The factors affecting peptide fragmentation have been extensively studied in the literature in order to better predict the fragment ion spectra of peptides and proteins. While there are countless influences to consider, metal cation binding in the gas-phase is particularly interesting. Herein, a comparison of fragmentation patterns of a model peptide series with various charge carriers (H+, Li+, Na+, K+, and Cu+) will assist in determining the location of the preferred binding site of the metal cation and in assessing differences in the fragmentation pattern as a result of this binding site. An interesting observation from these studies reveals abundant x-type fragment ions occurring from the fragmentation of alkali-metal cationized peptides. As these fragment ions have been observed in previous studies by others but not addressed, the factors affecting the formation of these x-type fragment ions are explored. Additionally, a home-built 193-nm photodissociation tandem time-of-flight mass spectrometer is utilized to study how peptide fragmentation kinetics affect the fragmentation pattern observed. Initially, the fragmentation timescales of various peptides are investigated. Results indicate that longer fragmentation timescales (~10 microseconds) result in an increased number of identified peaks with internal and ammonia loss fragment ions being the most common in comparison to 'prompt' fragmentation timescales (~1 microsecond). Furthermore, b-type fragment ion formation is also favored at longer timescales for the arginine containing peptides investigated. The fragmentation pattern of several proline containing peptides is examined by collision-induced dissociation and 193-nm photodissociation. Unique fragment ions are observed with each occurring at a proline residue. Few differences are detected between CID and 193-nm photodissociation spectra, indicating that the proline residues direct fragmentation rather than the dissociation method. In an effort to improve the performance of the photodissociation tandem TOF instrument, the addition of a second source and a dual-stage reflectron are incorporated. The modifications result in improved mass range, signal-to-noise, and increased fragment ion collection efficiencies. High quality mass spectra are acquired across a range of mass-to-charge ratios from ~600 to 1900. Furthermore, the modifications continue to allow investigation of various fragmentation timescales with the addition of an additional timeframe of ~3 microseconds.

Pagrindinis šio darbo tikslas buvo susintetinti vandenyje tirpius norimos struktūros bei skirtingo pakeitimo laipsnio skiepytuosius chitozano-polietilenglikolio (MPEG) kopolimerus bei katijonizuotus chitozano darinius ir ištirti jų savybes. „Klik“ chemijos reakcijų pagalba susintetinti nauji įvairaus pakeitimo laipsnio chitozano-MPEG skiepytieji kopolimerai, turintys triazolilliekaną. Pasiūlyti nauji chitozano-C(6)-MPEG bei N-2,3-epoksipropil-N,N,N-trimetilamonio chloridu C(6)-katijonizuoto chitozano sintezės būdai, chitozano aminogrupių apsaugai naudojant chitozano kompleksus su dodecilsulfatu. Dalinai N-katijonizuoto chitozano darinius papildomai katijonizuojant šarminėje terpėje, gauti N,O-katijonizuoti chitozano dariniai, turintys labai didelį katijonizavimo laipsnį. Pasiūlytas katijonizuoto chitozano fermentinės hidrolizės metodas, kurį naudojant chitozano darinio molekulinę masę lengvai galima sumažinti dešimtimis kartų. Chitozaną modifikuojant vyno, citrinų, adipo rūgštimis, susintetinti dalinai tinklinti chitozano dariniai. Prie chitozano ir karboksirūgštimis modifikuotų chitozano nanodalelių prijungus (4-cianpentano rūgšties)-4-ditiobenzenkarboksilatą, susintetintas makroiniciatorius gyvybingajai radikalinei polimerizacijai RAFT metodu. / The main aim of this work was to synthesize water-soluble chitosan-methoxy poly(ethylene glycol) (MPEG) graft copolymers and quaternized derivative of chitosans of various structure and desirable graft density and to study their properties. Novel chitosan-MPEG derivatives containing triazolyl moiety with different degree of substitution of chitosan were prepared for the first time by “click chemistry”. Several new schemes of the synthesis of chitosan-C(6)-TMPEG and C(6)-quaternized chitosan derivatives were suggested based on protection of amino functionality by using chitosan-dodecyl sulfate complexes. Additional cationization of cationic chitosan through its hydroxyl groups in alkaline medium enabled to prepare N,O-quaternized chitosans derivatives with very high degree of quaternization. It was suggested method of enzymatic hydrolysis of quaternized chitosans which allow a tenfold decrease the molecular weight of chitosans derivatives. Modification of chitosan by tartaric, citric or adipic acid yielded partially crosslinked chitosan derivatives. Modification of chitosan and further modification of chitosan nanoparticles by dithiobenzendicarboxylate resulted in RAFT macroinitiators which are precursors of functionalized nanoparticles.

