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Proteins have evolved over millions of years to serve a plethora of highly specialized functions in biological systems. Given the enormous diversity in structure and function, it is truly surprising that only 20 different amino acids are utilized as the building blocks of proteins. Furthermore, only a small set of metal cations that are biologically available are used as structural or catalytically active cofactors in proteins, whereas rare metal cations such as platinum, ruthenium or rhodium remain absent. In the 20th century myriad catalysts, based on non-biological transition metals, emerged that can facilitate numerous organic transformations. The goal of the thesis was to introduce new functions into proteins by attaching platinum metals and fluorescent metal sensors. Thus, semi-synthetic proteins for catalytic and analytical applications were generated. The replacement of organic solvents by environmentally benign solvents such as water is an imperative step towards achieving "green chemistry". The combination of small molecule catalysts with proteins may introduce new functions and take advantage of the benefits of "both worlds" while avoiding their potential drawbacks. Therefore semi-synthetic catalysts were developed for enantioselective organic reactions in aqueous medium. A suitable reaction, reaction conditions and catalytic system for later utilization in a semi-synthetic protein were designed, developed and characterized. Ruthenium porphyrins catalyzed cyclopropanation reactions with fair yields and high stereoselectivity in aqueous medium. The successful reaction in water was a crucial requirement for a catalytically active semi-synthetic protein. Mechanistic studies did not elucidate the actual catalytic species for the formation of the cyclopropanation product and the side-product diethyl maleate; however, new insights were gained from the analysis of potential reaction pathways. Moreover, studies of the influence of axial ligands, resembling likely residues coordinating to the ruthenium metal center in the active site of a semi-synthetic protein, on the carbene formation of ruthenium porphyrins illustrated that coordination of axial ligands may inhibit the catalytic activity. The generation of ruthenium porphyrin based semi-synthetic proteins and their subsequent catalysis of cyclopropanation reactions was carried out. Myoglobin and myoglobin mutants were successfully reconstituted with a heme-like ruthenium carbonyl porphyrin; however, none of the formed semi-synthetic proteins catalyzed the enantioselective cyclopropanation of styrene. Efforts to determine the reconstitution efficiency of the generated semi-synthetic were hampered by problems to purify the generated semi-synthetic proteins that are probably due to non-specific binding of the ruthenium porphyrin to the protein surface. The exploration of labile metal pools of the biologically relevant transition metals copper, iron and zinc in cells was the goal of developing semi-synthetic proteins for analytical applications. Combining fluorescent proteins with colored or fluorescent metal chelators by forming semi-synthetic proteins allows taking advantage of their beneficial properties while avoiding their downsides. This design offers an attractive platform for in vivo metal sensing. Plasmids encoding fluorescent proteins, targeting sequences and AGT or intein fusion domains (necessary for labeling) for eukaryotic and prokaryotic expression were generated. The targeting of intracellular compartments (mitochondria, nucleus and TGN) was successful (confirmed by light microscopy experiments with transfected mammalian cells). In vitro labeling experiments of expressed and purified fusion proteins with rhodamine derivatives succeeded with AGT based fusion proteins; however, labeling of fusion proteins by trans-splicing with split-inteins failed. A new Zinc(II)-chelator was attached to an AGT based protein and the resulting semi-synthetic protein exhibited strong changes of fluorescence in the presence of zinc(II). This represents an important step towards the goal of in vivo cell imaging of labile zinc(II) pools. Despite extensive efforts, all attempts failed to generate a chelator that forms Cu(I)-complexes with the 1:1 stochiometry (ligand:metal) that is necessary for metal sensing with semi-synthetic proteins.

