A new experimental set-up and a new simulated moving bed (SMB) operation are presented in this work. A desktop SMB unit developed as a modification of the commercial AKTA(TM) explorer working platform has been utilized for the separation of different mixtures of nucleosides. Both two fraction and three fraction SMB separations have been carried out, the latter made possible by the adoption of a new SMB configuration and operating mode (three fraction SMB, 3F-SMB, operation). Experiments demonstrate the feasibility of the 3F-SMB operation, and confirm the trends predicted based on considerations about retention of the components to be separated along the unit. 

The need for alternative cognitive enhancers has risen due to the fact that clinical trial results of the drugs currently approved for treating these disorders have not been satisfactory.

IRAP has become a possible drug target for treating cognitive impairment brought about by Alzheimer’s disease, head trauma or cerebral ischemia, among others. This came after the revelation that Angiotensin IV enhances memory and learning. Angiotensin IV, the endogenous ligand of IRAP has been structurally modified with the aim of producing potent IRAP inhibitors. However, the peptidic nature of these inhibitors restricts their use; they are not likely to cross the blood brain barrier.

Other strategies for generating IRAP inhibitors have been through structure-based design and receptor based virtual screening. These drug-like molecules have exhibited positive results in animal studies.

IRAP inhibitors have been identified via a HTS of 10500 low-molecular weight compounds to give the hit based on a spirooxindole dihydroquinazolinone scaffold, with an IC₅₀ value of 1.5 µM. In this project, some analogues to this hit compound have successfully been synthesized using a known method, whereas others have been synthesized after additional method development.

The application of the developed method was found to be limited, because poor yield was obtained when a compound with an electron withdrawing substituent on the aniline was synthesized. As a result of this, modification of this method may be required or new methods may have to be developed to synthesize these types of analogues.

Inhibition capability of 5 new spirooxindole dihydroquinazolinones was tested through a biochemical assay. Compound 6e emerged as the most potent inhibitor in the series, with an IC₅₀ value of 0.2 µM. This compound will now serve as a lead compound and should be used as a starting point for future optimization in order to generate more potent IRAP inhibitors.

The molecular interactions between the enzyme acetylcholinesterase (AChE) and two compound classes consisting of N-[2-(diethylamino)ethyl]benzenesulfonamides and N-[2-(diethylamino)ethyl]benzenemethanesulfonamides have been investigated using organic synthesis, enzymatic assays, X-ray crystallography, and thermodynamic profiling. The inhibitors' aromatic properties were varied to establish structure activity relationships (SAR) between the inhibitors and the peripheral anionic site (PAS) of AChE. The two structurally similar compound classes proved to have distinctly divergent SARs in terms of their inhibition capacity of AChE. Eight X-ray structures revealed that the two sets have different conformations in PAS. Furthermore, thermodynamic profiles of the binding between compounds and AChE revealed class-dependent differences of the entropy/enthalpy contributions to the free energy of binding. Further development of the entropy-favored compound class resulted in the synthesis of the most potent inhibitor and an extension beyond the established SARs. The divergent SARs will be utilized to develop reversible inhibitors of AChE into reactivators of nerve agent-inhibited AChE.

Protein inhibition by covalent modifications has been widely explored during the last decades. Despite the worries regarding the toxicity and suitability of irreversible covalent drug inhibitors, lately they have gained more and more attention in scientific community. Here we investigate covalent modifications of non-catalytic protein residues with small-molecules as the potential building blocks for future drug discovery. The intricacies of protein structure and the environment they exist in, usually complicate the understanding of the reactivity between the amino acids and compounds. In this study, we attempted to approach this subject from an analytical point of view. By applying recombinant DNA techniques, we expressed and purified proteins of interest; using liquid chromatography–mass spectrometry (LC–MS) we attempted to label a number of redesigned proteins with the ultimate goal to apply this to human protein kinases, few of which will be presented here. This may potentially assist in rationally target residues in proteins, ideally not ctalytic ones that can be covalently modified, which can serve in later drug design studies. Furthermore, it will optimistically lead us to new efforts in discovering alternative methods of cancer treatment. Ultimately, the combination of experimental techniques and computational models will broaden our knowledge of covalent modifications at allosteric positions in proteins.

