Macrocyclization and Backbone Modification in RiPP Biosynthesis.

The past decade has seen impressive advances in understanding the biosynthesis of ribosomally synthesized and posttranslationally modified peptides (RiPPs). One of the most common modifications found in these natural products is macrocyclization, a strategy also used by medicinal chemists to improve metabolic stability and target affinity and specificity. Another tool of the peptide chemist, modification of the amides in a peptide backbone, has also been observed in RiPPs. This review discusses the molecular mechanisms of biosynthesis of a subset of macrocyclic RiPP families, chosen because of the unusual biochemistry involved: the five classes of lanthipeptides (thioether cyclization by Michael-type addition), sactipeptides and ranthipeptides (thioether cyclization by radical chemistry), thiopeptides (cyclization by [4+2] cycloaddition), and streptide (cyclization by radical C-C bond formation). In addition, the mechanisms of backbone amide methylation, backbone epimerization, and backbone thioamide formation are discussed, as well as an unusual route to small molecules by posttranslational modification.

Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.

Production, purification, characterization, and safety evaluation of constructed recombinant D-psicose 3-epimerase.

BACKGROUND: D-psicose 3-epimerase (DPEase) is a potential catalytic enzyme for D-psicose production. D-psicose, also known as D-allulose, is a low-calorie sweetener that has gained considerable attention as a healthy alternative sweetener due to its notable physicochemical properties. This research focused on an in-depth investigation of the expression of the constructed DPEase gene from Agrobacterium tumefaciens in Escherichia coli for D-psicose synthesis. Experimentally, this research created the recombinant enzyme, explored the optimization of gene expression systems and protein purification strategies, investigated the enzymatic characterization, and then optimized the D-psicose production. Finally, the produced D-psicose syrup underwent acute toxicity evaluation to provide scientific evidence supporting its safety. RESULTS: The optimization of DPEase expression involved the utilization of Mn2+ as a cofactor, fine-tuning isopropyl ß-D-1-thiogalactopyranoside induction, and controlling the induction temperature. The purification process was strategically designed by a nickel column and an elution buffer containing 200 mM imidazole, resulting in purified DPEase with a notable 21.03-fold increase in specific activity compared to the crude extract. The optimum D-psicose conversion conditions were at pH 7.5 and 55 °C with a final concentration of 10 mM Mn2+ addition using purified DPEase to achieve the highest D-psicose concentration of 5.60% (w/v) using 25% (w/v) of fructose concentration with a conversion rate of 22.42%. Kinetic parameters of the purified DPEase were Vmax and Km values of 28.01 mM/min and 110 mM, respectively, which demonstrated the high substrate affinity and efficiency of DPEase conversion by the binding site of the fructose-DPEase-Mn2+ structure. Strategies for maintaining stability of DPEase activity were glycerol addition and storage at -20 °C. Based on the results from the acute toxicity study, there was no toxicity to rats, supporting the safety of the mixed D-fructose-D-psicose syrup produced using recombinant DPEase. CONCLUSIONS: These findings have direct and practical implications for the industrial-scale production of D-psicose, a valuable rare sugar with a broad range of applications in the food and pharmaceutical industries. This research should advance the understanding of DPEase biocatalysis and offers a roadmap for the successful scale-up production of rare sugars, opening new avenues for their utilization in various industrial processes.

Strategies for Accessing cis-1-Amino-2-Indanol.

cis-1-amino-2-indanol is an important building block in many areas of chemistry. Indeed, this molecule is currently used as skeleton in many ligands (BOX, PyBOX…), catalysts and chiral auxiliaries. Moreover, it has been incorporated in numerous bioactive structures. The major issues during its synthesis are the control of cis-selectivity, for which various strategies have been devised, and the enantioselectivity of the reaction. This review highlights the various methodologies implemented over the last few decades to access cis-1-amino-2-indanol in racemic and enantioselective manners. In addition, the various substitution patterns on the aromatic ring and their preparations are listed.

Erythrose and Threose: Carbonyl Migrations, Epimerizations, Aldol, and Oxidative Fragmentation Reactions under Plausible Prebiotic Conditions.

The prebiotic generation of sugars in the context of origins of life studies is of considerable interest. Among the important intramolecular processes of sugars are carbonyl migrations and accompanying epimerizations. Herein we describe the carbonyl migration-epimerization process occurring down the entire carbon chain of chirally pure d-tetroses sugars under mild conditions. Employing chirally pure 1-13 C-erythrose, 4-13 C-erythrose and 1-13 C-threose, we (1) identify all the species formed as the carbonyl migrates down the four-carbon chain and (2) assess the rates associated with the production of each of these species. Competing aldol reactions and oxidative fragmentation processes were also observed. Further observations of self-condensation of glycolaldehyde mainly yielding 2-keto-hexoses (sorbose and tagatose) and tetrulose also provides a basis for understanding the effect of carbonyl migrations on the product distribution in plausible prebiotic scenarios.