The main aim of this work was to synthesize water-soluble chitosan – methoxy poly(ethylene glycol) (MPEG) graft copolymers and cationized chitosan derivatives of various structure and desirable graft density, and to study their properties. Novel chitosan-MPEG derivatives with different degree of substitution of chitosan were prepared for the first time by “click” chemistry. Several new schemes of the synthesis of chitosan-MPEG and additionally cationized chitosan derivatives were suggested based on protection of amino functionality by using chitosan-dodecyl sulphate complexes. Additional cationization of chitosan through its hydroxyl groups in alkaline medium enabled to prepare chitosan derivatives with very high charge density. A method of enzymatic hydrolysis of the cationized chitosans was proposed which allowed a tenfold decrease of the molecular weight of chitosan derivatives. Modification of chitosan by tartaric, citric or adipic acid yielded chitosan nanoparticles. Further modification of chitosan nanoparticles by dithiobenzendicarboxylate resulted in RAFT macroinitiators used as precursors of functionalized nanoparticles. / Pagrindinis šio darbo tikslas buvo susintetinti vandenyje tirpius norimos struktūros bei pakeitimo laipsnio skiepytuosius chitozano – polietilenglikolio (MPEG) kopolimerus bei katijonizuotus chitozano darinius ir ištirti jų savybes. Įvairaus pakeitimo laipsnio chitozano-MPEG skiepytieji kopolimerai susintetinti vykdant „klik“ chemijos reakcijas. Pasiūlyti nauji chitozano-MPEG bei papildomai katijonizuoto chitozano sintezės būdai, chitozano aminogrupių apsaugai naudojant chitozano kompleksus su dodecilsulfatu. Dalinai katijonizuoto chitozano darinius papildomai katijonizuojant šarminėje terpėje, gauti chitozano dariniai, turintys labai didelį krūvio tankį. Pasiūlytas katijonizuoto chitozano fermentinės hidrolizės metodas, kurį naudojant chitozano darinio molekulinę masę lengvai galima sumažinti dešimtimis kartų. Chitozaną modifikuojant vyno, citrinų arba adipo rūgštimis, susintetintos nanodalelės. Prie chitozano nanodalelių prijungus (4-cianpentano rūgšties)-4-ditiobenzenkarboksilatą, susintetintas makroiniciatorius gyvybingajai radikalinei polimerizacijai RAFT metodu.

