A simple method for regiospecific analysis of triacylglycerols by gas chromatography

A simple method for regiospecific analysis of triglycerides was developed. It consists of partial deacylation of triglycerides by ethylmagnesium bromide followed by derivatization of monoglycerides with n-butyryl chloride, and direct analysis of dibutyrate derivatives of monoglycerides by gas chromatography. The chromatographic conditions were carried out with monoglycerides of C₁₂ to C₂₀ fatty acids and resulted in separation of dibutyrate derivatives between those bearing the medium- or long-chain fatty acid in the sn-1(3) and sn-2 positions of glycerol. Beef tallow and grapeseed and cotton seed oils were analyzed using this new method, and their regiospecific distributions were compared with literature data. The method does not require separation of products by thin-layer chromatography or special analytical equipment other than a standard gas chromatograph, and it can thus be used for routine regiospecific analysis of triglycerides.     

Regioselective analysis of triacylglycerols by lipase hydrolysis

A modified procedure for the regiospecific analysis of triacylglycerols (TAG) with a 1,3-specific lipase is described. After partial lipase hydrolysis of the triacylglycerol, the released free fatty acids (FFA) and 1,2(2,3)-diacylglycerols (DAG) were isolated by thin-layer chromatography (TLC) and converted to fatty acid methyl esters (FAME). The FAME were analyzed by gas-liquid chromatography (GLC). The 1,3-specific lipases used in this study included supported preparations from strains ofMucor miehei andRhizopus oryzae. The method also was applied to the regiospecific analyses of tung nut and Chinese melon seed oil triacyglycerols, both of which contain high proportions of α-elaeostearic acid. The TAG composition of the oils was substantiated in parallel analysis of the oils by highperformance liquid chromatography with chemical ionization mass spectrometric detection of intact TAG.     

Preparation of ethyl magnesium bromide for regiospecific analysis of triacylglycerols

This paper presents a procedure for preparation of a Grignard reagent, ethyl magnesium bromide, used for partial deacylation of triacylglycerols (TAG) in their regiospecific analysis. Magnesium turnings were reacted with ethereal solution of bromoethane in a screw-capped test tube to synthesize 2 mL of 1 M ethyl magnesium bromide. Continuously stirred with a vortex mixer, the reaction smoothly proceeded at room temperature. Regiospecific analysis of 1,3-distearoyl-2-oleoylglycerol using this product showed that fatty acid compositions of the sn-1(3) and sn-2 positions were contaminated by less than 2 mol% of fatty acids migrated from isomeric positions. The analyses of lard and cod liver/mackerel oil TAG showed typical distribution patterns of 16:0, 22:5n-3 and 22:6n-3 in pig and fish depot TAG. These results confirmed the view that the freshly prepared reagent is usable for regiospecific analysis of TAG.     

Rapid and direct quantitative analysis of positional fatty acids in triacylglycerols using 13C NMR

There is increasing evidence that the positional fatty acid composition (FAC) of TAG is more important than total FAC with regard to nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of positional fatty acids in TAG using ¹³C NMR was developed to overcome the tedious conventional methods which involve enzymatic hydrolysis, Grignard chemical degradation, and chromatography analysis. A set of NMR data acquisition parameters and processing methods had proven their excellent versatility and applicability on various types of oils and fats, with systematic error of 1.0 mol%. It was found that there are discrepancies between the regiospecific analysis results obtained by the current method and by conventional methods. Probable acyl migration occurring in the hydrolysis process in conventional methods is a noted problem. As the current ¹³C NMR method is a direct measurement and no hydrolysis of the sample is needed, acyl migration during the analysis is eliminated. As a result, the saturation level is always higher at sn‐1, 3 positions and lower at sn‐2 position in TAG structure of oils in the regiospecific data obtained from the current ¹³C NMR than that from conventional methods. In the absence of laborious chemical derivatization, this method is simple, accurate, and user‐friendly for researchers, especially for nutritionists to support their nutritional studies from the perspective of positional FAC of edible oils and fats.     

Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.     

Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by ¹³C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.     

