Introduction. 5-HMF and novel INN-compounds are left-shifting AEH, shown to have anti-sickling action by forming transiently covalent Schiff-base adducts with hemoglobin (Hb), thereby increasing the Hb O2-affinity. They are hypothesized to be substrates for aldehyde dehydrogenase (ALDH) in the liver and red blood cells (RBC). Methods. Biopharmaceutical assessments were made for AEH, using calculated physicochemical properties. Their in-vitro hepatic metabolism (mediated by ALDH) was characterized using hepatic cytosol, and in-vitro-in-vivo extrapolations (IVIVE) were made. Inter-species differences in hepatic cytosolic ALDH activity were investigated using acetaldehyde as a model substrate in different mammalian species. Time- and concentration-dependent in-vitro disposition of 5-HMF in human whole blood was fully characterized and quantitatively modeled. In-vitro time- and concentration-dependent pharmacodynamic (PD) profiling of AEH (0.5 – 5 mM) was carried out in normal whole blood. 5-HMF binding to (normal) HbA and (sickle) HbS was studied in systematic time- and concentration-dependency studies using isolated Hb solutions. Quantitative PK/PD models were developed to fit the experimental data by nonlinear regression (Scientist®). Results. 5-HMF and the two INN-compounds were classified as BCS-I and BCS-II, respectively. All AEH were substrates for hepatic ALDH, with predicted low/intermediate hepatic extraction. Intrinsic ALDH activity varied significantly between mammalian species. In whole blood, 5-HMF plasma concentrations declined rapidly (t1/2 of 0.8 – 4 hrs), with nonlinear kinetics, due to saturable Hb-binding. AEH showed a time-dependent, biphasic PD effect in whole blood, suggesting transiently covalent Hb binding, with slow recovery to the baseline, corresponding to dissociation from Hb and subsequent metabolism by RBC-ALDH. Binding studies with HbA and HbS demonstrated slight differences in binding affinity, but sustained adduct formation - with slow dissociation t1/2. A novel semi-mechanistic target-site drug disposition (TSDD)/PD model was developed, integrating the information, for simultaneous modeling of 5-HMF concentrations in plasma, and its effect in whole blood. Conclusions. This translational research investigated in detail the in-vitro PK/PD of AEH, and systematically compared findings with older generation compounds. A (generic) novel TSDD/PD model was developed for disposition of AEH, identifying k-1 (dissociation constant of AEH from Hb) and kmet (RBC-ALDH metabolism rate constant) as key properties for the time course of PD effect.

Currently, the major sources of fuel, energy, and chemicals are nonrenewable fossil resources such as petroleum, natural gas, and coal. Additionally, petroleum is used for the production of most transportation fuels and for the production of about 95% of organic chemicals. However, the production and use of non-renewable fossil fuels are unsustainable. For economic and environmental sustainability, there is a need to search for new and/or renewable resources and technologies for energy, fuels, and chemicals production that have the potential of effectively substituting fossil resources. In this context, lignocellulosic biomass is one of the candidates that meet these requirements due to its abundance and renewability. Lignocellulosic biomass-derived sugars can be chemically converted into 5-(hydroxymethyl)furfural (HMF), a versatile platform chemical that can used to generate intermediates for the production of biofuels and chemicals. In this dissertation novel catalytic processes for converting monosaccharides into HMF are described. In chapter one, a literature review of the significance of HMF and the chemical intermediates derived from HMF is presented. The chapter also describes some of recent developments in the catalytic production of HMF from lignocellulosic sugars using homogeneous and heterogeneous catalysts. Chapter two discusses the catalytic activity of dimethylaluminum complexes bearing (aminomethyl)phenolate ligands that I developed for converting glucose to HMF in ionic liquids. A systematic study on the effects of modification of the aluminum ancillary ligands on the efficiency of glucose conversion is presented. High HMF yield were obtained with substitution of an aryl substituent on the amino groups of the (aminomethyl)phenolate ligands. In an effort to improve HMF yield, the effects of modifying the ligands on the phenolate moiety of the (aminomethyl)phenolate ligands were investigated in chapter three. Using bulky ortho-phenoxide substituents achieved high HMF yields. The selectivity for HMF production with respect to fructose dehydration was also discussed, together with spectroscopic characterization of the polymeric humins produced from the dehydration reactions. In chapter four a study of the structural differences of poor vs. effective dimethylaluminum complexes bearing (aminomethyl)phenolate ligands is described. Insights from this study shows that different precatalyst intermediate could be formed depending on the aluminum complex used, which in turn affects HMF selectivity of dehydration reactions. The isomerization of glucose to fructose using aluminum complexes in N-methyl-2-pyrrolidone (NMP) is discussed in chapter five. Using NMR spectroscopy on isotopically labeled glucose, a mechanism for glucose isomerization to fructose is presented. Finally, chapter six gives a summary and describes potential future directions for the research detailed in this dissertation.

