University of Groningen Enzymatic Synthesis and Polymerization of Saccharide-Vinyl Monomers in Aqueous Systems Adharis, Azis

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University of Groningen

Enzymatic Synthesis and Polymerization of Saccharide-Vinyl Monomers in Aqueous Systems

Adharis, Azis

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Adharis, A. (2019). Enzymatic Synthesis and Polymerization of Saccharide-Vinyl Monomers in Aqueous

Systems. University of Groningen.

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Summary

Samenvatting

Acknowledgments

About the Author

List of Publications

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Summary

Carbohydrates are a renewable biomass in which they are formed regularly through photosynthetic reactions in plants. Carbohydrates are available almost everywhere on 1ÖũŶĞÖłùŶĞāƑÖũĢāŶĢāŭŋĕùĢƦāũāłŶóÖũðŋĞƘùũÖŶāŭĢłłÖŶŽũāĢŭÖðŽłùÖłŶ̍NŋƒāƑāũ̇ŶĞāłāāù for utilization of carbohydrates as polymeric materials only arose in the last few decades due to several reasons. First, most polymers are generated from fossil resources that are predicted to be exhausted in the next several hundred years. Therefore, carbohydrates ŋƦāũÖłÖķŶāũłÖŶĢƑāũāłāƒÖðķāĕāāùŭŶŋóĴĕŋũŶĞĢŭťŽũťŋŭā̍āóŋłù̇ŶĞāĞŽĿÖłÖƒÖũāłāŭŭŋĕ creating more sustainable polymers, that have less impact on the environment compared to the fossil-based polymers, was improved. Third, novel functional polymeric materials can be developed when resources with complex functionalities, like carbohydrates, are incorporated in the polymeric structures. For example, in recent years it was reported that glycopolymers which are comprised of carbohydrates as pendant moieties have been developed and are suitable for applications as disease inhibitors, biosensors, as well as drug delivery systems. Glycomonomers, the precursor of these glycopolymers, consist of saccharide units that are linked to some polymerizable groups of which vinyl groups are the most exploited ones.

This thesis discusses the synthesis of several glycomonomers and polymerization of the monomers via environmentally friendly methods. The synthesis of saccharide-vinyl (macro)monomers utilized carbohydrates as starting materials and enzymes as biocatalyst, which are both derived from renewable resources. In addition, the glycomonomers were successfully polymerized by reversible addition–fragmentation chain transfer (RAFT) polymerization and free radical polymerization (FRP) as well as by enzyme-mediated FRP in aqueous solvents. Double-hydrophilic and amphiphilic block glycopolymers were prepared and their self-assembly resulting in polymeric micelles was studied. An overview of the performed projects in this thesis is shown in Figure S1.

Chapter 1 of this thesis presents a brief introduction about carbohydrates as well as the role of enzymes in synthetic organic reactions. Additionally, a compact review of recent literature on biocatalytic synthesis of saccharide-vinyl (macro)monomers is given. Hydrolase enzymes like lipases, proteases, and glycosidases, were found to catalyze the synthesis of these monomers.

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Figure S1 Schematic representation of the synthesis of glycomonomers and glycopolymers in this

thesis.

Chapter 2 demonstrates the kinetically controlled enzymatic synthesis of novel glucosyl-(meth)acrylamide monomers using the Ⱦ-glucosidase derived from almonds. The aqueous transglycosylation reaction was performed on cellobiose and (hydroxy)alkyl (meth) acrylamide substrates which, respectively, worked as the glucosyl donor and acceptor. The obtained glycomonomers, namely N-(Ⱦ-glucosyloxy)ethyl acrylamide, N-(Ⱦ-glucosyloxy) ethyl methacrylamide, and N-(Ⱦ-glucosyloxy)butyl methacrylamide, are proven to be anomerically pure and monofunctional. Their respective structures were characterized by

1_{H NMR,}13_{C NMR, and mass spectrometry measurements. An improvement of the monomer}

yield from 16% to 68% was achieved by replacing cellobiose with activated glucose (p-nitrophenyl Ⱦ-D-glucopyranoside) and using an ionic liquid (BMIMPF₆)-water mixture

as the reaction medium. The synthesized glycomonomers were successfully polymerized ðƘÖŨŽāŋŽŭD¦ÖłùD̍1!ĿāÖŭŽũāĿāłŶŭóŋłƩũĿāùŶĞÖŶŶĞāėķƘóŋťŋķƘĿāũŭŋðŶÖĢłāù by RAFT polymerization showed molecular weights from 43 to 66 kg mol-1_{with a narrow}

dispersity (Ð ͟ ː̍ˑ˘̜̇ ƒĞĢķā ŶĞā ėķƘóŋťŋķƘĿāũŭ ťũāťÖũāù ðƘ D āƗĞĢðĢŶāù ĿŋķāóŽķÖũ weights of about 220 kg mol-1_{ƒĢŶĞÖðũŋÖùùĢŭťāũŭĢŶƘ̛ˑ̍˓˕͟Ð͟˒̍ˑ˘̜̍mŋũāŋƑāũ̇ŶĞā}