Most of the devastating diseases such as cancer are relatively incurable and have high risks of relapse. Therefore, persistent endeavors have been devoted to improve patient survival rate and quality of life. Drug delivery systems (DDS) based on polymeric nanoparticles (PNPs) have been demonstrated to increase the therapeutic index (efficacy/toxicity ratio) of chemotherapeutic agents. This thesis focuses on designing non-toxic and multifunctional biodegradable PNPs from preformed polymers for bioimaging and drug delivery applications. Multifunctional poly(lactide-co-glycolide) (PLGA) NPs were simultaneously loaded with imaging probes, superparamagnetic iron oxide nanoparticles (SPION) and manganese-doped zinc sulfide (Mn:ZnS) quantum dots (QDs), as well as an anti-cancer drug, busulfan (Bu), during the particle formation. The NPs were utilized to enhance magnetic resonance imaging (MRI) in vivo and controlled drug release in vitro (Paper I). Poly(ε-caprolactone) (PCL) was copolymerized with poly(ethylene glycol) (PEG) to achieve stealth property for in vivo purposes. Aluminum phthalocyanine, a photosensitizer and an anti-cancer drug, was encapsulated in the PEG-b-PCL NPs for photodynamic therapy during particle formation. The biodistribution of the prepared nanophotosensitizer showed targeted drug delivery toward lungs, liver and spleen as monitored by the intrinsic fluorescence of the photosensitizer (Paper II). The PEG-b-PCL NPs were loaded with SPION or surface functionalized with VivoTag 680XL fluorochrome and utilized for in vivo multimodal imaging, MRI and fluorescence imaging (Paper III). This thesis also presents stable and engineered PNPs obtained using reversible addition-fragmentation chain transfer (RAFT) mediated polymerization-induced self-assembly (PISA). Hydrophobic agents, nile red (NR) dye or doxorubicin (DOX) drug, were encapsulated in poly(N-[3- (dimethylamino) propyl] methacrylamide)-b-poly(methyl methacrylate) (PDMAPMA-b-PMMA) NPs via one-pot RAFT-mediated PISA in water (Paper IV). The PDMAPMA-b-PMMA NPs showed very monodisperse spheres and core-shell nanostructures. Stable and non-toxic poly(acrylic acid)-b-poly(butyl acrylate) (PAA-b-PBA) NPs, synthesized via RAFTmediated PISA in water, were surface engineered by allyl-functional groups prior to bio-conjugation for targeted drug delivery (Paper V). The engineered NPs retained their colloidal stability and size post-allyl functionalization. DOX was efficiently (90 %) encapsulated in the PAA-bPBA NPs during NPs formation. A controlled release pattern of DOX from PAA-b-PBA NPs was observed over 7 days.

Solvents play a crucial role in the Active Pharmaceutical Ingredient (API) manufacturing and are used in large quantities. Most of the industries incinerate the waste solvents or send it to waste management companies for destruction to avoid waste handling and cross-contamination. It is not a cost effective method and also hazardous to the environment. This study has been performed at AstraZeneca’s API manufacturing plant at Sodertalje, Sweden. In order to find a solution, a solvent recovery system is modeled and simulated using ASPEN plus and ASPEN batch modeler. The waste streams were selected based on the quantity and cost of the solvents present in them. The solvent mixture in the first waste stream was toluene-methanol in which toluene was the key-solvent whereas in the second waste stream, isooctane-ethyl acetate was the solvent mixture in which isooctane was the key-solvent. The solvents in the waste stream were making an azeotrope and hence it was difficult to separate them using conventional distillation techniques. Liquid-Liquid Extraction with water as a solvent followed by batch distillation was used for the first waste stream and Pressure Swing Distillation was used for the second waste stream. The design was optimized based on cost analysis and was successful to deliver 96.1% toluene recovery with 99.5% purity and 83.6% isooctane recovery with 99% purity. The purity of the solvents was decided based on the quality conventions used at AstraZeneca so that it can be recovered and recycled in the same system. The results were favorable with a benefit of €335,000 per year and preventing nearly one ton per year carbon dioxide emissions to the environment. A theoretical study for the recovery system of toluene-methanol mixture was performed. The proposed design was an integration of pervaporation to the batch distillation. A blend of polyurethane / poly(dimethylsiloxane) (PU / PDMS) membrane was selected for the separation of methanol and toluene mixture. The results of preliminary calculations show 91.4% toluene recovery and 72% methanol recovery with desired purity.

