Enzymatic synthesis of quercetin oleate esters using <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B

Objectives

To investigate the lipase-catalyzed acylation of quercetin with oleic acid using Candida antarctica lipase B.

Results

Three acylated analogues were produced: quercetin 4′-oleate (C₃₃H₄₂O₈), quercetin 3′,4′-dioleate (C₅₁H₇₄O₉) and quercetin 7,3′,4′-trioleate (C₆₉H₁₀₆O₁₀). Their identities were confirmed with UPLC–ESI–MS and ¹H NMR analyses. The effects of temperature, duration and molar ratio of substrates on the bioconversion yields varied across conditions. The regioselectivity of the acylated quercetin analogues was affected by the molar ratio of substrates. TLC showed the acylated analogues had higher lipophilicity (152% increase) compared to quercetin. Partition coefficient (log P) of quercetin 4′-oleate was higher than those of quercetin and oleic acid. Quercetin 4′-oleate was also stable over 28 days of storage.

Conclusions

Quercetin oleate esters with enhanced lipophilicity can be produced via lipase-catalyzed reaction using C. antarctica lipase B to be used in topical applications.

     

Synthesis,Protonation Behavior,Conformational Analysis,and Regioselective Enzymatic Acylation of the Novel Diamino Analogue of (E)-5-(2-Bromovinyl)-2′-deoxyuridine (BVDU)

Abstract

(E)-3′,5′-Diamino-5-(2-bromovinyl)-2′,3′,5′-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. The protonation behavior of 5 has been studied by means of pH-metric measurements and NMR spectroscopy. This study allows the determination of the basicity constants and the stepwise protonation sites. Thus, the main species at physiological pH is the monoprotonated form. The conformational analysis of this nucleoside analogue was also carried out through ¹H NMR spectroscopy. In addition, a convenient synthesis of N-3′ and N-5′ acylated derivatives was developed by regioselective enzymatic acylation. Thus, Candida antarctica lipase B (CAL-B) selectively acylated the 5′-amino group, thus furnishing nucleosides 8. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) exhibited the opposite selectivity, conferring acylation at the 3′-amino group, thus affording derivatives 9.     

Regioselective enzymatic acylation of ribavirin to give potential multifunctional derivatives

Lipase-catalyzed synthesis of potential multifunctional ribavirin derivatives was performed in acetone. Divinyl dicarboxylates with different chain lengths (C₄, C₆, C₉, C₁₀) were used as acyl donors and the reactions were catalyzed by lipase immobilized on acrylic resin from Candida antarctica (CAL-B). Ribavirin was regioselectivly acylated at the primary hydroxyl groups and the corresponding vinyl esters (C₄, C₆, C₉, C₁₀) were prepared in respective yields of 48%, 65%, 54%, 55%.     

Enzymatic regioselective and complete deacetylation of two arabinonucleosides

Candida antarctica lipase B (CAL-B)-catalysed regioselective deacetylation of 2′,3′,5′-tri-O-acetyl-1-β-d-arabinofuranosyluracil (1) and 2′,3′,5′-tri-O-acetyl-9-β-d-arabinofuranosyladenine (2) was studied. The choice of the reaction medium allowed the regioselective formation of products bearing different degree of acetylation: in isopropanol, CAL-B catalysed the formation of the corresponding 2′-O-acetylated arabinonucleosides, while hydrolyses afforded the 2′,3′-di-O-acetylated products. In particular, the procedure herein described allows a simple and efficient preparation of the reported vidarabine prodrug 2′,3′-di-O-acetyl-9-β-d-arabinofuranosyladenine, avoiding the utilisation of protective groups. Moreover, to achieve full deacetylation of the assayed substrates, a set of commercial hydrolases and fungal keratinases from Doratomyces microsporus (DMK) and Paecilomyces marquandii (PMK) were tested. While only PMK and DMK catalysed the quantitative complete deacetylation of 1, DMK accomplished full deacetylation of 2 in shorter time than the other assayed enzymes.     

Regioselective acylation of several polyhydroxylated natural compounds by Candida antarctica lipase B

Regioselective acylation of four polyhydroxylated natural compounds, deacetyl asperulosidic acid (1), asperulosidic acid (2), puerarin (3) and resveratrol (4) by Candida antarctica Lipase B in the presence of various acyl donors (vinyl acetate, vinyl decanoate or vinyl cinnamoate) was studied. Compounds 1, 2 and 4 were regioselectively acetylated with vinyl acetate to afford products, 3′-O-acetyl-10-O-deacetylasperulosidic acid (1a), 3′,6′-O-diacetyl-10-O-deacetylasperulosidic acid (1b), 3′-O-acetylasperulosidic acid (2a), 3′,6′-O-diacetylasperulosidic acid (2b), 4′-O-acetylresveratrol (4a), respectively, with yields of 22 to 50%, while reactions with vinyl decanoate and vinyl cinnamoate were slow with lower yields. Compound 3 was readily acylated with all three acyl donors and quantitatively converted to products 6″-O-acetylpuerarin (3a), 6″-O-decanoylpuerarin (3b), 6″-O-cinnamoylpuerarin (3c), respectively. The structures of these acylated products were determined by spectroscopic methods (MS and NMR).     

