Chalcone cyclase and flavonoid biosynthesis in grapefruit

A chalcone cyclase (CC), which acts unidirectionally upon the chalcone-flavanone equilibrium reaction, was isolated from immature grapefruit. The enzyme required neohesperidose at C-4′ of the chalcone A-ring and a free, unhindered hydroxyl group at C-4 of the B-ring for activity. The CC bound, but did not cyclize, prunin chalcone (K_i= 2.5 × 10^?5 M). The results suggest that the intermediates that form the B-ring of chalcones are hydroxylated prior to chalcone formation, that chalcones are glycosylated during their formation, and that methylation occurs after cyclization of the chalcones to flavanones.     

Microwave accelerated solvent-free synthesis and antifungal evaluations of flavanones

Microwave irradiation of 2-hydroxy chalcones under solvent-free conditions resulted in a “green-chemistry” procedure for the preparation of flavanones in good yields, using an unmodified household microwave oven and silica as solid support. By irradiation of 2-hydroxy chalcones with trifluoroacetic acid over silica gel, 11 known flavanones were prepared in high yields. The synthesised compounds were characterised using spectroscopic techniques, namely, ¹H NMR, ¹³C NMR and IR, and screened for their antifungal activity in vitro against Sclerotium rolfsii and Rhizoctonia solani by poisoned food technique. The compounds tested were found to be more active against R. solani, whereas against S. rolfsii, moderate activity was observed, as evident from LC₅₀ values. The most potent compound 2-(4-fluorophenyl)-2,3-dihydrochromen-4-one (4a) had LC₅₀ value of 12.0 mg L^?1 followed by 11, 11a, 3a, 9a, 8a, 10a and 10 having LC₅₀ values 18.21, 18.3, 32.9, 50.7, 88.8, 118.8 and 119.7 mg L^?1, respectively.     

Synthesis of stilbene-fused 2′-hydroxychalcones and flavanones

Synthesis of stilbene-fused chalcones and flavanones were successfully completed. Molecules were designed in a way to mimic the structural features of both “stilbene and chalcones” or “stilbene and flavanones” at the same time, and synthesized by three steps. Heck reactions of 3-bromobenzaldehyde with styrene derivatives gave corresponding (E)-stilbenes, which were reacted with acetophenones to furnish stilbene-fused 2′-hydroxychalcones under basic conditions. Finally, intramolecular cyclization reactions were performed to produce stilbene-fused flavanones.     

Phenols displaying tyrosinase inhibition from Humulus lupulus

Tyrosinase is the rate-limiting enzyme for the production of melanin and other pigments via the oxidation of l-tyrosine. The methanol extract from Humulus lupulus showed potent inhibition against mushroom tyrosinase. The bioactivity-guided fractionation of this methanol extract resulted in the isolation of seven flavonoids (1–7), identified as xanthohumol (1), 4′-O-methylxanthohumol (2), xanthohumol C (3), flavokawain C (4), xanthoumol B (5), 6-prenylnaringenin (6) and isoxanthohumol (7). All isolated flavonoids (1–7) effectively inhibited the monophenolase (IC₅₀s?=?15.4–58.4?µM) and diphenolase (IC₅₀s?=?27.1–117.4?µM) activities of tyrosinase. Kinetic studies using Lineweaver–Burk and Dixon-plots revealed that chalcones (1–5) were competitive inhibitors, whereas flavanones (6 and 7) exhibited both mixed and non-competitive inhibitory characteristics. In conclusion, this study is the first to demonstrate that the phenolic phytochemicals of H. lupulus display potent inhibitory activities against tyrosinase.     

Chalcone synthesis with enzyme extracts from tulip anther tapetum using a biphasic enzyme assay

The in vitro synthesis of chalcones has been demonstrated using a special biphasic enzyme assay. The highly viscous lower phase in this assay stems from a tapetum fraction of anthers of Tulipa cv. “Apeldoorn” which has been used an enzyme source. The upper phase of this system consists of a reaction mixture of the normal “flavanone synthase” assay. It is suggested that chalcone synthesis occurs at the boundary layer between the two phases. To prevent spontaneous as well as enzymatic cyclization of the chalcones formed (phloroglucinyl type), the pH of the upper phase must not be allowed to exceed pH 4.0. Under these pH conditions, chalcone formation by a reverse reaction of chalcone-flavanone isomerase can be excluded. The measured substrate specificity of the “chalcone synthase” corresponds to the conditions of chalcone formation in the natural system. Using p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA, respectively, as substrates, the enzyme system forms the correspondingly substituted chalcones which are also accumulated in the loculus of tulip anthers. It is suggested that this chalcone synthase is identical to the previously described “flavanone synthase”. The results can be further explained as follows. (i) Not flavanones, but rather chalcones are the first C₁₅ intermediates of flavonoid biosynthesis in tulip anthers. (ii) In this Tulipa system, the substitution pattern of three different hydroxycinnamic acids can be transferred unchanged into the flavonoid C₁₅ stage. (iii) The role of chalcone-flavanone isomerase is to cyclize chalcones to flavanones on the direct biosynthetic pathway to the further accumulated flavonol glycosides. (iv) The sensitivity of the reaction with regard to chalcone production points to the localization of chalcone synthase in a most unstable and, up to now, unknown tapetal compartment. Since purification of the enzyme results in exclusive production of flavanones, it is suggested that certain “chalcone stabilizing factors” must occur in the natural system. (v) The phenomenon of chalcone accumulation in tulip anthers, however, must be caused by a complex system, distinguished by cooperation of certain biochemical and physiological conditions, and, finally, by special compartmentation of the enzymes which are responsible for the biosynthesis of flavonoids.     

