-κB to the target DNA site in LPS-stimulated RAW 264.7 cells. Moreover, DDMG-1 had an inhibitory effect on the production of IL-8, which is produced in CD14⁺-THP-1 cells stimulated by LPS. DDMG-1 is thus a promising drug candidate lead compound for the treatment of chronic inflammatory diseases, such as rheumatoid arthritis.     

Magnificines A and B,Antimicrobial Marine Alkaloids Featuring a Tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione Backbone from the Red Sea Sponge Negombata magnifica

Investigation of the Red Sea sponge Negombata magnifica gave two novel alkaloids, magnificines A and B (1 and 2) and a new β-ionone derivative, (±)-negombaionone (3), together with the known latrunculin B (4) and 16-epi-latrunculin B (5). The analysis of the NMR and HRESIMS spectra supported the planar structures and the relative configurations of the compounds. The absolute configurations of magnificines A and B were determined by the analysis of the predicted and experimental ECD spectra. Magnificines A and B possess a previously unreported tetrahydrooxazolo[3,2-a]azepine-2,5(3H,6H)-dione backbone and represent the first natural compounds in this class. (±)-Negombaionone is the first β-ionone of a sponge origin. Compounds 1-3 displayed selective activity against Escherichia coli in a disk diffusion assay with inhibition zones up to 22 mm at a concentration of 50 µg/disc and with MIC values down to 8.0 µM. Latrunculin B and 16-epi-latrunculin B inhibited the growth of HeLa cells with IC₅₀ values down to 1.4 µM.     

First Identification of 12β-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12β-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC)

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12β-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12β-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12β-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.