identical glycopolymers synthesized by both techniques showed a similar glass transition ŶāĿťāũÖŶŽũā̛ÖũŋŽłùː˓ˑ̞ː˖ː΅!̜̇ÖŭĿāÖŭŽũāùðƘùĢƦāũāłŶĢÖķŭóÖłłĢłėóÖķŋũĢĿāŶũƘ̍ The synthesized glucosyl-(meth)acrylamide monomers in Chapter 2, as well as the glucosyl-(meth)acrylate monomers reported by Kloosterman et al. (see Figure S1), can act as precursors for polymerization reactions. For example, the polymerization of the glucosyl units of these monomers by cellodextrin phosphorylase yielded óāķķŋŋķĢėŋŭÖóóĞÖũĢùā̟ƑĢłƘķĿÖóũŋĿŋłŋĿāũŭÖŭŋŽŶķĢłāùĢł!ĞÖťŶāũ˒̍RłÖðŽƦāũŭŋķŽŶĢŋł̇ the enzyme catalyzed reverse phosphorolysis reaction was carried out with glucosyl-vinyl

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monomers and Ƚ-glucose 1-phosphate serving as the glucosyl acceptor and the glucosyl donor, respectively. The enzymatic synthesis was followed by thin layer chromatography ÖłùŶĞāĢŭŋķÖŶāùťũŋùŽóŶƘĢāķùŭƒāũāÖðŋŽŶ˕˔ͮ̍DĢƑāŶƘťāŭŋĕƒāķķ̟ùāƩłāù̇ƑĢłƘķ̟ðÖŭāù oligocelluloses were successfully prepared, namely (Ⱦ-oligocellulosyloxy)ethyl acrylate (OC-EA), (Ⱦ-oligocellulosyloxy)ethyl methacrylate (OC-EMA), (Ⱦ-oligocellulosyloxy) butyl acrylate (OC-BA), (Ⱦ-oligocellulosyloxy)ethyl acrylamide (OC-EAAm), and (Ⱦ-oligocellulosyloxy)ethyl methacrylamide (OC-EMAAm). These macromonomers possess an average number of repeating glucosyl units of 7.3–8.9 and show average molecular weights of 1310–1553 g mol-1_{, according to}1_{H NMR, MALDI-ToF MS, and SEC}

experiments. In addition, bond fragmentation at the Ƚ-position of (meth)acrylate units was observed in OC-EA, OC-EMA, and OC-BA during the course of the reaction; but, this phenomenon was absent in OC-EAAm and OC-EMAAm. According to WAXD experiments, the crystal type of the prepared macromonomers followed a cellulose II polymorph, the most thermodynamically stable form of crystalline cellulose with well-ordered structures. The availability of vinyl functionalities on the glycomonomer structures opens up another chance for these molecules to work as a precursor for polyaddition. Chapter 4 focuses on the green polymerization of glucosyl-(meth)acrylate monomers by an enzymatic method. Polymerization of the enzymatically synthesized glycomonomers was performed in a free radical manner using a horseradish peroxidase/H₂O₂/acetylacetone ternary initiating ŭƘŭŶāĿÖŶũŋŋĿŶāĿťāũÖŶŽũāĢłðŽƦāũŭŋķŽŶĢŋłŭ̍1_{H NMR spectroscopy was applied to}

determine the monomer conversion and the structure of the prepared glycopolymers. The acrylate-based glycomonomers were polymerized faster than the methacrylate ones due to the formation of less stable acrylate radicals during the propagation reaction. For comparison, synthesis of the identical glycopolymers using potassium persulphate as the chemical initiator for FRP was successful, but required an elevated reaction temperature (50 °C) as compared to the enzymatic reaction. The milder reaction conditions highlight the advantage of an enzyme as biocatalyst requiring less energy to catalyze the polymerization. Both glycopolymers prepared by enzymatic and chemical initiators possess similar structures, thermal, and degradation properties. The molecular weight of the resulting glycopolymers was up to 297 kg mol-1 _{and the glass transition temperature}

was in the range of 71–127 °C. Under a nitrogen atmosphere, the synthesized glycopolymers had three decomposition steps at 150 °C, 320 °C, and 413 °C.

Chapter 5 extends the utilization of the vinyl group of the glycomonomers for the synthesis of block glycopolymers via RAFT polymerization. One of the synthesized glycomonomers, namely 2-(Ⱦ-glucosyloxy)ethyl methacrylate (GEMA), was used as the starting material to create a hydrophilic PGEMA block that was combined with a hydrophilic

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hydroxyethyl methacrylate) (PHEMA) or a hydrophobic poly(ethyl methacrylate) (PEMA). The resulting double-hydrophilic and amphiphilic block glycopolymers were composed ŋĕŶƒŋùĢƦāũāłŶóĞÖĢłķāłėŶĞŭŋĕ̛N̜1mÖłùŭĢĿĢķÖũĿŋķāóŽķÖũƒāĢėĞŶŭŋĕF1m̍ The chemical structures of the obtained block glycopolymers were characterized by 1_H

