Formation of Toxic α,β-Unsaturated 4-Hydroxy-Aldehydes in Thermally Oxidized Fatty Acid Methyl Esters

To investigate the heat induced formation of α,β-unsaturated 4-hydroxy-aldehydes (4-hydroxy-2-trans-hexenal (HHE), 4-hydroxy-2-trans-octenal (HOE), 4-hydroxy-2-trans-nonenal (HNE) and 4-hydroxy-2-trans-decenal (HDE)) fatty acid methyl esters (FAMEs) of stearic, oleic, linoleic and linolenic acids were heated separately at 185°C for 0 to 6 hrs. The formation of 2,4-decadienal, a suspected intermediate in HNE formation, was also measured in these FAMEs. As expected methyl stearate (MS) and methyl oleate (MO) did not produce any of the α,β-unsaturated 4-hydroxy-aldehydes as a consequence of thermally induced lipid peroxidation. The formation of HHE was detected in both methyl linoleate (ML) and methyl linolenate (MLN), with concentration higher in MLN than in ML. The maximum HHE concentration was 3.99 μg HHE/g ML after 2 h and 50.78 μg HHE/g MLN after 4 h of heat treatment. HOE was detected in both ML and MLN, and the maximum concentration was 102.50 μg HOE/g ML after 6 h and 90.56 μg HOE/g MLN after 2 h of heating. HNE was found only in ML and its highest concentration was 84.82 μg HNE/g ML after 3 h of heating. HDE was not detected in any of the four heat treated FAMEs. 2,4-Decadienal was not found to be an intermediate in the formation of HNE in thermally induced oxidation of FAMEs.     

Formation of 4‐Hydroxy‐2‐Trans‐Nonenal,a Toxic Aldehyde,in Thermally Treated Olive and Sunflower Oils

4‐Hydroxy‐2‐trans‐nonenal (HNE) is a toxic aldehyde produced mostly in oils containing polyunsaturated fatty acid due to heat‐induced lipid peroxidation. The present study examined the effects of the heating time, the degree of unsaturation, and the antioxidant potential on the formation of HNE in two light olive oils (LOO) and two sunflower oils (one high oleic and one regular) at frying temperature. HNE concentrations in these oil samples heated for 0, 1, 3, and 5 hours at 185 °C were measured using high‐performance liquid chromatography. The fatty‐acid distribution and the antioxidant capacity of these four oils were also analyzed. The results showed that all oils had very low HNE concentrations (<0.5 μg g^?1 oil) before heating. After 5 hours of heating at 185 °C, HNE concentrations were increased to 17.98, 25.00, 12.51, and 40.00 μg g^?1 in the two LOO, high‐oleic sunflower oil (HOSO), and regular sunflower oil (RSO), respectively. Extending the heating time increased HNE formation in all oils tested. It is related to their fatty‐acid distributions and antioxidant capacities. RSO, which contained high levels of linoleic acid (59.60%), a precursor for HNE, was more susceptible to degradation and HNE formation than HOSO and LOO, which contained only 6–8% linoleic acid.     

4‐Hydroxynonenal (HNE), a Toxic Aldehyde in French Fries from Fast Food Restaurants

The toxic lipid peroxidation product, α,β,4‐hydroxy‐2‐trans‐nonenal (HNE) concentration, was measured in French fries (FF) from six local fast food restaurants. FF were purchased between 2 and 3 pm from all six restaurants. FF were also purchased at 12, 2, 4, 6 pm from one and at 1, 3, 5, 7 pm from another restaurant. Samples were analyzed for total fat, fatty acid distribution and for HNE by high performance liquid chromatography (HPLC). HNE was confirmed by HPLC/MS. HNE concentrations in FF from the 6 fast food restaurants were between 7.83 and 32.15 µg HNE/100 g FF and between 0.9 and 4.9 µg HNE/g extracted fat. HNE concentrations in FF purchased at 12, 2, 4, 6 pm were between 19.07 and 32.15 µg/g of FF and purchased at 1, 3, 5, 7 pm were between 7.47 and 10.21 µg HNE/100 g of FF. Differences in FA distribution were observed in the samples from some restaurants. FF which contained higher levels of linoleic acid (LA) also contained more HNE. It is clear that HNE is produced during the heating process of the frying oils and is incorporated into FF. Frequently consumed foods containing considerable amounts of HNE, a toxic aldehyde, may be a public health concern since HNE toxicity is related to a number of common pathological conditions.     

