Characterization and catalytic investigation of fungal single-module nonribosomal peptide synthetase in terpene-amino acid meroterpenoid biosynthesis.

Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single-module nonribosomal peptide synthetases (NRPSs) have been identified to be involved in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking, and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives. ONE-SENTENCE SUMMARY: The enzymatic basis and mechanistic insights into AneB, a single-module NRPS, highlight its capacity to generate various terpene-amino acid meroterpenoid derivatives.

Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun's lipoprotein from peptidoglycan.

IMPORTANCE: Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

Carbohydrate Ligands for COVID-19 Spike Proteins.

An outbreak of SARS-CoV-2 coronavirus (COVID-19) first detected in Wuhan, China, has created a public health emergency all over the world. The pandemic has caused more than 340 million confirmed cases and 5.57 million deaths as of 23 January 2022. Although carbohydrates have been found to play a role in coronavirus binding and infection, the role of cell surface glycans in SARS-CoV-2 infection and pathogenesis is still not understood. Herein, we report that the SARS-CoV-2 spike protein S1 subunit binds specifically to blood group A and B antigens, and that the spike protein S2 subunit has a binding preference for Lea antigens. Further examination of the binding preference for different types of red blood cells (RBCs) indicated that the spike protein S1 subunit preferentially binds with blood group A RBCs, whereas the spike protein S2 subunit prefers to interact with blood group Lea RBCs. Angiotensin converting enzyme 2 (ACE2), a known target of SARS-CoV-2 spike proteins, was identified to be a blood group A antigen-containing glycoprotein. Additionally, 6-sulfo N-acetyllactosamine was found to inhibit the binding of the spike protein S1 subunit with blood group A RBCs and reduce the interaction between the spike protein S1 subunit and ACE2.

Entropy-driven binding of gut bacterial ß-glucuronidase inhibitors ameliorates irinotecan-induced toxicity.

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of ß-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial ß-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 µM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.

Threshold of Thioglycoside Reactivity Difference Is Critical for Efficient Synthesis of Type I Oligosaccharides by Chemoselective Glycosylation.

Synthesis of type I LacNAc (Galß1 → 3GlcNAc) oligosaccharides usually suffers from low yields. We herein report the efficient synthesis of type I LacNAc oligosaccharides by chemoselective glycosylation. With 16 relative reactivity values (RRVs) measured thiotoluenyl-linked disaccharide donors and acceptors, chemoselective glycosylations were investigated to obtain optimal conditions. In these reactions, the RRV difference between the donors and acceptors had to be more than 6311 to obtain type I LacNAc tetrasaccharides in 72-86% yields, with minimal occurrence of aglycon transfer. The threshold of RRV difference was further applied to plan the synthesis of longer glycans. Because it is challenging to measure the RRVs of tetrasaccharides, anomeric proton chemical shifts were utilized to predict the corresponding RRVs, which consequently explained the outcome of glycosylations for the synthesis of type I LacNAc hexasaccharides. The result supported the idea that elongation of glycan chains has to proceed from the reducing to the nonreducing end for a better yield.

Substituent Position of Iminocyclitols Determines the Potency and Selectivity for Gut Microbial Xenobiotic-Reactivating Enzymes.

Selective inhibitors of gut bacterial ß-glucuronidases (GUSs) are of particular interest in the prevention of xenobiotic-induced toxicities. This study reports the first structure-activity relationships on potency and selectivity of several iminocyclitols (2-7) for the GUSs. Complex structures of Ruminococcus gnavus GUS with 2-7 explained how charge, conformation, and substituent of iminocyclitols affect their potency and selectivity. N1 of uronic isofagomine (2) made strong electrostatic interactions with two catalytic glutamates of GUSs, resulting in the most potent inhibition (Ki ≥ 11 nM). C6-propyl analogue of 2 (6) displayed 700-fold selectivity for opportunistic bacterial GUSs (Ki = 74 nM for E. coli GUS and 51.8 µM for RgGUS). In comparison with 2, there was 200-fold enhancement in the selectivity, which was attributed to differential interactions between the propyl group and loop 5 residues of the GUSs. The results provide useful insights to develop potent and selective inhibitors for undesired GUSs.

Dissecting the Structure-Activity Relationship of Galectin-Ligand Interactions.

