Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters.

Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the predicted BGCs revealed large gene cluster families, the vast majority uncharacterized. We experimentally characterized the most prominent family, consisting of two subfamilies of hundreds of BGCs distributed throughout the Proteobacteria; their products are aryl polyenes, lipids with an aryl head group conjugated to a polyene tail. We identified a distant relationship to a third subfamily of aryl polyene BGCs, and together the three subfamilies represent the largest known family of biosynthetic gene clusters, with more than 1,000 members. Although these clusters are widely divergent in sequence, their small molecule products are remarkably conserved, indicating for the first time the important roles these compounds play in Gram-negative cell biology.

High-throughput functional annotation of natural products by integrated activity profiling.

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.

High-Throughput, Nondestructive, and Biosafe Method to Accurately Quantify Water Content in Human Fecal Samples by Benchtop 1H TD-NMR Analysis for Downstream Bioanalytical and Clinical Uses.

Water or moisture content in human stool samples is an important parameter for bioanalytical and clinical purposes. For bioanalytical use, accurate quantitation of water content in stool can provide the extent of dilution within the stool sample which can further be used for absolute quantitation of various stool based biomarkers. For clinical use, water or moisture content in stool is an important indicator of gastrointestinal health, and its accurate determination can enable quantitative assessment of the Bristol Stool Form Scale. In general, accurate determination of water content of stool samples is cumbersome, low-throughput process and is prone to harmful stool pathogens biocontamination, sample cross-contamination using techniques such as gravimetry and karl fischer titration. Here, we report a novel user-friendly high-throughput method to quantitatively and accurately measure the overall water content in human fecal samples nondestructively and biocontained in a closed tube using benchtop a 1H time domain nuclear magnetic resonance analyzer. We used gravimetry and measurement of various bile acid metabolites in stool to verify the accuracy and robustness of the water content measurement using this technique.

The effect of tube quality on externally calibrated quantitative nuclear magnetic resonance analysis: How bad can it be?

Externally calibrated quantitative nuclear magnetic resonance (NMR) approaches offer practical means to simultaneously evaluate chemical identity and content without the addition of calibrants to the test sample. Despite continuous advances in external calibration over the last few decades, adoption of these approaches has been slower than expected. Variations in NMR tube geometry are a commonly overlooked factor that can have a substantial effect on externally calibrated quantitation methods. In this report, we investigate the extent to which tube-to-tube volume variability can affect quantitative NMR outcomes. The results highlight the importance of considering tube quality during the development stages of externally calibrated quantitative methods. In addition, we propose a simple, yet effective volume correction strategy using the residual protonated solvent signal that, based on experiments with mixed NMR tubes of varying quality, alleviates the effect of tube-to-tube variability.

A reliable external calibration method for reaction monitoring with benchtop NMR.

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.

Middle-out sequence confirmation of CRISPR/Cas9 single guide RNA (sgRNA) using DNA primers and ribonuclease T1 digestion.

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.

On-line Sequencing of CRISPR Guide RNAs and Their Impurities via the Use of Immobilized Ribonuclease Cartridges Attached to a 2D/3D-LC-MS System.

In this study, for the first time, the automated digestion and sequencing of an RNA molecule via the use of immobilized RNase cartridges attached to a multidimensional liquid chromatography (LC)-mass spectrometry (MS) system are presented. We first developed an on-line digestion-HILIC two-dimensional (2D)-LC-MS method in order to sequence CRISPR guide RNAs for gene editing. Three RNases (T1, A, and U2) were immobilized on polyetheretherketone cartridges, and their performance was evaluated. Ultrafast digestions were performed within 2.3 min with the on-line approach versus 30 min via the conventional off-line approach. The higher sequence coverage was achieved by the RNase T1 (71%), which is the same as the off-line mode. A 20-fold reduction in the gRNA sample amount was achieved with the on-line digestion approach (6.5 µg) in comparison to that with the off-line approach (130 µg). In the second step, a three-dimensional (3D)-LC-MS method was developed for the sequencing of fractions collected on-line across the main peak and the partially separated tail by the reference ion-pairing RPLC method. Additional insights were gained in order to better understand the cause of the main peak tailing.

Full Sequencing of CRISPR/Cas9 Single Guide RNA (sgRNA) via Parallel Ribonuclease Digestions and Hydrophilic Interaction Liquid Chromatography-High-Resolution Mass Spectrometry Analysis.