Synthesis,characterization and biological evaluation of novel 4′-fluoro-2′-hydroxy-chalcone derivatives as antioxidant,anti-inflammatory and analgesic agents

Abstract

In an effort to develop safe and potent anti-inflammatory agents, a series of novel 4′-fluoro-2′-hydroxychalcones 5a–d and their dihydropyrazole derivatives 6a–d was prepared. It was synthesized via aldol condensation of 4′-fluoro-2′-hydroxyacetophenone with appropriately substituted aldehydes followed by cyclization with hydrazine hydrate. All the synthesized compounds were evaluated for their antioxidant, anti-inflammatory, cyclooxygenase inhibition selectivity and analgesic activities. The dimethoxychalcone 5a and its dihydropyrazole derivative 6a showed the highest antioxidant activity, while the monomethoxychalcone 5d and its dihydropyrazole derivative 6d showed the highest analgesic and anti-inflammatory activities. It was also found that there is a close correlation between 4′-fluoro-2′-hydroxychalcones 5a–d and their dihydropyrazole derivatives 6a–d in the screened biological activities. To explain the correlation between the synthesized chalcones and their dihydropyrazole derivatives, especially for the anti-inflammatory activity, docking studies were performed.     

Simultaneous extraction and biotransformation process to obtain high bioactivity phenolic compounds from brazilian citrus residues

Recent studies have pointed to a reduction in the incidence of some cancers, diabetes, and neuro‐degenerative diseases as a result of human health benefits from flavanones. Currently, flavanones are obtained by chemical synthesis or extraction from plants, and these processes are only produced in the glycosylated form. An interesting environmentally friendly alternative that deserves attention regarding phenolic compound production is the simultaneous extraction and biotransformation of these molecules. Orange juice consumption has become a worldwide dietary habit and Brazil is the largest producer of orange juice in the world. Approximately half of the citrus fruit is discarded after the juice is processed, thus generating large amounts of residues (peel and pectinolytic material). Hence, finding an environmentally clean technique to extract natural products and bioactive compounds from different plant materials has presented a challenging task over the last decades. The aim of this study was to obtain phenolics from Brazilian citrus residues with high bioactivity, using simultaneous extraction (cellulase and pectinase) and biotransformation (tannase) by enzymatic process. The highest hesperetin, naringenin and ellagic acid production in the experiment were 120, 80, and 11,250 µg g^?1, respectively, at 5.0 U mL^?1 of cellulase and 7.0 U mL^?1 of tannase at 40°C and 200 rpm. Also, the development of this process generated an increase of 77% in the total antioxidant capacity. These results suggest that the bioprocess obtained innovative results where the simultaneous enzymatic and biotransformatic extracted flavanones from agro‐industrial residues was achieved without the use of organic solvents. The methodology can therefore be considered a green technology. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1273–1279, 2015     

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL^pro) and a papain-like protease (PL^pro) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1–9) and four coumarins (10–13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL^pro and PL^pro) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL^pro and PL^pro inhibitory activity with IC₅₀ values of 11.4 and 1.2?µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL^pro, whereas noncompetitive inhibition was observed with the SARS-CoV PL^pro.     

Chalcone isomerase in grape vine: gene expression and localization in the developing fruit

Chalcone isomerase (CHI, EC 5.5.1.6) is an entrance enzyme in the flavonoid biosynthesis, which catalyzes the conversion of chalcones to flavanones. In this study, the full-length CHI cDNA from grape vine (Vitis vinifera L.) was cloned, the recombinant protein was purified and the polyclonal antibody was prepared. Using these tools, the expression and tissue localization of CHI in developing grape berry was analyzed by RT-PCR, gel blot hybridization and immunohistochemical techniques. The expression of CHI was dependent on developmental stage, and CHI protein was mainly distributed in vascular bundles throughout all the stages of berry development, which suggested that flavonoids in the berry might have been partially synthesized in situ.     

