Antioxidative long chain alkylresorcinols. Synthesis and deuterium labelling of bioactive compounds present in whole grains

The first synthesis of long chain 5-n-alkylresorcinols (C15-C25) in whole grains and whole grain products by a novel modification of Wittig reaction is described. 5-n-Alkylresorcinols are phenolic lipids that have various effects on biological systems, such as antioxidant activity and interaction with biological membranes. These compounds are considered as biomarkers of whole grain intake, which is connected with reduced risk of cardiovascular diseases and certain cancers. Novel hapten derivatives of 5-n-alkylresorcinols, potential compounds for immunoanalytical techniques, are prepared by the same procedure utilizing microwave catalysed aqueous Wittig reaction as the key step. The synthesised analogues are required by various analytical, metabolism and bioactivity investigations. Four alternative strategies for producing deuterium polylabelled 5-n-alkylresorcinols are explored. Ring-labelled D3-alkylresorcinols were synthesized by acidic H/D exchange. Side chain -labelled D4-derivative was prepared by a total synthesis approach utilizing D2 deuterogenation of a D2-alkene derivative, and deuterogenation of alkynes was investigated in another total synthesis approach. An -D3-labelled alkylresorcinol is isotopically pure and completely stable under all relevant conditions encountered during analytical work. The labelling of another phenolic component of whole grains was explored. The preparation of D3-ferulic acid and related compounds by way of selective methylation of the precursors is described. The deuterated compounds are useful as standards in the quantification of these natural products in various substances, such as food and human fluids. The pure 5-n-alkylresorcinol analogues prepared were used in in vitro experiments on alkylresorcinol antioxidant activity and antigenotoxicity. The in vitro experiments show that alkylresorcinols act as antioxidants, especially when incorporated into biological systems, but possess lower activity in chemical tests (FRAP and DPPH assay). Whole grain alkylresorcinols are shown for the first time to have a protective effect against copper induced oxidation of LDL, and H2O2 or genotoxic faecal water induced damage on HT29 cells.

Autoxidation of conjugated linoleic acid methyl ester in the presence of α-tocopherol: the hydroperoxide pathway

The autoxidation of conjugated linoleic acid (CLA) is poorly understood in spite of increasing interest in the beneficial biological properties of CLA and growing consumption of CLA-rich foods. In this thesis, the autoxidation reactions of the two major CLA isomers, 9-cis,11-trans-octadecadienoic acid and 10-trans,12-cis-octadecadienoic acid, are investigated. The results contribute to an understanding of the early stages of the autoxidation of CLA methyl ester, and provide for the first time a means of producing and separating intact CLA methyl ester hydroperoxides as well as basic knowledge on lipid hydroperoxides and their hydroxy derivatives. Conjugated diene allylic monohydroperoxides were discovered as primary autoxidation products formed during autoxidation of CLA methyl esters in the presence and absence of α-tocopherol. This established that one of the autoxidation pathways of CLA methyl ester is the hydroperoxide pathway. Hydroperoxides were produced from the two major CLA methyl esters by taking advantage of the effect of α-tocopherol to promote hydroperoxide formation. The hydroperoxides were analysed and separated first as methyl hydroxyoctadecadienoates and then as intact hydroperoxides by HPLC. The isolated products were characterized by UV, GC-MS, and NMR techniques. In the presence of a high amount of α-tocopherol, the autoxidation of CLA methyl ester yields six kinetically-controlled conjugated diene monohydroperoxides and is diastereoselective in favour of one particular geometric isomer as a pair of enantiomers. The primary autoxidation products produced from the two major CLA isomers include new positional isomers of conjugated diene monohydroperoxides, the 8-, 10-, 12-, and 14-hydroperoxyoctadecadienoates. Furthermore, two of these new positional isomers have an unusual structure for a cis,trans lipid hydroperoxide where the allylic methine carbon is adjacent to the cis instead of the usual trans double bond. The 1H and 13C NMR spectra of nine isomeric methyl hydroxyoctadecadienoates and of ten isomeric methyl hydroperoxyoctadecadienoates including the unusual cis,trans hydroperoxides, i.e. Me 8-OOH-9c,11t and Me 14-OOH-10t,12c, were fully assigned with the aid of 2D NMR spectroscopy. The assigned NMR data enabled determination of the effects of the hydroxyl and hydroperoxyl groups on the carbon chemical shifts of CLA isomers, identification of diagnostic signals, and determination of chemical shift differences of the olefinic resonances that may help with the assignment of structure to as yet unknown lipid hydroperoxides either as hydroxy derivatives or as intact hydroperoxides. A mechanism for the hydroperoxide pathway of CLA autoxidation in the presence of a high amount of α-tocopherol was proposed based on the characterized primary products, their relative distribution, and theoretical calculations. This is an important step forward in CLA research, where exact mechanisms for the autoxidation of CLA have not been presented before. Knowledge of these hydroperoxide formation steps is of crucial importance for understanding the subsequent steps and the different pathways of the autoxidation of CLA. Moreover, a deeper understanding of the autoxidation mechanisms is required for ensuring the safety of CLA-rich foods. Knowledge of CLA oxidation and how it differs from the oxidation of nonconjugated polyunsaturated fatty acids may also be the key to understanding the biological mechanisms of CLA activity.

