Synthesis of Quillaic Acid through Sustainable C-H Bond Activations.

To meet the demand for quillaic acid, a multigram synthesis of quillaic acid was accomplished in 14 steps, starting from oleanolic acid, leading to an overall yield of 3.4%. Key features include C-H activation at C-16 and C-23. Through Pd-catalyzed C-H acetoxylation, the oxidation at C-23 was observed as the major product, as opposed to at C-24. A copper-mediated C-H hydroxylation using O2 successfully afforded the single isomer, 16ß-ol triterpenoid, followed by configuration inversion to the desired 16α-ol compound. In summary, with steps optimized and conducted on a multigram scale, quillaic acid could be feasibly acquired through C-H activation with inexpensive copper catalysts, promoting a more sustainable approach.

Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition.

In this study, a series of 2- and/or 3-substituted juglone derivatives were designed and synthesized. Among them, 9, 18, 22, 30, and 31 showed stronger inhibition activity against cell surface PDI or recombinant PDI and higher inhibitory effects on U46619- and/or collagen-induced platelet aggregation than juglone. The glycosylated derivatives 18 and 22 showed improved selectivity for inhibiting the proliferation of multiple myeloma RPMI 8226 cells, and the IC50 values reached 61 and 48 nM, respectively, in a 72 h cell viability test. In addition, 18 and 22 were able to prevent tumor cell-induced platelet aggregation and platelet-enhanced tumor cell proliferation. The molecular docking showed the amino acid residues Gln243, Phe440, and Leu443 are important for the compound-protein interaction. Our results reveal the potential of juglone derivatives to serve as novel antiplatelet and anticancer dual agents, which are available to interrupt platelet-cancer interplay through covalent binding to PDI catalytic active site.

Regulatory T cells in inflamed liver are dysfunctional in murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune disease characterized by immune-mediated destruction of intrahepatic small bile ducts. CD8 T cells play a critical role in biliary destruction. However, regulatory T cells (Tregs) have also been identified in the portal tracts of PBC patients. This study tested the hypothesis that hepatic Tregs in PBC were dysfunctional in suppressing immune responses in disease by using our human PBC-like autoimmune cholangitis (AIC) mouse model induced by 2-octynoic acid-conjugated ovalbumin (2-OA-OVA). Our results showed that female and male mice immunized with 2-OA-OVA developed AIC; however, female AIC mice had more severe liver inflammation and fibrosis than male AIC mice. Levels of functional effector CD8 T cells and their chemoattractants, CXCL9 and CXCL10, in the liver were markedly elevated in female AIC mice than in male AIC mice. These results reinforce that CD8 T cells are the primary effector cells in PBC. The number of hepatic Tregs in AIC mice was also higher than in saline-treated mice, but there was no difference between male and female AIC mice. The suppressive function of AIC Tregs was evident despite a discrepancy in the changes in their co-inhibitory receptors and inhibitory cytokines. However, the expansion of hepatic Tregs by low-dose IL-2 treatment did not reduce immune responses to AIC, which may be due to the dysfunction of Tregs in inhibiting T cells. In conclusion, the function of Tregs in the inflamed liver of PBC was insufficient, and low-dose IL-2 treatment could not restore their function to suppress pathological immune responses. Transferring normal Tregs or directly targeting effector CD8 T cells may be beneficial for treating PBC.

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Toward an Improved Triterpene 3-O-Glucuronidation: The Systematic Determination of the Relative Reactivities of Glucuronyl Donors and Acceptors.