Abstract Starch is one of the most abundant polysaccharides found in nature and is widely utilized in various fields of industry. Due to the complex structure of native starch it is insoluble in most organic solvents and needs modification prior utilization. In this study, ionic liquids (ILs), modern green chemistry alternatives for common solvents were used as reaction media in starch modification. At first various starch species were depolymerized in 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) with p-TsOH as a catalyst. Microwave activation or conventional bath heating were used as heating methods while HPLC-ELSD was used as an analytical method. All studied starch species depolymerized similarly into water-soluble starch oligomers while microwave activation shortened the depolymerization time considerably compared to oil bath heating. Barley starch was chosen for further experiments, in which various ILs were studied as potential media for starch dissolution and depolymerization. Results suggested that both the anion and the cation part of the IL had an effect on the dissolution and depolymerization of barley starch. After the depolymerization reactions, the depolymerized barley starch was further modified by cationization. [AMIM]Cl was used as the reaction media, microwave activation as the heating method while HPLC-ELSD, 1H NMR and elemental analysis were used as analytical methods. The modified products had DS values from 0.2 to 0.5 depending on the reaction conditions. The products were studied as potential binding agents for heavy metal ions which showed that moderately substituted modified starch (DS 0.4) could be used to bind Cu(II), Fe(III) and Zn(II) ions from an aqueous solution. Finally, potato peel waste was studied as an alternative starch source to produce cationized starch for wastewater purification. Peel waste was pre-treated by alkaline depolymerization after which it was cationized in a water solution to produce cationized products with DS from 0 to 0.35. The cationized peel waste products were studied preliminary as binding agents for Cu(II) ions from a water solution using ICP-OES as an analytical method. The results suggested that when the molar ratio between cationized waste starch and copper was 3:1, cationized waste starch was an effective binding agent for copper ions. / Tiivistelmä Tärkkelys on yksi yleisimmistä luonnossa esiintyvistä polysakkarideista. Sitä hyödynnetään useilla eri teollisuuden aloilla. Monimutkaisen rakenteensa vuoksi tärkkelys on liukenematon useimpiin orgaanisiin liuottimiin ja veteen, minkä vuoksi sitä täytyy modifioida ennen käyttöä. Tässä väitöstutkimuksessa tärkkelyksen modifioinnissa käytettiin ionisia nesteitä reaktioväliaineena. Tutkimuksen alussa eri tärkkelyslajeja depolymeroitiin 1-allyyli-3-metyyli-imidatsoliumkloridissa ([AMIM]Cl) katalyyttinä p-TsOH. Mikroaaltoaktivointia ja haudekuumennusta käytettiin vaihtoehtoisina lämmitysmenetelminä. Reaktion edistymistä ja tuotteiden muodostumista tutkittiin HPLC-ELSD -menetelmällä. Eri tärkkelyslajit depolymeroituivat samankaltaisesti vesiliukoisiksi, lyhytketjuisiksi tärkkelysoligomeereiksi. Mikroaaltoaktivointi lyhensi reaktioaikaa haudekuumennukseen verrattuna. Tutkimuksen seuraavassa vaiheessa tutkittiin ohratärkkelyksen liukoisuutta ja depolymeroitumista eri ionisissa nesteissä. Tulosten perusteella ionisen nesteen sekä anioni- että kationiosa vaikuttivat tärkkelyksen liukenemiseen. Depolymeroidun ohratärkkelyksen modifiointitutkimuksia jatkettiin [AMIM]Cl:ssa kationisoinnilla. Lämmitysmenetelmänä käytettiin mikroaaltoaktivointia. Tuotteet tutkittiin käyttäen alkuaineanalyysiä sekä HPLC-ELSD- että 1H NMR-tekniikoita. Kationisoitujen tuotteiden substituutioaste (DS) vaihteli reaktio-olosuhteista riippuen välillä 0.2–0.5. Saatuja tuotteita tutkittiin raskasmetalli-ionien sitomisessa vesiliuoksesta. Havaittiin, että kohtalaisesti substituoitu (DS 0.4) modifioitu tärkkelys sitoi Cu(II)-, Fe(III)- ja Zn(II)-ioneja vesiliuoksesta. Tutkimuksen loppuosassa tutkittiin perunan kuorijätettä vaihtoehtoisena tärkkelyslähteenä kationisoidun tärkkelyksen valmistamisessa. Kuorijäte esikäsiteltiin kuumentamalla se emäksisessä etanoliliuoksessa, minkä jälkeen sille suoritettiin kationisointi vesiliuoksessa. Kationisten tuotteiden substituutioasteet vaihtelivat välillä 0–0.35. Tuotteiden soveltuvuutta Cu(II)-ionien sitomiseen vesiliuoksesta tutkittiin ICP-OES -menetelmän avulla. Alustavien tulosten mukaan kationisoitu jätetärkkelys sitoi kupari-ioneja vedestä, kun tärkkelyksen ja kuparin moolisuhde oli 3:1.

