Steryl esters and phenylethanol esters from Syringa komarowii

Three new steryl esters and a new phenylethanol ester, together with 22 known compounds were isolated from the aqueous ethanolic extract of the whole plants of Syringa komarowii. The new compounds were elucidated as stigmastane-3beta,6alpha-diol 3-O-tetradecanoate (1), stigmastane-3beta,6alpha-diol 3-O-palmitate (2), stigmastane-3beta,6alpha-diol 3-O-stearate (3), and 2-(4-hydroxyphenyl)-ethyl dotriacontanoate (4) on the basis of extensive spectral data and chemical evidences.     

9-Fluorenylmethyl esters

N alpha-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by beta-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation. Deblocking was achieved also by catalytic hydrogenation.     

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3β-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3β-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estrodiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 and C-17β. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.     

Myristoyl esters of lactose

Whereas lactose did not undergo a base-catalyzed transesterification with methyl esters of fatty acids, methyl beta-lactoside reacted under identical conditions to give mono- and di-myristates. This difference in behavior is explained in terms of the formation of an unreactive, internally chelated potassium-lactose complex. Supporting evidence for this hypothesis is the observed change in the anomeric equilibrium of lactose in the presence of potassium carbonate. The monomyristates of methyl beta-lactoside were assigned the structures of 3' and 6' derivatives, and it is concluded that the diesters are the 3',6', and 6,6' derivatives.     

Microbial formation of esters

Small aliphatic esters are important natural flavor and fragrance compounds and have numerous uses as solvents and as chemical intermediates. Besides the chemical or lipase-catalyzed formation of esters from alcohols and organic acids, small volatile esters are made by several biochemical routes in microbes. This short review will cover the biosynthesis of esters from acyl-CoA and alcohol condensation, from oxidation of hemiacetals formed from aldehydes and alcohols, and from the insertion of oxygen adjacent to the carbonyl group in a straight chain or cyclic ketone by Baeyer–Villiger monooxygenases. The physiological role of the ester-forming reactions can allow degradation of ketones for use as a carbon source and may play a role in detoxification of aldehydes or recycling cofactors. The enzymes catalyzing each of these processes have been isolated and characterized, and a number of genes encoding the proteins from various microbes have been cloned and functionally expressed. The use of these ester-forming organisms or recombinant organisms expressing the appropriate genes as biocatalysts in biotechnology to make specific esters and chiral lactones has been studied in recent years.     

Steryl esters of liverworts

Fatty acid steryl esters have been isolated from two liverwort species, and the nature of both the sterols and the fatty acids has been determined by analysis using GLC and GC/MS methods.     

Methanolysis of cholesteryl esters: conditions for quantitative preparation of methyl esters.

We have investigated the conditions required to obtain a quantitative yield of methyl esters from cholesteryl esters by alkaline methanolysis. Methanolysis of the cholesteryl ester for 60 min at room temperature with 1 m methoxide reagent ensured complete reaction. Some ester hydrolysis always accompanied methanolysis and necessitated acid-catalyzed methylation of the resultant fatty acids after completion of the alcoholysis. Analysis of the composition of methyl ester product and remaining cholesteryl ester substrate before methanolysis had gone to completion showed selective hydrolysis of some fatty acid cholesteryl esters and illustrates the importance of obtaining a quantitative yield of methyl esters following methanolysis.     

Hydrolysis of dolichyl esters by oviduct membranes and characterization of endogenous dolichyl esters

The chick oviduct system has been employed to study whether dolichol esters might serve as a storage form of dolichol to be converted to dolichyl phosphate (Dol-P) during periods when Dol-P levels increase. Chicken oviduct membranes catalyze the hydrolysis of dolichyl-[14C]oleate; the reaction is dependent on detergent (0.04% NP-40 is optimal), is unaffected by divalent cations and EDTA, and exhibits a pH optimum of 6.0. Oviduct membranes also hydrolyze cholesteryl-[14C]oleate, which exhibits similar properties except the pH optimum is 5.0-5.5. Neither Dol-[14C]palmitate nor Chol-[14C]palmitate is hydrolyzed by membranes. Chol-ester hydrolysis is more sensitive to heat-denaturation than is Dol-ester hydrolysis. Esterase activity was assayed in membranes prepared from immature chicks, chicks treated with diethylstilbestrol, chicks withdrawn from diethylstilbestrol, and mature hens. The highest esterase specific activity was observed in membranes obtained from chicks withdrawn from hormone. In order to characterize the fatty acid composition of Dol-esters they were purified from mature hen oviducts by chromatography on DEAE-cellulose and Fractogel ORPVA-6000, reverse-phase HPLC, and TLC. About 15-25% of oviduct dolichol is in the esterified form. Fatty acid analysis revealed that approximately 85% of the dolichol was esterified to oleic acid. The fact that the highest esterase activity is found in membranes from chicks withdrawn from hormone and that only 20% of the dolichol is esterified argues against a role for Dol-esters as a reservoir of dolichol for conversion to Dol-P.     

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C₃₆, C₃₄ and C₃₂, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.     

Aromatic esters from Solidago decurrens

From Solidago decurrens several new benzyl benzoates and two cinnamyl angelates were variously isolated from stems, flower and root.     

Apiene esters from Ferula haussknechtii

Ten new apiene-type humulenoid esters were isolated from the root of Ferula haussknechtii. The structures were elucidated by spectroscopic methods.