The Synthesis of trans‐Perhydroindolic Acids and their Application in Asymmetric Domino Reactions of Aldehyde Esters with β,γ‐Unsaturated α‐Keto Esters

(2S,3aR,7aS)‐Perhydroindolic acid, the key intermediate in the synthesis of trandolapril, and its trans‐isomers, were readily prepared. These proline‐like molecules are unique in that they contain a rigid bicyclic structure, with two hydrogen atoms trans to each other at the bridgehead carbon atoms. These molecules were used successfully as chiral organocatalysts in asymmetric domino Michael addition/cyclization reactions of aldehyde esters with β,γ‐unsaturated α‐keto esters. They proved to have excellent catalytic behavior, allowing for the synthesis of multi‐substituted, enantiomerically enriched hemiacetal esters. Under optimal conditions (using 10 mol% catalyst loading), a series of β,γ‐unsaturated α‐keto esters was examined with up to 99% de, ee and yield, respectively. Additionally, the enantiomerically enriched hemiacetal esters could be readily transformed into their corresponding bioactive pyrano[2,3‐b]pyrans (possessing a multi‐substituted bicyclic backbone).     

Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.

     

Lipase‐mediated hydrolysis of flax seed oil for selective enrichment of α‐linolenic acid

Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation.     

The effect of intermittent and continuous heating of soybean oil at frying temperature on the formation of 4-hydroxy-2-trans-nonenal and other α-, β-unsaturated hydroxyaldehydes

4-Hydroxy-2-trans-nonenal (HNE) is a cytotoxic secondary lipid peroxidation product of linoleic acid. Previous investigations in this laboratory showed that HNE is formed in thermally oxidized soybean oil, which is high in linoleic acid. Continuous exposure of the oil to frying temperature (185°C) for up to 6 h graduallyincreased the formation of HNE and other polar lipophilic aldehydes. Additional investigations in this laboratory showed that HNE is absorbed into food fried in thermally oxidized oil in the same concentration as was found in the oil. In the present experiment, the effect of intermittent heating on the formation of HNE in soybean oil was compared with continuous heating. Soybean oil samples were heated either for 1 h each day for five sequential days or for 5 h continuously at 185±5°C. The thermally oxidized soybean oil samples were analyzed by HPLC for the presence of HNE and three other polar lipophilic α-,β-unsaturated hydroxyaldehydes: 4-hydroxy-2-trans-hexanal, 4-hydroxy-2-trans-octenal, and 4-hydroxy-2-trans-decenal. Under intermittent and continuous heating over a total of 5 h, the concentration of these compounds increased similarly. These results indicate that the formation of HNE and other hydroxyaldehydes at frying temperature is a cumulative result of oxidation of PUFA over time.     

Regio‐ and Enantioselective Copper‐Catalyzed 1,4‐Conjugate Addition of Trimethylaluminium to Linear α,β,γ,δ‐Unsaturated Alkyl Ketones

A regio‐ and enantioselective copper‐catalyzed 1,4‐conjugate addition of trimethylaluminium to linear δ‐aryl‐substituted α,β,γ,δ‐unsaturated alkyl ketones was developed. A series of γ,δ‐unsaturated alkyl ketones were obtained in good yields with high regio‐ and enantioselectivity (up to 88% ee and 96:4 dr). Expansion of the reaction scope to substrates containing aromatic heterocycles also afforded good yields and enantioselectivities (up to 91% ee) with very high regioselectivities, exclusively providing the single 1,4‐products.