Semi-synthetic β-lactam antibiotics are the most prescribed class of antibiotics in the world. Chemical coupling of a β-lactam moiety with an acyl side chain has dominated the industrial production of semi-synthetic β-lactam antibiotics since their discovery in the early 1960s. Enzymatic coupling of a β-lactam moiety with an acyl side chain can be accomplished in a process that is much more environmentally benign but also results in a much lower yield. The goal of the research presented in this dissertation is to improve the enzymatic synthesis of β-lactam antibiotics via reaction engineering, medium engineering and data-drive protein engineering. Reaction engineering was employed to demonstrate that the hydrolysis of penicillin G to produce the β-lactam nucleus 6-aminopenicillanic acid (6-APA), and the synthesis of ampicillin from 6-APA and (R)-phenylglycine methyl ester ((R)-PGME), can be combined in a cascade conversion. In this work, penicillin G acylase (PGA) was utilized to catalyze the hydrolysis step, and PGA and α-amino ester hydrolase (AEH) were both studied to catalyze the synthesis step. Two different reaction configurations and various relative enzyme loadings were studied. Both configurations present a promising alternative to the current two-pot set-up which requires intermittent isolation of the intermediate, 6-APA. Medium engineering is primarily of interest in β-lactam antibiotic synthesis as a means to suppress the undesired primary and secondary hydrolysis reactions. The synthesis of ampicillin from 6-APA and (R)-PGME in the presence of ethylene glycol was chosen for study after a review of the literature. It was discovered that the transesterification product of (R)-PGME and ethylene glycol, (R)-phenylglycine hydroxyethyl ester, is transiently formed during the synthesis reactions. This never reported side reaction has the ability to positively affect yield by re-directing a portion of the consumption of (R)-PGME to an intermediate that could be used to synthesize ampicillin, rather than to an unusable hydrolysis product. Protein engineering was utilized to alter the selectivity of wild-type PGA with respect to the substituent on the alpha carbon of its substrates. Four residues were identified that had altered selectivity toward the desired product, (R)-ampicillin. Furthermore, the (R)-selective variants improved the yield from pure (R)-PGME up to 2-fold and significantly decreased the amount of secondary hydrolysis present in the reactions. Overall, we have expanded the applicability of PGA and AEH for the synthesis of semi-synthetic β-lactam antibiotics. We have shown the two enzymes can be combined in a novel one-pot cascade, which has the potential to eliminate an isolation step in the current manufacturing process. Furthermore, we have shown that the previously reported ex-situ mixed donor synthesis of ampicillin for PGA can also occur in-situ in the presence of a suitable side chain acyl donor and co-solvent. Finally, we have made significant progress towards obtaining a selective PGA that is capable of synthesizing diastereomerically pure semi-synthetic β-lactam antibiotics from racemic substrates.

The implementation of sustainable solvents and processes is critical to new developments in reducing environmental impact, improving net efficiency, and securing economic profitability in the chemical and pharmaceutical industries. In order to address the challenge of sustainability, researchers have used switchable solvents for both reaction and separation by utilizing a built-in switch to undergo a step change in chemical and physical properties. This allows us to facilitate reactions in the solvent then activate the switch to enable separation and facile product recovery. Subsequently, we can recover the solvent for reuse and avoid energy- or waste-intensive separation processes; thus we are developing and using these switchable solvents as sustainable and environmentally benign alternatives to traditional processes. In this research, we enable the sustainable scale-up of a switchable solvent - piperylene sulfone - a "volatile" and recyclable DMSO replacement. In the development of this process, we improved the reaction performances and developed a green purification method. Furthermore, we enable and demonstrate the implementation of a Meerwein-Ponndorf-Verley (MPV) reduction, a pharmaceutically relevant reaction, into a continuous flow platform. The innovation of continuous flow processes can replace traditional batch reaction technology, and is indeed a key research area that has been acknowledged by the pharmaceutical industry. Additionally, we utilize the switchable sulfone solvents, piperylene and butadiene sulfone, for reaction and separation of HMF produced from monosaccharides as an alternative to a process which has been limited by an inefficient separation step.