Internal Addition of Ketocarbenes to Conjugated Double Bonds

The internal, copper-catalysed, addition of keto-carbenes (generated from a diazoketone group) to a double bond conjugated either to a ketone or carboxylic ester function has been found to be a feasible preparative reaction in selected cases. This, together with the facile and regiospecific hydrogenolysis of the resulting 1,2-dicarbonyl cyclopropane intermediate, appear to constitute a convenient route to multifunctional bridged-ring systems.     

Detection of olive oil adulteration using principal component analysis applied on total and regio FA content

Principal component analysis (PCA) has been used to establish a new method for the detection of olive oil adulteration. The data set, composed of values obtained from the determination of the mole percentage of total FA and their regiospecific distribution in positions 1 and 3 in TG of oils (pure or mixtures) by GC analysis, was subjected to PCA. 3-D scatter plots showed clearly that it is possible to distinguish the pure oils from the mixtures. Moreover it is possible to discriminate the different types of seed oil used for the adulteration.     

Synthesis of Acenaphtho[3, 4-b]thiophene: An Aceanthrylene Isoster

The synthesis of a novel thiophene isoster of aceanthrylene is reported. The sequence involves, a newly observed regiospecific condensation of oxalylchloride to naphtho[2,3-b]thiophene. Reduction of the quinone and dehydrogenation gave the title compound in 28% overall yield. The proton nmr signals of the title compounds were assigned on the basis of COSY and NOE difference spectroscopy. Its isolectronic nature with aceanthrylene is seen in the similarity of the UV spectra.     

Regiospecificity profiles of storage and membrane lipids from the gill and muscle tissue of atlantic salmon (Salmo salar L.) grown at elevated temperature

Regiospecific and traditional analysis, of both storage and membrane lipids, was performed on gill, white muscle, and red muscle samples taken from Atlantic salmon (Salmo salar) to gauge the effect of elevated water temperature. The fish, fed a commercial diet, were held at an elevated water temperature of 19°C. Total n-3 PUFA, total PUFA, and n-3/n-6 and unsaturated/saturated fatty acid (UFA/SFA) ratios in the FA profile of the total lipid extract in the white muscle were fairly low compared with fish grown at 15°C. Adaptation of structural and storage lipids at elevated temperatures was shown by a significant (P<0.01) reduction in PUFA especially in the percentage of EPA (6–8%). Further adaptation was indicated by the percentages of SFA, which were significantly (P<0.05) higher in gill (56%) and white muscle (58%) polar lipid fractions and coincided with lower percentages of n-3, n-6, and total PUFA. The regiospecific profiles indicated a high affinity of DHA to the sn-2 position in both the TAG (61–68%) and polar lipid (35–60%) fractions. The combination of detailed regiospecific and lipid analyses demonstrated adaptation of cell membrane structure in Atlantic salmon grown at an elevated water temperature.     

Functionalization of suluble polymers

Summary In a first example of the functionalization of soluble polymers the conversion of the hydroxyl groups of poly(oxyethylene) into primary amino groups has been studied. Starting from poly(oxyethylene), the preparation of the diphthalimido derivative via the disodium salt is described. Prom this regiospecific functionalized polymer the di(aminoethyl) poly(oxyethylene), which exhibits excellent solubility properties, can be obtained by polymeranalogous hydrazinolysis.     

Synthesis of 2‐monoglycerides by alcoholysis of palm oil and tuna oil using immobilized lipases

Commercial immobilized lipases were used for the synthesis of 2‐monoglycerides (2‐MG) by alcoholysis of palm and tuna oils with ethanol in organic solvents. Several parameters were studied, i.e., the type of immobilized lipases, water activity, type of solvents and temperatures. The optimum conditions for alcoholysis of tuna oil were at a water activity of 0.43 and a temperature of 60 °C in methyl‐tert‐butyl ether for ～12 h. Although immobilized lipase preparations from Pseudomonas sp. and Candida antarctica fraction B are not 1, 3‐regiospecific enzymes, they were considered to be more suitable for the production of 2‐MG by the alcoholysis of tuna oil than the 1, 3‐regiospecific lipases (Lipozyme RM IM from Rhizomucor miehei and lipase D from Rhizopus delemar). With Pseudomonas sp. lipase a yield of up to 81% 2‐MG containing 80% PUFA (poly‐unsaturated fatty acids) from tuna oil was achieved. The optimum conditions for alcoholysis of palm oil were similar as these of tuna oil alcoholysis. However, lipase D immobilized on Accurel EP100 was used as catalyst at 40 °C with shorter reaction times (<12 h). This lead to a yield of ～60% 2‐MG containing 55.0‐55.7% oleic acid and 18.7‐21.0% linoleic acid.     

