Biosynthesis of pentacyclic triterpenoids in leaves of Ilex aquifolium L.

Ilex leaves can utilize sucrose and mevalonate for the synthesis of triterpene esters. Mevalonate is also used for the synthesis of free triterpenoid alcohols, but sucrose is not. The selectivity of precursor utilization indicates separate sites for triterpenol and triterpenol-ester synthesis. The sites of synthesis are not found at the main locations of accumulation of triterpenols (the epicuticular wax) or triterpene esters (the cytoplasmatic lipid globules). Transport from the site of synthesis to the lipid globules, and especially towards the epicuticular wax, is slow.Abbreviations MVA mevalonic acid lactone - TLC thin layer chromatography     

Identification and characterization of an acyl-CoA:triterpene acyltransferase activity in rabbit and human tissues.

Rabbit and human tissues contain substantial amounts of an unusual lipid, a fatty acid ester of a pentacyclic triterpene, that is a potent in vitro inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). A possible origin of the triterpene ester is via dietary absorption of plant triterpenes (which have a similar structure to the triterpene moiety of the animal triterpene ester), followed by fatty acid esterification of the triterpene in animal tissues. To support this idea, homogenates of rabbit and human enterocytes and liver are now shown to contain an acyl-CoA:triterpene acyltransferase activity (ATAT) which esterifies triterpene to a fatty acid. The enzyme activity was stimulated by exogenous triterpene and required ATP and coenzyme A when fatty acid was used as substrate; ATP and coenzyme A were not required when fatty acyl-CoA was used. ATAT was not inhibited by two structurally different ACAT inhibitors, which may indicate that ACAT and ATAT are different enzymes. Rat enterocytes and liver contained very little ATAT activity, consistent with the finding that rat liver contained very little triterpene ester. To establish that triterpene esterification occurs in vivo, [3H]triterpene was shown to be incorporated into triterpene ester in several organs and tissues from a rabbit given a gastric bolus of the labeled triterpene. These data provide support for the hypothesis that triterpene esters in animal tissues arise from the dietary absorption of triterpenes followed by the esterification of the triterpenes by an enzymatic activity in the animal tissues.     

Rabbit and human liver contain a novel pentacyclic triterpene ester with acyl-CoA: cholesterol acyltransferase inhibitory activity

Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) catalyzes the intracellular fatty acid esterification of cholesterol and is thought to play a key role in lipoprotein metabolism and atherogenesis. Herein we describe the purification and characterization of a novel pentacyclic triterpene ester from rabbit liver that has ACAT inhibitory activity. The inhibitor was purified by a combination of silicic acid chromatography and preparative thin layer chromatography. The compound inhibited both rabbit and rat liver microsomal ACAT activity with an IC50 = 20 microM. The lipid did not inhibit fatty acid incorporation into triglycerides, diglycerides, monoglycerides, or phospholipids nor did it inhibit plasma lecithin:cholesterol acyltransferase activity. However, rat liver microsomal acyl-CoA:retinol acyltransferase activity was inhibited by the terpene ester. Kinetic data are consistent with a mechanism in which ACAT is inhibited by the compound in an irreversible manner. The subcellular fractionation pattern of both ACAT activity and the ACAT inhibitor were similar in rabbit liver (both were approximately equally distributed in membranes that pelleted at 10,000 X g and 100,000 X g). A lipid with similar properties to the rabbit liver inhibitor was found in many other rabbit tissues, including adrenal and spleen, as well as in human liver. Rat liver did not contain this lipid. Structural analysis by NMR, mass spectrometry, and x-ray crystallography indicated that the rabbit liver inhibitor was a fatty acid ester (mostly stearate) of a pentacyclic triterpene acid. The carbon skeleton of the triterpene moiety is a new member of the olean-12-ene triterpene family. Both the negatively charged carboxylic acid group of the triterpene moiety and the esterified fatty acid group were necessary for the ACAT-inhibitory activity of the triterpene ester. Lastly, we present preliminary data which, together with the structural homology of the rabbit triterpene with known plant compounds, suggest the hypothesis that the triterpene moiety of the rabbit ACAT inhibitor arises from dietary absorption of a plant triterpene.     