3-O-ß-Glucuronide triterpenes are plant-derived compounds. Some of them have been used as herbal medicine and in pharmaceuticals, such as chikusetsu saponins and Quillaja saponins. However, the demand for these materials has remained largely a challenge owing to their natural scarcity and low-yielding purification process. Therefore, a chemical triterpene 3-O-glucuronidation was conducted in this study to alleviate the surging demand on natural source. Various glucuronyl imidate donors and oleanane-type triterpene acceptors were synthesized, and the relative reactivity values (RRV) and acceptor nucleophilic constants (Aka) were systematically measured to study their influence on glucuronidation yield. As a result, applying donors in higher RRV value generally improved the production of 3-O-glucuronide triterpenes. Meanwhile, a bulky pivaloyl group was an ideal 2-O-protection to provide ß-selectivity and prevented side reactions, including orthoester formation and acyl-transfer reaction. Collectively, a positive correlation was observed between reactive donors/acceptors and improved glucuronidation yields. These findings offered insights on the influence of donors' and acceptors' reactivities on 3-O-ß-glucuronide triterpenes synthesis, and this knowledge would help to access saponins of interest to address future needs.

Design, Synthesis, and Evaluation of Glucose Transporter Inhibitor-SN38 Conjugates for Targeting Colorectal Cancer.

Irinotecan (1), a prodrug of SN38 (2) approved by the US Food and Drug Administration for treating colorectal cancer, lacks specificity and causes many side effects. To increase the selectivity and therapeutic efficacy of this drug, we designed and synthesized conjugates of SN38 and glucose transporter inhibitors (phlorizin (5) or phloretin (6)), which could be hydrolyzed by glutathione or cathepsin to release SN38 in the tumor microenvironment, as a proof of concept. These conjugates (8, 9, and 10) displayed better antitumor efficacy with lower systemic exposure to SN38 in an orthotopic colorectal cancer mouse model compared with irinotecan at the same dosage. Further, no major adverse effects of the conjugates were observed during treatment. Biodistribution studies showed that conjugate 10 could induce higher concentrations of free SN38 in tumor tissues than irinotecan at the same dosage. Thus, the developed conjugates exhibit potential for treating colorectal cancer.

Isolation of Anti-SARS-CoV-2 Natural Products Extracted from Mentha canadensis and the Semi-synthesis of Antiviral Derivatives.

Traditional herbal medicine offers opportunities to discover novel therapeutics against SARS-CoV-2 mutation. The dried aerial part of mint (Mentha canadensis L.) was chosen for bioactivity-guided extraction. Seven constituents were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Syringic acid and methyl rosmarinate were evaluated in drug combination treatment. Ten amide derivatives of methyl rosmarinate were synthesized, and the dodecyl (13) and 3-ethylphenyl (19) derivatives demonstrated significant improvement in the anti-SARS-CoV-2 plaque reduction assay, achieving IC50 of 0.77 and 2.70 µM, respectively, against Omicron BA.1 as compared to methyl rosmarinate's IC50 of 57.0 µM. Spike protein binding and 3CLpro inhibition assays were performed to explore the viral inhibition mechanism. Molecular docking of compounds 13 and 19 to 3CLpro was performed to reveal potential interaction. In summary, natural products with anti-Omicron BA.1 activity were isolated from Mentha canadensis and derivatives of methyl rosmarinate were synthesized, showing 21- to 74-fold improvement in antiviral activity against Omicron BA.1.

Design, structure-activity relationships, and enzyme kinetic studies of tricyclic and tetracyclic coumarin-based sulfamates as steroid sulfatase inhibitors.

Inhibition of steroid sulfatase (STS) decreases estrogen production and thus, suppresses tumor proliferation. Inspired by irosustat, the first STS inhibitor in clinical trials, we explored twenty-one tricyclic and tetra-heterocyclic coumarin-based derivatives. Their STS enzyme kinetic parameters, docking models, and cytotoxicity toward breast cancer and normal cells were evaluated. Tricyclic derivative 9e and tetracyclic derivative 10c were the most promising irreversible inhibitors developed in this study, with KI of 0.05 and 0.4 nM, and kinact/KI ratios of 28.6 and 19.1 nM-1min-1 on human placenta STS, respectively.

Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.

PD-L1 Enhanced by cis-Urocanic Acid on Langerhans Cells Inhibits Vγ4+ γδT17 Cells in Imiquimod-Induced Skin Inflammation.