NMR spectroscopy. The (H)EMA conversion was preserved below 60% to minimize the loss of dithiobenzoyl end groups during the synthesis of P(H)EMA macro-CTAs with a molecular weight up to 16.6 kg mol-1_{. In contrast, 99% conversion of GEMA was achieved}

in the preparation of block glycopolymers, resulting in molecular weights in the range of 36.6 to 45.3 kg mol-1_{. Both macro-CTAs and block glycopolymers were synthesized in a}

controlled fashion, as shown by a relatively narrow and monomodal distribution of the refractive index signals, as well as a moderately low dispersity (*͟ 1.5) based on SEC measurements. The synthesized double-hydrophilic and amphiphilic block glycopolymers exhibited an ability to self-assemble into micellar structures in aqueous solutions with the P(H)EMA blocks serving as the core and PGEMA blocks as the corona. A low critical micelle concentration of about 0.30 mg mL-1_{ƒÖŭùāŶāũĿĢłāùðƘƪŽŋũāŭóāłóāÖłùÁ̞ƑĢŭ}

spectroscopy measurements. Besides that, the hydrodynamic diameter of the formed micelles was around 9 to 21 nm according to DLS experiments and PHEMA-b-PGEMA micelles had a lower hydrodynamic diameter than PEMA-b-PGEMA micelles.

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Samenvatting

Koolhydraten zijn een hernieuwbare biomassa die doorgaans worden gevormd via fotosynthetische reacties in planten. Koolhydraten zijn bijna overal op aarde beschikbaar en verschillende variëteiten van koolhydraten zijn overvloedig in de natuur aanwezig. De noodzaak om koolhydraten te gaan gebruiken als polymere materialen is echter pas in de laatste paar decennia ontstaan. Hier zijn verschillende oorzaken voor te geven. Ten āāũŭŶāƒŋũùāłùāĿāāŭŶāťŋķƘĿāũāłėāĿÖÖĴŶŽĢŶėũŋłùŭŶŋƦāłÖĕĴŋĿŭŶĢėƑÖłĕŋŭŭĢāķā bronnen waarvan verwacht wordt dat zij de komende eeuwen uitgeput raken. Koolhydraten bieden hiervoor een alternatief als een hernieuwbare grondstof. Ten tweede is er een groeiende bewustwording voor het belang om duurzamere polymeren te produceren die een lagere impact hebben op het milieu dan polymeren uit fossiele bronnen. Ten derde kunnen er nieuwe functionele polymere materialen ontwikkeld worden wanneer ėũŋłùŭŶŋƦāłĿāŶóŋĿťķāƗāĕŽłóŶĢŋłÖķĢŶāĢŶāł̇ơŋÖķŭĴŋŋķĞƘùũÖŶāł̇ŋťėāłŋĿāłƒŋũùāłĢł de polymeerstructuur. Bijvoorbeeld, onlangs is aangetoond dat glycopolymeren waarbij de koolhydraten als zijgroepen aan de polymeerketen hangen, geschikt zijn voor het gebruik in verschillende toepassingen, waaronder ziekteremmers, biosensoren en medicijnafgifte ŭƘŭŶāĿāł̍FķƘóŋĿŋłŋĿāũāł̇ùāėũŋłùŭŶŋƦāłƑŋŋũėķƘóŋťŋķƘĿāũāł̇ðāŭŶÖÖłŽĢŶŭÖóĞÖũĢùā eenheden die gebonden zijn aan polymeriseerbare groepen, waarvan de vinyl groep het meeste word gebruikt.

Dit proefschrift beschrijft de synthese van verscheidene glycomonomeren en hun polymerisatie via milieuvriendelijke methoden. Voor de synthese van sacharide-vinyl (macro)monomeren werden koolhydraten als grondstof gebruikt en enzymen als biokatalysator. Beiden zijn afkomstig uit hernieuwbare bronnen. De glycomonomeren werden met succes gepolymeriseerd door middel van reversibele additie-fragmentatie ketenoverdracht (RAFT) polymerisatie, vrije radicaal polymerisatie (FRP) en enzym-ėāĿāùĢāāũùāDĢłƒÖŶāũ̍'Žððāķ̟ĞƘùũŋƩāķāāłÖĿƩƩāķāðķŋĴėķƘóŋťŋķƘĿāũāłƒāũùāł bereid en hun zelforderningsgedrag, resulterend in micellen, werd bestudeerd. Een overzicht van de uitgevoerde projecten beschreven in dit proefschrift is weergeven in Figuur S1.

Hoofdstuk 1 geeft een beknopte inleiding over koolhydraten en de rol van enzymen in synthetische organische reacties. Daarnaast wordt er een compact overzicht gegeven van de recente literatuur over biokatalytische synthese van sacharide-vinyl (macro) monomeren. Hydrolase-enzymen, zoals lipasen, proteasen en glycosidasen, bleken in staat de synthese van deze monomeren te katalyseren.

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Figuur S1 Schematische weergave van de synthese van glycomonomeren en glycopolymeren zoals

beschreven in dit proefschrift.