Cross-linked polyethylene has higher upper use temperature than normal polyethylene and is used as an insulating material for electricity carrying cables and hot water pipes. The most common method of inducing crosslinks is by reaction with silanes. After incorporation of silanes into polyethylene and upon hydrolysis with ambient moisture or with hot water, Si-O-Si crosslinks are formed between the various linear polyethylene chains. Industrially, this reaction is performed routinely. However, the efficiency of this reaction with respect to the silane is low and control of product distribution is difficult. A precise fundamental understanding is necessary to be able to manipulate the reactions and thus, allow for the facile processing of the polymers. Hydrocarbon models of polymers - heptane, dodecane - are being used to study this reaction in the laboratory. For the reaction, vinyltrimethoxysilane is used as the grafting agent along with di-tert-butyl peroxide as the radical initiator. MALDI, a mass spectrometric technique is used for the analysis of the product distribution after work-up. Advanced NMR techniques (COSY, HSQC, DEPT, APT, HMBC) are being conducted on the grafted hydrocarbon compounds to gain an in-depth understanding of the mechanism and regiochemistry of the grafting reaction. Scalable and cost effective methods to capture CO2 are important to counterbalance some of the global impact of the combustion of fossil fuels on climate change. The main options available now include absorption, adsorption and membrane technology. Amines, especially monoethanolamine, have been the most commercialized technology. However, it is not without disadvantages. House et al have investigated the energy penalty involved in the post-combustion CO2 capture and storage from coal-fired power plants and found that 15-20% reduction in the overall electricity usage is necessary to offset the penalty from capturing and storing 80% of United States coal fleet's CO2 emssions1. Novel non-aqueous amine solvents, developed by the Eckert Liotta group, react with CO2 to form ionic liquids. The ionic liquids readily desorb CO2 upon heating, regenerating the reactive amines and this cycle can be carried out multiple times. An iterative procedure is being adopted to develop amine solvents for CO2 capture. Thermodynamic information like reversal temperature and boiling point of the solvents are collected; they are then used to formulate structure property relationships which allow for new molecules to be engineered. On reaction with CO2, there is a sharp increase in viscosity which is unfavorable from a processing standpoint. Many approaches to mitigate and control viscosity are being studied as well. 1House et al, Energy Environ Sci, 2009, 2, 193-205

<p>The aim of our research was to investigate if oat is capable of extracting cadmium to such extent that it is usable in decontaminating polluted soils. We grew oat in a hydroponic culture during 28 days in a controlled environment and a total of 30 plants were used. The nutrient solutions were contaminated with cadmium of ten different concentrations after seven days. After harvesting the plants, the roots were separated from the shoots, placed in separate containers and then turned to ashes. The cadmium content was measured three times per sample in an atomic absorption spectrometer.</p><p>Our results indicate that the ability of oat to extract cadmium from a solution is linearly dependent of the cadmium concentration of the solution. We also noted that the resistivity to cadmium of oat is limited. When the concentration of accessible cadmium ions exceeded 0,06mM, a significant difference of the shoot growth appeared. The results we obtained from shoots and roots show cadmium amounts much higher in the roots than the shoots. Despite this difference there were sufficient amounts of cadmium in the shoots to call oat a hyper accumulator. This means that oat fulfills one of the criteria of a phytoextractor.</p>

<p>There are several incentives for using bottom ash from municipal solid waste incineration (MSWI bottom ash) as a construction material, such as for road construction. These incentives include decreased disposal of material on landfills and a reduced amount of raw material extracted for road building purposes. However, one of the main obstacles to utilising the material is uncertainties regarding its environmental properties. The overall objective of this thesis is to describe the potential environmental impacts of utilising MSWI bottom ash in constructions and to improve the tools for environmental assessments.</p><p>An environmental systems analysis (ESA) approach based on a life cycle perspective was outlined and used in a case study, with the aim of describing the differences in resource use and emissions that can be expected if crushed rock in the sub-base of a road in the Stockholm region in Sweden were to be substituted by MSWI bottom ash. The whole life cycle of the road was taken into account and the alternative disposal of the bottom ash was included. It was found that the studied alternatives would cause different types of potential environmental impact; whereas the conventional alternative with only crushed rock in the road’s sub-base would lead to larger use of energy and natural resources, the alternative with MSWI bottom ash in the sub-base would lead to larger contaminant leaching. It was concluded that a life cycle approach is needed in order to include both resource use and emissions in the comparison between the two alternative scenarios. The leaching of metals turned out to be the most important environmental aspect for the comparison and in particular the difference in copper (Cu) leaching was shown to be large.</p><p>However, a large amount of Cu may not pose an environmental threat if the Cu is strongly bound to dissolved organic carbon (DOC). In order to improve the basis for toxicity estimates and environmental risk assessments, and thereby provide better input values for ESAs, the speciation of Cu to DOC in MSWI bottom ash leachate was studied. It was found that Cu to a large extent was bound to DOC, which is consistent with previous research. The results also suggest that the hydrophilic fraction of the MSWI bottom ash DOC is important for Cu complexation and that the pH-dependence for Cu complexation to MSWI bottom ash DOC is smaller than for natural DOC. This implies that models calibrated for natural DOC may give inconsistent simulations of Cu-DOC complexation in MSWI bottom ash leachate.</p>

