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Highlights • Universal detectors that can detect all types of compounds and give uniform response are highly desirable. • Reviewed commonly used detectors in analytical characterization, with the focuses on the "universal" features. • UV, RI, ELSD, CAD, CLND, FID, VUV, MS, NMR, etc. and hyphenated detections are reviewed. Abstract It is highly desirable to have a universal detector that can detect all types of compounds and give a uniform response regardless of the physiochemical properties of the compounds. With such a universal detector, all components in a sample can be accurately quantified without the need for individual standards. This is especially needed for the characterization of unknowns and for non-targeted analysis, or for samples that have no isolated standards available for each component. Over the years, much effort has been put into seeking a universal detection technology. In this review, we discuss the commonly used detectors for analytical characterization, including UV, RI, ELSD, CAD, CLND, FID, VUV, MS, NMR, and hyphenated detection, with the focuses on the "universal" features of these detectors regarding the types of molecules they can detect and the uniformity of responses. [ABSTRACT FROM AUTHOR]

Cyanobacteria possess a unique capacity for the production of structurally novel secondary metabolites compared to the biosynthetic abilities of other environmental prokaryotes such as bacteria of the genus Streptomyces. Two different strategies to explore cyanobacteria-derived natural products have been explored previously: (1) cultivation of single cyanobacterial strains, in bioreactors for example; (2) bulk collections from the environment of so called ‘algal blooms’ that are dominated by cyanobacteria. In this study a new environmentally friendly collection technique for obtaining large quantities of algal bloom biomass was utilized. Algal biomass derived from eight million liters of lake water was concentrated using a novel continuous countercurrent filtration system. Analysis of this freshwater algal bloom from Grand Lake-Saint Marys, Ohio led to the discovery of anabaenopeptin 679 ( 1 ), as well as the known anabaenopeptins B, F, H and 908. Anabaenopeptin 679 is unusual in that it possesses the classical anabaenopeptin-like cyclic pentapeptide core, but lacks the typical sidechain attached to the constitutive ureido group. Screening of all anabaenopeptin derivatives in an enzymatic assay for inhibitory activity toward carboxypeptidase A identified anabaenopeptin 679 as a strong inhibitor of carboxypeptidase A with an IC 50 value of 4.6 μg/mL. This result defines a new minimal core structure for carboxypeptidase activity among the anabaenopeptin class, and provides further insight into the structure–activity relationship of anabaenopeptin-like carboxypeptidase A inhibitors. [ABSTRACT FROM AUTHOR]

In pharmaceutical development, structural elucidation of small molecules from process related impurities and degradation products is an essential component. As one of the most important methods in the toolbox, high resolution mass spectrometry (HRMS) and specifically tandem mass spectrometry (MS/MS) often provide fast and informative structural insights. However, many small molecule drugs containing certain biological relevant pharmacophores result in limited numbers of fragments when using traditional collision based fragmentation techniques, such as higher energy collisional dissociation (HCD), due to its inherent preference of cleaving the weakest bond first. As an alternative, ultraviolet photodissociation (UVPD), which irradiates the precursor ion with high energy photons, can lead to more extensive fragmentation from the readily UV absorbing small molecules. Here, we showcase the advantage of UVPD over HCD on pyrrolidine and piperidine containing molecules derivatized from a model compound, telmisartan. While HCD only yielded a single, highly abundant ion resulting from the pyrrolidine and pipieridine ring cleavage, UVPD generated rich and structurally informative fragment ions. UVPD is an attractive and powerful alternative for traditional fragmentation techniques for small molecule structural elucidation. [Display omitted] • Pyrrolidine and piperidine motifs are useful pharmacophores as they enhance physiochemical properties of small molecules. • Collisional activation of small molecules containing these –idene motifs leads to uninformative structural information. • Structural characterization of these derivitized molecules becomes more feasible when performing ultraviolet photodissociation. [ABSTRACT FROM AUTHOR]