Hoofdstuk 2 beschrijft de kinetisch gecontroleerde enzymatische synthese van nieuwe glucosyl-(meth)acrylamide monomeren met behulp van een Ⱦ-glucosidase verkregen uit amandelen. In de waterige tranglycosyleringsreactie trad cellobiose op als glucosyldonor en werden verscheidene (hydroxy)alkyl (meth)acrylamide substraten gebruikt als glucosylacceptor. De verkregen glycomonomeren, te weten N-(Ⱦ-glucosyloxy) ethyl acrylamide, N-(Ⱦ-glucosyloxy)ethyl methacrylamide en N-(Ⱦ-glucosyloxy) butyl methacrylamide, waren monofunctioneel en anomeerzuiver. De monomeren werden gekarakteriseerd met behulp van 1_{H NMR,}13_{C NMR en massaspectrometrie. De}

monomeeropbrengst werd verhoogd van 16% naar 68% door cellobiose te vervangen voor het geactiveerde p-nitrofenyl Ⱦ-D-glucopyranoside en door een ionische vloeistof

(BMIMPF₆)-water mengsel te gebruiken als reactiemedium. FRP en RAFT polymerisatie in waterige oplossingen werden gebruikt om de verkregen glycomonomeren te polymeriseren. De glycopolymeren gesynthetiseerd met RAFT toonden molaire massas tussen 43 en 66 kg mol-1_{ĿāŶāāłŭĿÖķķāùĢŭťāũŭĢŶāĢŶ̛*͟ː̍ˑ˘̜̇ŶāũƒĢıķùāėķƘóŋťŋķƘĿāũāłðāũāĢùĿāŶ}

FRP molaire massas lieten zien rond de 220 kg mol-1_{ĿāŶāāłðũāùāùĢŭťāũŭĢŶāĢŶ̛ˑ̍˓˕͟}

*͟˒̍ˑ˘̜̍złÖĕĞÖłĴāķĢıĴƑÖłƒāķĴāťŋķƘĿāũĢŭÖŶĢāŶāóĞłĢāĴƒāũùėāðũŽĢĴŶðķāĴāłėāķĢıĴā glycopolymeren een vergelijkbare glasovergangstemperatuur (rond 142–171 °C) te bezitten. De gesynthetiseerde glucosyl-(meth)acrylamide monomeren uit Hoofdstuk 2, evenals de glucosyl-(meth)acrylaat monomeren beschreven door Kloosterman et al. (zie Figuur S1), kunnen dienen als startmateriaal voor polymerisaties. Bijvoorbeeld, zoals beschreven in Hoofdstuk 3, werden cellooligosacharide-vinyl macromonomeren verkregen door de glucosyleenheden te polymeriseren met cellodextrinefosforylase. De enzym ėāĴÖŶÖķƘŭāāũùāŋĿėāĴāāũùāĕŋŭĕŋũƘķÖŭāũāÖóŶĢāƒāũùŽĢŶėāƑŋāũùĢłāāłðŽƦāũŋťķŋŭŭĢłėĿāŶ

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glucosyl-vinylmonomeren als glucosylacceptor en Ƚ-glucose 1-fosfaat als glucosyldonor. 'āƑŋŋũŶėÖłėƑÖłùāāłơƘĿÖŶĢŭóĞāŭƘłŶĞāŭāƒāũùėāƑŋķėùĿāŶùŽłłāķÖÖėóĞũŋĿÖŶŋėũÖƩā̍ De geïsoleerde productopbrengsten lagen rond de 65%. Vijf verschillende vinyl-groep houdende oligocellulosen werdenbereid, te weten (Ⱦ-oligocellulosyloxy)ethyl acrylaat (OC-EA), (Ⱦ-oligocellulosyloxy)ethyl methacrylaat (OC-EMA), (Ⱦ-oligocellulosyloxy) butyl acrylaat (OC-BA), (Ⱦ-oligocellulosyloxy)ethyl acrylamide (OC-EAAm) en (Ⱦ-oligocellulosyloxy)ethyl methacrylamide (OC-EMAAm). Uit verschillende analyses (1_{H NMR, MALDI-ToF MS en SEC) bleek dat deze macromonomeren een gemiddeld aantal}

glucosyleenheden bevatten van 7.3–8.9 en een gemiddelde molaire massa hadden van 1310– 1553 g mol-1_{. Tijdens de reactie werd er fragmentatie waargenomen van de Ƚ-binding in de}

(meth)acrylaat eenheden van OC-EA, OC-EMA en OC-BA. Voor OC-EAAm en OC-EMAAm was dit fenomeen afwezig. WAXD experimenten lieten zien dat de kristalstructuur van de verkregen macromonomeren overeenkwam met het cellulose II polymorf, de meest thermodynamisch stabiele vorm van kristallijn cellulose.

De beschikbaarheid van vinylgroepen in de structuur van de glycomonomeren biedt nog een andere mogelijkheid om deze moleculen als grondstof te gebruiken voor polyadditie. Hoofdstuk 4 is geweid aan de groene polymerisatie van glucosyl-(meth)acrylaat monomeren, daarbij gebruikmakend van enzymen. De enzymatisch gesynthetiseerde glycomonomeren werden gepolymeriseerd bij kamertemperatuur via ƑũĢıāũÖùĢóÖÖķťŋķƘĿāũĢŭÖŶĢāĢłāāłėāðŽƦāũùāŋťķŋŭŭĢłė̍NāŶŶāũłÖĢũāĢłĢŶĢÖŶĢāŭƘŭŶāāĿ mierikswortelperoxidase/H₂O₂/acetylaceton werd hierbij gebruikt. De monomeerconversie en de structuur van de verkregen glycopolymeren werden bepaald met 1_H