Galectins are ß-galactoside-binding proteins. As carbohydrate-binding proteins, they participate in intracellular trafficking, cell adhesion, and cell-cell signaling. Accumulating evidence indicates that they play a pivotal role in numerous physiological and pathological activities, such as the regulation on cancer progression, inflammation, immune response, and bacterial and viral infections. Galectins have drawn much attention as targets for therapeutic interventions. Several molecules have been developed as galectin inhibitors. In particular, TD139, a thiodigalactoside derivative, is currently examined in clinical trials for the treatment of idiopathic pulmonary fibrosis. Herein, we provide an in-depth review on the development of galectin inhibitors, aiming at the dissection of the structure-activity relationship to demonstrate how inhibitors interact with galectin(s). We especially integrate the structural information established by X-ray crystallography with several biophysical methods to offer, not only in-depth understanding at the molecular level, but also insights to tackle the existing challenges.

Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors.

Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3'-deoxy-3,3'-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A-E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from µM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.

Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence.

Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl and 6'-phosphatidyl α-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labelling method for characterising these derivatives qualitatively and quantitatively with a femtomolar detection limit. The development generated an MS/MS database of CGds, allowing for profiling of all the cholesterol-derived metabolites. The subsequent analysis led to the unprecedented information that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helps to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence. These findings demonstrate an important connection between the chain length of CAGs and the bacterial pathogenicity.

Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galß1-3/4GlcNAc.

Galectins represent ß-galactoside-binding proteins and are known to bind Galß1-3/4GlcNAc disaccharides (abbreviated as LN1 and LN2, respectively). Despite high sequence and structural homology shared by the carbohydrate recognition domain (CRD) of all galectin members, how each galectin displays different sugar-binding specificity still remains ambiguous. Herein we provided the first structural evidence of human galectins-1, 3-CRD and 7 in complex with LN1. Galectins-1 and 3 were shown to have higher affinity for LN2 than for LN1, while galectin-7 displayed the reversed specificity. In comparison with the previous LN2-complexed structures, the results indicated that the average glycosidic torsion angle of galectin-bound LN1 (ψ(LN1) ≈ 135°) was significantly differed from that of galectin-bound LN2 (ψ(LN2 )≈ -108°), i.e. the GlcNAc moiety adopted a different orientation to maintain essential interactions. Furthermore, we also identified an Arg-Asp/Glu-Glu-Arg salt-bridge network and the corresponding loop (to position the second Asp/Glu residue) critical for the LN1/2-binding preference.

Efficient Mapping of Sulfated Glycotopes by Negative Ion Mode nanoLC-MS/MS-Based Sulfoglycomic Analysis of Permethylated Glycans.

We have previously developed the enabling techniques for sulfoglycomics based on mass spectrometry (MS) analysis of permethylated glycans, which preserves the attractive features of more reliable MS/MS sequencing compared with that performed on native glycans, while providing an easy way to separate and hence enrich the sulfated glycans. Unlike LC-MS/MS analysis of native glycans in negative ion mode that has been more widely in use, the characteristics and potential benefits of similar applications based on permethylated sulfated glycans have not been fully investigated. We report here the important features of reverse phase-based nanoLC-MS/MS analysis of permethylated sulfated glycans in negative ion mode and demonstrate that complementary sets of diagnostic fragment ions afforded can allow rapid identification of various fucosylated, sialylated, sulfated glycotopes and definitive determination of the location of sulfate in a way difficult to achieve by other means. A parallel acquisition of both higher collision energy and trap-based MS(2) coupled with a product dependent MS(3) is conceivably the most productive sulfoglycomic workflow currently possible and the manually curated fragmentation characteristics presented here will allow future developments in automating data analysis.

Characterization of protein serotonylation via bioorthogonal labeling and enrichment.

Protein serotonylation is a transglutaminase-mediated phenomenon whose biological mechanism of protein serotonylation is not yet fully understood, as the complete profiling of serotonylation targets in a proteome remains a critical challenge to date. Utilizing an alkyne-functionalized serotonin derivative bioorthogonally coupled to a cleavable linker, we developed a method to selectively enrich serotonylated proteins in a complex sample. With online nanoflow liquid chromatography and LTQ-Orbitrap Velos hybrid mass spectrometer detection, we identified 46 proteins with 50 serotonylation sites at their glutamine residues. Mass spectrometric analysis also generated direct residue-level evidence of various biological processes such as transglutaminase-chaperon interactions as well as actin assembly. An enrichment workflow utilizing click chemistry and on-bead digestion allowed us to achieve site-specific identification of protein serotonylation by mass spectrometry, and results obtained hereby also provided a great foundation in the elucidation of the true roles of protein serotonylation in biological systems.

Synthesis and characterization of sulfated Gal-ß-1,3/4-GlcNAc disaccharides through consecutive protection/glycosylation steps.