Background

Maternal obesity is accompanied by maternal and fetal complications during and after pregnancy. The risks seem to increase with degree of obesity. Leptin has been suggested to play a role in the development of obesity related complications. Whether maternal leptin levels differ between obese and morbidly obese women, during and after pregnancy, have to our knowledge not been previously described. Neither has the association between maternal leptin levels and gestational weight gain in obese women. The aim was to evaluate if maternal plasma leptin levels were associated with different degrees of maternal obesity and gestational weight gain.

Methods

Prospective cohort study including women categorized as obesity class I-III (n = 343) and divided into three gestational weight gain groups (n = 304). Maternal plasma leptin was measured at gestational week 15, 29 and 10 weeks postpartum. Maternal Body Mass Index (BMI) was calculated from early pregnancy weight. Gestational weight gain was calculated using maternal weight in delivery week minus early pregnancy weight. The mean value and confidence interval of plasma-leptin were analysed with a two-way ANOVA model. Interaction effect between BMI and gestational weight gain group was tested with a two-way ANOVA model.

Results

The mean maternal leptin concentrations were significantly higher in women with obesity class III compared to women in obesity class I, at all times when plasma leptin were measured. The mean leptin concentrations were also significantly higher in women with obesity class II compared to women in obesity class I, except in gestational week 29. There was no difference in mean levels of plasma leptin between the gestational weight gain groups. No significant interaction between BMI and gestational weight gain group was found.

Conclusions

Plasma leptin levels during and after pregnancy were associated with obesity class but not with degree of gestational weight gain. These results are in concordance with epidemiological findings where the risk of obstetric complications increases with increased maternal obesity class. The effect on obstetric outcome by degree of gestational weight gain is less pronounced than the adverse effects associated with maternal obesity.

The escalating rate of cancer cases, together with treatment deficiencies and long-term side effects of currently used cancer drugs, has made this disease a global burden of the 21st century. The number of breast and lung cancer patients has sharply increased worldwide in the last few years. Presently, surgical treatment, radiotherapy, chemotherapy, and immunotherapy strategies are used to cure cancer, which cause severe side effects, toxicities, and drug resistance. In recent years, anti-cancer peptides have become an eminent therapeutic strategy for cancer treatment due to their high specificity and fewer side effects and toxicity. This review presents an updated overview of different anti-cancer peptides, their mechanisms of action and current production strategies employed for their manufacture. In addition, approved and under clinical trials anti-cancer peptides and their applications have been discussed. This review provides updated information on therapeutic anti-cancer peptides that hold great promise for cancer treatment in the near future.

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences.

This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities.

The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies.

The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

3-Benzylidene-indoline-2-ones play a prominent role in the pharmaceutical industry due to the diverse biomedical applications of oxindole heterocycles. Despite the extensive reports on their biological properties, these compounds have hardly been studied for their photochemical activity. Here, we present 3-benzylidene-indoline-2-ones as a promising class of photoswitches with high yields, robust photochemical switching with quantum yields reaching up to 50 % and potential for biological applications. 3-Benzylidene-indoline-2-ones are of great importance for the pharmaceutical industry. These compounds have been extensively reported for their biological properties, but have hardly been studied concerning their photochemical activity. Here, we present 3-benzylidene-indoline-2-ones as a promising class of photoswitches with high yields, robust photochemical switching, and quantum yields reaching up to 50 %.image