Synthesis of Isoflavone Conjugates

During this study different approaches were studied to obtain isoflavone sulphates, glucuronides and sulphoglucuronides. Three isoflavone disulphates (daidzein-di-O-sulphate, genistein-di-O-sulphate and glycitein-di-O-sulphate) and three isoflavonoid disulphates (dihydrodaidzein-di-O-sulphate, dihydrogenistein-di-O-sulphate and equol-di-O-sulphate) were synthesised in moderate yields by using in situ prepared pyridine sulphur trioxide complex, made from chlorosulphonic acid and pyridine. These disulphated compounds can be used to develop analytical procedures and study the biological activity of disulphated products. As the use of the HPLC-MS methods in the field of isoflavones has increased its popularity, deuterated isoflavone disulphates were synthesised. A new microwave assisted deuteration method, using CF3COOD, was developed for this purpose. Three polydeuterated isoflavone disulphates (daidzein-d6-di-O-sulphate, genistein-d4-di-O-sulphate and glycitein-d6-di-O-sulphate) were obtained in moderate yields with high isotopic purity. A synthetic method was developed for daidzein sulphoglucuronide (daidzein-7-O-b-D-glucuronide-4´-O-sulphate), which is a major metabolite in rat bile. By using protection/deprotection steps, the desired product was finally obtained in moderate yield. The method developed can be used in further studies of synthesis of isoflavonoid mixed conjugates. As a part of this study, the structure of naturally occurring daidzein-4´-O-b-glucoside was verified. Different glycosidation methods are reviewed and possible factors affecting the stereoselectivity are discussed. The study of the selective chlorination of isoflavones was a consequence of the observed unexpected chlorination during the synthesis of isoflavone acid chlorides by thionyl chloride. This fascinating phenomenon was investigated further with various isoflavones and as a result a method for producing isoflavone chlorides (8-chlorogenistein, 6,8-dichlorogenistein and 6,8-dichlorobiochanin A) was developed. Protecting groups played a great role during this study, which led to an intensive study on them. A regioselective protection method was developed by using direct introduction of the protecting group (Benzyl and Benzoyl) to positions 7-O or 4´-O in daidzein, genistein and glycitein with t-BuOK as a base in DMF in moderate yields. The possibility of exploiting the transesterification was also investigated. It was observed that by using K2CO3 as a base in DMF, daidzein, genistein and glycitein could be benzoylated at position 4´-O selectively, in the presence of the more acidic 7 hydroxy group. Transesterification also proved to be useful in the glycosidation of isoflavones at position 7-O, starting from 7-O-benzoylated isoflavones. Different carboxylic acid derivatives were synthesised for use either in the development of radioimmunoassay (7-O-carboxymethylglycitein and 4´-O-carboxymethylglycitein) or synthesis of daunorubicin isoflavone derivative for biological testing (7-O-carboxypropylbiochanin A and 7-O-carboxypropylgenistein).

