Universal Quantification of Structurally Diverse Natural Products Using an Evaporative Light Scattering Detector

A lack of good methods for absolute quantification of natural products has limited the accuracy of high-throughput screening. Many currently used methods for quantification are either too slow or not amenable to the structural diversity of natural products. Recent developments in low-temperature evaporative light scattering detectors (ELSD-LT) have overcome several historical limitations of ELSDs, including analyte decomposition and low sensitivity. Primarily, ELSDs have been used for relative quantification and detection of compounds that lack a UV chromophore. In this study, we employ an ELSD-LT for absolute quantification of natural products. Calibration curves were constructed using a weighted least-squares analysis for a diverse set of natural products and other compounds. An average calibration curve was evaluated for the “universal” quantification of natural products. Optimization of ELSD-LT hardware and parameters improved sensitivity and throughput and established the utility of ELSD-LT for quantification of large natural product libraries

Peptidolipins B–F, Antibacterial Lipopeptides from an Ascidian-Derived <i>Nocardia</i> sp.

A marine <i>Nocardia</i> sp. isolated from the ascidian <i>Trididemnum orbiculatum</i> was found to produce five new lipopeptides, peptidolipins B–F (<b>1</b>–<b>5</b>), which show distinct similarities to the previously reported l-Val­(6) analog of peptidolipin NA. Synthetic modification of peptidolipin E (<b>4</b>) was used to determine the location of an olefin within the lipid chain. The advanced Marfey’s method was used to determine the absolute configurations of the amino acids. Peptidolipins B (<b>1</b>) and E (<b>4</b>) demonstrated moderate antibacterial activity against methicillin-resistant <i>Staphylococcus aureus</i> and methicillin-sensitive <i>Staphylococcus aureus</i>

Probing the Regiospecificity of Enzyme-Catalyzed Steroid Glycosylation

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-<i>O</i>-β-d-glucoside

Marine Natural Product Libraries for High-Throughput Screening and Rapid Drug Discovery

There is a need for diverse molecular libraries for phenotype-selective and high-throughput screening. To make marine natural products (MNPs) more amenable to newer screening paradigms and shorten discovery time lines, we have created an MNP library characterized online using MS. To test the potential of the library, we screened a subset of the library in a phenotype-selective screen to identify compounds that inhibited the growth of BRCA2-deficient cells

Thalassosamide, a Siderophore Discovered from the Marine-Derived Bacterium <i>Thalassospira profundimaris</i>

Here we describe the rapid identification and prioritization of novel active marine natural products using an improved dereplication strategy. During the course of our screening of marine natural product libraries, a new cyclic trihydroxamate compound, thalassosamide, was discovered from the α-proteobacterium <i>Thalassospira profundimaris</i>. Its structure was determined by 2D NMR and MS/MS experiments, and the absolute configuration of the lysine-derived units was established by Marfey’s analysis, whereas that of C-9, 9′, and 9″ was determined via the circular dichroism data of the [Rh₂(OCOCF₃)₄] complex and DFT NMR calculations. Thalassosamide showed moderate in vivo efficacy against <i>Pseudomonas aeruginosa</i>

Heatmap and hierarchical clustering of select secondary metabolite peak volumes in LCMS runs on <i>L. patella</i> extracts (top left).

<p>Clustering based on secondary metabolites that are produced by the symbiotic bacteria <i>Prochloron didemni</i> and <i>Ca.</i> Endolissoclinum faulkneri closely follows the hosts' phylogeny as determined by COXI sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g002" target="_blank">Fig. 2</a>). The <i>P. didemni</i> compounds shown are all cyanobactins produced either by a patellamide-type pathway (red), or a trunkamide-type pathway (blue). These two types of are closely related ribosomal pathways that are highly tolerant to changes in the precursor peptide sequence. The patellazoles (magenta) are produced by another symbiont, <i>Candidatus</i> Endolissoclinum faulkneri, by a polyketide synthase pathway.</p

Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.

<p>Phylogenetic tree of 18S rRNA nucleotide sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona intestinalis</i> acting as an outgroup.</p

Phylogenetic tree of mitochondrial cytochrome <i>c</i> oxidase 1 (COXI) protein sequences from our collected <i>L. patella</i> animals and other Didemnidae, with <i>Ciona savignyi</i> acting as the outgroup.

<p>Note: the <i>Didemnum vexillum</i> clade is collapsed for space. The Didemnidae COXI genes found in the NCBI database cover two non-overlapping regions of the gene (see Main Text), and therefore two separate trees were constructed (for the other tree, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone-0095850-g004" target="_blank">Figure 4</a>).</p

Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Schematic representation of the draft mitochondrial genome of <i>L. patella</i> animal L2 (top, to scale).

<p>The hive plots <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095850#pone.0095850-Krzywinski1" target="_blank">[61]</a> at the bottom of the figure show that the L2 assembly is syntenic with contigs assembled of the mitochondrial genomes of L5 and L6. In these plots, protein-coding genes are represented as circles, and contig boundaries are represented as zigzag lines. Homologous genes are joined by curved lines colored according to the sequence identity of the gene relevant gene pair. Abbreviations: COX, cytochrome <i>c</i> oxidase; NADH, nicotinamide adenine dinucleotide dehydrogenase.</p