Lignans from Schisandra chinensis berries show various pharmacological activities, of which their antioxidative and cytoprotective properties are among the most studied ones. Here, the first report on antibacterial properties of six dibenzocyclooctadiene lignans found in Schisandra spp. is presented. The activity was shown on two related intracellular Gram-negative bacteria Chlamydia pneumoniae and Chlamydia trachomatis upon their infection in human epithelial cells. All six lignans inhibited C. pneumoniae inclusion formation and infectious progeny production. Schisandrin B inhibited C. pneumoniae inclusion formation even when administered 8 h post infection, indicating a target that occurs relatively late within the infection cycle. Upon infection, lignan-pretreated C. pneumoniae elementary bodies had impaired inclusion formation capacity. The presence and substitution pattern of methylenedioxy, methoxy and hydroxyl groups of the lignans had a profound impact on the antichlamydial activity. In addition our data suggest that the antichlamydial activity is not caused only by the antioxidative properties of the lignans. None of the compounds showed inhibition on seven other bacteria, suggesting a degree of selectivity of the antibacterial effect. Taken together, the data presented support a role of the studied lignans as interesting antichlamydial lead compounds.

In organic chemistry and especially process chemistry, there is a constant need to develop cost-effective ways to optimize different reaction conditions. With the increased development of Machine Learning (ML) combined with Data Mining (DM) new possibilities arise to reduce time and costs in the field of chemical science. In order to address the need for reduced time-/cost savings in process chemistry, the often-employed Suzuki-Miyaura reaction was studied by such ML and DM methods. A representative dataset containing molecular and structural properties of substrates and product were calculated with open-source toolkits Indigo, Chemistry Development Kit and RDKit available in KNIME. To predict any form of reaction outcomes, catalysts and reaction conditions were ranked based on several binary classification Machine Learning models designed with a Random Forest algorithm. On model lead to a binary classification model performing at a low computational cost. It showed an AUC of 98.5% predicting a reaction to a certain threshold of yield ( >=60% and<=40%). A second model encompassed six unique binary classification models and presented an average accuracy of 91.6% to predict a correct catalyst. These six different models were combined to later rank catalysts that are best suited for a new reaction and gave a probability result between 23.6% to 77.3%. The experimental validation was proven to highlight the uncertainty of the performance, were the least suitable (23.6%) catalyst demonstrated best performance. Overall, the models showed a promising correlation to support the synthesis optimization problem and with further adjustment there are great opportunities to obtain a model that can assist chemists in the future.

The aim with this project was to evaluate Waters AccQTag method which was ananalytical method that Waters Corporation provides for determination of aminoacids. The amino acid analysis was carried out by reversed-phase AQUITY UPLCH-Class, using a BEH C18-column and a PDA detector.Both primary and secondary amino acids were analyzed after precolumnderivatization with the AccQTag reagent kit by a manual procedure and, detected at260 nm. Fresenius Kabis existing products, Aminoven 10 % and Vamin 18EF wereused as samples. The evaluation was based on external calibration curves withprepared standard solutions from Fresenius Kabi, with the addition of the internalstandard L-Norvaline. Waters amino acid standard was used for the system suitabilitytest. A validation according to the ICH guidelines were performed with the followingparameters: specificity, linearity, precision, LOQ, LOD and robustness.Complementary parameters for system suitability test (SST) and stability of standardand reagent were also evaluated. The validation showed that the % RSD values forspecificity, system suitability test and robustness were within the acceptance criteriaof equal or less than 3 % for amino acids. Although the precision were between1.3-7.5 % which was higher than the acceptance criteria of equal or less than 3 %, thiswas likely due to sensitive sample preparation. The linearity was bad which made itimpossible to quantify the method since the calibration curves were below 0.999. Itwas concluded that the system was robust and can be used to analyze amino acids inAminoven 10 % and Vamin 18EF but the preparation step should if possible beautomatized to achieve better precision in the analytical work. Two amino acids,cysteine and histidine, needs to be studied further; cysteine gave three peaks insteadof one and histidine gave a peak split at higher injection volume than 0.5 uL.

This work investigates the fluid flow in a cylindrical millilitre vial stirred by a magnetic stirred bar using Computational Fluid Dynamic (CFD). Stirred millilitre vials are used to study nucleation phenomena and crystallization as an outline of literature study of nucleation and crystallization phenomena and the role of stirring in this process. The baffle free vial was meshed with around 500,000 cells. To simulate the stirring a rotary frame and moving walls were used. Stirring speeds were between 100 and 1000 rpm where considered, correspondently to a stirrer Reynolds number between 260 and 2600. For stirring speeds bellow 500 rpm, simulations by both the both laminar flow model and the k-ε model where run, while above 500 rpm only k-ε was used. Results of the two models were very similar indicative the adequacy of k-ε to simulate the flow even at low Reynolds. The flow shows expected circulation pattern with upwards pumping close to side walls and downwards pumping in the centre of cylindrical vial. At 1000 rpm circulation patterns expands up to the top of the vial while at 300 rpm and lower the upper half of the vial is poorly mixed. The average turbulent energy of the flow is very low comparing with the squared stirrer tip speed and the power number decrees with Reynolds number, indicating that the flow is not fully turbulent.

