Empirical Chiroptical Analyses of Vicinal Bromochloro Natural Products by van't Hoff's Principle of Optical Superposition: Assignment of the C-16 Configurations of Callophycols A and B.

A simple empirical method is described that allows the assignment of absolute configurations of natural products containing chiral vicinal bromochloro (VBC) units, including the bromochloro substituted isoprenyl units present in the structures of antiproliferative halomon (1a) and its halogen-swapped isomer iso-halomon (1b) from the red alga, Portieria hornemannii, and callophycols A (3) and B (4) from Callophycus serratus. The relative configurations of 3 and 4, published in 2007, were incomplete: C-16 was left unassigned. It is now shown that the additivity of molar rotations, [M]D (herein, abbreviated [M])─a consequence of van't Hoff's principle of optical superposition─could be used to deconvolute rotatory contributions, designated as [MX] and [MY] of the two remotely spaced chiral substructures within 3 and 4 using simple arithmetic. Input of proxy values, [M Y1] and [MY2], for the two different VBC units in two equations for [MX] and application of a "conditional test" returns the same value for [MX] only when a proxy with the correct configuration is included. It is revealed that 3 and 4 have opposite configurations at the C-16 stereocenter: 16S and 16R, respectively. Two important implications lie in these findings: 3 and 4 appear to qualify as paired-regioisomers, coupled through a putative dyotropic rearrangement (DR), and the biosyntheses of other Callophycus secondary metabolites, now numbering over 50, are tightly controlled by stereoelectronic considerations including neighboring group interactions of the DR. It now appears, counter to earlier suggestions, that the chirality of Callophycus secondary metabolites, despite their high chemodiversity, are surprisingly highly conserved. Enantiofacial halogenation additions to the C═C double bonds of precursor alkenes appear to direct the formation of the remaining stereocenters at both the halogenated benzoate-decalin core and the distal VBC of 3 and 4. A consistent hypothesis is proposed to account for macrolactonizations in other Callophycus natural products including bromophycolides A and B. The conditional test of molar rotations was applied in a different context to understand the chiroptical properties and trends observed in the highly iodinated meroditerpenes, iodocallophycols A-E, also from Callophycus sp., resulting in the revision of the configuration of callophycol E from (10R,14R) to (10S,14S).

Small molecule in situ resin capture provides a compound first approach to natural product discovery.

Culture-based microbial natural product discovery strategies fail to realize the extraordinary biosynthetic potential detected across earth's microbiomes. Here we introduce Small Molecule In situ Resin Capture (SMIRC), a culture-independent method to obtain natural products directly from the environments in which they are produced. We use SMIRC to capture numerous compounds including two new carbon skeletons that were characterized using NMR and contain structural features that are, to the best of our knowledge, unprecedented among natural products. Applications across diverse marine habitats reveal biome-specific metabolomic signatures and levels of chemical diversity in concordance with sequence-based predictions. Expanded deployments, in situ cultivation, and metagenomics facilitate compound discovery, enhance yields, and link compounds to candidate producing organisms, although microbial community complexity creates challenges for the later. This compound-first approach to natural product discovery provides access to poorly explored chemical space and has implications for drug discovery and the detection of chemically mediated biotic interactions.

DFT investigation of coupling constant anomalies in substituted ß-lactams.

ß-lactams are a chemically diverse group of molecules with a wide range of biological activities. Having recently observed curious trends in 2JHH coupling values in studies on this structural class, we sought to obtain a more comprehensive understanding of these diagnostic NMR parameters, specifically interrogating 1JCH, 2JCH, and 2JHH, to differentiate 3- and 4-monosubstituted ß-lactams. Further investigation using computational chemistry methods was employed to explore the geometric and electronic origins for the observed and calculated differences between the two substitution patterns.

Orthogonal Deprotection Strategy of Fmoc Provides Improved Synthesis of Sensitive Peptides: Application to Z-Arg-Lys-AOMK.