Determining mechanism of action (MOA) is one of the biggest challenges in natural products discovery. Here, we report a comprehensive platform that uses Similarity Network Fusion (SNF) to improve MOA predictions by integrating data from the cytological profiling high-content imaging platform and the gene expression platform Functional Signature Ontology, and pairs these data with untargeted metabolomics analysis for de novo bioactive compound discovery. The predictive value of the integrative approach was assessed using a library of target-annotated small molecules as benchmarks. Using Kolmogorov-Smirnov (KS) tests to compare in-class to out-of-class similarity, we found that SNF retains the ability to identify significant in-class similarity across a diverse set of target classes, and could find target classes not detectable in either platform alone. This confirmed that integration of expression-based and image-based phenotypes can accurately report on MOA. Furthermore, we integrated untargeted metabolomics of complex natural product fractions with the SNF network to map biological signatures to specific metabolites. Three examples are presented where SNF coupled with metabolomics was used to directly functionally characterize natural products and accelerate identification of bioactive metabolites, including the discovery of the azoxy-containing biaryl compounds parkamycins A and B. Our results support SNF integration of multiple phenotypic screening approaches along with untargeted metabolomics as a powerful approach for advancing natural products drug discovery. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique with the ability to acquire both quantitative and structurally insightful data for multiple components in a test sample. This makes NMR spectroscopy a desirable tool to understand, monitor, and optimize chemical transformations. While quantitative NMR (qNMR) approaches relying on internal standards are well‐established, using an absolute external calibration scheme is beneficial for reaction monitoring as resonance overlap complications from an added reference material to the sample can be avoided. Particularly, this type of qNMR technique is of interest with benchtop NMR spectrometers as the likelihood of resonance overlap is only enhanced with the lower magnetic field strengths of the used permanent magnets. The included study describes a simple yet robust methodology to determine concentration conversion factors for NMR systems using single‐ and multi‐analyte linear regression models. This approach is leveraged to investigate a pharmaceutically relevant amide coupling batch reaction. An on‐line stopped‐flow (i.e., interrupted‐flow or paused‐flow) benchtop NMR system was used to monitor both the 1,1′‐carbonyldiimidazole (CDI) promoted acid activation and the amide coupling. The results highlight how quantitative measurements in benchtop NMR systems can provide valuable information and enable analysts to make decisions in real time. [ABSTRACT FROM AUTHOR]

The skyllamycins are a family of highly functionalized non-ribosomal cyclic depsipeptide natural products which contain the extremely rare α-OH-glycine functionality. Herein the first total synthesis of skyllamycins A-C is reported, together with the biofilm inhibitory activity of the natural products. Linear peptide precursors for each natural product were prepared through an efficient solid-phase route incorporating a number of synthetic modified amino acids. A novel macrocyclization step between a C-terminal amide and an N-terminal glyoxylamide moiety served as a key transformation to install the unique α-OH-glycine unit and generate the natural products in the final step of the synthesis. [ABSTRACT FROM AUTHOR]

Accurate sequencing of single guide RNAs (sgRNAs) for CRISPR/Cas9 genome editing is critical for patient safety, as the sgRNA guides the Cas9 nuclease to target site-specific cleavages in DNA. An approach to fully sequence sgRNA using protective DNA primers followed by ribonuclease (RNase) T1 digestion was developed to facilitate the analysis of these larger molecules by hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-HRMS). Without RNase digestion, top-down mass spectrometry alone struggles to properly fragment precursor ions in large RNA oligonucleotides to provide confidence in sequence coverage. With RNase T1 digestion of these larger oligonucleotides, however, bottom-up analysis cannot confirm full sequence coverage due to the presence of short, redundant digestion products. By combining primer protection with RNase T1 digestion, digestion products are large enough to prevent redundancy and small enough to provide base resolution by tandem mass spectrometry to allow for full sgRNA sequence coverage. An investigation into the general requirements for adequate primer protection of specific regions of the RNA was conducted, followed by the development of a generic protection and digestion strategy that may be applied to different sgRNA sequences. This middle-out technique has the potential to expedite accurate sequence confirmation of chemically modified sgRNA oligonucleotides. [ABSTRACT FROM AUTHOR]

The genus Burkholderia is an emerging source of novel natural products chemistry, yet to date few methods exist for the selective isolation of strains of this genus from the environment. More broadly, tools to efficiently design selection media for any given genus would be of significant value to the natural products and microbiology communities. Using a modification of the recently published SMART protocol, we have developed a two-stage isolation protocol for strains from the genus Burkholderia. This method uses a combination of selective agar isolation media and multiplexed PCR profiling to derive Burkholderia strains from environmental samples with 95% efficiency. Creation of this new method paves the way for the systematic exploration of natural products chemistry from this important genus and offers new insight into potential methods for selective isolation method development for other priority genera. [ABSTRACT FROM AUTHOR]