Virtual screening has the potential to accelerate and reduce costs of probe development and drug discovery. To develop and benchmark virtual screening methods, validation data sets are commonly used. Over the years, such data sets have been constructed to overcome the problems of analogue bias and artificial enrichment. With the rapid growth of public domain databases containing high-throughput screening data, such as the PubChem BioAssay database, there is an increased possibility to use such data for validation. In this study, we identify PubChem data sets suitable for validation of both structure- and ligand-based virtual screening methods. To achieve this, high-throughput screening data for which a crystal structure of the bioassay target was available in the PDB were identified. Thereafter, the data sets were inspected to identify structures and data suitable for use in validation studies. In this work, we present seven data sets (MMP13, DUSP3, PTPN22, EPHX2, CTDSP1, MAPK10, and CDK5) compiled using this method. In the seven data sets, the number of active compounds varies between 19 and 369 and the number of inactive compounds between 59 405 and 337 634. This gives a higher ratio of the number of inactive to active compounds than what is found in most benchmark data sets. We have also evaluated the screening performance using docking and 3D shape similarity with default settings. To characterize the data sets, we used physicochemical similarity and 2D fingerprint searches. We envision that these data sets can be a useful complement to current data sets used for method evaluation.

The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique. 

Non-invasive methods for early diagnosis of skin cancer are highly valued. One possible approach is to monitor relevant biomarkers such as tryptophan (Trp) and kynurenine (Kyn), on the skin surface. The primary aim of this in vitro investigation was, therefore, to examine whether reverse iontophoresis (RI) can enhance the extraction of Trp and Kyn, and to demonstrate how the Trp/Kyn ratio acquired from the skin surface reflects that in the epidermal tissue. The study also explored whether the pH of the receiver medium impacted on extraction efficiency, and assessed the suitability of a bicontinuous cubic liquid crystal as an alternative to a simple buffer solution for this purpose. RI substantially enhanced the extraction of Trp and Kyn, in particular towards the cathode. The Trp/Kyn ratio obtained on the surface matched that in the viable skin. Increasing the receiver solution pH from 4 to 9 improved extraction of both analytes, but did not significantly change the Trp/Kyn ratio. RI extraction of Trp and Kyn into the cubic liquid crystal was comparable to that achieved with simple aqueous receiver solutions. We conclude that RI offers a potential for non-invasive sampling of low-molecular weight biomarkers and further investigations in vivo are therefore warranted.

Many skin disorders, including cancer, have inflammatory components. The non-invasive detection of related biomarkers could therefore be highly valuable for both diagnosis and follow up on the effect of treatment. This study targets the extraction of tryptophan (Trp) and its metabolite kynurenine (Kyn), two compounds associated with several inflammatory skin disorders. We furthermore hypothesize that lipid-based bicontinuous cubic liquid crystals could be efficient extraction matrices. They comprise a large interfacial area separating interconnected polar and apolar domains, allowing them to accommodate solutes with various properties. We concluded, using the extensively studied GMO-water system as test-platform, that the hydrophilic Kyn and Trp favored the cubic phase over water and revealed a preference for locating at the lipid-water interface. The interfacial area per unit volume of the matrix, as well as the incorporation of ionic molecules at the lipid-water interface, can be used to optimize the extraction of solutes with specific physicochemical characteristics. We also observed that the cubic phases formed at rather extreme water activities (>0.9) and that wearing them resulted in efficient hydration and increased permeability of the skin. Evidently, bicontinuous cubic liquid crystals constitute a promising and versatile platform for non-invasive extraction of biomarkers through skin, as well as for transdermal drug delivery.