Protecting groups (PGs) in peptide synthesis have inspired advanced design principles that incorporate "orthogonality" for selective C- and N-terminus and side-chain deprotections. The conventionally acid-stable 9-fluorenylmethoxycarbonyl (Fmoc) group is one of the most widely used N-protection groups in solid- and solution-phase synthesis. Despite the versatility of Fmoc, deprotection by the removal of the Fmoc group to unmask primary amines requires the use of a basic secondary amine nucleophile, but this stratagem poses challenges in sensitive molecules that bear reactive electrophilic groups. An expansion of PG versatility, a tunable orthogonality, in the late-stage synthesis of peptides would add flexibility to the synthetic design and implementation. Here, we report a novel Fmoc deprotection method using hydrogenolysis under mildly acidic conditions for the synthesis of Z-Arg-Lys-acyloxymethyl ketone (Z-R-K-AOMK). This new method is not only valuable for Fmoc deprotection in the synthesis of complex peptides that contain highly reactive electrophiles, or other similar sensitive functional groups, that are incompatible with traditional Fmoc deprotection conditions but also tolerant of N-Boc groups present in the substrate.

Petrosamine Revisited. Experimental and Computational Investigation of Solvatochromism, Tautomerism and Free Energy Landscapes of a Pyridoacridinium Quaternary Salt.

Petrosamine (1)-a colored pyridoacridine alkaloid from the Belizean sponge, Petrosia sp., that is also a potent inhibitor of acetylcholine esterase (AChE)-was investigated by spectroscopic and computational methods. Analysis of the petrosamine-free energy landscapes, pKa and tautomerism, revealed an accurate electronic depiction of the molecular structure of 1 as the di-keto form, with a net charge of q = +1, rather than a dication (q = +2) under ambient conditions of isolation-purification. The pronounced solvatochromism (UV-vis) reported for 1, and related analogs were investigated in detail and is best explained by charge delocalization and stabilization of the ground state (HOMO) of 1 rather than an equilibrium of competing tautomers. Refinement of the molecular structure 1 by QM methods complements published computational docking studies to define the contact points in the enzyme active site that may improve the design of new AChE inhibitors based on the pyridoacridine alkaloid molecular skeleton.

Small Molecule in situ Resin Capture - A Compound First Approach to Natural Product Discovery.

Microbial natural products remain an important resource for drug discovery. Yet, commonly employed discovery techniques are plagued by the rediscovery of known compounds, the relatively few microbes that can be cultured, and laboratory growth conditions that do not elicit biosynthetic gene expression among myriad other challenges. Here we introduce a culture independent approach to natural product discovery that we call the Small Molecule In situ Resin Capture (SMIRC) technique. SMIRC exploits in situ environmental conditions to elicit compound production and represents a new approach to access poorly explored chemical space by capturing natural products directly from the environments in which they are produced. In contrast to traditional methods, this compound-first approach can capture structurally complex small molecules across all domains of life in a single deployment while relying on Nature to provide the complex and poorly understood environmental cues needed to elicit biosynthetic gene expression. We illustrate the effectiveness of SMIRC in marine habitats with the discovery of numerous new compounds and demonstrate that sufficient compound yields can be obtained for NMR-based structure assignment. Two new compound classes are reported including one novel carbon skeleton that possesses a functional group not previously observed among natural products and a second that possesses potent biological activity. We introduce expanded deployments, in situ cultivation, and metagenomics as methods to facilitate compound discovery, enhance yields, and link compounds to producing organisms. This compound first approach can provide unprecedented access to new natural product chemotypes with broad implications for drug discovery.

Geobarrettin D, a Rare Herbipoline-Containing 6-Bromoindole Alkaloid from Geodia barretti.

