Grace à leur excellente propriété photochromique, les dérivés d’azobenzène trouvent de plus en plus d’applications pour le photocontrôle spatio-temporel de conformations moléculaires, de réactivités chimiques, des activités biologiques et pharmaceutiques. Dans ce contexte, introduire un motif photochromique dans les macrocycles contenant de glucides représente une approche excitante afin de moduler par la lumière les propriétés physicochimiques, chimiques et biologiques de cette unique classe de molécules qui attirent d’attention croissante en chimie médicinale et en science des matériaux.Deux séries de glycomacrocycles photocommutables ont été synthétisés dans cette thèse. La première est obtenue via une approche d’O-alkylation en one-pot, à partir 2,2’-dihydroxyazobenzène et di-O-bromoacétyl glycosides. Ces glycomacrocycles possèdent d’excellentes propriétés photochromiques, avec une haute résistance à la fatigue. Le transfert de chiralité de glucide à l’azobenzène a été observé. L’un des glycomacrocycles est capable de former d’organogels qui répond aux stimuli photo, thermique et mécanique. La seconde série de macrocycles est synthétisée via une glycosylation intramoléculaire, en utilisant dihydroxyazobenzène comme attache. Dans cette approche, l’efficacité et la stéréoselectivité de glycosylation sont étudiées en modulant la nature de liens et la configuration d’azobenzène. / As excellent photochromic molecules, azobenzene derivatives have increasing applications in the spatial and temporal photocontrol of molecular conformation, chemical reactivity, biological and pharmacological activities. In this context, an exciting attempt is introducing a photochromic motif into carbohydrate-containing macrocycles, a unique class of products with growing attention in medicinal and material science, so as to modulate their physicochemical, chemical and biologic properties by light.Two series of photoswitchable glycomacrocycles have been prepared in this thesis. The first one is obtained from 2,2’-dihydroxyazobenzene and di-O-bromoacetyl glycosides via one-pot O-alkylation approach. These macrocycles show excellent photophromic properties with high fatigue resistance. Chirality transfer from sugar to azobenzene moiety has been observed. One of the macrocycles can form organogel that responses to photo-, thermal- and mechanical stimulus. The second series of glycomacrocycles were prepared through intramolecular glycosylation by using dihydroxyazobenzene as tether. The efficiency and the stereoselective outcome of glycosylation have been investigated by modulating the nature of the linkers and the configuration of the azobenzene tether.

Cryopreservation is a method used to preserve the quality of various cell types over long periods of time (up to several years). Using this preservation method can vastly improve cellular therapies and regenerative medicine by allowing the creation of biobanks containing high-quality cell products. For example, biobanks of red blood cells (RBCs) would be beneficial for cellular therapies such as RBC transfusions, which are used to treat patients suffering from hemorrhages, anemias, and to replace blood loss after traumatic/surgical events. RBCs are currently preserved via hypothermic storage which limits their shelf life to 42 days. Similarly, biobanks of hematopoietic stem cells (HSCs) from umbilical cord blood would be beneficial for regenerative medicine therapies such as HSC transplantations, which offer treatment for blood- and immune-related diseases by reconstituting hematopoiesis. The outcome of these transplantations depends greatly on the quality of the cell product; therefore, it is important for preserved HSCs to have a minimum loss of viability and functionality. The cryopreservation of cells at low sub-zero temperatures (-80 to -196 degC) in a cryoprotectant solution allows for long-term storage. Common cryoprotectants used are 40% glycerol for the cryopreservation of RBCs and 10% dimethyl sulfoxide (DMSO) for the cryopreservation of HSCs. Before clinical use of cryopreserved products, DMSO and glycerol must be removed as they are severely toxic to patients upon infusion. The removal of 40% glycerol from RBCs is complicated, time consuming, and can result in a significant amount of cell damage. DMSO and glycerol also do not address the occurrence of ice recrystallization, which is the main cause of cellular damage during cryopreservation. Ice recrystallization describes the process of ice crystals growing larger and replacing smaller ice crystals, and significantly contributes to the damage of cells post-thaw. Therefore, methods to decrease the concentration of cryoprotectants to improve their removal process while also mitigating ice recrystallization is of interest. In nature, antifreeze proteins and glycoproteins (AF(G)Ps) are found in animals that can survive below-freezing temperatures. The Ben laboratory has used the structural components of AF(G)Ps to design several small-molecule carbohydrates that exhibit ice recrystallization (IRI) activity. O-aryl-b-D-glucosides and N-aryl-D-gluconamides are two classes of IRIs developed that have been used as supplemental additives to DMSO and glycerol to improve the post-thaw viabilities and functionalities of RBCs and HSCs. While many structure-activity relationship studies have been performed amongst these classes, one area of improvement is their solubilities to facilitate their use as cryoprotectants. This thesis focuses on the design of a new class of effective IRIs that have high solubilities (>100 mM in phosphate-buffered saline). Previous studies on the structure of small-molecule IRIs have demonstrated the importance of balancing the hydrophobicity and hydrophilicity within a molecule, making it difficult to achieve high solubilities. This thesis further explores this point by the design and synthesis of IRIs with polar functional groups possessing an overall molecular charge. N-aryl-D-gluconamides bearing amino- and azido-substituents were designed, however their synthesis was unsuccessful. Instead, this work revealed a synthetically facile route towards N-xylo-L-furanosyl amide and ester compounds. Phosphonate-substituted carbohydrates were also designed and synthesized as a means to obtain highly soluble IRIs. All of these compounds displayed high solubilities, however the majority of the compounds exhibited moderate IRI activities. While there are many assays used to measure IRI activity, this thesis also evaluates two of the most common IRI assays and their effect on IRI activity. In addition, the effect that cryoprotective agents (CPAs) like DMSO and glycerol have on IRI activity was also evaluated. In both cases, the type of assay used and the addition of CPAs affected the quantitative values describing IRI activity. Notably, DMSO and glycerol, had an antagonistic effect on the IRI activity of N-aryl-D-gluconamides and antifreeze protein type I. This was a significant observation since these IRIs are sufficient cryoprotectants in the presence of DMSO or glycerol. Lastly in this thesis, phosphonate-substituted IRIs and antifreeze (glyco)proteins (AF(G)Ps) were evaluated as cryoprotectants for the cryopreservation of RBCs and/or HSCs. These studies showed that phosphonate IRIs and AF(G)Ps were not toxic to RBCs and/or HSCs, however the concentrations evaluated were unable to improve the post-thaw viability and/or functionality of these cell types.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12936号 / 論農博第2816号 / 新制||農||1033(附属図書館) / 学位論文||H27||N4895(農学部図書室) / 32146 / (主査)教授 伏木 亨, 教授 保川 清, 教授 安達 修二 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19644号 / 人健博第36号 / 新制||人健||3(附属図書館) / 32680 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 三谷 章, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