` Background There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis. Methodology/Principal Findings Patients (20 females, 15 males, age range 44-78 years, BMI range 24-42) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography - mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman's. -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin. Conclusions/Significance In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.

In the first part of this thesis, palladium(0)-catalyzed and -mediated carbonylations are discussed. Paper I describes a new method for the safe, efficient use of a solid carbon monoxide source in the synthesis of primary and secondary benzamides. In total, 35 benzamides were synthesized from aryl iodides (20 examples, 69-97% yield) and aryl bromides (15 examples, 32-93% yield). Reduction-prone groups were used successfully in the reactions. In paper II, the same protocol was adopted for the palladium(0)-catalyzed synthesis of N-cyanobenzamides from aryl iodides/bromides, carbon monoxide and cyanamide. In total, 22 N-cyanobenzamides were synthesized (42-88% yield). The radiosynthesis of [¹¹C]N-cyanobenzamides is discussed in paper III. In total, 22 compounds were synthesized from various aryl halides in 28-79% decay corrected radiochemical yield. The protocol was then applied to the radiosynthesis of [¹¹C]N-cyanobenzamide analogs of flufenamic acid and dazoxibene.

In the second part of this thesis, compounds of interest in relation to amyloid diseases are discussed. Paper IV describes the solid-phase synthesis of BACE-1 enzyme inhibitors containing secondary and tertiary hydroxyl as the transition state isostere. In total, 22 inhibitors were synthesized. The most potent compound (IC₅₀= 0.19 µM) was co-crystallized at the active site of the enzyme to reveal a new binding mode. In paper V, the evaluation of a potent BACE-1 inhibitor as a potential radiotracer for use in PET is described. The radiolabeled [¹¹C]BSI-IV was obtained in 29±12% decay corrected radiochemical yield by a three-component palladium(0)-mediated aminocarbonylation. Its properties as a potential PET tracer were investigated in vitro by autoradiography and in vivo in rats using small animal PET-CT. A new class of amyloid-binding PET ligands is described in paper VI. Three polythiophenes were labeled with carbon-11 or fluorine-18 (26-43% decay-corrected radiochemical yield). The in vitro studies showed that these ligands bind specifically to amyloid deposits. In vivo PET showed low uptake in the organs of interest in healthy rats and a monkey. These results suggest the labeled thiophenes derivatives could be useful as PET tracers for the study of amyloid diseases.

Peptide drugs are used to treat a broad spectrum of diseases such as cancer and HIV and have many more promising applications, such as new vaccines against SARS-CoV-2. The most popular manufacturing method for peptides is solid-phase peptide synthesis (SPPS). The main drawback of SPPS is that it is a costly and wasteful process. 

SpinChem is a company that provides technology solutions for chemical processes. Recently, SpinChem has started investigating if their Rotating Bed Reactor (RBR) is suitable for peptide synthesis. The goal of this project is to investigate how the RBR can make processes like SPPS more resource-efficient. The idea is that the RBR-system can maximize the solid-phase to liquid ratio (STL). The STL is the ratio of the volume of solid-phase material and the volume of liquid. By maximizing the STL, it is possible to manufacture peptides using less solvents and chemicals. The main quest of the project is formulated into a single question: 

How does a high STL affect the efficiency of the RBR-system? 

To answer the question, Minitab's statistical software and design of experiments (DOE) will be used to plan and perform experiments in both lab- and industrial scales. DOE factorial experiments are used to gain as much information as possible about the new RBR-system. The results are analyzed and summarized to make a solid foundation for the continued work on the new RBR application. 

Peptide synthesis efficiency in the RBR-system is measured using ionic adsorption. The ionic adsorption rate is measured in both lab-scale and industrial-scale experiments. In the lab-scale experiments, the decrease of ions was on average 86,5% after just 15 s with an average STL of 0,936. The industrial-scale experiments showed a similar result where the average decrease in ions was 92,9% after 20 s with an average STL of 0,947. It was concluded that the RBR-system can reduce the consumption of washing-solvent in SPPS by up to 82%.