Microbial synthesis of dihydrochalcones using Rhodococcus and Gordonia species

Chalcones are important compounds in food and cosmetics industry, and in food chemistry research. We have developed a method of synthesis of dihydrochalcones from flavanone and α,β-unsaturated chalcones by microbial hydrogenation. It has been found that bacterial strains of Rhodococcus sp. and Gordonia sp. can be successfully used in the key step of dihydrochalcones synthesis. This kind of activity has not been previously examined.Twelve microorganisms were initially screened for their ability to catalyze biotransformation reactions of selected flavonoid compounds. Of these, Rhodococcus sp. and Gordonia sp. transformed flavanone and chalcones to hydrogenation products in good isolated yield of 13–94%.     

Synthesis,cyclooxygenase inhibition and anti-inflammatory evaluation of new 1,3,5-triaryl-4,5-dihydro-1H-pyrazole derivatives possessing methanesulphonyl pharmacophore

A new series of 1,3,5-triaryl-4,5-dihydro-1H-pyrazole derivatives 13a–p were synthesized via aldol condensation of 3/4-nitroacetophenones with appropriately substituted aldehydes followed by cyclization of the formed chalcones with 4-methanesulfonylphenylhydrazine hydrochloride. All the synthesized compounds were evaluated for their cyclooxygenase (COX) inhibition, anti-inflammatory activity and ulcerogenic liability. All compounds were more potent inhibitors for COX-2 than COX-1. While most compounds showed good anti-inflammatory activity, compounds 13d, 13f, 13k and 13o were the most potent derivatives (ED₅₀?=?66.5, 73.4, 79.8 and 70.5?μmol/kg, respectively) in comparison with celecoxib (ED₅₀?=?68.1?μmol/kg). Compounds 13d, 13f, 13k and 13o (ulcer index?=?3.89, 4.86, 4.96 and 3.92, respectively) were 4–6 folds less ulcerogenic than aspirin (ulcer index?=?22.75) and showed approximately ulceration effect similar to celecoxib (ulcer index?=?3.35). In addition, molecular docking studies were performed for compounds 13d, 13f, 13k and 13o inside COX-2 active site which showed acceptable binding interactions (affinity in kcal/mol ?2.1774, ?6.9498) in comparison with celecoxib (affinity in kcal/mol ?6.5330).     

THE FLAVONOIDS OF THE DECIDUOUS RHODODENDRON OF NORTH AMERICA (ERICACEAE)

The native Azaleas of North America (Rhododendron: subgenus Pentanthera) produce 58 flavonoids in five aglycone classes: flavonols, dihydroflavonols, flavanones, dihydrochalcones and chalcones. A comparison of the flavonoids of selected samples of these species indicated that the compounds generally occur as species specific ensembles. Species were grouped into “alliances” based on common flavonoid constituents and a phylogenetic treatment of the group was developed. Evolutionary trends of flavonoids within subgenus Pentanthera are not well-defined but appear to be associated with a loss of the ability to synthesize some compounds and a decrease in diversity of glycosides and methoxylated flavonoids. Intraspecific variation in flavonoids was found in Rhododendron canescens, R. alabamense and R. austrinum when these were sampled on a population basis.     

Anthelmintic activity of 3,6-dibenzyl-2,5-dioxopiperazine, cyclo(L-Phe-L-Phe)

Summary After oral administration, dipeptide Phe-Phe-OMe1 exhibits anthelmintic activity againstEchinococcus multilocularis larvae, cestoda, in mongolian gerbils (intraperitoneal localization), but not againstHymenolepis nana, cestoda, in fasted mice (gastro-intestinal localization). This compound rapidly provides its cyclization product dioxopiperazine2 in pH 7.4 buffer at 37°C, but was stable at pH 2.4 during 16h at 30°C. It was postulated that dipeptide1 could act as a prodrug of2. Initial pharmacokinetics studies were carried out in mice and dogs. After oral administration, biotransformation of1 into2 occurred to some extent in mice but not in fasted dogs. Results of these studies did not allow to ascertain that1 is a prodrug of2. Compound2 has been tested in mice againstH. nana andSchistosoma mansoni, a gastrointestinal trematoda. Albeit less active than the reference compound praziquantel,2 has shown a good activity against both worms. 2,5dioxopiperazines represent therefore a new class of anthelmintics.     

Microbial transformation of hydrocortisone by two fungal species Fusarium fujikuroi PTCC 5144 and Rhizomucor pusillus PTCC 5134

Abstract

This paper examines the biotransformation of hydrocortisone (1) by Fusarium fujikuroi and Rhizomucor pusillus. These species have not previously been tested for hydrocortisone biotransformation. The metabolites produced during hydrocortisone biotransformation by these two fungi were 11β,17α,20β,21-tetrahydroxypregn-4-en-3-one (2) and 11 β-hydroxyandrost-4-en-3,17-dione (3). Chemical structures were determined by spectroscopic methods. A time course study revealed that the disappearance of hydrocortisone was accompanied by the formation of metabolites 2 and 3. Metabolite 2 was produced as the major metabolite with high yield but the transformation to metabolite 3 was considerably lower, as determined by HPLC.     