No description available.

Deriving electrical energy from glucose and other carbohydrates under mild conditions is an important research objective because these biomolecules are abundant, renewable, and can provide 12 to 24 electrons per molecule, yielding substantial electrical power. It was previously observed that disubstituted viologens, salts of N,N'-disubstituted 4,4'-bipyridine, are able to oxidize glucose under alkaline conditions. Building on that initial result, the objective of this work was to understand and quantify the effectiveness and utility of viologens as catalysts for use in direct carbohydrate fuel cells.The extent that viologens oxidize carbohydrates, the conditions under which that oxidation occurs, and the mechanism for the oxidation were examined using oxygen-uptake and other methods. Viologens were found to catalytically oxidize carbohydrates extensively in alkaline solution. Viologens were also found to react with the enediol form of the carbohydrate, initiating carbohydrate oxidation with subsequent reduction of the viologen. If the viologen/carbohydrate ratio is low, electron transfer from the carbohydrate to the viologen becomes limiting and the carbohydrates undergoing oxidation rearrange into unreactive intermediates such as carboxylic acids and alcohols. At high catalyst ratios, excess viologen more rapidly oxidizes the carbohydrate and minimizes formation of unreactive intermediates. We also found that viologen polymers were more efficient than an equivalent concentration of monomers, suggesting that the higher localized concentration in polymeric viologen acts to efficiently oxidize carbohydrates and simulates high viologen/carbohydrate ratios.Monoalkyl viologens, aminoviologens, indigo carmine, and methylene blue were investigated by the method of cyclic voltammetry to inform their use as catalysts in the oxidation of carbohydrates. Redox potentials, diffusion coefficients, and heterogeneous electron-transfer rate constants were determined. Stability in alkaline solution and aqueous solubility were also examined in a semi-quantitative fashion. A comparison between the catalysts was made and viologens were found to be superior based on the examined parameters.The catalytic oxidation of carbohydrates by viologen was also examined using a fuel cell-like device. For the conditions in which a test cell was operated, oxidation efficiencies of up to 33% were observed, compared to previously reported values from about 2.5% to 80%. Anode polarization curves were obtained and used to determine the behavior of the viologen-controlled anode as a function of pH, viologen and carbohydrate concentration, and carbohydrate identity. pH was found to have a stronger effect on the performance at the anode for carbohydrates with a higher number of carbons than those with a lower number.