Unraveling the Antioxidant Activity of 2R,3R-dihydroquercetin.

It has been reported that in an oxidative environment, the flavonoid 2R,3R-dihydroquercetin (2R,3R-DHQ) oxidizes into a product that rearranges to form quercetin. As quercetin is a very potent antioxidant, much better than 2R,3R-DHQ, this would be an intriguing form of targeting the antioxidant quercetin. The aim of the present study is to further elaborate on this targeting. We can confirm the previous observation that 2R,3R-DHQ is oxidized by horseradish peroxidase (HRP), with H2O2 as the oxidant. However, HPLC analysis revealed that no quercetin was formed, but instead an unstable oxidation product. The inclusion of glutathione (GSH) during the oxidation process resulted in the formation of a 2R,3R-DHQ-GSH adduct, as was identified using HPLC with IT-TOF/MS detection. GSH adducts appeared on the B-ring of the 2R,3R-DHQ quinone, indicating that during oxidation, the B-ring is oxidized from a catechol to form a quinone group. Ascorbate could reduce the quinone back to 2R,3R-DHQ. No 2S,3R-DHQ was detected after the reduction by ascorbate, indicating that a possible epimerization of 2R,3R-DHQ quinone to 2S,3R-DHQ quinone does not occur. The fact that no epimerization of the oxidized product of 2R,3R-DHQ is observed, and that GSH adducts the oxidized product of 2R,3R-DHQ on the B-ring, led us to conclude that the redox-modulating activity of 2R,3R-DHQ quinone resides in its B-ring. This could be confirmed by chemical calculation. Apparently, the administration of 2R,3R-DHQ in an oxidative environment does not result in 'biotargeting' quercetin.

Diastereoselective Synthesis of Dispiro[Imidazothiazolotriazine-Pyrrolidin-Oxindoles] and Their Isomerization Pathways in Basic Medium.

Highly diastereoselective methods for the synthesis of two series of regioisomeric polynuclear dispyroheterocyclic compounds with five or six chiral centers, comprising moieties of pyrrolidinyloxindole and imidazo[4,5-e]thiazolo[3,2-b]-1,2,4-triazine of linear structure or imidazo[4,5-e]thiazolo[2,3-c]-1,2,4-triazine of angular structure, have been developed on the basis of a [3+2] cycloaddition of azomethine ylides to functionalized imidazothiazolotriazines. Depending on the structure of the ethylenic component, cycloaddition proceeds as an anti-exo process for linear derivatives, while cycloaddition to angular ones resulted in a syn-endo diastereomer. Novel pathways of isomerization for the synthesized anti-exo products upon treatment with sodium alkoxides have been found, which resulted in two more series of diastereomeric dispiro[imidazothiazolotriazine-pyrrolidin-oxindoles] inaccessible with the direct cycloaddition reaction. For the first series, the inversion of the configuration of one stereocenter, i.e., C-4' atom of the pyrrolidine cycle, (epimerization) was established. For the second one, configuration of the obtained diastereomer formally corresponded to the syn-endo approach of the azomethine ylide in the case of cycloaddition to the ethylenic component.

Copper-Catalyzed Radical Enantioselective Synthesis of γ-Butyrolactones with Two Non-vicinal Carbon Stereocenters.

Chiral heterocycles with two or more carbon stereocenters are quite important skeletons in many fields. However, powerful strategies for the construction of such synthetically valuable heterocycles, especially with two or more remote carbon stereocenters, have largely lagged behind. We report here a powerful method for the synthesis of chiral γ-butyrolactones with two non-vicinal carbon stereocenters from readily available chemical feedstocks under mild conditions. Both of the two diastereoisomers can be obtained with good to excellent enantioselectivities. The well-designed copper/PyBox catalytic system overrides the intrinsic stereoinduction of the close chirality center generated by the previous innocent radical addition step. Nevertheless, this work has the power to selectively provide one single diastereoisomer by taking advantage of the epimerization effect but also to synthesize all four diastereoisomers with the pair of chiral ligands L2 and L2' having opposite chirality. The obtained useful chiral γ-butyrolactones can be synthetically transformed into acyclic or cyclic molecules with two non-vicinal carbon stereocenters. Mechanistic studies reveal that this radical reaction follows a linear relationship and can be well performed with a less loading amount of ligand compared to that of the copper catalyst.

Stereochemical Editing.

Stereochemical editing has recently risen to prominence, allowing the direct editing of organic molecules with stereocenter(s) to adjust their relative stereochemistry at a late-stage. Several seminal light-driven stereochemical editing reactions such as deracemization and epimerization have been successively developed. Recently, Wendlandt and co-workers reported a versatile photochemical epimerization of unactivated tertiary stereogenic centers to rapidly prepare the stereoisomers that were previously challenging to access.