Human exposure to harmful compounds in the environment, from intake via food, occupational exposures or other sources, could have health implications. Exposure to reactive compounds/metabolites can be identified and quantified as hemoglobin (Hb) adducts by mass spectrometry. This thesis aimed at improved synthetic pathways for reference standards, and improved analytical methods for adducts to N-terminal valine in Hb from a range of reactive compounds; isocyanates, aldehydes, methyl vinyl ketone (MVK), and diepoxybutane (DEB). Isocyanates form urea adducts with N-terminal valine by carbamoylation, which are detachable as hydantoins by hydrolysis. A new synthetic pathway for reference standards of adducts from isocyanates and a method for their analysis by liquid chromatography/mass spectrometry (LC/MS) were developed. Aldehydes form reversible imines (Schiff bases) with N-termini in Hb. After stabilisation by reduction and detachment by isothiocyanates using modified Edman methods, these adducts could be analysed by gas chromatography/mass spectrometry (GC/MS) or LC/MS. 5-Hydroxymethylfurfural, its metabolites, and other aldehydes related to exposure via food, were studied with regard to analysis by these methods with synthesised standard references. A considerably improved analytical method for imines was developed. Many of the studied adducts are too short-lived in vivo or in vitro to be used for long-term biomonitoring. However, different approaches for the analysis were evaluated. Through synthesised reference standards, an observed unknown adduct in blood was verified as the adduct from MVK. There exist both natural and anthropogenic sources for MVK. DEB, metabolite of butadiene, forms a cyclic adduct to valine-N. A new approach using hydrazinolysis of protein and enrichment by molecularly imprinted solid-phase extraction was tested on synthesised reference DEB-adduct and gave promising results. Synthesised standards were characterized by NMR, LC/MS and GC/MS. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.</p>

A cadeia sucroenergética tem uma grande influência para economia nacional. Ao longo da safra 2013/2014, a cadeia movimentou cerca de US$ 100 bilhões, sendo que somente o açúcar gerou uma arrecadação de cerca de US$ 5.55 bilhões as usinas. O Produto Interno Bruto chegou a US$ 43 bilhões, representando assim aproximadamente 2,0% do PIB Nacional. Todavia, o setor está atravessando uma crise econômica que foi potencializada pela queda no valor da saca de açúcar e por uma severa seca que atingiu em especial o estado de São Paulo. A diminuição de perdas se torna essencial em momentos de dificuldades e uma maneira de atingir esse objetivo é melhorando o monitoramento do processo. Nesse cenário, o presente pesquisa monitorou a degradação de sacarose ao coletar amostras em uma usina da região de Piracicaba-SP, associando como causador não somente as elevadas temperaturas, como outros fatores e medidas utilizadas pela indústria. Conforme esperado aumentou a concentração de sacarose ao longo do processo, iniciando com média de 137,69 g/L na moenda e finalizando com 812,91 g/L no açúcar que saiu do secador. Todavia também apresentou um aumento de açúcares redutores (182,38 g/L no caldo da moenda para 245,81 g/L no xarope), indicando a degradação de sacarose. Inclusive, percebe-se uma razão de inversão maior durante da evaporação do que no tratamento (0,46 e 0,28, respectivamente). A justificativa pode estar associada a possível queda no pH, a evidente utilização de maior quantidade de calor em especial no primeiro efeito, a um tempo de residência maior do que o normal nos evaporadores ou mesmo através da entrada de vapor nos corpos, sendo o último mais raro. Os outros parâmetros levantados da degradação térmica de sacarose, 5-hidroximetilfurfural, furfural e as melanoidinas apresentaram uma alta correlação em função da concentração de açúcares redutores (0,99 para o HMF, 0,98 furfural e 0,96 melanoidinas), confirmando a tendência de perdas da molécula de interesse. Também foram levantadas as populações de bactérias mesófilas totais, cujas