<p>The existence of trihalomethanes (THMs) in a drinking water plant of Nicaragua has been investigated in order to see whether the concentration exceeded the maximum contaminant level recommended by the environmental protection agency of the United States (USEPA) and the Nicaragua guidelines. The influence of pH, temperature, chlorine dose and contact time on the formation of THMs were studied. The contents of organic matter measured by surrogate parameters such as total organic carbon, dissolved organic carbon, ultraviolet absorbance and specific ultraviolet absorbance were also determined in order to show which type of organic matter is most reactive with chlorine to form THMs. Models developed by other researchers to predict the formation of trihalomethanes were tested to see whether they can be used to estimate the trihalomethane concentration. In addition, empirical models were development to predict the THM concentration of the drinking water plant analysed. The raw water was treated by conventional and enhanced coagulation and these processes were compared with regard to the removal of natural organic matter (NOM). The significance of the results was assessed using statistic procedures.</p><p>The average concentration of THMs found at the facility is below the USEPA and Nicaragua guideline values. Nevertheless the maximum contaminant level set by USEPA is sometimes exceeded in the rainy season when the raw water is rich in humic substances. Comparison between the water treated by conventional and enhanced coagulation shows that enhanced coagulation considerably diminished the trihalomethane formation and the value after enhanced coagulation never exceeded the guidelines. This is because enhanced coagulation considerably decreases the organic matter due to the high coagulant dose applied. The study of the trihalomethane formation when varying pH, time, temperature and chlorine dose using water treated by conventional and enhanced coagulation showed that higher doses of chlorine, higher pH, higher temperature and a longer time increases the formation of THMs. However, combinations of two and three factors are the opposite. The predicted THM formation equations cannot be used for the water at this facility, since the results shown that the measured THM differs significantly from the THM concentration predicted. Two empirical models were developed from the data for enhanced coagulation, using linear and non-linear regression. These models were tested using the database obtained with conventional coagulation. The non-linear model was shown to be able to predict the formation of THMs in the Boaco drinking water plant.</p>

In the selection of candidates for CO2 absorption, solvent thermal degradation has become a general concern due to the significant impact on operational cost and the intention to use thermal compression from high temperature stripping to minimize the overall process energy. In this research, the impact of flue gas contaminants on Monoethanolamine (MEA) thermal degradation was investigated at elevated temperatures consistent with those in the CO2 stripper. Nitrite, fly ash, sulfate and thiosulfate were each added to 5.0 M MEA and the contaminant-containing MEA solutions were degraded at 125 °C, 135 °C and 145 °C. MEA degrades significantly more in the presence of nitrite (5000 ppm) than MEA alone at the same amine molar concentration for all three temperatures. MEA degradation activation energy of MEA-nitrite solution is approximately one-seventh of that of MEA solution without nitrite. Fly ash was observed to inhibit nitrite-induced MEA degradation and greatly increase the MEA degradation activation energy of MEA-nitrite solution. Fly ash, sodium sulfate and sodium thiosulfate by themselves were not shown to impact MEA thermal degradation rate. Sodium salts of glycine, sarcosine, alanine and ß-alanine were thermally degraded at 125 °C, 135 °C and 145 °C, respectively, to discover the structural reasons for their thermal stability. These four amino acids have enhanced thermal degradation rates compared to MEA. The stability order for amino acid salts tested to date is: sarcosinate > alaninate > ß-alaninate. Calculated activation energies for the degradation processes are lower than that of MEA. ß-Alaninate (ß-Ala) thermal degradation generates ß-Ala dimer (major degradation product), ß-Ala dimer carbamate and tetrahydro-1,3-oxazin-6-one. Functional groups, amine orders and steric effect were investigated for their impact on amine thermal degradation. Primary amines with chain structures showed a thermal stability trend as diamine > alkanolamine > amino acid salt. For alknolamine and diamine structural isomers, the primary amines are more stable than the secondary amines. Steric hindrance around the amine group plays a global positive role in protecting amines against thermal degradation.

