3-hydroxyl Group Research Articles

Selective Anomeric Acetylation of Unprotected Sugars with Acetic Anhydride in Water.

Unprotected sugars are selectively acetylated simply by stirring in aqueous solution in the presence of acetic anhydride and a weak base such as sodium carbonate. The reaction is selective for acetylation of the anomeric hydroxyl group of mannose, 2-acetamido, and 2-deoxy sugars and can be performed on a large scale. Competitive intramolecular migration of the 1-O-acetate to the 2-hydroxyl group when these two substituents are cis causes over-reaction and the formation of product mixtures.

Comparative analysis of the reactivity of anthocyanidins, leucoanthocyanidins, and flavonols using a quantum chemistry approach.

Anthocyanidins, leucoanthocyanidins, and flavonols are natural compounds mainly known due to their reported biological activities, such as antiviral, antifungal, anti-inflammatory activities, and antioxidant activity. In the present study, we performed a comparative structural, conformational, electronic, and nuclear magnetic resonance analysis of the reactivity of the chemical structure of primary anthocyanidins, leucoanthocyanidins, and flavonoids. We focused our analysis on the following molecular questions: (i) differences in cyanidin catechols ( +)-catechin, leucocyanidin, and quercetin; (ii) the loss of hydroxyl presents in the R1 radical of leucoanthocyanidin in the functional groups linked to C4 (ring C); and (iii) the electron affinity of the 3-hydroxyl group (R7) in the flavonoids delphinidin, pelargonidin, cyanidin, quercetin, and kaempferol. We show unprecedented results for bond critical point (BCP) of leucopelargonidin and leucodelphirinidin. The BCP formed between hydroxyl hydrogen (R2) and ketone oxygen (R1) of kaempferol has the same degrees of covalence of quercetin. Kaempferol and quercetin exhibited localized electron densities between hydroxyl hydrogen (R2) and ketone oxygen (R1). Global molecular descriptors showed quercetin and leucocyanidin are the most reactive flavonoids in electrophilic reactions. Complementary, anthocyanidins are the most reactive in nucleophilic reactions, while the smallest gap occurs in delphinidin. Local descriptors indicate that anthocyanidins and flavonols are more prone to electrophilic attacks, while in leucoanthocyanidins, the most susceptible to attack are localized in the ring A. The ring C of anthocyanidins is more aromatic than the same found in flavonols and leucoanthocyanidins. METHODS: For the analysis of the molecular properties, we used the DFT to evaluate the formation of the covalent bonds and intermolecular forces. CAM-B3LYP functional with the def2TZV basis set was used for the geometry optimization. A broad analysis of quantum properties was performed using the assessment of the molecular electrostatic potential surface, electron localization function, Fukui functions, descriptors constructed from frontier orbitals, and nucleus independent chemical shift.

Benzophenone-1 and -2 UV-filters potently inhibit human, rat, and mouse gonadal 3β-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis

Benzophenone (BP) ultraviolet (UV) -filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3β-hydroxysteroid dehydrogenases (3β-HSD) catalyse the conversion of pregnenolone to progesterone. This study explored the effect of 12 BPs on human, rat, and mouse 3β-HSD isoforms, and analysed the structure-activity relationship (SAR) and underlying mechanisms. The inhibitory potency was BP-1 (IC50, 5.66 ± 0.95 μM) > BP-2 (5.84 ± 2.22 μM) > BP-6 (185.8 ± 115.2 μM) > BP3-BP12 on human KGN 3β-HSD2, BP-2 (5.90 ± 1.02 μM) > BP-1 (7.55 ± 1.26 μM) > BP3-B12 on rat testicular 3β-HSD1, and BP-1 (15.04 ± 5.20 μM) > BP-2 (22.64 ± 11.81 μM) > BP-6（125.1 ± 34.65 μM）> BP-7 (161.1 ± 102.4 μM) > other BPs on mouse testicular 3β-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3β-HSDs, and BP-2 is a mixed inhibitor of human and rat 3β-HSDs and a noncompetitive inhibitor of mouse 3β-HSD6. 4-Hydroxyl substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, and mouse gonadal 3β-HSDs. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at ≥ 10 μM. Docking analysis revealed that the 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bonds with residue Ser123 of human 3β-HSD2 and residue Asp127 of rat 3β-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, and mouse gonadal 3β-HSDs and that there is a significant SAR difference.