     

Asymmetric α‐Hydroxylation of β‐Indanone Esters and β‐Indanone Amides Catalyzed by C‐2′ Substituted Cinchona Alkaloid Derivatives

The highly catalytic asymmetric α‐hydroxylation of β‐indanone esters and β‐indanone amides using peroxide as the oxidant was realized with a new C‐2′ substituted Cinchona alkaloid derivatives. The two enantiomers of α‐hydroxy‐β‐indanone esters could be obtained by simply changing the oxidant. This protocol allows a convenient access to the corresponding α‐hydroxy‐β‐indanone esters and α‐hydroxy‐β‐indanone amides with up to 99% yield and 98% ee.

     

Geometrical isomerization of polyunsaturated fatty acids in physically refined rapeseed oil during plant‐scale deodorization

The effect of the operating temperature (between 220 and 270 °C) on the formation of trans isomers of linoleic and linolenic acids in physically refined rapeseed oil during deodorization in a plant‐scale semicontinuous tray‐type deodorizer (capacity 10 t/h) was investigated. The industrial procedures of physical refining consisted of a two‐step bleaching and deodorization process. The degree of isomerization of linoleic acid ranged from 0.33 to 4.77% and that of linolenic acid from 4.43 to 45.22% between 220 and 270 °C, respectively. A relation between the logarithm of the degree of isomerization and the deodorization temperature can be approximated by statistically highly significant linear functions for both linoleic and linolenic acids. Oleic acid was resistant to the heat‐induced geometrical isomerization. The values found for the ratio between the degrees of isomerization of linolenic and linoleic acids, slightly decreasing with increasing temperature, were equal to 13.6 and 12.9 at 230 and 240 °C, respectively. Two trans isomers of linoleic acid, exclusively with one double bond isomerized into trans configuration, and four trans isomers of linolenic acid, mostly with one double bond isomerized into trans configuration, were determined in deodorized rapeseed oils. Linolenic acid was observed to be the main source responsible for the formation of nearly all trans fatty acids in physically refined rapeseed oil. At 235 °C, a deodorization temperature considered as a reasonable technological compromise, the content of trans fatty acids in plant‐scale physically refined rapeseed oil was less than 1% of total fatty acids, which would be acceptable for further application.     

Monitoring the oxidation of unsaturated oils and formation of oxygenated aldehydes by proton NMR

The oxidation process of rapeseed, walnut and linseed oils, rich sources of linolenic acyl groups, was studied by means of ¹H nuclear magnetic resonance spectroscopy. The visible changes in some spectrum signals, especially in those of methylic, allylic and bis‐allylic hydrogen atoms, provide information about the degradation of the different acyl groups throughout the oxidation process. The rate of the changes produced in the three oils was compared, not only in relation to the rate of degradation of the different acyl groups, but also to both the formation and degradation of primary as well as to the formation of secondary oxidation products. Among the secondary oxidation products, the study was focused on the formation of aldehydes, their nature and evolution throughout the oxidation process of these three oils. The formation of n‐alkanals, trans‐2‐alkenals, trans,trans‐2,4‐alkadienals, as well as of the well known geno‐ and cytotoxic 4,5‐epoxy‐, 4‐hydroperoxy‐ and 4‐hydroxy‐trans‐2‐alkenals, was proved to take place.     

A Simple Organocatalytic Enantioselective Cyclopropanation of α,β‐Unsaturated Aldehydes

A highly chemo‐ and enantioselective organocatalytic cyclopropanation of α,β‐unsaturated aldehydes with bromomalonate and 2‐bromoacetoacetate esters is presented. The reaction is catalyzed by chiral amines and gives access to 2‐formylcyclopropanes in high yields and up to 99 % ee.     