presenças estão associadas com as condições sanitárias em que o alimento foi processado e fungos (bolores e leveduras) que também diminuem o valor do produto. O tratamento de caldo mostrou-se eficiente na diminuição dos micro-organismos. Considerando a média dos dois meses, a queda foi de 4,10 x 106 UFC/mL para 6,62x102 UFC/mL para bactérias mesófilas, já para fungos a redução foi de 1,40 x 104 UFC/mL para 1,80 x 101 UFC/mL. Todavia, as altas concentrações encontradas no início e no final (bactérias apresentaram em média 3,00 x 103 UFC/g no xarope e 2,26 x 103 UFC/g no açúcar do secador, enquanto que para os fungos foram encontrados 3,50 x 101 UFC/g no xarope e 1,49 x 103 UFCmL no açúcar do secador) são indicativos de descuidos com a assepsia, aproveitando a falta de exigência de qualidade no açúcar VHP. A alta concentração do caldo da moenda apresentou potencial de estar relacionado com a perda de sacarose. Por fim, ao avaliar a situação e considerando que investimento em equipamentos novos é algo fora da realidade financeira da usina, sugere-se trabalhar em aprimorar as operações realizadas dentro e fora da indústria para se evitar as perdas. / The sugar-energy industry chain has a great influence on the Brazilian economy. Along the 2013/2014 crop, the industry accounted for about US$ 100 billion, and sugar alone yielded about US$ 5.55 billion to sugar mills. The industry yielded US$ 43 billion, accounting for approximately 2,0% of the Brazilian GDP. Nevertheless, the industry is facing an economic crisis aggravated by the drop prices of sugar bag and a severe drought that hit particularly the state of São Paulo in Brazil. Minimizing losses becomes essential in times of difficulties and a way to achieve it is to improve process monitoring. In this scenario, the present project monitored sucrose degradation in samples collected at sugar mill in the region of Piracicaba-SP, associating the degradation to high temperatures as well as other factors and measures used by the mill. As expected, sucrose concentration increased throughout the process, starting with an average of 137,69 g/L at milling and finishing with 812,91 g/L in sugar out of the dryer. However, there was also an increase of reducing sugars (182,38 g/L in milling juice to 245,81 g/L in syrup), indicating sucrose degradation. There was a larger reversal rate during evaporation than in the treatment (0,46 and 0,28, respectively). This is possibly attributed to a pH decrease, an evident use of greater amount of heat in particular in the first effect, a longer time than normal in evaporators or even due to steam inlet into the bodies, which is rarer. The other parameters arising from sucrose thermal degradation, 5-hydroxymethylfurfural, furfural and melanoidins, showed a high correlation with the concentration of reducing sugars (0,99 for HMF, 0,98 for furfural and 0,96 for melanoidins), confirming the trend for loss of the molecule of interest. We also investigated the total populations of mesophilic bacteria, whose presence is associated with health conditions where the food was processed, as well as fungi (molds and yeasts), which also decreases the value of the final product, may produce mycotoxins. The syrup treatment was effective to reduce microorganisms. Considering the average, the population of mesophilic bacteria decrease from 4,10 x 106 cfu/mL to 6,62 x102 cfu/mL. For fungi, the reduction was 1,40 x 104 cfu/mL to 1,80 x 101 cfu/mL. However, the high concentrations found at the beginning and end (Bacteria on average 3,00 x 103 cfu/g in syrup and 2,26 x 103 cfu/g in sugar at the dryer. Fungi 3,50 x 101 cfu/g in syrup and 1,49 x 103 cfu/mL in sugar at the dryer) of the process are indicative of oversights with asepsis, using the lack of demand for quality VHP sugar. The high concentration of juice at milling showed potential to be related sucrose loss. Finally, assessing the scenario and considering that investment in new equipment is something out of the financial reality of the sugar mill, improving the operations inside and outside the mill to avoid losses is suggested.