Psoriasis is an IL-23/IL-17-mediated inflammatory autoimmune dermatosis, and UVB may contribute to immunosuppression and ameliorate associated symptoms. One of the pathophysiology underlying UVB therapy is the production of cis-urocanic acid (cis-UCA) by keratinocytes. However, the detailed mechanism is yet to be fully understood. In this study, we found FLG expression and serum cis-UCA levels were significantly lower in patients with psoriasis than in healthy controls. We also noted that cis-UCA application inhibited psoriasiform inflammation through the reduction of Vγ4+ γδT17 cells in murine skin and draining lymph nodes. Meanwhile, CCR6 was downregulated on γδT17 cells, which would suppress the inflammatory reaction at a distal skin site. We revealed that the 5-hydroxytryptamine receptor 2A, the known cis-UCA receptor, was highly expressed on Langerhans cells in the skin. cis-UCA also inhibited IL-23 expression and induced PD-L1 on Langerhans cells, leading to the attenuated proliferation and migration of γδT-cells. Compared to the isotype control, α-PD-L1 treatment in vivo could reverse the antipsoriatic effects of cis-UCA. PD-L1 expression on Langerhans cells was sustained through the cis-UCA-induced mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. These findings uncover the cis-UCA-induced PD-L1-mediated immunosuppression on Langerhans cells, which facilitates the resolution of inflammatory dermatoses.

Structures of the xyloglucans in the monocotyledon family Araceae (aroids).

MAIN CONCLUSION: The xyloglucans of all aquatic Araceae species examined had unusual structures compared with those of other non-commelinid monocotyledon families previously examined. The aquatic Araceae species Lemna minor was earlier shown to have xyloglucans with a different structure from the fucogalactoxyloglucans of other non-commelinid monocotyledons. We investigated 26 Araceae species (including L. minor), from five of the seven subfamilies. All seven aquatic species examined had xyloglucans that were unusual in having one or two of three features: < 77% XXXG core motif [L. minor (Lemnoideae) and Orontium aquaticum (Orontioideae)]; no fucosylation [L. minor (Lemnoideae), Cryptocoryne aponogetonifolia, and Lagenandra ovata (Aroideae, Rheophytes clade)]; and > 14% oligosaccharide units with S or D side chains [Spirodela polyrhiza and Landoltia punctata (Lemnoideae) and Pistia stratiotes (Aroideae, Dracunculus clade)]. Orontioideae and Lemnoideae are the two most basal subfamilies, with all species being aquatic, and Aroideae is the most derived. Two terrestrial species [Dieffenbachia seguine and Spathicarpa hastifolia (Aroideae, Zantedeschia clade)] also had xyloglucans without fucose indicating this feature was not unique to aquatic species.

The Design, Structure-Activity, and kinetic studies of 3-Benzyl-5-oxa-1,2,3,4-Tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates as Steroid sulfatase inhibitors.

Steroid sulfatase inhibitors block the local production of estrogenic steroids and are attractive agents for the treatment of estrogen-dependent cancers. Inspiration of coumarin-based inhibitors, we synthesized thirty-two 5-oxa-1,2,3,4-tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates, focusing on the substitution derivatives on the adjacent phenyl ring and evaluated their abilities to block STS from human placenta and MCF-7 cells. SAR analysis revealed that the incorporation of chlorine at either meta and/or para position of the adjacent phenyl ring of the tricyclic skeleton enhanced STS inhibition. Di-substitutions at the adjacent phenyl ring were superior to mono and tri-substitutions. Further kinetic analysis of these compounds revealed that chloride-bearing compounds, such as 19m, 19v, and 19w, had KI of 0.02 to 0.11 nM and kinact/KI ratios of 8.8-17.5 nM-1min-1, a parameter indicated for the efficiency of irreversible inhibition. We also used the docking model to illustrate the difference in STS inhibitory potency of compounds. Finally, the safety and anti-cancer activity of selected compounds 19m, 19v, and 19w were also studied, showing the results of low cytotoxicity on NHDF cell line and being more potent than irosustat on ZR-75-1 cell, which was a hormone-dependent cancer cell line with high STS expression.

Structural compositions and biological activities of cell wall polysaccharides in the rhizome, stem, and leaf of Polygonatum odoratum (Mill.) Druce.