The binding mechanism of benzophenone-type UV filters and human serum albumin: The role of site, number, and type of functional group substitutions

Benzophenone-type UV filters (BPs) are common in natural aquatic environments. They can cause endocrine disruption or other adverse effects once they enter the human body via the food chain or drinking water. The primary cause of BPs accumulation and toxicity is the transport of BPs into the human body. Functional group substitutions can have a significant impact on the interactions of BPs and transporters, resulting in a variety of impact effects. Therefore, we explored the interaction between human serum albumin (HSA, a typical transporter) and ten typical BPs [benzophenone (BP1), 2-hydroxybenzophenone (BP2), 4-hydroxybenzophenone (BP3), 2,2′-dihydroxybenzophenone (BP4), 2,4-dihydroxybenzophenone (BP5), 4,4′-dihydroxybenzophenone (BP6), 2,4,4′-trihydroxybenzophenone (BP7), 2,2′,4,4′-tetrahydroxybenzophenone (BP8), 2-hydroxy-4-methoxybenzophenone (BP9), and 2,2′-dihydroxy-4-methoxybenzophenone (BP10)] to study the role of functional group substitutions in binding. The results showed that BPs could bind to HSA at site 2, with binding constants ranging from 2.01 × 103 to 4.57 × 105 L/mol. Compared to BP1, hydroxyl and methoxy substitutions enhanced the BPs-HSA binding. The combined effect of the number and site of hydroxyl substitution at BPs determined the binding strength between BPs and HSA. It was more accessible to bind HSA when BPs were substituted with para-hydroxyl (4-hydroxyl) groups than with ortho-hydroxyl (2-hydroxyl) groups. Moreover, the additional para-methoxy (4-methoxy) group increased the BP-HSA binding strength by approximately 47 times under the same hydroxyl substitution conditions. Theoretical calculations revealed that functional group substitutions increased the intermolecular binding force by increasing the negative electrostatic potential surface area of BPs, which significantly increased the electrostatic and dispersion forces between the BPs and HSA. This BPs-HSA binding decreased the α-helix of HSA and influenced the ratio of other secondary structures, including β-sheet, β-turn, and random coil of HSA. This study provides a theoretical and experimental foundation for understanding the human health risks associated with BPs.

An Unusual Acceleration of Amination Reactivity by the Proximal Ester Groups in Catechol Diesters: An Efficient Way for Peptide Synthesis

Peptide bond formation has drawn more and more attention due to its importance in medicinal chemistry and drug discovery. Using active esters to construct peptide bonds is an indispensable strategy in this field. We herein report a fast and efficient amination reaction to synthesize peptides from catechol diesters. Catechol monoesters are good active esters in peptide synthesis due to the anchimeric assistance effect. In previously reported work, the amination reaction was inactivated if the 2-hydroxyl group in the catechol esters was functionalized. However, we found an unusual acceleration of the amination reactivity when the 2-hydroxyl group was functionalized as an ester group. The reaction can be complete in 2–3 min in some cases. This acceleration was found through experimental studies to not result from the anchimeric assistance effect. We proposed a π*−π* interaction pathway between the two proximal ester groups. Some computational studies were made to support this assumption.

SnCl4 Promoted Efficient Cleavage of Acetal/Ketal Groups with the Assistance of Water in CH2Cl2

Acetalization and deacetalation are a pair of routine manipulations to protect and deprotect the 4- and 6-hydroxyl groups of glycosides in the synthesis of glycosyl building blocks. In this study, we found that treatment of SnCl4 with various carbohydrates containing acetal/ketal groups with the assistance of water in CH2Cl2 led to deacetalization/deketalization products in almost quantitative yields. In addition, for substrates containing both acetal/ketal and p-methoxylbenzyl groups, we also found that the p-methoxylbenzyl group was selectively cleaved by the use of a catalytic amount of SnCl4, while the acetal/ketal groups remained. Furthermore, based on this, 4,6-benzylidene glycosides can be conveniently converted to 4,6-OAc or 4-OH, 6-OAc glycosides.