NMR-spectroscopie. De glycomonomeren met een acrylaatfunctionaliteit vertoonden hogere polymerisatiesnelheden dan monomeren met een methacrylaatgroep, dit als gevolg van de lagere stabiliteit van acrylaatradicalen tijdens de propagatiestap. Ter vergelijking, de synthese van dezelfde glycopolymeren via FRP, maar geïnitieerd met kaliumpersulfaat, waren ook succesvol, alhoewel een hogere temperatuur (50 °C) nodig was. De mildere reactieomstandigheden benadrukken het voordeel van het gebruik van enzymen als biokatalysator die minder energie nodig hebben om de polymerisatie te katalyseren. Beide glycopolymeren verkregen via enzymatische of chemische initiatoren hebben vergelijkbare structuren en vergelijkbare thermische eigenschappen. Molecuulgewichten van de gesynthetiseerde glycopolymeren liepen op tot 297 kg mol-1 _en

de glasovergangstemperatuur lag in het bereik van 71–127 °C. Onder een stikstofatmosfeer werden drie ontledingstappen voor de glycopolymeren waargenomen, namelijk bij 150°C, 320 °C en 413 °C.

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In Hoofdstuk 5 wordt het gebruik van de vinylgroep in de glycomonomeren uitgebreid naar de synthese van blokglycopolymeren met behulp van RAFT polymerisatie. Het gesynthetiseerde glycomonomeer, 2-(Ⱦ-glucosyloxy)ethyl methacrylaat (GEMA), werd ėāðũŽĢĴŶ Öķŭ ŽĢŶėÖłėŭĿÖŶāũĢÖÖķ Ƒŋŋũ ùā ŭƘłŶĞāŭā ƑÖł āāł ĞƘùũŋƩāķ F1m ðķŋĴ ùĢā ƑāũƑŋķėāłŭƒāũùėāóŋĿðĢłāāũùĿāŶāāłÖłùāũĞƘùũŋƩāķðķŋĴ̇łÖĿāķĢıĴťŋķƘ̛ˑ̟ĞƘùũŋƗƘāŶĞƘķ methacrylaat) (PHEMA), of een hydrofoob blok, namelijk poly(ethyl methacrylaat) ̛1m̜̍'āƑāũĴũāėāłùŽððāķ̟ĞƘùũŋƩāķāāłÖĿƩƩāķāðķŋĴėķƘóŋťŋķƘĿāũāłĞÖùùāłŶƒāā verschillende ketenlengtes voor het P(H)EMA blok, maar vergelijkbare molecuulgewichten voor het PGEMA blok. De chemische structuur van de blokglycopolymeren werd bestudeerd met 1_{H NMR-spectroscopie. Voor de synthese van de P(H)EMA-macro-CTAs (CTA =}

chain transfer agent; Nederlands: ketenoverdrachtsmiddel) met een molaire massa tot 16.6 kg mol-1_{, werd de (H)EMA conversie beneden de 60% gehouden om verlies van de}

dithiobenzoyl eindgroup tot een minimum te beperken. Daarentegen werd een 99% conversie bereikt voor GEMA tijdens de synthese van de blokglycopolymeren, resulterend in molecuulgewichten van 36.6 tot 45.3 kg mol-1_{. De synthese van beide macro-CTAs en}

blokglycopolymeren geschiedde op een gecontroleerde manier, hetgeen afgeleid kon worden uit de relatief smalle en monomodale verdeling van het brekingsindexsignaal āł ùā ũāùāķĢıĴ ķÖėā ùĢŭťāũŭĢŶāĢŶ ̛* ͟ ː̍˔̜ ơŋÖķŭ ėāĿāŶāł ĿāŶ 1!̍ 'ā ėāŭƘłŶĞāŶĢŭāāũùā ùŽððāķĞƘùũŋƩāķāāłÖĿƩƩāķāðķŋĴėķƘóŋťŋķƘĿāũāłðāơÖŶāłĞāŶƑāũĿŋėāłŋĿĿĢóāķķÖĢũā structuren te vormen in waterige oplossingen waarbij de P(H)EMA blokken de kern vormen en de PGEMA blokken de corona. Een lage kritische micelconcentratie van 0.30 mg mL-1

ƒÖŭðāťÖÖķùĿāŶƪŽŋũāŭóāłŶĢāāłÁ̟ÁĢŭŭťāóŶũŋŭóŋťĢā̍'āĞƘùũŋùƘłÖĿĢŭóĞāùĢÖĿāŶāũ van de gevormde micellen werd gemeten met DLS en lag tussen de 9 en 21 nm. De micellen gevormd door PHEMA-b-PGEMA vertoonden een lagere hydrodynamische diameter dan

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Acknowledgments

Alhamdulillahirabbil’alamin… My praise and gratitude are expressed to Allah subhanahu wa ta’alȧŶĞāĿŋŭŶðāłāƩóāłŶÖłùŶĞāĿŋŭŶĿāũóĢĕŽķ̍RŶĞÖłĴŶĞāķĿĢėĞŶƘķķÖĞĕŋũėĢƑĢłė me the strength and patience to work through all these years.

RŶĢŭùāƩłĢŶāķƘÖķŋłėıŋŽũłāƘĕŋũĿāŶŋƩłĢŭĞŶĞĢŭĞ'̍RłŶĞāťÖŶĞŋĕóŋĿťķāŶĢłėŶĞĢŭ journey, I was supported and helped by many good people around me. Therefore, I would like to dedicate these pages to those who contributed to this thesis.

