Genetic origin of homopyrones, a rare type of hybrid phenylpropanoid- and polyketide-derived yellow pigments from Aspergillus homomorphus.

In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a C-methylation domain, which however seems to be promiscuous since only 2 is C-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi. KEY POINTS: • Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA. • CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.

Spectroscopic Fingerprinting of Organic Material Extracted from Tight Chalk Core Samples of the North Sea.

The detailed chemical composition of crude oil in subsurface reservoirs provides important information about reservoir connectivity and can potentially play a very important role for the understanding of recovery processes. Relying on studying produced oil samples alone to understand the rock-fluid and fluid-fluid interactions is insufficient as the heavier polar components may be retained by tight reservoirs and not produced. These heavy and polar compounds that constitute the resin and asphaltene fractions of crude oil are typically present in low concentrations and yet are determining for the physical-chemical properties of the oil because of their polarity. In order to obtain a fingerprint analysis of oils including polar compounds from different wells, the oil content of drill cores has been extracted and analyzed. Infrared spectroscopy has been used to perform chemical fingerprinting of the oil extracted from drill cores sampled in different geographical locations of the Danish North Sea. Statistical analysis has been employed to identify the chemical differences within the sample set and explore the link between chemical composition and geographic location of the sample. A principal component analysis, based on spectral peak fitting in the 1800-1400 cm-1 range, has allowed for statistical grouping of the samples and identified the primary chemical feature characteristic of these groups. Statistically significant differences in the quantities of polar oxygen- and nitrogen-containing compounds were found between the oil wells. The results of this analysis have been used as guidelines and reference to establish an express statistical approach based on the full-range infrared spectra for a further expansion of the sample set. The chemical information presented in this work is discussed in relation to oil fingerprinting and geochemical analysis.

Improved NOE fitting for flexible molecules based on molecular mechanics data - a case study with S-adenosylmethionine.

The use of molecular dynamics (MD) calculations to derive relative populations of conformers is highly sensitive to both timescale and parameterisation of the MD. Where these calculations are coupled with NOE data to determine the dynamics of a molecular system, this can present issues if these populations are thus relied upon. We present an approach that refines the highly accurate PANIC NMR methodology combined with clustering approaches to generate conformers, but without restraining the simulations or considering the relative population distributions generated by MD. Combining this structural sampling with NOE fitting, we demonstrate, for S-adenosylmethionine (aqueous solution at pH 7.0), significant improvements are made to the fit of populations to the experimental data, revealing a strong overall preference for the syn conformation of the adenosyl group relative to the ribose ring, but with less discrimination for the conformation of the ribose ring itself.

Karmitoxin: An Amine-Containing Polyhydroxy-Polyene Toxin from the Marine Dinoflagellate Karlodinium armiger.

Marine algae from the genus Karlodinium are known to be involved in fish-killing events worldwide. Here we report for the first time the chemistry and bioactivity of a natural product from the newly described mixotrophic dinoflagellate Karlodinium armiger. Our work describes the isolation and structural characterization of a new polyhydroxy-polyene named karmitoxin. The structure elucidation work was facilitated by use of 13C enrichment and high-field 2D NMR spectroscopy, where 1H-13C long-range correlations turned out to be very informative. Karmitoxin is structurally related to amphidinols and karlotoxins; however it differs by containing the longest carbon-carbon backbone discovered for this class of compounds, as well as a primary amino group. Karmitoxin showed potent nanomolar cytotoxic activity in an RTgill-W1 cell assay as well as rapid immobilization and eventual mortality of the copepod Acartia tonsa, a natural grazer of K. armiger.

S3 HMBC hetero: Spin-State-Selective HMBC for accurate measurement of long-range heteronuclear coupling constants.

A novel method, Spin-State-Selective (S3) HMBC hetero, for accurate measurement of heteronuclear coupling constants is introduced. The method extends the S3 HMBC technique for measurement of homonuclear coupling constants by appending a pulse sequence element that interchanges the polarization in 13C-1H methine pairs. This amounts to converting the spin-state selectivity from 1H spin states to 13C spin states in the spectra of long-range coupled 1H spins, allowing convenient measurement of heteronuclear coupling constants similar to other S3 or E.COSY-type methods. As usual in this type of techniques, the accuracy of coupling constant measurement is independent of the size of the coupling constant of interest. The merits of the new method are demonstrated by application to vinyl acetate, the alkaloid strychnine, and the carbohydrate methyl ß-maltoside.