Synthesis of 5- and 7-hydroxy indolizidin-2-one prostaglandin-F2α modulators and activity on myometrial contractility towards development of agents for delaying preterm birth.

In pursuit of more effective-labor delaying tocolytic agents, the prostaglandin F2α (PGF2α) receptor (FP) modulator PDC113.824 [(6S)-2] represents a potent lead for developing therapy to treat preterm birth. Derivatives of FP modulator (6S)-2 were synthesized, possessing respectively 5- and 7-hydroxyl groups on the indolizidin-2-one amino acid (I2 aa) residue. The effects of the alcohol substituents were examined in a PGF2α-induced myometrial contraction assay. Based on knowledge of dihedral angle values of model I2 aa peptides from X-ray analyses, the results of the study indicate respectively encouraging and limited potential for creating improved tocolytic agents by modifications at the 5- and 7-positions.

Cassaine diterpenoid glycosides from the seeds of Erythrophleum fordii Oliv. and their antiviral and anti-inflammatory activities

Seven new cassaine diterpenoids (1–7) along with four known ones (8–11) were isolated from the seeds of Erythrophleum fordii Oliv. (Leguminosae). Their chemical structures were elucidated by extensive spectroscopic data interpretation and chemical methods. Compound 1 is a rare unsymmetrical dimer, which is formed by the linking of another cassaine diterpenoid acid glycoside to the 6-hydroxyl group of the sugar unit in a cassaine amide glycoside through an ester bond. Compound 2 is a cassaine diterpenoid acid derivative featuring an unusual Z double bond at C-13 and C-15. The in vitro antiviral and anti-inflammatory activities of 1–11 were evaluated. The results showed that compounds 1, 2 and 3 showed significant antiviral activities against human respiratory syncytial virus (RSV) with IC50s of 6.3, 7.8, and 9.4 μM, respectively. Compound 9 significantly suppressed the expression of nuclear factor-kappa B (NF-κB) with an IC50 value of 2.6 μM.

Characterization of a novel natural tetracenomycin reveals crucial role of 4-hydroxy group in ribosome binding

The aromatic polyketides tetracenomycins were recently found to be potent inhibitors of protein synthesis. Their binding site is located in a unique locus within the tunnel of the large ribosomal subunit. Here we report the isolation and structure elucidation of a novel natural tetracenomycin congener – O4–Me-tetracenomycin C (O4–Me-TcmC). This compound is isomeric to tetracenomycin X (TcmX), however, in contrast to TcmX, O4–Me-TcmC exhibited no antimicrobial activity and was unable to inhibit protein synthesis in vitro. Structural alignment of tetracenomycins to the binding locus from cryo-EM TcmX-70S ribosome data revealed the crucial role of the 4-hydroxyl group. These findings are important for further development of semi-synthetic tetracenomycins as potential antibacterials.

Chemical Synthesis of the Nonreducing Hexasaccharide Fragment of Axinelloside A Based on a Stepwise Glycosylation Approach.

An expedient synthesis of the nonreducing hexasaccharide fragment of axinelloside A has been completed via a linear stepwise glycosylation approach. Challenges involved in the synthesis include the highly stereoselective construction of five consecutive 1,2-cis-glycosidic linkages and the formation of a sterically crowded 2,3-disubstituted l-fucoside subunit. Protecting group-directing glycosylation strategies such as the remote participation effect of the benzoyl substituent and the stereocontrolling effect of the 4,6-O-benzylidene group were employed for the synthesis of the desired 1,2-cis-glycosidic linkages. Moreover, the 2,3-branched l-fucoside framework was established through a 3-O and then 2-O glycosylation sequence in which the 3-hydroxyl group of the core l-fucose unit was glycosylated first and then the 2-hydroxyl. The synthetic hexasaccharide is properly protected, so it can be employed as a precursor to synthesize its natural form.

Halogenated Flavonoid Derivatives Display Antiangiogenic Activity.