Les protéines extraites des végétaux sont des matériaux relativement peu coûteux, non toxiques, biocompatibles et biodégradables. Elles représentent une bonne alternative aux protéines d’origine animale et aux polymères dérivés du pétrole. Dans le cadre de cette étude, les protéines extraites de graines de soja et de tournesol ont été utilisées en tant que matériaux enrobants pour la microencapsulation de la matière active hydrophobe (α-tocophérol) ou hydrophile (acide ascorbique) par le procédé d’atomisation. Les protéines de soja sont largement utilisées dans les applications alimentaires et non-alimentaires, notamment en microencapsulation. Elles sont donc étudiées dans ce travail comme matériau enrobant de référence. Les protéines de tournesol n’ont quant à elles pas d’application industrielle concrète, si ce n’est sous la forme de tourteaux dans l’alimentation animale. C’est pourquoi il nous semble pertinent de trouver des nouvelles voies de valorisation pour ce coproduit d’origine agricole. Plusieurs modifications des protéines, telles que l’hydrolyse enzymatique, l’acylation, la réticulation enzymatique et la cationisation ont été étudiées dans le but d’améliorer les propriétés encapsulantes du matériau enrobant. Dans le contexte de la chimie verte, toutes les modifications ont été effectuées sans utilisation de solvants organiques ni de catalyseurs chimiques. L’influence des modifications chimiques et enzymatiques des protéines, et des paramètres du procédé (pression d’homogénéisation, ratio matériau enrobant/matière active et concentration en protéines) sur les différentes caractéristiques des préparations liquides et des microparticules (viscosité, taille des gouttelettes dans le cas des émulsions, morphologie et taille des microparticules), ainsi que sur les paramètres liés au procédé d’atomisation (rendement et efficacité de microencapsulation) a été particulièrement étudiée au cours de ce travail. Les résultats obtenus confirment que l’extrait protéique de tournesol est tout à fait pertinent comme matériau enrobant et permet d’obtenir des efficacités de microencapsulation significativement plus élevées par rapport à celles obtenues avec l’extrait protéique de soja. / Proteins extracted from vegetables are relatively low-cost, non-toxic, biocompatible and biodegradable raw materials. They represent a good alternative to animal-based proteins and petroleum-extracted polymers. In this study, proteins derived from soybean and sunflower seeds were used as wall materials for microencapsulation of hydrophobic (-tocopherol) or hydrophilic (ascorbic acid) active material by spray-drying technique. Soybean proteins are widely used in food and non-food applications, especially in microencapsulation. They were studied in this work as wall material of reference. Sunflower proteins are not actually used in industrial application, but only in the form of oil-cake for animal feeding. That’s why new ways of valorization of this agricultural by-product should be investigated. Several proteins’ modifications such as enzymatic hydrolysis, acylation, cross-linking and cationization were studied in order to improve encapsulating properties of wall material. In the context of green chemistry, all the modifications and preparations were performed without use of organic solvents and chemical catalysts. The effect of protein chemical and enzymatic modifications, and process parameters (homogenization pressure, wall/core ratio and protein concentration) on different characteristics of liquid preparations and microparticles (viscosity, emulsion droplet size, microparticle size and morphology) and on parameters related to the spray-drying process (yield and efficiency of microencapsulation) was particularly investigated in this study. The obtained results confirmed that sunflower proteins are quite suitable as encapsulating agent and provide the microencapsulation efficiencies significantly higher compared to those obtained with soy proteins.