Highly Enantioselective Phase‐Transfer Catalytic α‐Alkylation of α‐tert‐Butoxycarbonyllactams: Construction of β‐Quaternary Chiral Pyrrolidine and Piperidine Systems

A new enantioselective α‐alkylation of α‐tert‐butoxycarbonyllactams for the construction of β‐quaternary chiral pyrrolidine and piperidine core systems is reported. α‐Alkylations of N‐methyl‐α‐tert‐butoxycarbonylbutyrolactam and N‐diphenylmethyl‐α‐tert‐butoxycarbonylvalerolactam under phase‐transfer catalytic conditions (solid potassium hydroxide, toluene, −40 °C) in the presence of (S,S)‐3,4,5‐trifluorophenyl‐3,3′,5,5′‐tetrahydro‐2,6‐bis(3,4,5‐trifluorophenyl)‐4,4′‐spirobi[4H‐dinaphth[2,1‐c:1′,2′‐e]azepinium] bromide [(S,S)‐NAS Br] (5 mol%) afforded the corresponding α‐alkyl‐α‐tert‐butoxycarbonyllactams in very high chemical (up to 99%) and optical yields (up to 98% ee). Our new catalytic systems provide attractive synthetic methods for pyrrolidine‐ and piperidine‐based alkaloids and chiral intermediates with β‐quaternary carbon centers.     

Enantioselective Biooxidation of Racemic trans‐Cyclic Vicinal Diols: One‐Pot Synthesis of Both Enantiopure (S,S)‐Cyclic Vicinal Diols and (R)‐α‐Hydroxy Ketones

Highly regio‐ and enantioselective alcohol dehydrogenases BDHA (2,3‐butanediol dehydrogenase from Bacillus subtilis BGSC1A1), CDDHPm (cyclic diol dehydrogenase from Pseudomonas medocina TA5), and CDDHRh (cyclic diol dehydrogenase from Rhodococcus sp. Moj‐3449) were discovered for the oxidation of racemic trans‐cyclic vicinal diols. Recombinant Escherichia coli expressing BDHA was engineered as an efficient whole‐cell biocatalyst for the oxidation of (±)‐1,2‐cyclopentanediol, 1,2‐cyclohexanediol, 1,2‐cycloheptane‐diol, and 1,2‐cyclooctanediol, respectively, to give the corresponding (R)‐α‐hydroxy ketones in >99% ee and (S,S)‐cyclic diols in >99% ee at 50% conversion in one pot. Escherichia coli (BDHA‐LDH) co‐expressing lactate dehydrogenase (LDH) for intracellular regeneration of NAD⁺ catalyzed the regio‐ and enantioselective oxidation of (±)‐1,2‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene to produce the corresponding (R)‐α‐hydroxy ketone in >99% ee and (S,S)‐cyclic diol in 96% ee at 49% conversion. Preparative biotransformations were also demonstrated. Thus, a novel and useful method for the one‐pot synthesis of both vicinal diols and α‐hydroxy ketones in high ee was developed via highly regio‐ and enantioselective oxidations of the racemic vicinal diols.

     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Synthesis and characterization of degradable copoly(ester–amide)s: Poly(trans‐4‐hydroxy‐L‐proline‐co‐ε‐caprolactam)

Novel copolyesteramides were synthesized by reacting trans‐4‐hydroxy‐N‐benzyloxycarbonyl‐L ‐proline (N‐CBz‐Hpr) with ε‐caprolactam (CLM) in the presence of stannous octoate [Sn(II) Oct.] as a catalyst. Various techniques, including ¹H‐NMR, IR, DSC, and viscosity, were used to elucidate structural characteristics and thermal properties of the resulting copolymers. Data showed that the optimal reaction condition for the synthesis of the copolymers was obtained by using 3 wt % Sn(II) Oct. at 170°C for 24 h. The DSC analysis demonstrated amorphous structure for most of the copolymers. The glass‐transition temperature of the copolymers shifts to a higher temperature with increasing Hpr/CLM molar ratio. In vitro degradation of these poly(N‐CBz‐Hpr‐co‐CLM)s was evaluated by weight loss measurements. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1615–1621, 2002     