Insight into substrate-assisted catalytic mechanism and stereoselectivity of bifunctional nocardicin thioesterase.

The inversion from L- to D-stereochemistry endows peptides improved bioactivity and enhanced resistance to many proteases and peptidases. To strengthen the biostability and bioavailability of peptide drugs, enzymatic epimerization becomes an important way to incorporate D-amino acid into peptide backbones. Recently, a bifunctional thioesterase NocTE, which is responsible for the epimerization and hydrolysis of the C-terminal (p-hydroxyphenyl)glycine residue of ß-lactam antibiotic nocardicin A, exclusively directs to the generation of D-diastereomers. Different from other epimerases, NocTE exhibits unique stereochemical selectivity. Herein, we investigated the catalytic mechanism of NocTE via molecular dynamic (MD) simulations and quantum mechanical/molecular mechanics (QM/MM) calculations. Through structural analyses, two key water molecules around the reaction site were found to serve as proton mediators in epimerization. The structural characteristics inspired us to propose a substrate-assisted mechanism for the epimerization, where multi-step proton transfers were mediated by water molecules and ß-lactam ring, and the free energy barrier was calculated to be 20.3 kcal/mol. After that, the hydrolysis of D-configured substrate was energetically feasible with the energy barrier of 14.3 kcal/mol. As a comparison, the energy barrier for the direct hydrolysis of L-configured substrate was obtained to be 24.0 kcal/mol. Our study provides mechanistic insights into catalytic activities of bifunctional thioesterase NocTE, uncovers more clues to the molecular basis for stereochemical selectivity and paves the way for the directed biosynthesis of novel peptide drugs with various stereostructural characteristics by enzyme rational design.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14ß-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(ß→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Late-Stage Modification of Aminoglycoside Antibiotics Overcomes Bacterial Resistance Mediated by APH(3') Kinases.

The continuous emergence of antimicrobial resistance is causing a threat to patients infected by multidrug-resistant pathogens. In particular, the clinical use of aminoglycoside antibiotics, broad-spectrum antibacterials of last resort, is limited due to rising bacterial resistance. One of the major resistance mechanisms in Gram-positive and Gram-negative bacteria is phosphorylation of these amino sugars at the 3'-position by O-phosphotransferases [APH(3')s]. Structural alteration of these antibiotics at the 3'-position would be an obvious strategy to tackle this resistance mechanism. However, the access to such derivatives requires cumbersome multi-step synthesis, which is not appealing for pharma industry in this low-return-on-investment market. To overcome this obstacle and combat bacterial resistance mediated by APH(3')s, we introduce a novel regioselective modification of aminoglycosides in the 3'-position via palladium-catalyzed oxidation. To underline the effectiveness of our method for structural modification of aminoglycosides, we have developed two novel antibiotic candidates overcoming APH(3')s-mediated resistance employing only four synthetic steps.

Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase.

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.

Synthesis of Four Orthogonally Protected Rare l-Hexose Thioglycosides from d-Mannose by C-5 and C-4 Epimerization.

l-Hexoses are important components of biologically relevant compounds and precursors of some therapeuticals. However, they typically cannot be obtained from natural sources and due to the complexity of their synthesis, their commercially available derivatives are also very expensive. Starting from one of the cheapest d-hexoses, d-mannose, using inexpensive and readily available chemicals, we developed a reaction pathway to obtain two orthogonally protected l-hexose thioglycoside derivatives, l-gulose and l-galactose, through the corresponding 5,6-unsaturated thioglycosides by C-5 epimerization. From these derivatives, the orthogonally protected thioglycosides of further two l-hexoses (l-allose and l-glucose) were synthesized by C-4 epimerization. The preparation of the key intermediates, the 5,6-unsaturated derivatives, was systematically studied using various protecting groups. By the method developed, we are able to produce highly functionalized l-gulose derivatives in 9 steps (total yields: 21-23%) and l-galactose derivatives in 12 steps (total yields: 6-8%) starting from d-mannose.

Synthesis of L-Hexoses: An Update.

Over the years, carbohydrates have increasingly become an important class of compounds contributing significantly to the target specific drug discovery and vaccine development. Several oligosaccharides contain L-hexoses that are biologically relevant as therapeutic and diagnostic tools. Since, L-hexoses and deoxy L-hexoses are not readily available in large amount and pure form, attention is drawn towards development of cost effective and high yielding synthetic routes for their procurement. In this review we give an update on the recent developments in strategies for synthesis of L-hexoses and deoxy L-hexoses.

Unambiguous Stereochemical Assignment of Cyclo(Phe-Pro), Cyclo(Leu-Pro), and Cyclo(Val-Pro) by Electronic Circular Dichroic Spectroscopy.