Differentiation of Fish Oils According to Species by <Superscript>13</Superscript>C-NMR Regiospecific Analyses of Triacyglycerols

The aim of this study was to use ¹³C-nuclear magnetic resonance (NMR) regiospecific analyses of triacylglycerols to distinguish fish oils from different fish species for authentication purposes. ¹³C-NMR data of muscle lipids from Atlantic salmon (Salmo salar L.), mackerel (Scomber scombrus) and herring (Clupea harengus) were obtained, and the distribution of omega-3 polyunsaturated fatty acids between the sn-1,3 and sn-2 glycerol chains calculated from the carbonyl region. The results show that there were significant differences in the sn-2 position specificity of the fatty acids 22:6n-3, 20:5n-3 and 18:4n-3 among the species investigated. The most pronounced difference was that herring had a higher proportion of its 22:6n-3 in the sn-2 position compared to the two other species. Principal component analysis of data points in the carbonyl-region showed that there were also differences in the level and regiospecific distribution of monounsaturated/saturated fatty acids, which made it possible to distinguish oils of these three species solely from the carbonyl region of ¹³C-NMR spectra.     

1,3-Dipolar Cycloadditions of 1,1-Difluoroallene,Nitrones, Nitrile-Oxides and Carbonyl Ylides

The 1,3-dipolar cycloadditions of C,N-diphenylnitrone, C-phenyl-N-methylnitrone, triphenylnitrone, mesitylnitrile oxide and tetracyanocarbonyl ylide with 1,1-difluoroallene (DFA) are reported and discussed with respect to regiochemistry. Each of the dipoles adds exclusively to the C₂—C₃, non-fluorine-substituted π-bond of DFA. The nitrones all undergo regiospecific reactions while mesitylnitrile oxide does not. The results are discussed with respect to FMO theory and other factors which can give rise to regiochemical variation.     

Synthesis by post-polymerization functionalization of sensitive polythiophenes for selective chemo-recognition purposes

The synthesis of new regioregular polyalkylthiophenes derivatives with different substituents inserted in the terminal position of a hexamethylenic side chain is described here. The new polymers were prepared by simple and effective post-polymerization functionalization of a soluble regioregular precursor, namely poly[3-(6-bromohexyl)thiophene] that was obtained by regiospecific organometallic coupling. The new materials were characterized by NMR, FT-IR, elemental analysis and their molecular weights determined by gel permeation chromatography. High-precision UV–Vis analyses (solvatochromism and affinitychromism) were conducted in solution and in film state, and the results are discussed in relation to the different inserted functional groups. Thanks to the good sensitivity and chromic response recorded for the prepared materials to different analytes and in some solvating systems, they appear to have great potential for use as electronic sensors in chemo-recognition applications.     

Viable synthesis of a self-assembling regioregular thiophenic copolymer for second-order non-linear optics

Regioregular azobenzene-substituted polythiophenic copolymers were prepared by a regiospecific organometallic polycondensation procedure based on a Grignard metathesis reaction. Copolymers with a high content of chromophoric groups in the side chains and a high degree of configurational order of the backbones were obtained. A sample was also functionalized with hydroxyalkyl chains capable of setting up ordered supramolecular structures through hydrogen bonds. The final materials were fully characterized via NMR, IR, elemental analysis, GPC, DSC and X-ray diffraction. Moreover, measurements of NLO activity on polymer films showed the synthetic strategies adopted and monomers used to be correct. Values obtained by the in situ deprotection of the interacting functional groups after the alignment of the chromophorized side chains were very promising, proving the procedure to be suitable for industrial and commercial applications.     