A cadeia sucroenergética tem uma grande influência para economia nacional. Ao longo da safra 2013/2014, a cadeia movimentou cerca de US$ 100 bilhões, sendo que somente o açúcar gerou uma arrecadação de cerca de US$ 5.55 bilhões as usinas. O Produto Interno Bruto chegou a US$ 43 bilhões, representando assim aproximadamente 2,0% do PIB Nacional. Todavia, o setor está atravessando uma crise econômica que foi potencializada pela queda no valor da saca de açúcar e por uma severa seca que atingiu em especial o estado de São Paulo. A diminuição de perdas se torna essencial em momentos de dificuldades e uma maneira de atingir esse objetivo é melhorando o monitoramento do processo. Nesse cenário, o presente pesquisa monitorou a degradação de sacarose ao coletar amostras em uma usina da região de Piracicaba-SP, associando como causador não somente as elevadas temperaturas, como outros fatores e medidas utilizadas pela indústria. Conforme esperado aumentou a concentração de sacarose ao longo do processo, iniciando com média de 137,69 g/L na moenda e finalizando com 812,91 g/L no açúcar que saiu do secador. Todavia também apresentou um aumento de açúcares redutores (182,38 g/L no caldo da moenda para 245,81 g/L no xarope), indicando a degradação de sacarose. Inclusive, percebe-se uma razão de inversão maior durante da evaporação do que no tratamento (0,46 e 0,28, respectivamente). A justificativa pode estar associada a possível queda no pH, a evidente utilização de maior quantidade de calor em especial no primeiro efeito, a um tempo de residência maior do que o normal nos evaporadores ou mesmo através da entrada de vapor nos corpos, sendo o último mais raro. Os outros parâmetros levantados da degradação térmica de sacarose, 5-hidroximetilfurfural, furfural e as melanoidinas apresentaram uma alta correlação em função da concentração de açúcares redutores (0,99 para o HMF, 0,98 furfural e 0,96 melanoidinas), confirmando a tendência de perdas da molécula de interesse. Também foram levantadas as populações de bactérias mesófilas totais, cujas presenças estão associadas com as condições sanitárias em que o alimento foi processado e fungos (bolores e leveduras) que também diminuem o valor do produto. O tratamento de caldo mostrou-se eficiente na diminuição dos micro-organismos. Considerando a média dos dois meses, a queda foi de 4,10 x 106 UFC/mL para 6,62x102 UFC/mL para bactérias mesófilas, já para fungos a redução foi de 1,40 x 104 UFC/mL para 1,80 x 101 UFC/mL. Todavia, as altas concentrações encontradas no início e no final (bactérias apresentaram em média 3,00 x 103 UFC/g no xarope e 2,26 x 103 UFC/g no açúcar do secador, enquanto que para os fungos foram encontrados 3,50 x 101 UFC/g no xarope e 1,49 x 103 UFCmL no açúcar do secador) são indicativos de descuidos com a assepsia, aproveitando a falta de exigência de qualidade no açúcar VHP. A alta concentração do caldo da moenda apresentou potencial de estar relacionado com a perda de sacarose. Por fim, ao avaliar a situação e considerando que investimento em equipamentos novos é algo fora da realidade financeira da usina, sugere-se trabalhar em aprimorar as operações realizadas dentro e fora da indústria para se evitar as perdas. / The sugar-energy industry chain has a great influence on the Brazilian economy. Along the 2013/2014 crop, the industry accounted for about US$ 100 billion, and sugar alone yielded about US$ 5.55 billion to sugar mills. The industry yielded US$ 43 billion, accounting for approximately 2,0% of the Brazilian GDP. Nevertheless, the industry is facing an economic crisis aggravated by the drop prices of sugar bag and a severe drought that hit particularly the state of São Paulo in Brazil. Minimizing losses becomes essential in times of difficulties and a way to achieve it is to improve process monitoring. In this scenario, the present project monitored sucrose degradation in samples collected at sugar mill in the region of Piracicaba-SP, associating the degradation to high temperatures as well as other factors and measures used by the mill. As expected, sucrose concentration increased throughout the process, starting with an average of 137,69 g/L at milling and finishing with 812,91 g/L in sugar out of the dryer. However, there was also an increase of reducing sugars (182,38 g/L in milling juice to 245,81 