Aspartame precursor synthesis in water miscible cosolvent catalysed by thermolysin

Summary The synthesis of the dipeptideN-benzyloxycarbonyl-L- aspartyl-phenylalanine methyl ester, aspartame precursor, catalysed by thermolysin in aqueous and aqueous methanolic solutions was studied. Thermolysin with concentration as low as 10 M in 25% methanol can catalyse the synthetic reaction. The optimum methanol compositions at 4°C and 37°C were 50% and 25% respectively where an increase in peptide yield of 85% was obtained for both conditions as compared to that in water.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-aspartic acid - L-Phe-OMe L-phenylalanine methyl ester - N-cbz-L-Asp-Phe-OMe N-benzyloxycarbonyl-L-aspartyl-phenylalanine methyl ester All the % of methanol is a volume % in water unless otherwise specified.     

Effects of methanol on the synthesis of aspartame precursor catalysed byPseudomonas aeruginosa elastase

Summary Pseudomonas elastase was found to be efficient in catalysing the reaction betweenN-benzyloxycarbonyl-L-aspartic acid and L-phenylalanine methyl ester producing the aspartame precursor in aqueous and aqueous methanolic solutions. 25% (v/v) methanol was most favourable for the synthesis where about 100% increase in yield was obtained compared to that in aqueous solution.Abbreviations N-cbz-L-Asp N-benzyloxycarbonyl-L-Aspartic acid - L-Phe-OMe L-phenyl alanine methyl ester - HPLC high performance liquid chromatography. All the % of methanol is a volume % in water unless otherwise specified     

Oleanane saponins from Gymnema sylvestre

Six oleanane-type saponins, along with two known triterpene saponins, were isolated from the leaves of Gymnema sylvestre. The structures of the oleanane triterpene glycosides were characterized as longispinogenin 3-O-beta-D-glucuronopyranoside, 21 beta-benzoylsitakisogenin 3-O-beta-D-glucuronopyranoside, 3-O-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester, oleanolic acid 3-O-beta-D-xylopyranosyl(1-->6)-beta-D-glucopyranosyl(1-->6)-beta-D- glucopyranoside, 3-O-beta-D-xylopyranosyl(1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl oleanolic acid 28-O-beta-D-glucopyranosyl ester and 3-O-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl oleanolic acid 28-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl ester on the basis of hydrolysis and spectral evidence, including 1D- and 2D-NMR (TOCSY, ROESY, HMQC and HMBC) and FABMS analyses.     

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, β-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.     

Biosynthesis of Leaf Alcohol Formation of 3Z-Hexenal from Linolenic Acid in Chloroplasts of Thea sinensis Leaves

The synthetic activity for 3Z-hexenal, an important precursor of 3Z-hexenol (leaf alcohol), was localized in chloroplasts of Thea sinensis leaves. 3Z-Hexenal, which is easily isomerized to 2E-hexenal (leaf aldehyde), was formed from linolenic acid in the presence of oxygen. 13-l-Hydroperoxy-linolenic acid also served as a precursor, but the triglyceride and methyl ester of linolenic acid did not. This enzyme system appeared to be tightly bound to the lamellae membranes of chloroplasts.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ-glutamyltranspeptidase activity

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.     

Subcellular distribution of free and ester-bound triterpene triols in Calendula officinalis flowers

In cellular subfractions obtained from Calendula officinalis ligulate flowers, the contents of individual free and ester-bound triterpene triols, as well as fatty acid components of the ester form, were determined. It was shown that triterpene triols are localized only in the chromoplast fraction, almost exclusively in a free form and in a very small amount as monoesters. The compositions of fatty acids esterifying triols were similar to those esterifying diols in chromoplasts.     

Triterpenoids from Desfontainia spinosa

The leaves and stem of Desfontainia spinosa were found to contain eleven urs-12-ene triterpene compounds. The major compound present was ursolic acid. Four other known compounds were isolated, i.e. ursolic acid acetate, pomolic acid, 23-hydroxytormentic acid and nigaichigoside Fl, its ester glucoside. The other six compounds are novel and have been identified as 24-hydroxytormentic acid, 7α-hydroxytormentic acid, 7α,23-dihydroxytormentic acid and their corresponding ester glucosides. It is unlikely that these compounds contribute to the reputed narcotic properties of this plant.     

Synthesis of BC-ring model of globostellatic acid X methyl ester, an anti-angiogenic substance from marine sponge

Concise synthesis of BC-ring model compounds of 13E,17E-globostellatic acid X methyl ester, an anti-angiogenic triterpene derivative from Indonesian marine sponge, was achieved through ynolate olefination and allylic oxidation as key steps. The model compound 5, which was synthesized within 10 reaction steps from commercially available Hajos-Parrish ketone, showed anti-proliferative activity against HUVECs with moderate selectivity.     