Cinnamic acid intermediates as precursors to mesembrine and some observations on the late stages in the biosynthesis of the mesembrine alkaloids

Experiments with various labelled cinnamic acid derivatives establish, in conjunction with previous work, that the incorporation of phenylalanine into the 3a-aryl octahydroindole ring system of the mesembrine alkaloids occurs via the intermediacy of cinnamic acid and 4′-hydroxycinnamic acid. The major pathway to the 3′,4′-di-oxyaryl substituted alkaloids proceeds via 4′-hydroxydihydrocinnamic acid (4′-phloretic acid), and 3′4′-dioxy-genated cinnamic acids are not involved as intermediates on this major pathway. In accord with this latter finding, the 3′-aryl oxygen substituent is introduced at a late state in the biosynthesis as evidenced by the bioconversion in S. strictum of sceletenone to mesembrenol and other related alkaloids. The late stages in the biosynthesis of the alkaloids are shown to involve the sequence: sceletenone, 4′-O-demethylmesembrenone, mesembrenone. Mesembrenone is converted to mesembrine, mesembrenol and mesembranol.     

Studies Towards the Large Scale Chemical Synthesis of the Precursors of Ribonucleosides-3′,4′,5′,5″- 2H 4 and -2′,3′,4′,5′,5″- 2H 5

Abstract

A summary delineating the large scale synthetic studies to prepare labeled precursors of ribonucleosides-3′,4′,5′,5″- ²H ₄ and -2′,3′,4′,5′,5″- ²H ₅ from D-glucose is presented. The recycling of deuterium-labeled by-products has been devised to give a high overall yield of the intermediates and an expedient protocol has been elaborated for the conversion of 3-O-benzyl-α,β-D-allofuranose-3,4-d ₂ 6 to 1-O-methyl-3-O-benzyl-2-O-t-butyldimethylsilyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 16 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ) or to 1-O-methyl-3,5-di-O-benzyl-α,β-D-ribofuranose-3,4,5,5′-d ₄ 18 (precursor of ribonucleosides-3′,4′,5′,5″- ²H ₄ ).     

Yellow Pigments of Aspergillus niger and Aspergillus awamori

Alkaline degradation of Aurasperone A, C₃₂H₂₆O₁₀, gave a binaphthyl (IIa), m.p. 255°C and acetone. (IIa) afforded a tetraacetate (IIb), C₃₂H₃₀O₁₂ m.p. 219°C and a tetramethyl ether (IId), C₂₈H₃₀O₈, m.p. 188°C. These facts along with the NMR spectra of aurasperone A and (IIb) confirm that aurasperone A is a dimeric 2-methyl-5-hydroxy-6,8-dimethoxy-4H-naphtho[2,3-b]pyran-4-one with asymmetric C-C linkage (7-10′ or 9-10′). The ether (IId) is not identical with 1,1′ ,3,3′ ?6,6′ ,8,8′-octamethoxy-4,4′-binaphthyl. Thus, it follows that (IId) is a 2,4′-binaphthyl and hence aurasperone A is 2,2′-dimethyl-5,5′- dihydroxy-6,6′,8,8′-tetrahydroxy-7,10′-bi[4H-naphtho[2,3-b]pyran-4-one] (I).     

Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition of Exogenous IAA in Arabidopsis thaliana

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D-glucopyranose, [2′,2′-²H₂]-2-oxoindole-3-acetic acid, and 1-O-([2′,2′-²H₂]-2-oxoindole-3-acetyl)-β-D-glucopyranose were also successfully synthesized from deuterated IAA, and effectively utilized as internal standards in the quantitative analysis of IAA and its metabolites in Arabidopsis thaliana by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The use of this technique shows that these metabolites were accumulated in the roots of Arabidopsis seedlings. Dynamic changes in the metabolites of IAA were observed in response to exogenous IAA, revealing that each metabolic action was regulated differently to contribute to the IAA homeostasis in Arabidopsis.     

Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.     