First of all, I would like to express my deepest gratitude to my supervisor, Prof. Katja Loos. bÖŶıÖ̇RŭŶĢķķũāĿāĿðāũŋŽũƩũŭŶĿāāŶĢłėĢłƘŋŽũŋƧóāŶŋùĢŭóŽŭŭŶĞāťķÖłłāùťũŋıāóŶĕŋũ my PhD research. Afterward, I really appreciate your prompt response concerning the required documents needed for the scholarship application. Thank you for providing me ŶĞāĕũāāùŋĿŶŋóÖũũĢāùŋŽŶŶĞāũāŭāÖũóĞ̍RÖĿėķÖùŶĞÖŶƘŋŽũŋƧóāùŋŋũĢŭÖķƒÖƘŭŋťāł̇ therefore, whenever I had doubt about my results or I had short questions, you always have time for that and give me encouraging feedback. Your help and advice were also very useful ÖŶŶĞāĿŋĿāłŶRĞÖùÖťũŋðķāĿƒĢŶĞŶĞāŶÖƗŋƧóā̍RÖĿƑāũƘŶĞÖłĴĕŽķĕŋũƘŋŽũāƦŋũŶŶŋũāÖù and assess my thesis within a relatively short period, so the thesis can be defended before the summer break. It is such a great experience to work with you and I hope to continue collaborating with you in the future.

I would also like to thank my supervisor Prof. Cynthia L. Radiman from Institut Teknologi Bandung, Indonesia. Ibu Cynthia, thank you for introducing me to Katja and accepting the position as one of the supervisors during my PhD program. I am grateful for your swift approval concerning my thesis manuscript and propositions in Hora Finita system. It would not be possible for me to continue the study at doctoral level abroad without the ƩłÖłóĢÖķŭŽťťŋũŶĕũŋĿLembaga Pengelola Dana Pendidikan (LPDP), Ministry of Finance, Republic of Indonesia. LPDP is greatly acknowledged for granting me the PhD scholarship. I would like to sincerely thank the members of the assessment committee: Prof. Katalin

Barta from the University of Groningen, Prof. Alessandro Gandini from the University of

São Paulo, and Prof. Robert Liska from Vienna University of Technology, for their valuable ŶĢĿāÖłùāƦŋũŶŶŋāƑÖķŽÖŶāŶĞĢŭŶĞāŭĢŭ̍

The administrative and technical help given by several people in the group provides good working environment that also contributes to my research. I would like to thank Karin

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Woudstra for constantly helping me with the paperwork. I would also like to thank Albert Woortman. Albert, thank you for the assistance with the SEC measurements. You have

patiently explained the results to me and answered my questions about the SEC. Besides that, whenever I had problems with the equipment in the lab, you were always willing to help me. Thank you for the nice collaboration on writing the paper in Chapter 3, as well as for the proofreading of the Dutch summary. Gert Alberda van Ekenstein and Jur van

Dijken, thank you for introducing and helping me with the DSC and TGA measurements.

During my PhD time, I managed to do research together with several colleagues and students. I would like to acknowledge 'āıÖłāŶũŋƑĢô. Dejan, thank you for being a nice ŋƧóāĿÖŶāÖłùťũŋƑĢùĢłėĿāāłŋŽėĞÖĿŋŽłŶŋĕāłơƘĿāŭŶŋóŋĿťķāŶāŶĞāāƗťāũĢĿāłŶ for Chapter 3 of this thesis. I really appreciate it since I don’t have any experiences with isolating enzyme from bacterial culture. You taught me about MALDI-ToF MS and gave great feedback for our paper. We also managed to assist one student (Jasper) for his internship in the group. I would like to thank my students from MBO Noorderpoort:

Nick, Dennis, and Thomas. Thank you for your contribution to my projects that make our

enjoyable work can be published in the top journals. I would also like to thank some guest researchers in the lab: Jessica from the University of the Basque Country, Ibrahim from ŋĘÖơĢöĢłĢƑāũŭĢŶƘ̇ÖłùGiovanni from the University of Campinas. Ibrahim, thank you for your hospitality and guidance to my family when we visited you in Istanbul 4 years ago. Even though you stayed in our group for just 3 months, our collaboration work (also with Dejan) has resulted in one paper.

Next, I would like to gratefully thank the current and former members of the research group of Macromolecular Chemistry and New Polymeric Materials. Martijn, thank you for helping me with the Samenvatting and the proofreading of my paper in Chapter 2. Judith, thank you for your kindness to help me with the proofreading of Chapter 1 and 4 of this thesis. Jingjin, thank you for the nice conversation during Christmas dinner in the last two years and also being a good roommate during Dutch Polymer Days. Teh Eryth, thank you ĕŋũĞāķťĢłėĿāėāŶŶĢłėÖùÖťŶāùùŽũĢłėŶĞāƩũŭŶĿŋłŶĞŭRÖũũĢƑāùĢłŶĞāėũŋŽť̍Anton, thank you for teaching me about RAFT polymerization and the proofreading of my propositions.