Previous studies of age-related differences in substrate oxidation over 24 hours and during exercise have provided divergent results, leaving it unclear how and if substrate oxidation is influenced with aging. The purpose of this study was to evaluate age-related differences in substrate oxidation during distinct periods of a 24-hr stay in a room calorimeter: over the entire 24 hours, during activities of daily living (ADLs), and during a 30-min treadmill walk (30MTW, at 1.3 m·s-1). Further, oxygen consumption transients were quantified in response to the 30MTW. Gas exchange measures were obtained during an incremental treadmill test in 9 young (28 + 3 yr) and 6 older (71 + 5 yr) healthy males to quantify VO2peak. On a separate visit, oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured throughout a 24-hr stay in a whole-room calorimeter, and used to determine substrate oxidation from carbohydrate (CHO) and fat for the selected periods. Energy expenditure (EE), CHO oxidation, and fat oxidation were normalized to lean mass (kJ∙kg LM-1), determined by dual energy x-ray absorptiometry. Oxygen consumption on- (oxygen deficit, mL) and off- (rate constant for VO2 decay) transients were determined in response to the 30MTW. As expected, VO2peak (ml∙kg-1 LM∙min-1) was ~24% lower in older compared to young (p=0.021). EE (kJ∙kg LM-1) did not differ significantly by group for any period (p>0.099). Compared with young, older oxidized less CHO over 24 hr (p0.053). In contrast, during the 30MTW CHO oxidation did not differ by group (p>0.148), but older oxidized more fat per LM than young (p=0.002). The contrast in substrate selection between the lower-intensity ADLs (~16 and 20% VO2peak in young and older) and the 30MTW (~28 and 39% VO2peak in young and older) is consistent with a crossover from predominantly fat to CHO at a relatively higher VO2peak (relative to LM and %peak) in this group of relatively fit older males. Notably, when expressed as a percent of total substrate oxidation, older oxidized relatively more fat and less CHO over 24 hr (p=0.012), ADLs (p

Carbohydrates are central components of biological systems, with roles ranging from metabolism to immune signaling, and are utilized as antibiotics, anti-coagulants, and biomaterials. Carbohydrate polymers with ionic functionality, such as alginic acid and chitosan, are used in hydrogels, tissue engineering, drug delivery, and as nucleic acid vectors. The clinical translation of polysaccharide biomaterials is hindered by the poor chemical definition, poor batch-to-batch consistency, and demanding purification process of naturally-obtained material. Additionally, there are few synthetic methods yielding enantiopure, water-soluble carbohydrate polymers with high molecular weight. To address the need for translatable carbohydrate biomaterials, our group recently introduced bioinspired Poly-Amido-Saccharides (PASs) as enantiopure, water-soluble, and well- defined carbohydrate polymers. These previously reported PAS polymers, however, mimic polysaccharides with primarily metabolic roles due to the lack of charged functional groups important for biomaterial applications. In this thesis, I describe the synthetic methodology of a regioselectively amine-functionalized β-lactam carbohydrate monomer, the subsequent synthesis of enantiopure, water-soluble amine-functional PASs (AmPAS), an evaluation of AmPAS biocompatibility and mucoadhesivity for pharmaceutical formulations, and the use of AmPAS for biocompatible nanoparticulate delivery of nucleic acids. Protecting group choices and regioselective modification are key to the synthesis of the AmPAS monomer via [2+2] cycloaddition with electron-deficient isocyanates. The results of a combined experimental and theoretical study indicate that bulky protecting groups of the 6’-OH enforce a 5H4 glycal conformation and favorable overlap of ring σC-O* with the glycal allyloxocarbenium system that enhances negative hyperconjugation effects due to electron withdrawing protecting groups. These data inform AmPAS monomer synthesis, where bulky, electron-withdrawing groups are required for regioselective glycal functionalization and intermediate protecting group stability is necessary to obtain cationic, water-soluble AmPAS. These polymers exhibit minimal cytotoxicity and immunogenicity, and, through single molecule force spectroscopy and ex vivo methods, significant mucoadhesivity important for pharmaceutical application. AmPAS are obtained with tunable molecular weight distributions to allow for nanoscale size- and charge-matched supramolecular assemblies with single stranded RNA and DNA oligonucleotides. These nanoparticles are stable in high serum conditions, exhibit high cell uptake, and are shown to successfully deliver anti-miR-21 oligonucleotides to mediate miR-21 knockdown in vitro. These promising results motivate the future application of AmPAS in small molecule and antisense oligonucleotide delivery formulations. / 2022-01-28T00:00:00Z