Antiangiogenic agents attenuate tumours’ growth and metastases and are therefore beneficial as an adjuvant or standalone cancer regimen. Drugs with dual antiproliferative and antiangiogenic activities can achieve anticancer efficacy and overcome acquired resistance. In this study, synthetic flavones (5a,b) with reported anticancer activity, and derivatives (4b and 6a), exhibited significant inhibition of endothelial cell tube formation (40–55%, 12 h) at 1 µM, which is comparable to sunitinib (50% inhibition at 1 µM, 48 h). Flavones (4b, 5a,b and 6a) also showed 25–37% reduction in HUVECs migration at 10 µM. In a Western blotting assay, 5a and 5b subdued VEGFR2 phosphorylation by 37% and 57%, respectively, suggesting that VEGFR2 may be their main antiangiogenic target. 5b displayed the best docking fit with VEGFR2 in an in silico study, followed by 5a, emphasizing the importance of the 7-hydroxyl group accompanied by a 4−C=S for activity. Conversely, derivatives with a 4-carbonyl moiety fitted poorly into the target’s binding pocket, suggesting that their antiangiogenic activity depends on a different target. This study provides valuable insight into the Structure Activity Relationships (SAR) and modes of action of halogenated flavones with VEGFR2 and highlights their therapeutic potential as antiangiogenic/anticancer lead compounds.

Discovery of a novel 2-spiroproline steroid mimetic scaffold for the potent inhibition of 11β-HSD1

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues, resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11β-HSD1, particularly in adipose tissues, has been associated with a variety of ailments including metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11β-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a 3-point pharmacophore for 11β-HSD1 that was utilized to design a 2-spiroproline derivative as a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of several leads, such as compounds 39 and 51. Importantly, deleterious hERG inhibition and pregnane X receptor induction were mitigated by the introduction of a 4-hydroxyl group to the proline ring system.

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

Synthesis of alkoxy-isoflavones as potential α-glucosidase inhibitors

The aim of the present study was to synthesize isoflavone-enaminones 3a-c and 7-alkoxy-isoflavones 4a-c, evaluate their inhibition of α-glucosidase, and analyze the bioisosteric effect of the presence versus absence of aromatic moieties in these benzopyran derivatives. All the test compounds exhibited greater inhibition of α-glucosidase than the positive control acarbose. The series of isoflavones 3a-c and 4a-c showed higher inhibitory activity (IC50 = 6.3–87.6 µM) than the parental 7-hydroxyisoflavones 2a-c (IC50 = 109.4–173.2 µM), suggesting that the attachment of a 4’-chloroacetophenone moiety to the 7-hydroxyl group of 2a-c is an efficient way to increase the inhibition of α-glucosidase. Furthermore, the aromatic moieties of the series of compounds 3 and 4 enhance inhibitory activity by hydrophobic effects, according to docking calculations. Graphical abstract

Antimicrobial action of phenolic acids combined with violet 405-nm light for disinfecting pathogenic and spoilage fungi.

The aim of this study is to investigate the fungicidal spectrum of six phenolic-cinnamic and -benzoic acid derivatives using four fungi, Aspergillus niger, Cladosporium cladosporioides, Trichophyton mentagrophytes and Candida albicans, in a photocombination system with violet 405-nm light. This is the first study to examine the fungicidal mechanism involving oxidative damage using the conidium of A. niger, as well as an assessment of cellular function and chemical characteristics. The results of the screening assay indicated that ferulic acid (FA) and vanillic acid (VA), which possess 4-hydroxyl and 3-methoxy groups in their phenolic acid structures, produced synergistic activity with 405-nm light irradiation. FA and VA (5.0mM) significantly decreased the viability of A. niger by 2.4 to 2.6-logs under 90-min irradiation. The synergistic effects were attenuated by the addition of the radical scavenger dimethyl sulfoxide. Generation of reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, were confirmed in the phenolic acid solutions tested after irradiation with colorimetric and electron spin resonance analyses. Adsorption of FA and VA to conidia was greater than other tested phenolic acids, and produced 1.55- and 1.85-fold elevation of intracellular ROS levels, as determined using an oxidant-sensitive probe with flow cytometry analysis. However, cell wall or membrane damage was not the main mechanism by which the combination-induced fungal death was mediated. Intracellular ATP was drastically diminished (5% of control levels) following combined treatment with FA and light exposure, even under a condition that produced negligible decreases in viability, thereby resulting in pronounced growth delay. These results suggest that the first stage in the photofungicidal mechanism is oxidative damage to mitochondria or the cellular catabolism system associated with ATP synthesis, which is a result of the photoreaction of phenolic acids adsorbed and internalized by conidia. This photo-technology in combination with food-grade phenolic acids can aid in developing alternative approaches for disinfection of pathogenic and spoilage fungi in the fields of agriculture, food processing and medical care.