Csaba, thank you for assisting me with the cover letter and point-to-point answer on my

ƑāũƘƩũŭŶťÖťāũ̍ÈŋŽũĴłŋƒķāùėāŋłāłơƘĿÖŶĢóťŋķƘĿāũĢơÖŶĢŋłŋĕƑĢłƘķĿŋłŋĿāũŭĢŭƑāũƘ ðāłāƩóĢÖķĕŋũĿāŋłũāÖķĢơĢłėŶĞāāƗťāũĢĿāłŶŭĕŋũ!ĞÖťŶāũ˓ŋĕŶĞĢŭŶĞāŭĢŭ̍Ivan, thank you for showing me the TEM facilities. I like your critical questions during the group meeting that often helped me understand my work better. Peter, thank you for your willingness to proofread Chapter 1 of this thesis. Yi, thank you for being a good travel companion for our weekly trip to Utrecht in 2014 to attend the RPK course. Jin, thank you for the time

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you spent on our conversation in the lab. Even though our work is completely unrelated, somehow we always manage to talk about something. I also thank for your help during the tutorial of the MMC course. MahȯŶĞÖłĴƘŋŽĕŋũðāĢłėÖėŋŋùŋƧóāĿÖŶāÖłùRũāķĢŭĞāù our conversation about family life. I would also like to thank the rest of the current and former group members for presenting good working atmosphere at the 3rd_{ƪŋŋũŋĕðŽĢķùĢłė}

5118: Prof. Marleen Kamperman, Dr. Giuseppe Portale, Niels, Chongnan, Aldo, Qi, Milad,

Maryam, Marco, Apostolos, Federico, Renato, Laura, Annemarie, Larissa, Yassaroh, Crystal, Feni, Pia, Indra, Qiuyan, Jakob, Abednego, Inge, Vincent, Kamlesh, Zheng, Salomeh, and others I forgot to mention.

A very special thank goes to my paranymphs, Dina and Masyitha̍'ĢłÖ̇ƘŋŽÖũāùāƩłĢŶāķƘ a talented graphic designer! I owe you a huge debt for your artistic expertise on designing the cover for my thesis as well as my paper in Biomacromolecules. Pardon me for often disturbing you with the layout of the data plots, the powerpoint slides, and the layout of ĿƘťŋŭŶāũŭ̍RÖĿŭŋķŽóĴƘÖłùƑāũƘėũÖŶāĕŽķŶŋĞÖƑāƘŋŽÖŭĿƘŋƧóāĿÖŶā̍mÖƘ̇ŶĞÖłĴƘŋŽ for keeping our lab in an organized condition and also providing some snacks. I wish both ŋĕƘŋŽėŋŋùķŽóĴŋłƩłĢŭĞĢłėƘŋŽũŶĞāŭĢŭÖłùėũÖùŽÖŶĢłėŭŋŋł̍

There is a frustrating and stressful period where I (and my wife) have to deal with the ŶÖƗŋƧóā̍RłŶĞÖŶťāũĢŋù̇RÖĿƑāũƘŶĞÖłĴĕŽķŶŋŭāƑāũÖķťāŋťķāŶĞÖŶĞāķťāùŽŭėŋŶĞũŋŽėĞ this “adventure”. Marco van der Vinne, I really appreciate your priceless assistance on this tax matter. Without your help, I would have just given up from the early stage of the process since I really had no idea how to solve this problem. Jos van Griensven, thank you for your guidance, advice, and support until the end of this journey. Bea Zand Scholten and Anmara Kuitert, thank you for helping us contacting several people to explain our situation and always gave us tremendous support.

Groningen is like a second hometown for me. This is the place where I start a family and even my daughter was born and raised there. Nevertheless, being far from families can make us homesick and Indonesian community in Groningen always provides warmness and kindness that makes us feel like home. I would like to thank my Indonesian friends and families: Keluarga Mas Lana, Keluarga Mas Kuswanto, Keluarga Ali Syariati, Keluarga

Ali Abdurrahman, Keluarga Kang Bino, Keluarga Teh Inda, Keluarga Mas Latif, Keluarga Mas Zainal, Keluarga Mas Romi, Keluarga Mas Krisna, Keluarga Kang Izul, Keluarga Mas Didik, Keluarga Fika, Keluarga Didin, Keluarga Mas Ega, Keluarga Zaki, Keluarga Kang Iqbal, Keluarga Irfan, Keluarga Surya, Keluarga Pak Asmoro, Keluarga Bli Kadek, Keluarga Kang Bintoro, Keluarga Mas Habibie, Keluarga Mas Akbar, Keluarga Fajar, Keluarga Mas Pandji, Keluarga Mas Donny, Keluarga Pak Tatang, Keluarga Mas Archi,

