Regioselective Annulation of 6-Carboxy-Substituted Pyrones as a Two-Carbon Unit in Formal [4 + 2] Cycloaddition Reactions.

Heterocycles serve as a critical motif in chemistry, but despite being present in more than 85% of pharmaceuticals, there are limited methods for their construction. Here, we describe the incorporation of intact pyrone (2H-pyran-2-one) into larger ring systems via annulation. In a formal [4 + 2] cycloaddition, the pyrone regioselectively accepts a benzylic anion as a nucleophile in a conjugate addition fashion, with the subsequent pyrone-derived enolate attaching to a pendant ester on the initial nucleophile. Subsequent base-driven enolate formation and elimination establish aromaticity of the newly formed ring. After optimization of this process using an NMR-based assessment to overcome solubility and separation challenges, the reaction was successfully applied to a library of 6-ester and -amide-substituted pyrones and using a phenyl ester and other substituted sulfoxides. This technology enables the incorporation of intact pyrone rings into more complex systems, such as for the total synthesis of the natural product thermorubin.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.

Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis.

Hapalindole alkaloids are a structurally diverse class of cyanobacterial natural products defined by their varied polycyclic ring systems and diverse biological activities. These complex metabolites are generated from a common biosynthetic intermediate by the Stig cyclases in three mechanistic steps: a rare Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution. Here we report the structure of HpiC1, a Stig cyclase that catalyzes the formation of 12-epi-hapalindole U in vitro. The 1.5-Å structure revealed a dimeric assembly with two calcium ions per monomer and with the active sites located at the distal ends of the protein dimer. Mutational analysis and computational methods uncovered key residues for an acid-catalyzed [3,3]-sigmatropic rearrangement, as well as specific determinants that control the position of terminal electrophilic aromatic substitution, leading to a switch from hapalindole to fischerindole alkaloids.

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases.

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non-native polyketide substrates. Reactions pairing wildtype PKS modules with non-native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non-cognate TE domains was severely reduced. In contrast, non-native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor-Induced Combinatorial Pairing of Stig Cyclases.

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three-step cascade involving Cope rearrangement, 6-exo-trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+ , to cooperatively control the stereochemistry of hapalindole natural products.

Engineering the Substrate Specificity of a Modular Polyketide Synthase for Installation of Consecutive Non-Natural Extender Units.

There is significant interest in diversifying the structures of polyketides to create new analogues of these bioactive molecules. This has traditionally been done by focusing on engineering the acyltransferase (AT) domains of polyketide synthases (PKSs) responsible for the incorporation of malonyl-CoA extender units. Non-natural extender units have been utilized by engineered PKSs previously; however, most of the work to date has been accomplished with ATs that are either naturally promiscuous and/or located in terminal modules lacking downstream bottlenecks. These limitations have prevented the engineering of ATs with low native promiscuity and the study of any potential gatekeeping effects by domains downstream of an engineered AT. In an effort to address this gap in PKS engineering knowledge, the substrate preferences of the final two modules of the pikromycin PKS were compared for several non-natural extender units and through active site mutagenesis. This led to engineering of the methylmalonyl-CoA specificity of both modules and inversion of their selectivity to prefer consecutive non-natural derivatives. Analysis of the product distributions of these bimodular reactions revealed unexpected metabolites resulting from gatekeeping by the downstream ketoreductase and ketosynthase domains. Despite these new bottlenecks, AT engineering provided the first full-length polyketide products incorporating two non-natural extender units. Together, this combination of tandem AT engineering and the identification of previously poorly characterized bottlenecks provides a platform for future advancements in the field.

Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids.

The formation of C-C bonds in an enantioselective fashion to create complex polycyclic scaffolds in the hapalindole- and fischerindole- type alkaloids from Stigonematales cyanobacteria represents a compelling and urgent challenge in adapting microbial biosynthesis as a catalytic platform in drug development. Here we determine the biochemical basis for tri- and tetracyclic core formation in these secondary metabolites, involving a new class of cyclases that catalyze a complex cyclization cascade.

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization.

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

Hapalindole/Ambiguine Biogenesis Is Mediated by a Cope Rearrangement, C-C Bond-Forming Cascade.

Hapalindoles are bioactive indole alkaloids with fascinating polycyclic ring systems whose biosynthetic assembly mechanism has remained unknown since their initial discovery in the 1980s. In this study, we describe the fam gene cluster from the cyanobacterium Fischerella ambigua UTEX 1903 encoding hapalindole and ambiguine biosynthesis along with the characterization of two aromatic prenyltransferases, FamD1 and FamD2, and a previously undescribed cyclase, FamC1. These studies demonstrate that FamD2 and FamC1 act in concert to form the tetracyclic core ring system of the hapalindoles from cis-indole isonitrile and geranyl pyrophosphate through a presumed biosynthetic Cope rearrangement and subsequent 6-exo-trig cyclization/electrophilic aromatic substitution reaction.

Forgotten Natural Products: Semisynthetic Development of Blasticidin S As an Antibiotic Lead.