Reaction mechanism of chalcone isomerase. pH dependence, diffusion control, and product binding differences.

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are approximately 90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(a) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'-tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are approximately 50% diffusion-limited, whereas the reactions of both dihydroxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 A resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction.     

Elucidation of biosynthetic pathway of a plant hormone abscisic acid in phytopathogenic fungi

ABSTRACT

Abscisic acid (ABA) is one of the plant hormones that regulates physiological functions in various organisms, including plants, sponges, and humans. The biosynthetic machinery in plants is firmly established, while that in fungi is still unclear. Here, we elucidated the functions of the four biosynthetic genes, bcABA1-bcABA4, found in Botrytis cinerea by performing biotransformation experiments and in vitro enzymatic reactions with putative biosynthetic intermediates. The first-committed step is the cyclization of farnesyl diphosphate to give α-ionylideneethane catalyzed by a novel sesquiterpene synthase, BcABA3, which exhibits low amino acid sequence identities with sesquiterpene synthases. Subsequently, two cytochrome P450s, BcABA1 and BcABA2, mediate oxidative modifications of the cyclized product to afford 1?,4?-trans-dihydroxy-α-ionylideneacetic acid, which undergoes alcohol oxidation to furnish ABA. Our results demonstrated that production of ABA does not depend on the nucleotide sequence of bcABA genes. The present study set the stage to investigate the role of ABA in infections.     

A Simple Total Synthesis of (±)-(δ-Cadinene

This paper reports on a total synthesis of racemic δ-cadinene (2), which had been obtained previously in optically active form by acid-catalyzed cyclization of (–)-germacrene D. The Robinson annelation using cyclohexenone enamine (12) proceeded stereoselectively to form δ-cadinenone (3), whose oxygen was removed by the thioketal–Raney Ni method to produce δ-cadinene.     

Structure-activity relationships of chalcone analogs as potential inhibitors of ADP- and collagen-induced platelet aggregation

In an effort to develop potent antiplatelet agents, 12 O-prenylated (2–13) and 10 O-allylated (14–23) chalcones were synthesized and screened for in vitro inhibitory effects on aggregation of washed rabbit platelets induced by ADP (20 μM) and collagen (10 μg/mL). In addition, the platelet aggregation activity of previously synthesized Mannich bases of heterocyclic chalcones (MBHC) (24–62) was evaluated. The preliminary structure–activity relationships suggested that the antiplatelet activity was governed to a great extent by the presence of a pyridyl ring-B and a hydroxy group at position C-3′ in ring-A of the MBHC templates.     

Niacin esters of chalcones with tumor-selective properties

Novel series of niacin esters of chalcones 2, 4 and 6 were designed as antineoplastic agents that have the potential to release the chemoprotectant niacin. These enones are cytotoxic to human CD₄^+?T-lymphocyte Molt 4/C8 and CEM and murine leukemia L1210 cells. Quantitative structure–activity relationship (QSAR) studies of the biodata in series 4 revealed that cytotoxic potency was enhanced by placing electron-repelling groups in one of the aryl rings. The compounds are lethal to HL-60, HSC-2, HSC-3 and HSC-4 neoplasms but less toxic to nonmalignant hepatocyte growth factor, hematopoietic progenitor cell and human periodontal ligament fibroblast cells. Hence, the compounds display tumor-selective toxicity. These chalcones are well tolerated in mice and no overt toxicity was noted. The results establish that in general the compounds in series 2, 4 and 6 have positive characteristics which warrant further studies.     

Biotransformation of the antitumor intermediate 5-fluorouridine by recombinant Escherichia coli with high pyrimidine nucleoside phosporylase activity

Abstract

5-Fluorouridine (5-FUrd) is a precursor of the widely used antitumor drug doxifluridine. We have produced 5-FUrd by biotransformation by cloning the gene encoding pyrimidine nucleoside phosphorylase (PyNPase) from Enterobacter aero-genes CMCC (B) 45103 and expression in Escherichia coli BL21 (DE3), resulting in recombinant E. coli BL21 (DE3)/ pET28a-PyNPase. After medium optimization, the PyNPase activity in the fermentation broth was 1613 U mg^–1, which was 54-fold that of E. aerogenes. Under optimal conditions (cell concentration, 0.5 g L^–1; uridine, 10 mM; 5-fluorouracil, 45 mM; temperature, 50°C; pH, 7.8), more than 90% of uridine was converted to 5-FUrd, suggesting that this is a valuable tool for application in the preparation of antiviral and antitumor drugs.