Specific Electrostatic Molecular Recognition in Water.

The identification of pairs of small peptides that recognize each other in water exclusively through electrostatic interactions is reported. The target peptide and a structure-biased combinatorial ligand library consisting of ≈78 125 compounds were synthesized on different sized beads. Peptide-peptide interactions could conveniently be observed by clustering of the small, fluorescently labeled target beads on the surface of larger ligand-containing beads. Sequences of isolated hits were determined by MS/MS. The interactions of the complex showing the highest affinity were investigated by a novel single-bead binding assay and by 2D NMR spectroscopy. Molecular dynamics (MD) studies revealed a putative mode of interaction for this unusual electrostatic binding event. High binding specificity occurred through a combination of topological matching and electrostatic and hydrogen-bond complementarities. From MD simulations binding also seemed to involve three tightly bound water molecules in the interface between the binding partners. Binding constants in the submicromolar range, useful for biomolecular adhesion and in nanostructure design, were measured.

S(3) HMBC: Spin-State-Selective HMBC for accurate measurement of homonuclear coupling constants. Application to strychnine yielding thirteen hitherto unreported J(HH).

A novel method, Spin-State-Selective (S(3)) HMBC, for accurate measurement of homonuclear coupling constants is introduced. As characteristic for S(3) techniques, S(3) HMBC yields independent subspectra corresponding to particular passive spin states and thus allows determination of coupling constants between detected spins and homonuclear coupling partners along with relative signs. In the presented S(3) HMBC experiment, spin-state selection occurs via large one-bond coupling constants ensuring high editing accuracy and unequivocal sign determination of the homonuclear long-range relative to the associated one-bond coupling constant. The sensitivity of the new experiment is comparable to that of regular edited HMBC and the accuracy of the J/RDC measurement is as usual for E.COSY and S(3)-type experiments independent of the size of the homonuclear coupling constant of interest. The merits of the method are demonstrated by an application to strychnine where thirteen J(HH) coupling constants not previously reported could be measured.

Methyl effect in azumamides provides insight into histone deacetylase inhibition by macrocycles.

Natural, nonribosomal cyclotetrapeptides have traditionally been a rich source of inspiration for design of potent histone deacetylase (HDAC) inhibitors. We recently disclosed the total synthesis and full HDAC profiling of the naturally occurring azumamides ( J. Med. Chem. 2013 , 56 , 6512 ). In this work, we investigate the structural requirements for potent HDAC inhibition by macrocyclic peptides using the azumamides along with a series of unnatural analogues obtained through chemical synthesis. By solving solution NMR structures of selected macrocycles and combining these findings with molecular modeling, we pinpoint crucial enzyme-ligand interactions required for potent inhibition of HDAC3. Docking of additional natural products confirmed these features to be generally important. Combined with the structural conservation across HDACs 1-3, this suggests that while cyclotetrapeptides have provided potent and class-selective HDAC inhibitors, it will be challenging to distinguish between the three major class I deacetylases using these chemotypes.

Dereplication guided discovery of secondary metabolites of mixed biosynthetic origin from Aspergillus aculeatus.

Investigation of the chemical profile of the industrially important black filamentous fungus Aspergillus aculeatus by UHPLC-DAD-HRMS and subsequent dereplication has led to the discovery of several novel compounds. Isolation and extensive 1D and 2D NMR spectroscopic analyses allowed for structural elucidation of a dioxomorpholine, a unique okaramine, an aflavinine and three novel structures of mixed biosynthetic origin, which we have named aculenes A-C. Moreover, known analogues of calbistrins, okaramines and secalonic acids were detected. All novel compounds were tested for antifungal activity against Candida albicans, however all showed only weak or no activity. Aspergillus aculeatus IBT 21030 was additionally shown to be capable of producing sclerotia. Examination of the sclerotia revealed a highly regulated production of metabolites in these morphological structures.