Forgotten natural products offer value as antimicrobial scaffolds, providing diverse mechanisms of action that complement existing antibiotic classes. This study focuses on the derivatization of the cytotoxin blasticidin S, seeking to leverage its unique ribosome inhibition mechanism. Despite its complex zwitterionic properties, a selective protection and amidation strategy enabled the creation of a library of blasticidin S derivatives including the natural product P10. The amides exhibited significantly increased activity against Gram-positive bacteria and enhanced specificity for pathogenic bacteria over human cells. Molecular docking and computational property analysis suggested variable binding poses and indicated a potential correlation between cLogP values and activity. This work demonstrates how densely functionalized forgotten antimicrobials can be straightforwardly modified, enabling the further development of blasticidin S derivatives as lead compounds for a novel class of antibiotics.

Semisynthetic blasticidin S ester derivatives show enhanced antibiotic activity.

A rich potential source of new antibiotics are undeveloped natural product cytotoxins, provided they can be derivatized to restrict their activity to bacteria. In this work, we describe modification of one such candidate, the broad-spectrum, translation termination inhibitor, blasticidin S. By semisynthetically modifying blasticidin S, we produced a series of ester derivatives of this highly polar, zwitterionic compound in a single step. These derivatives showed a marked increase in activity against Gram-positive bacteria and an increase in selectivity index for pathogenic bacteria over human cells. The results of this study suggest that semisynthetic derivatization of blasticidin S and other neglected natural product antimicrobials has the potential to increase their activity against and selectivity for bacteria, an approach that can be leveraged for the development of leads against antimicrobial resistant pathogens.

Data Driven Computational Design and Experimental Validation of Drugs for Accelerated Mitigation of Pandemic-like Scenarios.

Emerging pathogens are a historic threat to public health and economic stability. Current trial-and-error approaches to identify new therapeutics are often ineffective due to their inefficient exploration of the enormous small molecule design space. Here, we present a data-driven computational framework composed of hybrid evolutionary algorithms for evolving functional groups on existing drugs to improve their binding affinity toward the main protease (Mpro) of SARS-CoV-2. We show that combinations of functional groups and sites are critical to design drugs with improved binding affinity, which can be easily achieved using our framework by exploring a fraction of the available search space. Atomistic simulations and experimental validation elucidate that enhanced and prolonged interactions between functionalized drugs and Mpro residues result in their improved therapeutic value over that of the parental compound. Overall, this novel framework is extremely flexible and has the potential to rapidly design inhibitors for any protein with available crystal structures.

Functionalized low-density lipoprotein nanoparticles for in vivo enhancement of atherosclerosis on magnetic resonance images.

To allow visualization of macrophage-rich and miniature-sized atheromas by magnetic resonance (MR) imaging, we have converted low-density lipoprotein (LDL) into MR-active nanoparticles via the intercalation of a 1,4,7,10-tetraazacyclodecane-1,4,7-triacetic acid (DO3A) derivative and the subsequent coordination reaction with Gd(3+). After careful removal of nonchelated Gd(3+), an MR-active LDL (Gd(3+)-LDL) with a remarkably high payload of Gd(3+) (in excess of 200 Gd(3+) atoms per particle) and a high relaxivity (r(1) = 20.1 s(-1) mM(-1) per Gd(3+) or 4040 s(-1) mM(-1) per LDL) was obtained. Dynamic light-scattering photon correlation spectroscopy (DLS) and cryo transmission electron microscope (cryoTEM) images showed that Gd(3+)-LDL particles did not aggregate and remained of a similar size (25-30 nm) to native LDL. Intravenous injection of Gd(3+)-LDL into an atherosclerotic mouse model (ApoE(-/-)) resulted in an extremely high enhancement of the atheroma-bearing aortic walls at 48 h after injection. Free Gd(3+) dissociation from Gd(3+)-LDL was not detected over the imaging time window (96 h). Because autologous LDL can be isolated, modified, and returned to the same patient, our results suggest that MR-active LDL can potentially be used as a noninfectious and nonimmunogenic imaging probe for the enhancement of atheroplaques presumably via the uptake into macrophages inside the plaque.

Thermorubin Biosynthesis Initiated by a Salicylate Synthase Suggests an Unusual Conversion of Phenols to Pyrones.

Thermorubin is a tetracyclic naphthoisocoumarin natural product that demands investigation due to its novel mechanism of bacterial protein synthesis inhibition and its unusual structural features. In this work, we describe the identification of the biosynthetic cluster responsible for thermorubin from the sequenced Laceyella sacchari producer species and its confirmation via heterologous production in Escherichia coli. Based on an in-depth annotation of the cluster, we propose a biosynthetic pathway that accounts for the formation of the unique, nonterminal pyrone. Additionally, the expression and use of salicylate synthase TheO enabled testing of the stability properties of this extremophile-derived enzyme. TheO displayed rapid kinetics and a remarkably robust secondary structure, converting chorismate to salicylate with a KM of 109 ± 12 µM, kcat of 9.17 ± 0.36 min-1, and catalytic efficiency (kcat/KM) of 84 ± 9 nM-1 min-1, and retained significant activity up to 50 °C. These studies serve as the basis for continued biosynthetic investigations and bioinspired synthetic approaches.