3′-Deoxy-3′-C-trifluoromethyl Nucleosides: Synthesis and Antiviral Evaluation

Abstract

2′,3′-Dideoxy-3′-C-trifluoromethylthymidine 9a and -uridine 9b and 3′-C-trifluoromethyl-d4T 11 were prepared in a few steps from 3′-deoxy-3′-C-trifluoromethyl-D-ribose, which synthesis was recently reported. The biological assessment of these nucleoside analogues did not reveal interesting antiviral properties against HIV-1, HSV-1, CMV, Vaccine, and Cox B4.     

Direct Regioselective Enzymatic Acylation of Nucleosides: Building-Blocks for the Solution Phase Synthesis of Oligonucleotides

Abstract

The synthesis of 3′- and 5′-O-levulinyl nucleosidic monomers through enzymatic acylation with acetonoxime levulinate is demonstrated. The acylation process takes place in one-step and use of expensive reagents, such as DMTrCl is avoided. The regioselectivity of the procedure makes it very convenient for acylated monomers required for solution phase synthesis of oligonucleotides.     

Synthesis of fisetin and 2′,4′,6′-trihydroxydihyrochalcone 4′-O-β-neohesperidoside based on site-selective deacetylation and deoxygenation

Fisetin and 2′,4′,6′-trihydroxydihyrochalcone 4′-O-β-neohesperidoside were synthesized from commercially available quercetin and naringin in five steps. The key steps are site-selective deacetylation and subsequent deoxygenation. The target molecules were obtained in 37% and 23% yields from the starting materials, respectively.     

Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries

A high-throughput method (≥ 10⁶ of clones can be analysed on a single agar plate) for the selection of ester-hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′-O-tri-acetyluridine and 2′,3′,5′-O-tri-hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.     

Lipophilic C-methylflavonoids with no B-ring oxygenation in Metrosideros species (Myrtaceae)

Five unusual C-methylflavonoids lacking B-ring oxygenation (2′,4′-dihydroxy-3′,5′-dimethyl-6′-methoxychalcone, 2′,4′-dihydroxy-3′-methyl-6′-methoxychalcone, 2′,6′-dihydroxy-3′-methyl-4′-methoxychalcone, 2′-hydroxy-3′-methyl-4′,6′-dimethoxychalcone and 5,7-dihydroxy-6,8-dimethylflavanone) were found for the first time in Metrosideros excelsa. The flavanone was the major constituent in leaves, whereas 2′,6′-dihydroxy-3′-methyl-4′-methoxychalcone dominated all other aerial plant parts studied. Other Metrosideros species were investigated for these five flavonoids. C₁₉–C₃₆ aldehydes and C₂₂–C₃₂ alcohols were also identified from the dried seed capsules of M. excelsa.     

Synthesis of New Potential Lipophilic Co‐Drugs of 2‐Chloro‐2′‐deoxyadenosine (Cladribine, 2‐CdA,Mavenclad®, Leustatin®) and 6‐Azauridine (z6U) with Valproic Acid

2‐Chloro‐2′‐deoxyadenosine (cladribine, 1 ) was acylated with valproic acid ( 2 ) under various reaction conditions yielding 2‐chloro‐2′‐deoxy‐3′,5′‐O‐divalproyladenosine ( 3 ) as well as the 3′‐O‐ and 5′‐O‐monovalproylated derivatives, 2‐chloro‐2′‐deoxy‐3′‐O‐valproyladenosine ( 4 ) and 2‐chloro‐2′‐deoxy‐5′‐O‐valproyladenosine ( 5 ), as new co‐drugs. In addition, 6‐azauridine‐2′,3′‐O‐(ethyl levulinate) ( 8 ) was valproylated at the 5′‐OH group (→ 9 ). All products were characterized by ¹H‐ and ¹³C‐NMR spectroscopy and ESI mass spectrometry. The structure of the by‐product 6 (N‐cyclohexyl‐N‐(cyclohexylcarbamoyl)‐2‐propylpentanamide), formed upon valproylation of cladribine in the presence of N,N‐dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X‐ray crystallography. Cladribine as well as its valproylated co‐drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS‐3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol‐12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages. The most important result of these experiments is the finding that only the 3′‐O‐valproylated derivative 4 exhibits a significant antitumor activity while the 5′‐O‐ as well as the 3′,5′‐O‐divalproylated cladribine derivatives 3 and 5 proved to be inactive.     