Diels-Alder Reactions of in situ generated N-benzoylindolo-2,3-quinodimethane with carbodienophiles

The indolo-2,3-quinodimethane ( 5 ), generated from 2,3-bis(bromomethyl)indole ( 4 ), was trapped with unsymmetrical carbodienophiles or N, N′-p-phenylenedimaleimide to furnish the 1,2,3,4-tetrahydrocarbazoles ( 6 – 9 ). [4 + 2]-Cycloaddition of 5 with tetracyanoethylene gave rise to the tetracyanocarbazole ( 10 ) and a subsequent product 11a or 11b . The Diels-Alder reaction of 5 with divinyl sulfone was regiospecific within the detection limits of HPLC analysis. Similarly, the reaction of 5 with N, N′-p-phenylenedimaleimide yielded solely the stereoisomer 9 .     

硝苯柳胺合成方法的研究

研究了硝苯柳胺的三种合成方法 ,分析了其影响因素 ,比较了其优缺点并提出了各自的适用范围。其中氯气氯代法可在一个反应器内进行 ,不需分离中间体 ,收率可达 90 % ;次氯酸叔丁酯对水杨酸的氯代有很强的区域专一性反应特性 ,用其作为氯代剂合成硝苯柳胺收率为 81% ,产品纯度 97 9% ;用对硝基苯胺与二氯亚矾在吡啶存在下生成亚硫酰胺 ,再与 5 氯水杨酸反应合成硝苯柳胺 ,收率为 78% ,纯度可达 99%     

Physicochemical Properties of Interesterified Blends of Fully Hydrogenated Crambe abyssinica Oil and Soybean Oil

The chemical interesterification of blends of soybean (SO) and fully hydrogenated crambe oil (FHCO) in the ratios of 80:20, 75:25, 70:30, 65:35, and 60:40 (w/w), respectively, was investigated. FHCO is a source of behenic acid. The blends and the interesterified fats were analyzed for fatty acid and triacylglycerol composition, regiospecific distribution, slip melting point, solid profile, and consistency. The regiospecific analysis of the TAG indicated random insertion of saturated fatty acids at sn-2 of the glycerol of the interesterified blends with more significant alterations at sn-2 than at sn-1 and sn-3. The gradual addition of FHCO increased the solid fat content and the slip melting point. The chemical interesterification formed new TAG facilitating the miscibility between SO and FHCO. The 70:30 interesterified blend was suitable for general use, 60:40 for use as a base stock. At 35 °C, the 65:35 interesterified blend showed suitable plasticity for use in products with fat contents below 80 %. FHCO, rich in behenic acid, is not associated with increased total cholesterol and LDL cholesterol, and it can be used as a low trans fat. FHCO is not associated with increased total cholesterol and LDL cholesterol, and it can be used as a low trans fat alternative.     

Molecular Characterisation of Flavanone O-methylation in Eucalyptus

Flavonoids are ubiquitous polyphenolic compounds in plants, long recognised for their health-promoting properties in humans. Methylated flavonoids have received increasing attention due to the potential of methylation to enhance medicinal efficacy. Recently, Eucalyptus species with high levels of the O-methylated flavanone pinostrobin have been identified. Pinostrobin has potential commercial value due to its numerous pharmacological and functional food benefits. Little is known about the identity or mode of action of the enzymes involved in methylating flavanones. This study aimed to identify and characterise the methyltransferase(s) involved in the regiospecific methylation of pinostrobin in Eucalyptus and thereby add to our limited understanding of flavanone biosynthesis in plants. RNA-seq analysis of leaf tips enabled the isolation of a gene encoding a flavanone 7-O-methyltransferase (EnOMT1) in Eucalyptus. Biochemical characterisation of its in vitro activity revealed a range of substrates upon which EnOMT1 acts in a regiospecific manner. Comparison to a homologous sequence from a Eucalyptus species lacking O-methylated flavonoids identified critical catalytic amino acid residues within EnOMT1 responsible for its activity. This detailed molecular characterisation identified a methyltransferase responsible for chemical ornamentation of the core flavanone structure of pinocembrin and helps shed light on the mechanism of flavanone biosynthesis in Eucalyptus.