Polygonatum odoratum is a perennial rhizomatous medicinal plant and different plant parts have been used in the treatment of various ailments. Herein, we have investigated the structural compositions of rhizome, leaf, and stem cell walls. We found 30-44% of polysaccharides in these wall preparations were cyclohexanediaminetetraacetic acid (CDTA) extractable, the proportion of heteromannans (HMs) in the rhizome is nearly three-fold compared to that of the leave and stem. The pectic polysaccharides of the rhizome are also structurally more diverse, with arabinans and type I and type II arabinogalactans being richest as shown by linkage study of the sodium carbonate (Na2CO3) extract. In addition, the 2-linked Araf was rhizome-specific, suggesting the cell walls in the rhizome had adapted to a more complex structure compared to that of the leaf and stem. Water-soluble polysaccharide fractions were also investigated, high proportion of Man as in 4-linked Manp indicated high proportion of HMs. The 21.4 kDa pectic polysaccharides and HMs derived from rhizome cell walls induced specific immune response in mice macrophage cells producing IL-1α and hematopoietic growth factors GM-CSF and G-CSF in vitro.

Design and Synthesis of 1-O- and 6'-C-Modified Heparan Sulfate Trisaccharides as Human Endo-6-O-Sulfatase 1 Inhibitors.

The extracellular human endo-6-O-sulfatases (Sulf-1 and Sulf-2) are responsible for the endolytic cleavage of the 6-sulfate groups from the internal D-glucosamine residues in the highly sulfated subdomains of heparan sulfate proteoglycans. A trisaccharide sulfate, IdoA2OS-GlcNS6S-IdoA2OS, was identified as the minimal size of substrate for Sulf-1. In order to study the complex structure with Sulf-1 for developing potential drugs, two trisaccharide analogs, IdoA2OS-GlcNS6OSO2NH2-IdoA2OS-OMe and IdoA2OS-GlcNS6NS-IdoA2OS-OMe, were rationally designed and synthesized as the Sulf-1 inhibitors with IC50 values at 0.27 and 4.6 µM, respectively.

Novel Two-Step Process in Cellulose Depolymerization: Hematite-Mediated Photocatalysis by Lytic Polysaccharide Monooxygenase and Fenton Reaction.

To transform cellulose from biomass into fermentable sugars for biofuel production requires efficient enzymatic degradation of cellulosic feedstocks. The recently discovered family of oxidative enzymes, lytic polysaccharide monooxygenase (LPMO), has a high potential for industrial biorefinery, but its energy efficiency and scalability still have room for improvement. Hematite (α-Fe2O3) can act as a photocatalyst by providing electrons to LPMO-catalyzed reactions, is low cost, and is found abundantly on the Earth's surface. Here, we designed a composite enzymatic photocatalysis-Fenton reaction system based on nano-α-Fe2O3. The feasibility of using α-Fe2O3 nanoparticles as a composite catalyst to facilitate LPMO-catalyzed cellulose oxidative degradation in water was tested. Furthermore, a light-induced Fenton reaction was integrated to increase the liquefaction yield of cellulose. The innovative approach finalized the cellulose degradation process with a total liquefaction yield of 93%. Nevertheless, the complex chemical reactions and products involved in this system require further investigation.

Design, synthesis and biological evaluations of niclosamide analogues against SARS-CoV-2.

Niclosamide, a widely-used anthelmintic drug, inhibits SARS-CoV-2 virus entry through TMEM16F inhibition and replication through autophagy induction, but the relatively high cytotoxicity and poor oral bioavailability limited its application. We synthesized 22 niclosamide analogues of which compound 5 was found to exhibit the best anti-SARS-CoV-2 efficacy (IC50 = 0.057 µ M) and compounds 6, 10, and 11 (IC50 = 0.39, 0.38, and 0.49 µ M, respectively) showed comparable efficacy to niclosamide. On the other hand, compounds 5, 6, 11 contained higher stability in human plasma and liver S9 enzymes assay than niclosamide, which could improve bioavailability and half-life when administered orally. Fluorescence microscopy revealed that compound 5 exhibited better activity in the reduction of phosphatidylserine externalization compared to niclosamide, which was related to TMEM16F inhibition. The AI-predicted protein structure of human TMEM16F protein was applied for molecular docking, revealing that 4'-NO2 of 5 formed hydrogen bonding with Arg809, which was blocked by 2'-Cl in the case of niclosamide.