Recovery responses of acidified Finnish lakes under declining acid deposition

The present work provides a regional-scale assessment of the changes in acidifying deposition in Finland over the past 30 years and the current pattern in the recovery of acid-sensitive lakes from acidification in relation to changes in sulphate deposition. This information is needed for documenting the ecosystem benefits of costly emission reduction policies and further actions in air pollution policy. The development of sulphate deposition in Finland reflects that of European SO2 emissions. Before the 1990s, reductions in sulphur emissions in Europe had been relatively small and sulphate deposition showed no consistent trends. Due to emission reduction measures that were then taken, sulphate deposition started to clearly decline from the late 1980s. The bulk deposition of sulphate has declined 40-60% in most parts of the country during 1990-2003. The decline in sulphate deposition exceeded the decline of base cation deposition, which resulted in a decrease in acidity and acidifying potential of deposition over the 1990s. Nitrogen deposition also decreased since the late 1980s, but less than that of sulphate, and levelling off during the 1990s. Sulphate concentrations in all types of small lakes throughout Finland have declined from the early 1990s. The relative decrease in lake sulphate concentrations (average 40-50%) during 1990-2003 was rather similar to the decline in sulphate deposition, indicating a direct response to the reduction in deposition. There are presently no indications of elevated nitrate concentrations in forested headwater lakes. Base cation concentrations are still declining in many lakes, especially in south Finland, but to a lesser extent than sulphate allowing buffering capacity (alkalinity) to increase, being significant in 60% of the study lakes. Chemical recovery is resulting in biological recovery with populations of acid-sensitive fish species increasing. The recovery has been strongest in lakes in which sulphate has been the major acidifying agent, and recovery has been the strongest and most consistent in lakes in south Finland. The recovery of lakes in central Finland and north Finland is not as widespread and strong as observed in south. Many catchments, particularly in central Finland, have a high proportion of peatlands and therefore high TOC concentrations in lakes, and runoff-induced surges of organic acids have been an important confounding factor suppressing the recovery of pH and alkalinity in these lakes. Chemical recovery is progressing even in the most acidified lakes, but the buffering capacity of many lakes is still low and still sensitive to acidic input. Further reduction in sulphur emissions are needed for the alkalinity to increase in the acidified lakes. Increasing total organic carbon (TOC) concentrations are indicated in small forest lakes in Finland. The trends appear to be related to decreasing sulphate deposition and improved acid-base status of the soil, and the rise in TOC is integral to recovery from acidification. A new challenge is climate change with potential trends in temperature, precipitation and runoff, which are expected to affect future chemical and biological recovery from acidification. The potential impact on the mobilization and leaching of organic acids may become particularly important in Finnish conditions. Long-term environmental monitoring has evidently shown the success of international emission abatement strategies. The importance and value of integrated monitoring approach including physical, chemical and biological variables is clearly indicated, and continuous environmental monitoring is needed as a scientific basis for further actions in air pollution policy.

Genistein and 17β-estradiol fatty acid esters in blood and adipose tissue and the structure-related antioxidant activity of estrogens on lipoproteins