g/L in syrup), indicating sucrose degradation. There was a larger reversal rate during evaporation than in the treatment (0,46 and 0,28, respectively). This is possibly attributed to a pH decrease, an evident use of greater amount of heat in particular in the first effect, a longer time than normal in evaporators or even due to steam inlet into the bodies, which is rarer. The other parameters arising from sucrose thermal degradation, 5-hydroxymethylfurfural, furfural and melanoidins, showed a high correlation with the concentration of reducing sugars (0,99 for HMF, 0,98 for furfural and 0,96 for melanoidins), confirming the trend for loss of the molecule of interest. We also investigated the total populations of mesophilic bacteria, whose presence is associated with health conditions where the food was processed, as well as fungi (molds and yeasts), which also decreases the value of the final product, may produce mycotoxins. The syrup treatment was effective to reduce microorganisms. Considering the average, the population of mesophilic bacteria decrease from 4,10 x 106 cfu/mL to 6,62 x102 cfu/mL. For fungi, the reduction was 1,40 x 104 cfu/mL to 1,80 x 101 cfu/mL. However, the high concentrations found at the beginning and end (Bacteria on average 3,00 x 103 cfu/g in syrup and 2,26 x 103 cfu/g in sugar at the dryer. Fungi 3,50 x 101 cfu/g in syrup and 1,49 x 103 cfu/mL in sugar at the dryer) of the process are indicative of oversights with asepsis, using the lack of demand for quality VHP sugar. The high concentration of juice at milling showed potential to be related sucrose loss. Finally, assessing the scenario and considering that investment in new equipment is something out of the financial reality of the sugar mill, improving the operations inside and outside the mill to avoid losses is suggested.

Den nära förestående bristen på fossila resurser i kombination med deras associerade miljöfarlighet betonar behovet av utveckling av alternativa, mer hållbara kemikalier. I denna studie utvecklades en enzymatisk kaskad för förädling av 5-(hydroxymetyl)furfural (HMF) till 5-(aminometyl)-2-furfuraldehyd (AMFA). Kaskaden omfattar transaminering av HMF till 5-(hydroxymetyl)furfurylamin (HMFA) följt av oxidation av HMFA till AMFA. Transaminas från Silicibacter pomeroyi (SpATA) immobiliserades via his6-taggar på EziG-proteinbärare från EnginZyme AB. Proteinbärarna placerades i spin-kolonner under transamineringen vilket möjliggjorde omhändertagande av SpATA efter transamineringen av HMF. För oxidationen utvärderades alkoholdyhydrogenas från Thermoanaerobacter brockii och hästlever samt galaktosoxidas från Dactylium dendroides (GOase). Omsättning och produktbildning analyserades med HPLC. Resultaten indikerar att SpATA effektivt katalyserar transamineringen av HMF, att alkohol dehydrogenasen inte förmår katalysera oxidationen av HMF till HMFA och att galaktosoxidaset kan oxidera HMFA med hög omsättning vilket leder oss att tro att den föreslagna kaskaden för förädling av HMF till AMFA är möjlig. / The imminent shortage of fossil resources coupled with their associated environmental hazards stresses the need for the development of alternative, more sustainable chemicals. In this study an enzymatic cascade was developed for the valorisation of 5-(hydroxymethyl)furfural (HMF) into 5-(aminomethyl)-2-furfuraldehyde (AMFA). The cascade involves the transamination of HMF into 5-(hydroxymethyl)furfurylamine (HMFA) followed by the oxidation of HMFA into AMFA. Transaminases from Silicibacter pomeroyi (SpATA) was immobilised via his6-tags onto EziG-protein carriers from EnginZyme AB. The protein carriers were placed in spin-columns during the transamination which allowed for salvaging of the SpATA after the transamination of HMF. For the oxidation, alcohol dehydrogenases from Thermoanaerobacter brockii and horse liver as well as galactose oxidase from Dactylium dendroides (GOase) were evaluated. The conversion and product formation were analysed by HPLC. The results indicate that the SpATA efficiently catalyses the transamination of HMF, that the alcohol dehydrogenases are not able to catalyse the oxidation of HMF nor HMFA and that the GOase can oxidize HMFA with high conversion which leads us to believe that the proposed cascade for the valorisation of HMF to AMFA is feasible.