Targeting Colorectal Cancer with Conjugates of a Glucose Transporter Inhibitor and 5-Fluorouracil.

Overexpression of glucose transporters (GLUTs) in colorectal cancer cells is associated with 5-fluorouracil (1, 5-FU) resistance and poor clinical outcomes. We designed and synthesized a novel GLUT-targeting drug conjugate, triggered by glutathione in the tumor microenvironment, that releases 5-FU and GLUTs inhibitor (phlorizin (2) and phloretin (3)). Using an orthotopic colorectal cancer mice model, we showed that the conjugate exhibited better antitumor efficacy than 5-FU, with much lower exposure of 5-FU during treatment and without significant side effects. Our study establishes a GLUT-targeting theranostic incorporating a disulfide linker between the targeting module and cytotoxic payload as a potential antitumor therapy.

Structures, Biosynthesis, and Physiological Functions of (1,3;1,4)-ß-D-Glucans.

(1,3;1,4)-ß-D-Glucans, also named as mixed-linkage glucans, are unbranched non-cellulosic polysaccharides containing both (1,3)- and (1,4)-ß-linkages. The linkage ratio varies depending upon species origin and has a significant impact on the physicochemical properties of the (1,3;1,4)-ß-D-glucans. (1,3;1,4)-ß-D-Glucans were thought to be unique in the grasses family (Poaceae); however, evidence has shown that (1,3;1,4)-ß-D-glucans are also synthesized in other taxa, including horsetail fern Equisetum, algae, lichens, and fungi, and more recently, bacteria. The enzyme involved in (1,3;1,4)-ß-D-glucan biosynthesis has been well studied in grasses and cereal. However, how this enzyme is able to assemble the two different linkages remains a matter of debate. Additionally, the presence of (1,3;1,4)-ß-D-glucan across the species evolutionarily distant from Poaceae but absence in some evolutionarily closely related species suggest that the synthesis is either highly conserved or has arisen twice as a result of convergent evolution. Here, we compare the structure of (1,3;1,4)-ß-D-glucans present across various taxonomic groups and provide up-to-date information on how (1,3;1,4)-ß-D-glucans are synthesized and their functions.

Production of Structurally Defined Chito-Oligosaccharides with a Single N-Acetylation at Their Reducing End Using a Newly Discovered Chitinase from Paenibacillus pabuli.

Partially acetylated chito-oligosaccharides (paCOSs) are bioactive compounds with potential medical applications. Their biological activities are largely dependent on their structural properties, in particular their degree of polymerization (DP) and the position of the acetyl groups along the glycan chain. The production of structurally defined paCOSs in a purified form is highly desirable to better understand the structure/bioactivity relationship of these oligosaccharides. Here, we describe a newly discovered chitinase from Paenibacillus pabuli (PpChi) and demonstrate by mass spectrometry that it essentially produces paCOSs with a DP of three and four that carry a single N-acetylation at their reducing end. We propose that this specific composition of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) residues, as in GlcN(n)GlcNAc1, is due to a subsite specificity toward GlcN residues at the -2, -3, and -4 positions of the partially acetylated chitosan substrates. In addition, the enzyme is stable, as evidenced by its long shelf life, and active over a large temperature range, which is of high interest for potential use in industrial processes. It exhibits a kcat of 67.2 s-1 on partially acetylated chitosan substrates. When PpChi was used in combination with a recently discovered fungal auxilary activity (AA11) oxidase, a sixfold increase in the release of oligosaccharides from the lobster shell was measured. PpChi represents an attractive biocatalyst for the green production of highly valuable paCOSs with a well-defined structure and the expansion of the relatively small library of chito-oligosaccharides currently available.

Identification of existing pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection.

The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 µM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti-SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.