2,5-diketopiperazines (DKPs) are cyclic dipeptides ubiquitously found in nature. In particular, cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) are frequently detected in many microbial cultures. Each of these DKPs has four possible stereoisomers due to the presence of two chirality centers. However, absolute configurations of natural DKPs are often ambiguous due to the lack of a simple, sensitive, and reproducible method for stereochemical assignment. This is an important problem because stereochemistry is a key determinant of biological activity. Here, we report a synthetic DKP library containing all stereoisomers of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro). The library was subjected to spectroscopic characterization using mass spectrometry, NMR, and electronic circular dichroism (ECD). It turned out that ECD can clearly differentiate DKP stereoisomers. Thus, our ECD dataset can serve as a reference for unambiguous stereochemical assignment of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) samples from natural sources. The DKP library was also subjected to a biological screening using assays for E. coli growth and biofilm formation, which revealed distinct biological effects of cyclo(D-Phe-L-Pro).

Reactivity of flavanols: Their fate in physical food processing and recent advances in their analysis by depolymerization.

Flavanols, a subgroup of polyphenols, are secondary metabolites with antioxidant properties naturally produced in various plants (e.g., green tea, cocoa, grapes, and apples); they are a major polyphenol class in human foods and beverages, and have recognized effect on maintaining human health. Therefore, it is necessary to evaluate their changes (i.e., oxidation, polymerization, degradation, and epimerization) during various physical processing (i.e., heating, drying, mechanical shearing, high-pressure, ultrasound, and radiation) to improve the nutritional value of food products. However, the roles of flavanols, in particular for their polymerized forms, are often underestimated, for a large part because of analytical challenges: they are difficult to extract quantitatively, and their quantification demands chemical reactions. This review examines the existing data on the effects of different physical processing techniques on the content of flavanols and highlights the changes in epimerization and degree of polymerization, as well as some of the latest acidolysis methods for proanthocyanidin characterization and quantification. More and more evidence show that physical processing can affect content but also modify the structure of flavanols by promoting a series of internal reactions. The most important reactivity of flavanols in processing includes oxidative coupling and rearrangements, chain cleavage, structural rearrangements (e.g., polymerization, degradation, and epimerization), and addition to other macromolecules, that is, proteins and polysaccharides. Some acidolysis methods for the analysis of polymeric proanthocyanidins have been updated, which has contributed to complete analysis of proanthocyanidin structures in particular regarding their proportion of A-type proanthocyanidins and their degree of polymerization in various plants. However, future research is also needed to better extract and characterize high-polymer proanthocyanidins, whether in their native or modified forms.

An effective method to produce 7-epitaxol from taxol in HCO3.

It is known that 7-epitaxol has much stronger cytotoxicity than taxol does. However, the content of 7-epitaxol in yew is much less than taxol, which makes it more costly to obtain. We describe here a method to effectively convert taxol to 7-epitaxol. The key condition for reaction needs NaHCO3 in solvent acetonitrile (ACN). The conversion rate can be over 82%.

Synthesis of chlorophyll-a homologs by C132-substitutions and their physico- and biochemical properties.

A mixture of pheophytins-a/a', metal-free forms of photosynthetically active chlorophyll(Chl)s-a/a' bearing the 132-methoxycarbonyl group, was substituted at the C132-position by bimolecular nucleophilic substitution with methyl bromoacetate or Michael addition with methyl acrylate, followed by C132-demethoxycarbonylation and magnesium insertion at the central position, to afford Chl-a/a' homologs possessing a methoxycarbonylmethyl or 2-methoxycarbonylethyl group at the C132-position, respectively. These C132-methylene- and ethylene-inserted homologs were characterized by 1D/2D 1H NMR spectroscopy, and the optical properties of their C132-epimerically pure samples are investigated using visible absorption, fluorescence emission, and circular dichroism spectroscopies. The stereochemistry at the C132-chiral center of these Chl-a/a' homologs was not inverted in a basic solution, and the Chl-a homologs were effective for the substrates for the chlorophyllase reaction, hydrolysis of the phytyl ester.

Degradation Pathway of a Taxane Derivative DS80100717 Drug Substance and Drug Product.

The degradation pathway of a taxane derivative and anticancer agent, DS80100717, was investigated. Several degradants were generated under acidic, basic, and oxidative stress conditions in solution. The chemical structures of eight degradants of DS80100717 were elucidated using MS and NMR. The major degradant of the DS80100717 drug substance derived by heating in solid-state was the N-oxide form via oxidation and C2'-epimer of the side chain via acid hydrolysis. We proposed previously unreported degradation pathways of DS80100717 with taxane derivatives such as paclitaxel, docetaxel, and cabazitaxel.