The use of animal experiments can be minimized with computational models capable of reflecting the simulated environments. One such environment is intestinal fluid and the colloids formed in it. In this study we used molecular dynamics simulations to investigate solubilization patterns for three model drugs (carvedilol, felodipine and probucol) in dog intestinal fluid, a lipid-based formulation, and a mixture of both. We observed morphological transformations that lipids undergo due to the digestion process in the intestinal environment. Further, we evaluated the effect of bile salt concentration and observed the importance of interindividual variability. We applied two methods of estimating solubility enhancement based on the simulated data, of which one was in good qualitative agreement with the experimentally observed solubility enhancement. In addition to the computational simulations, we also measured solubility in i) aspirated dog intestinal fluid samples and ii) simulated canine intestinal fluid in the fasted state, and found there was no statistical difference between the two. Hence, a simplified dissolution medium suitable for in vitro studies provided physiologically relevant data for the systems explored. The computational protocol used in this study, coupled with in vitro studies using simulated intestinal fluids, can serve as a useful prescreening tool in the process of drug delivery strategies development.

Bioavailability of oral drugs often depends on how soluble the active pharmaceutical ingredient is in the fluid present in the small intestine. For efficient drug discovery and development, computational tools are needed for estimating this drug solubility. In this paper, we examined human intestinal fluids collected in the fed state, with coarse-grained molecular dynamics simulations. The experimentally obtained concentrations in aspirated duodenal fluids from five healthy individuals were used in three simulation sets to evaluate the importance of the initial distribution of molecules and the presence of glycerides in the simulation box when simulating the colloidal environment of the human intestinal fluid. We observed self-assembly of colloidal structures of different types: prolate, elongated, and oblate micelles, and vesicles. Glycerides were important for the formation of vesicles, and their absence was shown to induce elongated micelles. We then simulated the impact of digestion and absorption on the different colloidal types. Finally, we looked at the solubilization of three model compounds of increasing lipophilicity (prednisolone, fenofibrate, and probucol) by calculating contact ratios of drug–colloid to drug–water. Our simulation results of colloidal interactions with APIs were in line with experimental solubilization data but showed a dissimilarity to solubility values when comparing fasted-/fed-state ratios between two of the APIs. This work shows that coarse-grained molecular dynamics simulation is a promising tool for investigation of the intestinal fluids, in terms of colloidal attributes and drug solubility.

The present invention provides, inter alia, methods, models, and systems for selecting an effector having specificity for a target molecule. The methods and systems of the present invention involve several steps, including compiling a database containing structural data for a library of molecules and a population of ligands and activity data, establishing structure-based equivalence of sequence elements in the library of molecules, determining likely spatial orientations of population ligands in library molecules, calculating interaction energies for each ligand-molecule pair, generating statistical models that are predictive of sequence elements likely to contribute to a differential effect of ligands on molecules, selecting an effector that is likely to have a desired specificity for the target molecule, experimentally determining activity data for effector-library molecule pairs, and at least once repeating the steps listed above wherein the effector is a member of the population of ligands.

The thioether motif is found in numerous pharmaceuticals. The simplest form of this motif, the methyl-thioether is similarly found in many biologically active compounds and has exhibited many advantageous properties. The installation of the thiomethyl moiety can be troublesome due to the toxicity and malodor of small sulfur reagents. To avoid these issues, many alternative sources and methods have been developed, although these often require metal catalysis, or operate via electrophilic species as the sulfur source.

The first section of this thesis covers the discovery and development of BF₃SMe₂ as a Lewis acid and non-malodorous source for nucleophilic installation of the thiomethyl moiety. In paper I, this reagent is leveraged for the Lewis acid activation and the nucleophilic thiomethylation of electron-deficient haloarenes. The reagent was also found to be a selective reductant for nitropyridines and could in some instances perform concomitant thiomethylation via C-H substitution. The nucleophilic installation of the thiomethyl moiety was continued in paper II, where BF₃SMe₂ was developed for the conversion of aromatic aldehydes into methyl-dithioacetals. The dithioacetal is an important protecting group, and a useful intermediate, and since there are only a handful of reported strategies for the synthesis of the methyl-analog, this method represents an important addition to existing methods. In addition, BF₃SMe₂ was able to promote Friedel-Crafts reactions between aldehydes and electron-rich arenes, resulting in unsymmetrical, thiomethylated diarylmethanes. 