Hydroxycinnamoyltransferase and CYP98 in phenolic metabolism in the rosmarinic acid-producing hornwort Anthoceros agrestis

Main conclusionAnthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered.Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.

PI3K Isoforms in Cell Signalling and Innate Immune Cell Responses.

Phosphoinositide-3-kinases (PI3Ks) are enzymes involved in signalling and modification of the function of all mammalian cells. These enzymes phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositol, resulting in lipid products that act as second messengers responsible for coordinating many cellular functions, including activation, chemotaxis, proliferation and survival. The identification of the functions that are mediated by a specific PI3K isoform is complex and depends on the specific cell type and inflammatory context. In this chapter we will focus on the role of PI3K isoforms in the context of innate immunity, focusing on the mechanisms by which PI3K signalling regulates phagocytosis, the activation of immunoglobulin, chemokine and cytokines receptors, production of ROS and cell migration, and how PI3K signalling plays a central role in host defence against infections and tissue injury.

Discovery of novel cholic acid derivatives as highly potent agonists for G protein-coupled bile acid receptor

In this study, fourteen new cholic acid (CA) derivatives were designed and synthesized, and the GloSensor cAMP accumulation assay indicated that all derivatives could activate the Takeda G protein-coupled receptor 5 (TGR5). Methylation of 7- and 12-hydroxyl groups in CA significantly increased TGR5 agonism for the new derivatives. For example, 7,12-dimethoxy derivative B1 exhibited 78-fold higher potency for TGR5 than the 7,12-dihydroxyl derivative A1 and 258-fold higher potency than CA itself. On the other hand, A1 positively modulated chenodeoxycholic acid (CDCA) functional activity in TGR5, whereas B1 did not show similar activity. Molecular docking experiments indicated that A1 formed a hydrogen bond between the 12-OH and amino acid Thr131 of TGR5, which is significant for its allosteric property. However, methylation at the 12-hydroxyl group in CA (derivative B1) disrupted this pivotal H-bond. Therefore, the free 12-hydroxyl group is essential for the CA derivatives in TGR5 allosteric agonism. Overall, we discovered a highly potent TGR5 agonist, B1, which can be used as lead compound for further study.

Structure basis of the caffeic acid O-methyltransferase from Ligusiticum chuanxiong to understand its selective mechanism

Caffeic acid O-methyltransferase from Ligusticum chuanxiong (LcCOMT) showed strict regiospecificity despite a relative degree of preference. Compared with caffeic acid, methyl caffeate was the preferential substrate by its low Km and high Kcat. In this study, we obtained the SAM binary (1.80 Å) and SAH binary (1.95 Å) complex LcCOMT crystal structures, and established the ternary complex structure with methyl caffeate by molecular docking. The active site of LcCOMT included phenolic substrate pocket, SAM/SAH ligand pocket and conserved catalytic residues as well. The regiospecificity of LcCOMT that permitted only 3-hydroxyl group to be methylated arise from the interactions between the active site and the phenyl ring. However, the propanoid tail governed the relative preference of LcCOMT. The ester group in methyl caffeate stabilized the anionic intermediate caused by His268-Asp269 pair, whereas caffeic acid was unable to stabilize the anionic intermediate due to the adjacent carboxylate anion in the propanoid tail. Ser183 residue formed an additional hydrogen bond with SAH and its role was identified by S183A mutation. Ile318 residue might be a potential site for determination of substrate preference, and its mutation led to the change of tertiary conformation. The results supported the selective mechanism of LcCOMT.