Considering the worldwide growing discharge of minor actinides and the current need for geological disposal facilities for radioactive waste, this work provides a contribution to the safety case concerning Np transport if it would be released from deep repository sites and moving from alkaline cement conditions (near-field) to more neutral environmental conditions (far-field). The reducing conditions in a nuclear waste repository render neptunium tetravalent, which is assumed to be immobile in aqueous environment due to the low solubility solution of Np(IV). For tetravalent actinide nuclides, the most significant transport should occur via colloidal particles. This work demonstrates the formation of intrinsic neptunium dioxide nanocrystals and amorphous Np(IV) silica colloids under environmentally relevant conditions. The dissociation of the initial soluble Np(IV) complex (i.e. [Np(IV)(CO3)5]6-) induces the intrinsic formation of nanocrystalline NpO2 in the solution phase. The resulting irregularly shaped nanocrystals with an average size of 4 nm exhibit a face-centered cubic (fcc), fluorite-type structure (space group ). The NCs tend to agglomerate under ambient conditions due to the weakly charged hydrodynamic surface at neutral pH (zetapotential ~0 mV). The formation of micron-sized agglomerates, composed of nanocrystals of 2-5 nm in size, and the subsequent precipitation cause immobilization of the major amount of Np(IV) in the Np carbonate system. Agglomeration of NpO2 nanocrystals in dependence on time was indicated by PCS and UV-vis absorption spectroscopy with the changes of baseline characteristics and absorption maximum at 742 nm. Hitherto, unknown polynuclear species as intermediate species of NpO2 nanocrystal formation were isolated from solution and observed by HR-TEM. These polynuclear Np species appear as dimers, trimers and hexanuclear compounds in analogy with those reported for other actinides. Intrinsic formation of NpO2 (fcc) nanocrystals under ambient environmental conditions is prevented by admixing silicic acid: amorphous Np(IV) silica colloids are formed when silicate is present in carbonate solution. Herein, the initial molar ratio of Si to Np in solution lead to the formation of Np(IV) silica particles of different composition and size where Si content determines the structure and stability of resulting colloids. Implications for different electronic structures of Np(IV) in dependence on Si content in the solid phase are given by the shift of the absorption maximum at 742 nm characteristic for Np(IV) colloids, silica excess of 5 times the magnitude of Si to Np reveal a redshift up to 6 nm in the colloidal UV-vis spectrum. Precipitation of Np(IV) particles in the ternary system results in a different coordination sphere of Np(IV) compared to the binary system, and the incorporation of Si into internal structure of Np(IV) silica colloids in coffinite-like structure is confirmed by EXAFS. TEM confirms different kinds of particle morphologies in dependence on the silica content. Silica-poor systems reveal porous particles in the micron-range which consist of irregular cross-linked hydrolyzed Np(IV) silica compartments with pores <15 nm. In contrast, long-term stabilized and silica-enriched systems are characterized by isolated particles with an average particle size of 45 nm. Agglomerates of such isolated Np(IV) silica particles appear as consolidated amorphous solids with a densely closed surface and exhibit no internal fractures. The latter mentioned morphology of Np(IV) silica particles might facilitate the migration behavior of Np(IV) in a stabilized colloidal form under environmental conditions. The silica-enriched particles with densely closed surface are long-term stabilized as colloidal dispersion (>1 year) due to repulsion effects caused by significant surface charge. Particles synthesized from Si/Np = 9/1 carry exclusively negative surface charge in nearly the whole pH range from pH 3 to pH 10 with zetapotential = (-) 5 to (-) 30 mV. The zeta potentials of all particle systems containing silica are significantly shifted to more negative values below pH 7 where the isoelectrical point shifts from pH = 8.0 to 2.6 effecting negative charge under ambient conditions which supports electrostatic stabilization of Np(IV) particles. Particle surface charge at the slipping plane, particle size and shape necessarily depend on the initial magnitude of Si content in solution during particle formation. Particular changes of the morphology and internal structure of different Np(IV) silica colloids by aging are indicated by TEM and XPS. The composition and the crystallinity state of the initially formed amorphous phases partially changed into well-ordered nanocrystalline units characterized with fcc structure. The presence of silicate under conditions expected in a nuclear waste repository significantly influences the solubility of Np(IV) and provoke the stabilization of waterborne Np(IV) up to concentrations of 10-3 M, exceeding Np´s solubility limit by a factor of up 10.000. Neptunium and silicate significantly interact with each other, and thereby changing their individual hydrolysis and polymerization behavior. Silicate prevents the intrinsic formation of NpO2 NCs in fcc-structure, and at the same time, Np(IV) prevents the polymerization of silicate. Both processes result in the formation of Np(IV) silica colloids which possibly influence the migration behavior and fate of Np in the waste repositories and surrounding environments. For tetravalent actinides in general, the most significant transport in the environment would occur by colloidal particles. Therefore, Np(IV) silica colloids could have a significant implication in the migration of Np, the important minor actinide in the waste repositories, via colloidal transport.