Free and ester-bound triterpene alcohols and sterols in cellular subfractions of Calendula officinalis flowers

In the chromoplast fraction and in the chromoplast-free fraction, obtained from Calendula officinalis ligulate flowers, the contents of individual free and ester-bound triterpene alcohols and sterols as well as the fatty acid components of the ester form were determined. It was shown that all sterols and triterpene monols in both forms occur in the two subtractions investigated, whereas all diols are localized only in the chromoplast fraction. The compositions of the fatty acids esterifying monols and sterols were similar to those esterifying diols in the chromoplasts. However, the fatty acids esterifying extra-chromoplast monols and sterols were different. This result indicates that triterpene monol esters are substrates for the biosynthesis of 3-monoesters of diols.     

A METHOD FOR MEASURING BRAIN PROTEIN SYNTHESIS RATES IN YOUNG AND ADULT RATS

The injection of large quantities of radioactive amino acid precursor is proposed as a technique for determining rates of cerebral protein synthesis in vivo. In this way the specific radioactivity of the amino acid precursor in the brain is maintained at a relatively constant level for at least 2 h. Injections of 10–15 μ mol of valine per g body weight result in nearly constant rates of incorporation of radioactivity and do not appear to inhibit cerebral protein synthesis in adult or young (2–6 day old) rat brain. Similar rates were obtained in young rat brain with lysine and histidine. Rates of protein synthesis in cerebral hemisphere were for 2-day-olds 2·1 per cent replacement of protein bound amino acid per h and for adult 0·62 per cent per h. Advantages and disadvantages of the procedure are discussed.     

Short chain flavour esters synthesis by microbial lipases

Summary The peparative synthesis of 35 short chain flavour esters by lipases fromMucor miehi, Aspergillus sp.,Candida rugosa andRhizopus arrhizus was investigated in organic media. Acetic, propionic, butyric, valeric and caproic acids, as well as methanol, ethanol, butanol, i-pentanol, hexanol, citronellol and geraniol were used as substrates. Most of the esters were synthesized in good yield by at least one of the lipase preparations tested. Different conversion yields were observed according to the lipase specificity toward the acid or the alcohol moiety of the ester. Methyl- and ethyl acetates were also produced by changing the organic solvent. Enzymatic catalysis in organic solvent is thought to be a valuable method for preparative synthesis of flavour esters.     

Saponins in tissue culture of <Emphasis Type="Italic">Primula veris</Emphasis> L.

Roots of Primula veris L. contain considerable amounts of triterpene saponins, which are used in medicine as expectorants. P. veris is in many places an endangered plant, and its production in the field is laborious and a low yielding process. Plant tissue culture provides an alternative means for producing secondary metabolites. Shoot apex, callus, suspension, and root cultures of P. veris were developed for saponin production. In these cultures, the content of triterpene saponins, with focus on primula acid I, the most dominant saponin in Primula species, was determined and compared to that in soil-grown plants. The highest content of primula acid I was observed in root cultures, on average 29.5 mg/g dry weight. Some culture lines contained higher amounts of primula acid I (62.6 mg/g dry weight) than the roots of plants grown in soil.     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

The structure and stereochemistry of lantanilic acid,the β,β-dimethylacryloyl ester of lantaninilic acid,isolated from Lantana camara

The structure of lantanilic acid, a new triterpene isolated from the leaves of Lantana camara, has been determined as the β,β-dimethylacryloyl ester of lantaninilic acid.     

Factors affecting the synthesis of penicillins by the immobilized penicillin acylase from Escherichia coli

The effect of pH, temperature, reactant concentration, and reaction time has been investigated for the synthesis of N-benzhydryl-N′-acetamidopiperazyl-6-penicillanic acid and N-benzyl-N′-acetamidopiperazyl-6-penicillanic acid from 6-aminopenicillanic acid by the immobilized penicillin acylase from Escherichia coli. The synthesis of penicillins from carboxylic acids proceeds most rapidly at pH 5; with ethyl ester derivatives of carboxylic acids the pH optimum is higher (6–7). The most rapid synthesis of penicillins was obtained with ethyl ester derivatives of carboxylic acids. The optimum temperatures were 25–35°C.