Soy-derived phytoestrogen genistein and 17β-estradiol (E2), the principal endogenous estrogen in women, are also potent antioxidants protecting LDL and HDL lipoproteins against oxidation. This protection is enhanced by esterification with fatty acids, resulting in lipophilic molecules that accumulate in lipoproteins or fatty tissues. The aims were to investigate, whether genistein becomes esterified with fatty acids in human plasma accumulating in lipoproteins, and to develop a method for their quantitation; to study the antioxidant activity of different natural and synthetic estrogens in LDL and HDL; and to determine the E2 esters in visceral and subcutaneous fat in late pregnancy and in pre- and postmenopause. Human plasma was incubated with [3H]genistein and its esters were analyzed from lipoprotein fractions. Time-resolved fluoroimmunoassay (TR-FIA) was used to quantitate genistein esters in monkey plasma after subcutaneous and oral administration. The E2 esters in women s serum and adipose tissue were also quantitated using TR-FIA. The antioxidant activity of estrogen derivatives (n=43) on LDL and HDL was assessed by monitoring the copper induced formation of conjugated dienes. Human plasma was shown to produce lipoprotein-bound genistein fatty acid esters, providing a possible explanation for the previously reported increased oxidation resistance of LDL particles during intake of soybean phytoestrogens. Genistein esters were introduced into blood by subcutaneous administration. The antioxidant effect of estrogens on lipoproteins is highly structure-dependent. LDL and HDL were protected against oxidation by many unesterified, yet lipophilic derivatives. The strongest antioxidants had an unsubstituted A-ring phenolic hydroxyl group with one or two adjacent methoxy groups. E2 ester levels were high during late pregnancy. The median concentration of E2 esters in pregnancy serum was 0.42 nmol/l (n=13) and in pre- (n=8) and postmenopause (n=6) 0.07 and 0.06 nmol/l, respectively. In pregnancy visceral fat the concentration of E2 esters was 4.24 nmol/l and in pre- and postmenopause 0.82 and 0.74 nmol/l. The results from subcutaneous fat were similar. In serum and fat during pregnancy, E2 esters constituted about 0.5 and 10% of the free E2. In non-pregnant women most of the E2 in fat was esterified (the ester/free ratio 150 - 490%). In postmenopause, E2 levels in fat highly exceeded those in serum, the majority being esterified. The pathways for fatty acid esterification of steroid hormones are found in organisms ranging from invertebrates to vertebrates. The evolutionary preservation and relative abundance of E2 esters, especially in fat tissue, suggest a biological function, most likely in providing a readily available source of E2. The body s own estrogen reservoir could be used as a source of E2 by pharmacologically regulating the E2 esterification or hydrolysis.

Quantum Chemical Studies on Tropospheric Nucleation Mechanisms Involving Sulfuric Acid

Nucleation is the first step of the process by which gas molecules in the atmosphere condense to form liquid or solid particles. Despite the importance of atmospheric new-particle formation for both climate and health-related issues, little information exists on its precise molecular-level mechanisms. In this thesis, potential nucleation mechanisms involving sulfuric acid together with either water and ammonia or reactive biogenic molecules are studied using quantum chemical methods. Quantum chemistry calculations are based on the numerical solution of Schrödinger's equation for a system of atoms and electrons subject to various sets of approximations, the precise details of which give rise to a large number of model chemistries. A comparison of several different model chemistries indicates that the computational method must be chosen with care if accurate results for sulfuric acid - water - ammonia clusters are desired. Specifically, binding energies are incorrectly predicted by some popular density functionals, and vibrational anharmonicity must be accounted for if quantitatively reliable formation free energies are desired. The calculations reported in this thesis show that a combination of different high-level energy corrections and advanced thermochemical analysis can quantitatively replicate experimental results concerning the hydration of sulfuric acid. The role of ammonia in sulfuric acid - water nucleation was revealed by a series of calculations on molecular clusters of increasing size with respect to all three co-ordinates; sulfuric acid, water and ammonia. As indicated by experimental measurements, ammonia significantly assists the growth of clusters in the sulfuric acid - co-ordinate. The calculations presented in this thesis predict that in atmospheric conditions, this effect becomes important as the number of acid molecules increases from two to three. On the other hand, small molecular clusters are unlikely to contain more than one ammonia molecule per sulfuric acid. This implies that the average NH3:H2SO4 mole ratio of small molecular clusters in atmospheric conditions is likely to be between 1:3 and 1:1. Calculations on charged clusters confirm the experimental result that the HSO4- ion is much more strongly hydrated than neutral sulfuric acid. Preliminary calculations on HSO4- NH3 clusters indicate that ammonia is likely to play at most a minor role in ion-induced nucleation in the sulfuric acid - water system. Calculations of thermodynamic and kinetic parameters for the reaction of stabilized Criegee Intermediates with sulfuric acid demonstrate that quantum chemistry is a powerful tool for investigating chemically complicated nucleation mechanisms. The calculations indicate that if the biogenic Criegee Intermediates have sufficiently long lifetimes in atmospheric conditions, the studied reaction may be an important source of nucleation precursors.