Cellulose ethers are valuable matrices for drug-delivery systems (DDS), namely amorphous solid dispersions (ASD). ASD are efficient vehicles that can solubilize and stabilize poorly soluble drugs by increasing the time that it takes for drugs to crystallize, thereby allowing higher drug concentrations and providing increased bioavailability. However, most commercially available cellulose derivatives were not specifically designed for this application, leading to gaps in understanding the key mechanisms by which ASD operate. This creates the need for polysaccharide derivatives specifically conceptualized for ASD and for elucidating structure-property relationships. In this dissertation, I successfully demonstrated regioselective and chemoselective techniques to functionalize cellulose to prepare new ASD as well as smart tracking devices. I efficiently and successfully create complex structures via appending bile salt substituents using olefin cross-metathesis. I ascertained that high performance crystallization inhibitors can be achieved with enhanced hydrophilicity by the marriage of two classes crystallization inhibitors (cellulose and bile salts), as illustrated with the commercial, fast crystallizing prostate cancer drug, enzalutamide. I obtained ketone-functionalized cellulose derivatives using oxidation chemistry to produce fluorescent poly- and oligosaccharides (hydroxypropyl cellulose, hydroxypropyl methylcellulose, and hydroxypropyl beta cyclodextrin). Schiff-base chemistry was then explored to append a commercially available fluorescent label, Nile Blue. Due to the dynamic nature and hydrolytic lability of Schiff-bases, I applied reductive-amination chemistry with either one pot, or two-step techniques and evaluated the efficiency of these approaches. I characterized the new fluorescent polymers, and with the objective of elucidating ASD mechanisms, I investigated their response in solvents of different polarities to probe environment-sensitivity. Flavonoids are interesting drug candidates; they have been explored for many biomedical applications, including as inducers of apoptosis and functioning as antioxidants by radical scavenging. I prepared high-performance ASD polymer candidates, then prepared and characterized ASDs with different loadings of the flavonoids, genistein and quercetin. I explored the performance of polymers with different functionalities, hydrophilicity/hydrophobicity, and carboxylic acid content (cellulose acetate glutarate, 5-carboxypentyl hydroxypropyl cellulose, and hydroxypropyl methyl cellulose acetate succinate as positive control) by using in vitro dissolution studies. In this screening process, I determined that cellulose acetate glutarate provides the most advantageous enhancement, possessing the appropriate amphiphilicity to increase drug concentration in this study, supported by the similarity of the polymer and drug solubility parameters. I was further able to confirm via polarized light microscopy that advantageous nanodroplet formation occurs during the drug-release process. / Doctor of Philosophy / As sources for future ecofriendly materials, derivatives from nature offer fertile ground. One group of natural materials that attracts increasing attention to fulfill both performance and sustainability are polysaccharides, long chains of carbohydrates, that can be found in plant cell walls, exoskeletons of bugs or oceanic bottom feeders, algae, and indeed in all living things. Cellulose derivatives provide biologically safe materials that are biomedically relevant, including in the field of oral drug delivery. While most orally administered drugs are not 100% effective or absorbable, a class of drug delivery systems named amorphous solid dispersions can improve drug absorption with the aid of polysaccharide derivatives. Although amorphous solid dispersions are highly effective, there is still much room for improvement, and important opportunities to learn about the precise mechanisms that make such systems work. With fluorescent markers, I can also explore the surrounding environment of the drug delivery systems in preliminary studies. By understanding the environment of such polysaccharides, I determined important insight into how they improve oral drug availability and performance. Herein, I explored new amorphous solid dispersion polysaccharide derivatives, and how I have attached fluorescent labels to track them to learn how they work.

Eight competitive cyclists performed two cycling trials, one following a high carbohydrate diet (H-CHO) and the other following a low carbohydrate diet (L-CHO). Trials consisted of repeated 60s maximal effort sprints to fatigue at a workload designed to elicit 125-135% VO_2peak at 90rpm. Three min of recovery separated sprints. Muscle biopsies taken at rest (biopsy 1), 85% max interval rpm (biopsy 2), and 70% max interval rpm (biopsy 3) revealed a main effect of diet on muscle glycogen levels: 609 ± 38 HCHO vs. 390 ± 42 mmol/kgdw L-CHO at biopsy 1, 383 ± 29 vs. 252 ± 28 mmol/kgdw at biopsy 2, and 346 ± 29 vs. 196 ± 18 mmol/kgdw at biopsy 3 (p<0.01). Similar decreases in muscle glycogen (45%), creatine phosphate (CP) (35%), and sarcoplasmic reticulum (SR) Ca²⁺-uptake (56%) were shown in both trials from biopsy 1 to 3. SR Ca²⁺-release decreased by 53% in H-CHO subjects and 36% in L-CHO subjects. Total exercise time tended to be longer in H-CHO than L-CHO subjects (57.5 ± 10 vs. 42.0 ± .89min) (p=0.09). H-CHO subjects exercised significantly longer than L-CHO subjects from biopsy 2 to 3 (33.6 ± 10 vs. 18 ± 3.6min) (p< 0.05). Results suggest that fatigue from 40- 60min of intermittent 60s high intensity cycling intervals is associated with reductions in muscle glycogen, CP, and SR function, and that the latter part of performance is impaired by low muscle glycogen. These data do not support a relationship between muscle glycogen status and SR function in intermittent high intensity exercise. / Master of Science