Geobarrettin D (1), a new bromoindole alkaloid, was isolated from the marine sponge Geodia barretti collected from Icelandic waters. Its structure was elucidated by 1D, and 2D NMR (including 1H-15N HSQC, 1H-15N HMBC spectra), as well as HRESIMS data. Geobarrettin D (1) is a new 6-bromoindole featuring an unusual purinium herbipoline moiety. Geobarrettin D (1) decreased secretion of the pro-inflammatory cytokine IL-12p40 by human monocyte derived dendritic cells, without affecting secretion of the anti-inflammatory cytokine IL-10. Thus, compound 1 shows anti-inflammatory activity.

Spiroisoxazoline Inhibitors of Acetylcholinesterase from Pseudoceratina verrucosa. Quantitative Chiroptical Analysis of Configurational Heterogeneity, and Total Synthesis of (±)-Methyl Purpuroceratate C.

Examination of the MeOH extract of the sponge, Pseudoceratina cf. verrucosa, Berquist 1995 collected near Ningaloo Reef, Western Australia for selective acetylcholinesterase (AChE) inhibitors, yielded five new bromotyrosine alkaloids, methyl purpuroceratates A and B (1b and 2b), purpuroceratic acid C (3a), and ningalamides A and B (4 and 5). The structures of 1-4 share the dibromo-spirocyclohexadienyl-isoxazoline (SIO) ring system found in purealidin-R, while ketoxime 5 is analogous to ianthelline and purpurealidin I. The planar structures of all five compounds were obtained from analysis of MS, 1D and 2D NMR data, and the absolute configuration of the spiroisoxazoline (SIO) unit was assigned by electronic circular dichroism (ECD) and comparison with standards prepared by total synthesis of methyl purpuroceratate C, (±)-3b. Compound 4 is the most complex SIO described, to date. The configuration of the homoserine module (C) in 4 was ascertained, after acid hydrolysis, by derivatization of an l-tryptophanamide derivative based on Marfey's reagent. Chiral-phase HPLC, with comparison to synthetic standards, revealed that most SIOs isolated from P. cf. verrucosa were configurationally heterogeneous; some, essentially racemic. Chiral-phase HPLC, with UV-ECD detection, is demonstrated as a superlative method for configurational assignment and quantitation of the enantiomeric composition of SIOs. Two SIOs─aerophobin-1 and aplysinamisine II─emerged as selective inhibitors of AChE over butyrylcholinesterase (BuChE, IC50 ratio >10), while aplysamine-2 moderately inhibited both cholinesterases (ChEs, IC50, (AChE) 0.46 µM; IC50, (BuChE) 1.03 µM). SIO alkaloids represent a potential new structural manifold for lead-discovery of new therapeutics for treatment of Alzheimer's disease.

Bioactive Bromotyrosine Alkaloids from the Bahamian Marine Sponge Aiolochroia crassa. Dimerization and Oxidative Motifs.

Nine bromotyrosine alkaloids (BTAs), including debromoianthelline (1), pseudoceratinic acid (2a), methyl pseudoceratinate (2b), 13-oxo-ianthelline (3), aiolochroiamides A-D (4a,b and 5a,b), and 7-hydroxypurealidin J (6), were isolated from a Bahamian Aiolochroia crassa (Hyatt; previously, Pseudoceratina crassa). The structures of 1-6 were established from 1H, 13C, and 2D NMR spectra, IR, and mass spectrometry data. Compounds 2-4 comprise an O-methyl-2,6-dibromotyrosyl ketoxime (subunit A) amide linked to variable groups (subunit B). Compound 1 is debromoianthelline, and 2a and 2b are amides of 3-aminopropanoic acid and methyl 3-aminopropanoate, respectively. BTAs 3 and 4 are linked to 5-(2-aminoethyl)-2-iminoimidazolidin-4-one and a hexahydropyrrolo[2,3-d]imidazol-2(1H)-imine nucleus, respectively, whereas 5 is a self-dimerization motif of an aryl pyruvamide. Alkaloid 6 contains a spirocyclohexadienyl-isoxazoline-carboxamide amide coupled to 2-aminohistamine similar to that found in purealidin J and aerophobin-1 but with hydroxylation at C-7. The 2,4-diaminobutanoic acid residue in 3 was determined to be a 2:1 L- and D- mixture based on hydrolysis followed by derivatization with L-FDTA and LCMS. Diastereomeric pairs, 4a,b and 5a,b, were racemic. The relative configurations of 4a, 4b, 5a, and 5b were assigned by comparison of 1H and 13C chemical shifts with those calculated by DFT. Compounds 5a,b, ningalamide B (9), and ianthelline (7) moderately inhibited butyrylcholinesterase and Candida and Cryptococcus spp.