Abstract: Yellowjackets in the genera Vespula and Dolichovespula are prevalent eusocial insects of great ecological and economic significance, but the chemical signals of their sexual communication systems have defied structural elucidation. Herein, we report the identification of sex attractant pheromone components of virgin bald‐faced hornet queens (Dolichovespula maculata). We analyzed body surface extracts of queens by coupled gas chromatographic–electroantennographic detection (GC‐EAD), isolated the compounds that elicited responses from male antennae by high‐performance liquid chromatography (HPLC), and identified these components by GC mass spectrometry (MS), HPLC‐MS, and NMR spectroscopy. In laboratory olfactometer experiments, synthetic (2Z,7E)‐3,7‐dimethyldeca‐2,7‐diendioic acid (termed here maculatic acid A) and (2Z,7E)‐10‐methoxy‐3,7‐dimethyldeca‐10‐oxo‐deca‐2,7‐dienoic acid (termed here maculatic acid C) in binary combination significantly attracted bald‐faced hornet males. These are the first sex attractant pheromone components identified in yellowjackets. [ABSTRACT FROM AUTHOR]

Abstract: Yellowjackets in the genera Vespula and Dolichovespula are prevalent eusocial insects of great ecological and economic significance, but the chemical signals of their sexual communication systems have defied structural elucidation. Herein, we report the identification of sex attractant pheromone components of virgin bald‐faced hornet queens (Dolichovespula maculata). We analyzed body surface extracts of queens by coupled gas chromatographic–electroantennographic detection (GC‐EAD), isolated the compounds that elicited responses from male antennae by high‐performance liquid chromatography (HPLC), and identified these components by GC mass spectrometry (MS), HPLC‐MS, and NMR spectroscopy. In laboratory olfactometer experiments, synthetic (2Z,7E)‐3,7‐dimethyldeca‐2,7‐diendioic acid (termed here maculatic acid A) and (2Z,7E)‐10‐methoxy‐3,7‐dimethyldeca‐10‐oxo‐deca‐2,7‐dienoic acid (termed here maculatic acid C) in binary combination significantly attracted bald‐faced hornet males. These are the first sex attractant pheromone components identified in yellowjackets. [ABSTRACT FROM AUTHOR]

• Development of a RPLC NPLC two-dimensional liquid chromatography system. • Simultaneous achiral-chiral analysis to support high-throughput experimentation. • Empirical rules and limitations defined to mitigate solvent incompatibility. Typical chromatographic analysis of chiral compounds requires the use of achiral methods to evaluate impurities or related substances along with separate methods to evaluate chiral purity. The use of two-dimensional liquid chromatography (2D-LC) to support simultaneous achiral-chiral analysis has become increasingly advantageous in the field of high-throughput experimentation where low reaction yields or side reactions can lead to challenging direct chiral analysis. Advancements in multi-dimensional chromatography have led to the development of robust 2D-LC instrumentation with reversed phase solvent systems (RPLC-RPLC) enabling this simultaneous analysis, eliminating the need to purify crude reaction mixtures to determine stereoselectivity. However, when chiral RPLC cannot separate a chiral impurity from the desired product, there are few viable commercial options. The coupling of NPLC to RPLC (RPLC-NPLC) continues to remain elusive due to solvent immiscibility between the two solvent systems. This solvent incompatibility leads to lack of retention, band broadening, poor resolution, poor peak shapes, and baseline issues in the second dimension. A study was conducted to understand the effect of various water-containing injections on NPLC and applied to the development of robust RPLC-NPLC methods. Following thoughtful consideration and modifications to the design of a 2D-LC system in regards to mobile phase selection, sample loop sizing, targeted mixing, and solvent compatibility, proof of concept has been demonstrated with the development of reproducible RPLC-NPLC 2D-LC methods to perform simultaneous achiral-chiral analysis. Second dimension NPLC method performance proved comparable to corresponding 1D-NPLC methods with excellent percent difference in enantiomeric excess results ≤ 1.09% and adequate limits of quantitation down to 0.0025 mg/mL for injection volumes of 2 µL, or 5 ng on-column. [ABSTRACT FROM AUTHOR]

Analysis of substrates directly on solid phase resins without the need for separate cleavage conditions remains an outstanding challenge in the field of solid phase synthesis. We now present the first example of simultaneous cleavage and mass spectrometric analysis of peptides from solid supports using direct analysis in real time (DART) mass spectrometry. We have shown that this method is compatible with a diverse array of solid phase resins and is suitable for analysis of both peptides and organic substrates. [ABSTRACT FROM AUTHOR]