In the final part of this section, BF₃SMe₂ was used for the activation of trifluoromethylarenes for defluorination and sulfur incorporation, resulting in the methyl-dithioester moiety. This functional group is a useful intermediate, but current methodology for its synthesis suffers from several drawbacks, including multistep reactions. The method developed in paper III is a convenient one-step approach to reach the methyl-dithioester, and was also expanded to one-pot synthesis of thioamides and different heterocyclic systems starting from the trifluoromethyl moiety.

In the second section of this thesis, an in situ method for diazomethane release and consumption was investigated and applied. Diazomethane is a useful reagent with a unique reactivity profile, including mild and selective O-methylation of carboxylic acids. The reactivity of this gaseous reagent however comes with hazards, such as high toxicity. Therefore, earlier efforts have been made for in situ generation of diazomethane, although with limitations such as specific solvent requirements or alkaline conditions. The method developed in paper IV however is an improvement upon these with base-free conditions, and wide solvent compatibility, and was successfully applied to the methylation of carboxylic acids, solvent-mediated deuterium labeling without any a priori deuterium incorporation, and synthesis of diazoketones.

Proteolysis targeting chimeras (PROTACs) represent a new modality in drug design. They are bifunctional molecules; with one end, they bind to a protein of interest, and with the other end, they bind to endogenous E3 ligases. The E3 ligases will then attach ubiquitin to the protein of interest, which marks it for degradation by endogenous cellular mechanisms. This can be used to silence a certain protein, which is useful if the protein plays an important role in a certain disease. The protein NUDT5 was recently identified as playing a major role in breast cancer, for which reason silencing it could provide new treatment opportunities for the disease. PROTAC candidates were synthesised using two different NUDT5 ligands previously identified by researchers at Karolinska Institute, two commercially available E3 ligase ligands and five different linker designs. These designs were chosen to ensure a wide coverage of chemical space, resulting most importantly in varying lengths and hydrophobicities and using a variety of chemical techniques. 16 compounds were obtained, characterized and sent to Karolinska Institute for biological testing. All of the tested compounds were binders to NUDT5 according to a differential scan fluorimetry assay, and further testing indicated that one of them induces degradation of endogenous NUDT5. This is a very promising result, and for this reason, said compound should be optimized to elucidate its structure-activity relationship and take further steps towards using it to treat breast cancer.

Since the outbreak of the HIV/AIDS pandemic in the 1980s, the disease has cost the lives of over 30 million people, and a further 33 million are currently living with the HIV infection. With the appropriate treatment, HIV/AIDS can today be regarded as a chronic but manageable disease. However, treatment is not available globally and UNAIDS still estimates that there are currently 5000 AIDS-related deaths worldwide per day.

HIV protease inhibitors (PIs) constitute one of the fundaments of HIV treatment, and are commonly used in so-called highly active antiretroviral therapy (HAART), together with reverse transcriptase inhibitors. Although there are ten PIs on the market, there is still a need for novel structures. The rapid development of resistant strains, due to the high frequency of mutations, together with the commonly observed adverse effects of the drugs available, illustrate the need to develop new potent structures.

Two novel scaffolds were investigated in this work. A tertiary alcohol-containing scaffold comprising a three-carbon tether, and a β-hydroxy γ-lactam-based scaffold were designed, synthesized and evaluated using enzyme- and cell-based assays. X-ray analyses of inhibitors from each class provided information on inhibitor–protease interactions. The inhibitors containing the tertiary alcohol provided at best an enzymatic inhibition (K_i) of 2.3 nM, and an inhibition in the cell-based assay (EC₅₀) of 0.17 µM. The γ-lactam-based inhibitors exhibited better inhibition than the first series; the best values being K_i = 0.7 nM and EC₅₀ = 0.04 µM.

The second part of these studies involved the evaluation of a novel non-resonance continuous-flow microwave instrument. The instrument was validated regarding heating capacity, temperature stability and temperature homogeneity. A number of model reactions were performed with low- and high-microwave-absorbing solvents. It was found that the microwave heating source allowed rapid temperature adjustment, together with easily regulated, flow-dependent reaction times, providing an efficient tool for reaction optimisation.