Chlororesistoflavins A and B, Chlorinated Benzopyrene Antibiotics Produced by the Marine-Derived Actinomycete Streptomyces sp. Strain EG32.

As part of a collaborative biomedical investigation of actinomycete bacteria isolated from sediments collected along the northern coast of Egypt (Mediterranean Sea), we explored the antibacterial metabolites from a bacterium identified as a Streptomyces sp., strain EG32. HPLC analysis and antibacterial testing against methicillin-resistant Staphylococcus aureus (MRSA) resulted in the identification of six compounds related to the resistoflavin and resistomycin class. Two of these metabolites were the chlorine-containing analogues chlororesistoflavins A (1) and B (2). The absolute configurations of the lone stereogenic center (C-11b) in these metabolites were assigned by analysis of their ECD spectra. Interestingly, the ECD spectrum of chlororesistoflavin A (1) shows a Cotton effect of the n-π* transition antipodal to that of the parent natural product, a consequence of 1,3-allylic strain induced by the adjacent bulky chlorine atom that distorts the coplanarity of the carbonyl group with the π-system. The chiroptical analysis thus resolves the paradox and uniformly aligns the configuration of all analogues as identical to that reported for natural resistoflavin. Chlororesistoflavins A (1) and B (2) exhibited antibacterial activity against MRSA with a minimum inhibitory concentration of 0.25 and 2.0 µg/mL, respectively.

Oceanalin B, a Hybrid α,ω-Bifunctionalized Sphingoid Tetrahydroisoquinoline ß-Glycoside from the Marine Sponge Oceanapia sp.

Oceanalin B (1), an α,ω-bipolar natural product belonging to a rare family of sphingoid tetrahydoisoquinoline ß-glycosides, was isolated from the EtOH extract of the lyophilized marine sponge Oceanapia sp. as the second member of the series after oceanalin A (2) from the same animal. The compounds are of particular interest due to their biogenetically unexpected structures as well as their biological activities. The structure and absolute stereochemistry of 1 as a α,ω-bifunctionalized sphingoid tetrahydroisoquinoline ß-glycoside was elucidated using NMR, CD and MS spectral analysis and chemical degradation. Oceanalin B exhibited in vitro antifungal activity against Candidaglabrata with a MIC of 25 µg/mL.

Oceanapiside, a Marine Natural Product, Targets the Sphingolipid Pathway of Fluconazole-Resistant Candida glabrata.

Oceanapiside (OPS), a marine natural product with a novel bifunctional sphingolipid structure, is fungicidal against fluconazole-resistant Candida glabrata at 10 µg/mL (15.4 µM). The fungicidal effect was observed at 3 to 4 h after exposure to cells. Cytological and morphological studies revealed that OPS affects the budding patterns of treated yeast cells with a significant increase in the number of cells with single small buds. In addition, this budding morphology was found to be sensitive in the presence of OPS. Moreover, the number of cells with single medium-sized buds and cells with single large buds were decreased significantly, indicating that fewer cells were transformed to these budding patterns, suggestive of inhibition of polarized growth. OPS was also observed to disrupt the organized actin assembly in C. glabrata, which correlates with inhibition of budding and polarized growth. It was also demonstrated that phytosphingosine (PHS) reversed the antifungal activity of oceanapiside. We quantified the amount of long chain-bases (LCBs) and phytoceramide from the crude extracts of treated cells using LC-ESI-MS. PHS concentration was elevated in extracts of cells treated with OPS when compared with cells treated with miconazole and amphotericin B. Elevated levels of PHS in OPS-treated cells confirms that OPS affects the pathway at a step downstream of PHS synthesis. These results also demonstrated that OPS has a mechanism of action different to those of miconazole and amphotericin B and interdicts fungal sphingolipid metabolism by specifically inhibiting the step converting PHS to phytoceramide.