CRISPR/Cas9 is a powerful genome editing approach in which a Cas9 enzyme and a single guide RNA (sgRNA) form a ribonucleoprotein complex effectively targeting site-specific cleavages of DNA. Accurate sequencing of sgRNA is critical to patient safety and is the expectation by regulatory agencies. In this paper, we present the full sequencing of sgRNA via parallel ribonuclease (RNase) T1, A, and U2 digestions and the simultaneous separation and identification of the digestion products by hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS). When using RNase T1 digestion alone, a maximal sequence coverage of 81% was obtained excluding the nonunique fragments. Full sgRNA sequencing was achieved using unique fragments generated by RNase T1, A, and U2 parallel digestions. Thorough optimization of sgRNA digestions was performed by varying the nuclease-to-sgRNA ratio, buffer conditions, and reaction times. A biocompatible ethylene-bridged hybrid amide column was evaluated for the separation of RNase digestion products. To our knowledge, it is the first time that (i) RNA digests are separated and identified by HILIC-HRMS and (ii) chemically modified sgRNAs are directly sequenced via a bottom-up approach.

Isolation, Structure Elucidation, and Total Synthesis of Dolichovespulide, a Sesquiterpene from Dolichovespula Yellowjackets.

As part of an ongoing program to identify sex attractant pheromone components that mediate sexual communication in yellowjacket wasps, a novel sesquiterpene was isolated from body surface extracts of virgin bald-faced hornet queens, Dolichovespula maculata. The gross structure of this sesquiterpene was proposed through microscale spectroscopic analyses, and the configuration of the central olefin was subsequently confirmed by total synthesis. This new natural product (termed here dolichovespulide) represents an important addition to the relatively small number of terpenoids reported from the taxonomic insect family Vespidae.

A selective genome-guided method for environmental Burkholderia isolation.

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.

Synthetic Studies Toward the Skyllamycins: Total Synthesis and Generation of Simplified Analogues.

Herein, we report our synthetic studies toward the skyllamycins, a highly modified class of nonribosomal peptide natural products which contain a number of interesting structural features, including the extremely rare α-OH-glycine residue. Before embarking on the synthesis of the natural products, we prepared four structurally simpler analogues. Access to both the analogues and the natural products first required the synthesis of a number of nonproteinogenic amino acids, including three ß-OH amino acids that were accessed from the convenient chiral precursor Garner's aldehyde. Following the preparation of the suitably protected nonproteinogenic amino acids, the skyllamycin analogues were assembled using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. To access the natural products (skyllamycins A-C) the synthetic route used for the analogues was modified. Specifically, linear peptide precursors containing a C-terminal amide were synthesized via solid-phase peptide synthesis. After cleavage from the resin the N-terminal serine residue was oxidatively cleaved to a glyoxyamide moiety. The target natural products, skyllamycins A-C, were successfully prepared via a final step cyclization with concomitant formation of the unusual α-OH-glycine residue. Purification and spectroscopic comparison to the authentic isolated material confirmed the identity of the synthetic natural products.

Integration of high-content screening and untargeted metabolomics for comprehensive functional annotation of natural product libraries.

Traditional natural products discovery using a combination of live/dead screening followed by iterative bioassay-guided fractionation affords no information about compound structure or mode of action until late in the discovery process. This leads to high rates of rediscovery and low probabilities of finding compounds with unique biological and/or chemical properties. By integrating image-based phenotypic screening in HeLa cells with high-resolution untargeted metabolomics analysis, we have developed a new platform, termed Compound Activity Mapping, that is capable of directly predicting the identities and modes of action of bioactive constituents for any complex natural product extract library. This new tool can be used to rapidly identify novel bioactive constituents and provide predictions of compound modes of action directly from primary screening data. This approach inverts the natural products discovery process from the existing "grind and find" model to a targeted, hypothesis-driven discovery model where the chemical features and biological function of bioactive metabolites are known early in the screening workflow, and lead compounds can be rationally selected based on biological and/or chemical novelty. We demonstrate the utility of the Compound Activity Mapping platform by combining 10,977 mass spectral features and 58,032 biological measurements from a library of 234 natural products extracts and integrating these two datasets to identify 13 clusters of fractions containing 11 known compound families and four new compounds. Using Compound Activity Mapping we discovered the quinocinnolinomycins, a new family of natural products with a unique carbon skeleton that cause endoplasmic reticulum stress.