Kinetic study of β‐carotene and lutein degradation in oils during heat treatment

The kinetics of trans‐β‐carotene and trans‐lutein degradation were individually investigated in palm olein and Vegetaline®, at four temperatures ranging from 120 to 180 °C. HPLC‐DAD analysis was carried out to monitor trans and cis carotenoid variations over the heating time at each temperature. In both oils, initial trans‐β‐carotene and trans‐lutein degradation rates increased with temperature. Trans‐lutein was found to degrade at a slower rate than trans‐β‐carotene, suggesting a higher thermal resistance. The isomers identified were 13‐cis‐ and 9‐cis‐β‐carotene, and 13‐cis‐, 9‐cis‐, 13'‐cis‐, and 9'‐cis‐lutein. In spite of the higher number of lutein cis isomers, their total amount was lower than that of β‐carotene cis isomers. Trans and cis carotenoids were involved in degradation reactions at rates that increased with temperature. All degradation rates were generally found to be lower in Vegetaline® than in palm olein. These results were explained by the initial composition of the two oils and especially their peroxide and vitamin E contents.     

Organocatalytic Asymmetric Mannich Reactions of 5H‐Oxazol‐4‐ones: Highly Enantio‐ and Diastereoselective Synthesis of Chiral α‐Alkylisoserine Derivatives

The first organocatalytic Mannich reaction of 5H‐oxazol‐4‐ones with various readily prepared aryl‐ and alkylsulfonimides has been developed. Two commercially available pseudoenantiomeric Cinchona alkaloids‐derived tertiary amine/ureas have been demonstrated as the most efficient catalysts to access the opposite enantiomers of the Mannich products with equally excellent enantio‐ and diastereoselectivities. From the Mannich adducts, important α‐methyl‐α‐hydroxy‐β‐amino acid derivatives, such as the α‐methylated C‐13 side chain of taxol and taxotere, can be conveniently prepared.     

Asymmetric Aza‐Morita–Baylis–Hillman‐Type Reactions: The Highly Enantioselective Reaction between Unmodified α,β‐ Unsaturated Aldehydes and N‐Acylimines by Organo‐co‐catalysis

The highly enantioselective organo‐co‐catalytic aza‐Morita–Baylis–Hillman (MBH)‐type reaction between N‐carbamate‐protected imines and α,β‐unsaturated aldehydes has been developed. The organic co‐catalytic system of proline and 1,4‐diazabicyclo[2.2.2]octane (DABCO) enables the asymmetric synthesis of the corresponding N‐Boc‐ and N‐Cbz‐protected β‐amino‐α‐alkylidene‐aldehydes in good to high yields and up to 99% ee. In the case of aza‐MBH‐type addition of enals to phenylprop‐2‐ene‐1‐imines, the co‐catalytic reaction exhibits excellent 1,2‐selectivity. The organo‐co‐catalytic aza‐MBH‐type reaction can also be performed by the direct highly enantioselective addition of α,β‐unsaturated aldehydes to bench‐stable N‐carbamate‐protected α‐amidosulfones to give the corresponding β‐amino‐α‐alkylidene‐aldehydes with up to 99% ee. The organo‐co‐catalytic aza‐MBH‐type reaction is also an expeditious entry to nearly enantiomerically pure β‐amino‐α‐alkylidene‐amino acids and β‐amino‐α‐alkylidene‐lactams (99% ee). The mechanism and stereochemistry of the chiral amine and DABCO co‐catalyzed aza‐MBH‐type reaction are also discussed.     

N,N′‐Dioxide‐Magnesium Ditriflate Complex‐Catalyzed Asymmetric α‐Hydroxylation of β‐Keto Esters and β‐Keto Amides

The highly catalytic asymmetric α‐hydroxylation of 1‐tetralone‐derived β‐keto esters and β‐keto amides using tert‐butyl hydroperoxide (TBHP) as the oxidant was realized by a chiral N,N′‐dioxide‐magnesium ditriflate [Mg(OTf)₂] complex. A series of corresponding chiral α‐hydroxy dicarbonyl compounds was obtained in excellent yields (up to 99%) with excellent enantioselectivities (up to 98% ee). The products were easily transformed into useful building blocks and the precursor of daunomycin was achieved in an asymmetric catalytic way for the first time.