Bromotryptamine and Imidazole Alkaloids with Anti-inflammatory Activity from the Bryozoan Flustra foliacea.

Chemical investigation of the marine bryozoan Flustra foliacea collected in Iceland resulted in isolation of 13 new bromotryptamine alkaloids, flustramines Q-W (1-7) and flustraminols C-H (8-13), and two new imidazole alkaloids, flustrimidazoles A and B (14 and 15), together with 12 previously described compounds (16-27). Their structures were established by detailed spectroscopic analysis using 1D and 2D NMR and HRESIMS. Structure 2 was verified by calculations of the 13C and 1H NMR chemical shifts using density functional theory. The relative and absolute configurations of the new compounds were elucidated on the basis of coupling constant analysis, NOESY, [α]D, and ECD spectroscopic data, in addition to chemical derivatization. The compounds were tested for in vitro anti-inflammatory activity using a dendritic cell model. Eight compounds (1, 3, 5, 13, 16, 18, 26, and 27) decreased dendritic cell secretion of the pro-inflammatory cytokine IL-12p40, and two compounds (4 and 14) increased secretion of the anti-inflammatory cytokine IL-10. Deformylflustrabromine B (27) showed the most potent anti-inflammatory effect (IC50 2.9 µM). These results demonstrate that F. foliacea from Iceland expresses a broad range of brominated alkaloids, many without structural precedents. The potent anti-inflammatory activity in vitro of metabolite 27 warrants further investigations into its potential as a lead for inflammation-related diseases.

Synchronous bond molecular dynamics of conjugated chlorocyclopropyl alk-yn-enes revealed by ECD and UV-vis.

Chlorocyclopropanes (CCPs) conjugated to alk-yn-enes occur in a unique family of polyketide natural products from marine sponges. Synthesis of several optically enriched analogs of CCPs and measurement of their UV-vis spectra and electronic circular dichroism (ECD) spectra reveal unusually strong hyperconjugation that constrains and aligns the cyclopropyl C-C bond with the π-plane of the distal ene-bond. This alignment imposes a barrier to rotation of at least 5.0 kcal·mol-1 . Comparison of red-shifted Cotton effects in chiral CCPs show the barrier is independent of alkene substituent and establishes an empirical rule for assignment of other CCP-containing natural products.

Bromo-spiroisoxazoline Alkaloids, Including an Isoserine Peptide, from the Caribbean Marine Sponge Aplysina lacunosa.

Three new bromotyrosine spiroisoxazoline alkaloids, lacunosins A and B (1 and 2) and desaminopurealin (3), were isolated from a MeOH extract of the marine sponge Aplysina lacunosa that showed modest α-chymotrypsin inhibitory activity. The structures of 1-3 share the spirocyclohexadienyl-isoxazoline ring system found in purealidin-R and several other Verongid sponge secondary metabolites. Compounds 1 and 2 are coupled to a glycine and an isoserine methyl ester, respectively. Alkaloid 3 is linked, contiguously, to an O-1-aminopropyl 3,5-dibromotyrosyl ether and, finally, to histamine through an amide bond. The planar structures of all three compounds were obtained from analysis of MS and 1D and 2D NMR data. The absolute configuration of the SIO unit of 1-3 was assigned by electronic circular dichroism (ECD). The isoserine amino acid residue in 2 was found to be a 1:1 mixture of epimers using a new Marfey's type reagent, derived from Trp-NH2. Allylic O-naphthoylation of the SIO subunit enhances the ECD spectrum of SIOs and improves discrimination of enantiomorphs. A unifying hypothesis is proposed that links the biosynthesis of several of the new compounds with previously reported analogues.