Alternative spiroketalization methods toward purpuromycin: a hemiketal conjugate addition strategy and use of an electron-rich isocoumarin precursor.

Two methods are presented that were designed to circumvent the persistent problem of benzofuran formation and instead yield a spiroketal of the rubromycin family type. First, using an alternative disconnection, a hemiketal conjugate addition to a naphthaquinone electrophile was investigated. Synthesis of the requisite electrophile provided insight into the selective oxidation and functionalization of the naphthalene portion. Second, the electronic features of the isocoumarin ring system were adjusted, and the corresponding reactivity further supports the hypothesis that electron-rich isocoumarins are capable of spiroketalization. Robust, flexible syntheses from simple precursors were developed that allowed multiple reduced isocoumarins to be generated. Combined, the data presented herein give insight into the sensitivities of this family and illuminate other potential methods of spiroketalization. In addition, the convergent assembly of substrates containing different naphthaquinone and isocoumarin subunits highlights the utility of our 1,3-dipolar cycloaddition approach to generate analogs of these structures for SAR, as well as chemical reactivity studies.

Alternative spiroketalization methods toward purpuromycin: a diketone approach to prevent benzofuran formation.

The central portion of purpuromycin has been assembled via a classical spiroketalization reaction. Key to promoting this reaction mode versus benzofuran formation was the oxidation state of the spiroketal core. With a higher oxidation state, even the electron-deficient isocoumarin found in purpuromycin could be employed directly in the spiroketalization. The two halves of the spiroketalization precursor were joined via a nitrile oxide/styrene 1,3-dipolar cycloaddition. A very mild selenium dioxide oxidation was used to introduce the required oxidation state of the spiroketal core.

Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase.

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

Structural diversification of hapalindole and fischerindole natural products via cascade biocatalysis.

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonemataceae order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, L-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process including a Cope rearrangement, 6-exo-trig cyclization and electrophilic aromatic substitution to create a polycyclic core. Formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to the wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward core structural diversification of this family of indole alkaloids. Through synthesis of cis-indole isonitrile subunit derivatives, and aided by protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives, and gained insights into the basis for substrate flexibility in this system.

Multicomponent Microscale Biosynthesis of Unnatural Cyanobacterial Indole Alkaloids.

Genome sequencing and bioinformatics tools have facilitated the identification and expression of an increasing number of cryptic biosynthetic gene clusters (BGCs). However, functional analysis of all components of a metabolic pathway to precisely determine biocatalytic properties remains time-consuming and labor intensive. One way to speed this process involves microscale cell-free protein synthesis (CFPS) for direct gene to biochemical function analysis, which has rarely been applied to study multicomponent enzymatic systems in specialized metabolism. We sought to establish an in vitro transcription/translation (TT)-assay to assess assembly of cyanobacterial-derived hapalindole-type natural products (cNPs) because of their diverse bioactivity profiles and complex structural diversity. Using a CFPS system including a plasmid bearing famD2 prenyltransferase from Fischerella ambigua UTEX 1903, we showed production of the central prenylated intermediate (3GC) in the presence of exogenous geranyl-pyrophosphate (GPP) and cis-indole isonitrile. Further addition of a plasmid bearing the famC1 Stig cyclase resulted in synthesis of both FamD2 and FamC1 enzymes, which was confirmed by proteomics analysis, and catalyzed assembly of 12-epi-hapalindole U. Further combinations of Stig cyclases (FamC1-C4) produced hapalindole U and hapalindole H, while FisC identified from Fischerella sp. SAG46.79 generated 12-epi-fischerindole U. The CFPS system was further employed to screen six unnatural halogenated cis-indole isonitrile substrates using FamC1 and FisC, and the reactions were scaled-up using chemoenzymatic synthesis and identified as 5- and 6-fluoro-12-epi-hapalindole U, and 5- and 6-fluoro-12-epi-fischerindole U, respectively. This approach represents an effective, high throughput strategy to determine the functional role of biosynthetic enzymes from diverse natural product BGCs.

Larger Substituents on Amide Cavitands Induce Bigger Cavities.

A series of quinoxaline cavitands bearing pendant amide groups with various substituent sizes (Et, iPr, tBu) were synthesized, and their cavity size/structure were investigated by X-ray and NMR analyses. In the case of the Et or iPr amide cavitand, the conformation of the molecule was in the vase form, while the bulky tBu amide cavitand gave the kite conformation at room temperature. X-ray crystal structures of Et and iPr cavitands clearly showed the intramolecular H-bondings to influence the conformation and the cavity sizes dependent on the bulkiness of functional groups. The 1H NMR spectrum revealed that the Et cavitand can encapsulate an adamantane guest compound with slow exchange.