We have developed an expeditious procedure to yield large amounts of orthogonally protected Gal-ß1,3/4-GlcNAc, which allowed for the systematic introduction of a sulfate group onto the C3/C6 positions of Gal and/or the C6 position of GlcNAc. In particular, the disaccharide precursors were prepared in five or six steps and high overall yield from para-tolyl-6-O-tert-butyldiphenylsilyl-1-thio-ß-D-galactopyranoside. After deprotection and sulfation steps, the final products were characterized by using several NMR methods to unambiguously confirm the location of each introduced sulfate group and they were examined for their binding specificity of human galectin-1 and galectin-8.

Development of fucosyltransferase and fucosidase inhibitors.

L-Fucose-containing glycoconjugates are essential for a myriad of physiological and pathological activities, such as inflammation, bacterial and viral infections, tumor metastasis, and genetic disorders. Fucosyltransferases and fucosidases, the main enzymes involved in the incorporation and cleavage of L-fucose residues, respectively, represent captivating targets for therapeutic treatment and diagnosis. We herein review the important breakthroughs in the development of fucosyltransferase and fucosidase inhibitors. To demonstrate how the synthesized small molecules interact with the target enzymes, i.e. delineation of the structure-activity relationship, we cover the reaction mechanisms and resolved X-ray crystal structures, discuss how this information guides the design of enzyme inhibitors, and explain how the molecules were optimized to achieve satisfying potency and selectivity.

Rapid synthesis of oligomannosides with orthogonally protected monosaccharides.

We developed a facile synthesis to yield orthogonally protected mannose building blocks with high overall yields. The protection/glycosylation steps can be carried out in a successive manner without purification of intermediate products. This developed synthesis led to formation of linear/branched tri-, penta- and heptasaccharides.

Galectin-1 and galectin-8 have redundant roles in promoting plasma cell formation.

Galectin (Gal) family members are a type of soluble lectin, and they play important roles in immunomodulation. Their redundant roles have been proposed. We previously found that Gal-1 promotes the formation of Ab-secreting plasma cells, but B cells from Gal-1-deficient and control animals produce comparable amounts of Abs. In the current study, we used synthetic sulfomodified N-acetyllactosamine (LacNAc) analogs and short hairpin RNAs for Gal-8 to demonstrate a redundancy in the effects of Gal-1 and Gal-8 on plasma cell formation. Gal-1 and Gal-8 were both expressed during plasma cell differentiation, and both Gals promoted the formation of plasma cells. Gal-1 and Gal-8 bound better to mature B cells than to plasma cells, and the expression of glycosyltransferase enzymes changed during differentiation, with a decrease in mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetyl-glucosaminyltransferase and N-acetylglucosaminyltransferase-1 mRNAs in plasma cells. Synthetic sulfomodified Galß1-3GlcNAc disaccharides (type 1 LacNAcs) selectively prevented Gal-8 binding, leading to a blockade of Ab production in Gal-1-deficient B cells. Furthermore, synthetic type 1 LacNAcs that were able to block the binding of both Gals greatly reduced the effect of exogenously added recombinant Gal-1 and Gal-8 on promoting Ab production. These results reveal a novel role for Gal-8 in collaboration with Gal-1 in plasma cell formation, and suggest the possibility of using distinct LacNAc ligands to modulate the function of Gals.

Stereoselective three-component synthesis of trans-endo-decahydroquinolin-4-one derivatives from aldehydes, aniline, and acetylcyclohexene.

The diastereoselective synthesis of trans-endo-decahydroquinolin-4-one (4) via a three-component reaction of aldehydes (1), anilines (2), and 1-acetylcyclohexene (3) in the presence of iodine in a one-pot reaction at room temperature is described. The short reaction time, easy workup, excellent yield, and mild reaction conditions make this novel annulation strategy both practical and attractive.

Efficient conversion of nitronate into nitrile oxide using cyanuric chloride. One-pot synthesis of bicyclic isoxazolines and isoxazoles from nitroalkenes.

Bicyclic isoxazolines and isoxazoles are obtained in good yields by proceeding through a convenient one-pot, two-step procedure utilizing 2,4,6-trichloro-1,3,5-triazine (TCT) as a dehydrating agent.

An investigation of the reaction of 2-aminobenzaldehyde derivatives with conjugated nitro-olefins: an easy and efficient synthesis of 3-nitro-1,2-dihydroquinolines and 3-nitroquinolines.

2-Aryl-3-nitro-1,2-dihydroquinolines 3 were prepared from the reaction of beta-nitrostyrenes 2 and 2-aminobenzaldehyde 1 in the presence of DABCO. Not only beta-nitrostyrenes but other alkyl nitro olefins also can be used in this reaction as well. When DDQ or silica gel was added to a solution of 3-nitro-1,2-dihydroquinolines 3, 3-nitro-2-substituted-quinolines 4 were obtained. When 2-aminobenzaldehyde derivatives 7 and 12 were reacted with beta-nitrostyrenes 2, unique rearrangement products were produced.