The Configuration of Distaminolyne A is S: Quantitative Evaluation of Exciton Coupling Circular Dichroism of N, O- Bis-arenoyl-1-amino-2-alkanols.

The 2 S configuration of the marine natural product distaminolyne A was recently disputed based upon total synthesis, yet paradoxically supported by a second independent total synthesis from a different research group. We now verify the 2 S configuration of distaminolyne A by extensive chiroptical studies and support the veracity of the EC ECD method originally used to prove it. The origin of the apparent paradox appears to lie in the limits of precision of polarimetry in the context of weakly rotatory molecules, which strikes a cautionary note on the reliability of "reassignment" of natural product configurations based solely on specific rotation.

Synergistic Anti-Candida Activity of Bengazole A in the Presence of Bengamide A †.

Bengazoles A⁻G from the marine sponge Jaspis sp. exhibit potent in vitro antifungal activity against Candida spp. and other pathogenic fungi. The mechanism of action (MOA) of bengazole A was explored in Candida albicans under both liquid culture and surface culture on Mueller-Hinton agar. Pronounced dose-dependent synergistic antifungal activity was observed with bengazole A in the presence of bengamide A, which is also a natural product from Jaspis sp. The MOA of bengazole A was further explored by monitoring the sterol composition of C. albicans in the presence of sub-lethal concentrations of bengazole A. The GCMS of solvent extracts prepared from liquid cultures of C. albicans in the presence of clotrimazole-a clinically approved azole antifungal drug that suppresses ergosterol biosynthesis by the inhibition of 14α-demethylase-showed reduced cellular ergosterol content and increased concentrations of lanosterol and 24-methylenedihydrolanosterol (a shunt metabolite of ergosterol biosynthesis). No change in relative sterol composition was observed when C. albicans was cultured with bengazole A. These results eliminate an azole-like MOA for the bengazoles, and suggest that another as-yet unidentified mechanism is operative.

The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Covering: up to 2018With contributions from the global natural product (NP) research community, and continuing the Raw Data Initiative, this review collects a comprehensive demonstration of the immense scientific value of disseminating raw nuclear magnetic resonance (NMR) data, independently of, and in parallel with, classical publishing outlets. A comprehensive compilation of historic to present-day cases as well as contemporary and future applications show that addressing the urgent need for a repository of publicly accessible raw NMR data has the potential to transform natural products (NPs) and associated fields of chemical and biomedical research. The call for advancing open sharing mechanisms for raw data is intended to enhance the transparency of experimental protocols, augment the reproducibility of reported outcomes, including biological studies, become a regular component of responsible research, and thereby enrich the integrity of NP research and related fields.

Correction: The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research.

Correction for 'The value of universally available raw NMR data for transparency, reproducibility, and integrity in natural product research' by James B. McAlpine et al., Nat. Prod. Rep., 2018, DOI: .

Enantiomeric Variability of Distaminolyne A. Refinement of ECD and NMR Methods for Determining Optical Purity of 1-Amino-2-Alkanols.

Sample configurations of distaminolyne A (1a); isolated from the ascidians Pseudodistoma opacum and P. cereum, and collected at different sites in New Zealand, were investigated by two methods: Exciton coupled electronic circular dichroism (EC ECD) of the corresponding N,O-dibenzoyl derivative 1b; and chiral reagent derivatization of 1a with (S)- and (R)-α-methoxyphenylacetic acid (MPA), followed by ¹H-NMR analysis. Configuration and optical purity of 1a (%ee) was found to vary depending on the geographic distribution of ascidian colonies. An improved method for preparing N,O-diarenoyl derivatives of 1a was optimized. The EC ECD method was found to be complementary to the MPA-NMR method at different ranges of %ee.