LC‐ESI‐QTOF‐MS for the Profiling of the Metabolites and in Vitro Enzymes Inhibition Activity of Bryophyllum pinnatum and Oxalis corniculata Collected from Ramechhap District of Nepal

The objective of this study was to profile the chemical components and biological activity analysis of crude extract of Bryophyllum pinnatum and Oxalis corniculata. Results revealed that the analyzed plant materials encompass the high amount of total phenolic and flavonoids content and have significant antioxidant activities. Furthermore, methanol extracts are the potential source of α‐amylase, α‐glucosidase, lipase, tyrosinase and elastase inhibitors. High resolution mass spectrometry revealed the presence of diverse metabolites such as quercetin 3‐O‐α‐L‐rhamnopyranoside, myricetin 3‐rhamnoside, bersaldegenin 1,3,5‐orthoacetate, bryophyllin C, syringic acid, caffeic acid, p‐coumaric acid, and quercetin in B. pinnatum and isoorientin, swertisin, apigenin 7,4′‐diglucoside, vitexin, 4‐hydroxybenzoic acid, vanillic acid, ethyl gallate, 3,3′,4′‐trihydroxy‐5,7‐dimethoxyflavone, and diosmetin‐7‐O‐β‐D‐glucopyranoside in O. corniculata. Our finding suggested that these two plant species have high medicinal importance and are potential source of inhibitors for modern pharmaceuticals, nutraceuticals and cosmetics industries.     

Isolation and Characterization of PDE-I and II,the Inhibitors of Cyclic Adenosine-3′,5′-monophosphate Phosphodiesterase

In the screening for inhibitors of cyclic adenosine-3′,5′-monophosphate phosphodiesterase, two compounds, PDE-I (C₁₃H₁₃N₃O₅) and PDE-II (C₁₄H₁₄N₂O₅), were isolated from culture filtrates of a Streptomyces. Concentrations for 50% inhibitions of PDE-I and PDE-II against the high Km enzyme were 15 µm and 13 µm, and those against the low Km enzyme were 65 µm and 130 µm, respectively. Production, isolation and characterization of these compounds are described.     

Inhibition of the β-carbonic anhydrases from Mycobacterium tuberculosis with C-cinnamoyl glycosides: Identification of the first inhibitor with anti-mycobacterial activity

A small series of C-cinnamoyl glycoside containing the phenol moiety was tested for the inhibition of the three Mycobacterium tuberculosis β-carbonic anhydrases (CAs, EC 4.2.1.1) with activities in the low micromolar range detected. The compounds were also tested for the inhibition of growth of M. tuberculosis H₃₇Rv strain, leading to the identification of (E)-1-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4-(3-hydroxyphenyl)but-3-en-2-one (1) as the first carbonic anhydrase inhibitor with anti-tubercular activity.     

Hydroxyl Radical-Mediated Oxidation of Serotonin: Potential Insights into the Neurotoxicity of Methamphetamine

Abstract: When incubated with a hydroxyl radical (HO^?)-generating system (ascorbic acid/Fe²⁺-EDTA/O₂/H₂O₂), 5-hydroxytryptamine (5-HT; serotonin) is rapidly oxidized initially to a mixture of 2,5-, 4,5-, and 5,6-dihydroxytryptamine (DHT). The major reaction product is 2,5-DHT, which at physiological pH exists as its keto tautomer, 5-hydroxy-3-ethylamino-2-oxindole (5-HEO). Rapid autoxidation of 4,5-DHT gives tryptamine-4,5-dione (T-4,5-D), which reacts with the C(3)-centered carbanion of 5-HEO to give 3,3′-bis(2-aminoethyl)-5-hydroxy-[3,7′-bi-1H-indole]-2,4′,5′-3H-trione (7). The latter slowly cyclizes to 3′-(2-aminoethyl)-1′,6′,7′,8′-tetrahydro-5-hydroxyspiro[3H-indole-3,9′-[9H]pyrrolo[2,3-f]quinoline]-2,4′,5′(1H)- trione (9). A minor amount of T-4,5-D dimerizes to give 7,7′-bi-(5-hydroxytryptamine-4-one) (7,7′-D). In the presence of GSH, the reaction of T-4,5-D with 5-HEO is diverted and, in the presence of sufficient concentrations of this tripeptide, completely blocked. This is because GSH preferentially reacts with T-4,5-D to give 7-S-glutathionyltryptamine-4,5-dione (11). The results of this investigation suggest that 5,6-DHT, 5-HEO, 7, and 9 are products unique to the HO^?-mediated oxidation of 5-HT. Thus, the observation of other investigators that 5,6-DHT is formed in the brains of rats following a large dose of methamphetamine (MA) suggests that this drug might evoke HO^? formation. However, the present in vitro study indicates that 5,6-DHT is a rather minor, unstable product of the HO^?-mediated oxidation of 5-HT and suggests that detection of 5-HEO, 7/9, and 11 in rat brain following MA administration could provide additional support for HO^? formation. Furthermore, one or more of the intermediates and major products of oxidation of 5-HT by HO^? might, in addition to 5,6-DHT, contribute to the MA-induced degeneration of serotonergic neurons.