Maculatic Acids-Sex Attractant Pheromone Components of Bald-Faced Hornets.

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.

Total Synthesis of Skyllamycins†A-C.

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.

Discovery of anabaenopeptin 679 from freshwater algal bloom material: Insights into the structure-activity relationship of anabaenopeptin protease inhibitors.

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 IC50 value of 4.6µg/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.

Connecting phenotype and chemotype: high-content discovery strategies for natural products research.

In recent years, the field of natural products has seen an explosion in the breadth, resolution, and accuracy of profiling platforms for compound discovery, including many new chemical and biological annotation methods. With these new tools come opportunities to examine extract libraries using systematized profiling approaches that were not previously available to the field and which offer new approaches for the detailed characterization of the chemical and biological attributes of complex natural products mixtures. This review will present a summary of some of these untargeted profiling methods and provide perspective on the future opportunities offered by integrating these tools for novel natural products discovery.

Investigating the Origin of Epimerization Attenuation during Pd-Catalyzed Cross-Coupling Reactions.

Palladium-catalyzed cross-couplings remain among the most robust methodologies to form carbon-carbon and carbon-heteroatom bonds. In particular, carbon-nitrogen (C-N) couplings (Buchwald-Hartwig aminations) find widespread use in fine chemicals industries. The use of base in these reactions is critical for catalyst activation and proton sequestration. Base selection also plays an important role in process design, as strongly basic conditions can impact sensitive stereocenters and result in erosion of stereochemical purity. Herein we investigate the role of a Pd catalyst in suppressing base-mediated epimerization of a sultam stereocenter during a C-N cross-coupling reaction to access the RORγ inhibitor GDC-0022. Online high-performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to acquire reaction time course profiles and to delineate epimerization behavior, identify decomposition pathways, and monitor Pd-containing species. Our ability to monitor organopalladium complexes in real time by HPLC-MS provided strong evidence that the degree of epimerization was correlated to the Pd speciation in solution. Specifically, Pd(II) complexes were associated with mitigating epimerization of six-membered sultams. Additional studies showed that the suppression of epimerization in the presence of Pd(II) can impact Pd-catalyzed reactions of other substrates such as enolizable ketones, thus providing practical insight on the execution and optimization of such processes.

Automated online deconjugation of antibody-drug conjugate for small molecule drug profiling.

Antibody-drug conjugates (ADCs) are designed by chemically linking highly potent cytotoxic small molecule drugs to monoclonal antibodies of unique specificity for targeted destruction of cancer cells. This innovative class of molecules incurs unique developmental challenges due to its structural complexity of having both small molecule and protein components. The stability of the small molecule payload on the ADC is a critical attribute as it directly relates to product efficacy and patient safety. This study describes the use of an end-to-end automated workflow for effective and robust characterization of the small molecule drug while it is conjugated to the antibody. In this approach, online deconjugation was accomplished by an autosampler user defined program and 1D size exclusion chromatography was utilized to provide separation between small molecule and protein species. The small molecule portion was then trapped and sent to the 2D for separation and quantification by reversed-phase liquid chromatography with identification of impurities and degradants by mass spectrometry. The feasibility of this system was demonstrated on an ADC with a disulfide-based linker. This fully automated approach avoids tedious sample preparation that may lead to sample loss and large assay variability. Under optimized conditions, the method was shown to have excellent specificity, sensitivity (LOD of 0.036 µg/mL and LOQ of 0.144 µg/mL), linearity (0.04-72.1 µg/mL), precision (system precision %RSD of 1.7 and method precision %RSD of 3.4), accuracy (97.4 % recovery), stability-indicating nature, and was successfully exploited to analyze the small molecule drug on a panel of stressed ADC samples. Overall, the workflow established here offers a powerful analytical tool for profiling the in-situ properties of small molecule drugs conjugated to antibodies and the obtained information could be of great significance for guiding process/formulation development and understanding pharmacokinetic/pharmacodynamic behavior of ADCs.

Two-dimensional reversed phase-normal phase liquid chromatography for simultaneous achiral-chiral analysis to support high-throughput experimentation.

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.

Ultraviolet photodissociation facilitates mass spectrometry-based structure elucidation with pyrrolidine and piperidine containing compounds.

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.