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Keluarga Bang Ade, Keluarga Kang Dimas, Keluarga Mas Azzam, Keluarga Mas Naufal, Keluarga Kang Zakiyullah, Keluarga Mas Adhyat, Keluarga Kang Ivan, Keluarga Mas Amak, Keluarga Mas Joko, Keluarga Mas Khairul, Keluarga Mas Agung, Keluarga Ibu Elvira, Keluarga Mas Riswandi, Keluarga Mba Titis, Keluarga Mas Chalis, Keluarga Mba Atikah, Keluarga Yudi, Keluarga Mas Rully, bāķŽÖũėÖÖĴ¦ÖŽƩĴ, Keluarga Mas Ristiono, Keluarga Kang Hegar, Keluarga Mba Erna, Keluarga Kang Iging, Keluarga Mba Ria, Keluarga Bude Nunung, Keluarga Uwak Asiyah dari Delfzijl, Keluarga Bude Arie dari Hoogezand, Kang Wahono, Mas Yusuf, Teh Astri, Insan, Reren, Azkario, Azka, Rai, Salva, Yusran, Adityo, Guntur, Mba Frita, Mba Nur, Mba Nuril, Mba Ira, Panji, Adjie, Ibu Ima, Kang Fean, Ʃĕ, Mas Ury, Mas Fajri, Mas Tri, Retha, Fandi, Mba Tania, Mba Vera,

and others that I unintentionally forgot to mention. I also thanks to Duhita, Niken, Yovi,

Dedes, Dasha, ŋƩ, Novika, Risa, Nisa, Vania, Asa, and Erin, for being good housemates

and playing with my daughter in your free time.

Finally, I would like to thank my dear family. Ema dan Bapa, terima kasih untuk doa yang tak henti-hentinya kalian panjatkan, untuk semangat yang selalu kalian berikan, serta keyakinan kalian bahwa saya dapat menyelesaikan sekolah di sini. Alhamdulillah, perjuangan Azis, Nna, dan Dinara selama 5 tahun ini akan segera berakhir. Untuk Hendri,

Nunik, dan Erik, terima kasih untuk doa-doanya selama ini dan juga sudah membantu

Kaka menjaga Ema dan Bapa. Insha Allah kita semua bisa segera berkumpul bersama lagi. Terima kasih juga buat Bapa Deden, Mamah Dini, Kaka Amalia, Mizan, Aiko, dan Kiki, untuk segala bantuan, dukungan, dan doa kalian yang senantiasa dialamatkan untuk kami di sini.

Lastly, I owe thanks to a very special person, my beloved wife Amalina. Thank you for being a good wife, mother, and PhD student at the same time! I know it is never easy to do all of them but you have successfully managed it. Thank you for your unconditional ķŋƑā̇ óŋłŶĢłŽŋŽŭ ŭŽťťŋũŶ̇ Öłù óŋŽłŶķāŭŭ ŭÖóũĢƩóāŭ ùŽũĢłė ŋŽũ ťŽũŭŽĢŶ ŋĕ Ğ' ùāėũāā ƒĞĢóĞĞāķťŭŽŭėāŶŶŋŶĞĢŭťŋĢłŶ̍RėũāÖŶķƘƑÖķŽāƘŋŽũóŋłŶũĢðŽŶĢŋłŶŋŶĞāƪŽŋũāŭóāłóā ŭťāóŶũŋŭóŋťƘāƗťāũĢĿāłŶŭĢł!ĞÖťŶāũ˔ŋĕŶĞĢŭŶĞāŭĢŭ̍¦ĞāũāŭŽķŶŭŭĢėłĢƩóÖłŶķƘùāƩłāŶĞā ŭÖĿťķāóĞÖũÖóŶāũĢŭŶĢóŭŶĞÖŶĿÖĴāĿāóŋłƩùāłŶŶŋŭŽðĿĢŶŶĞāĿÖłŽŭóũĢťŶĢłŋłāŋĕŶĞā leading polymer science journals. Amazingly, our paper is even highlighted on the cover of the issue of that journal! I appreciate my dearest daughter, Dinara, for your cheerful personality and sweet smiles that deliver a joyful life. Words would never enough to ŭÖƘĞŋƒĢłùāðŶāùRÖĿŶŋðŋŶĞŋĕƘŋŽ̍ÂāŭŶÖũŶŶĞĢŭıŋŽũłāƘŶŋėāŶĞāũ̇ƒāƒĢķķƩłĢŭĞŶĞĢŭ together too, Insha Allah.

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About the Author

Azis Adharis was born on 3 August 1987 in Cilacap, Indonesia. He obtained his bachelor degree from the Department of Chemistry, Institut Teknologi Bandung (ITB), Indonesia, in July 2009. Shortly after that, he continued his study in a double degree master program in the Department of Chemistry at ITB and Chemical Engineering at the University of Twente, The Netherlands, and graduated in July 2011. His master research entitled "Polyurethane Nanocomposite Foams:

Preparation, Characterization, and Foam Structure" was performed in the research group of Materials Science and Technology of Polymers at the latter university. In December 2011, he worked as a research scientist at Nanoscience Innovation Pte Ltd in Singapore for one year.

Since September 2013, he started his PhD program in the research group of Macromolecular Chemistry and New Polymeric Materials, University of Groningen, The Netherlands, under the supervision of Prof. Katja Loos. The aim of his project is to prepare saccharide-based monomers using enzymatic approaches and to polymerize the synthesized monomers in eco-friendly systems. The results of his PhD project are presented in this thesis.

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List of Publications

1. A. Adharis, D. Vesper, N. Koning and K. Loos, “Synthesis of (meth)acrylamide-based

glycomonomers using renewable resources and their polymerization in aqueous systems”, Green Chemistry, 2018, 20, 476–484.

2. A. Adhariṡ'̍m̍āŶũŋƑĢô̇R̍ơùÖĿÖũ̇̍`̍`̍ÂŋŋũŶĿÖłÖłùb̍dŋŋŭ̦̇1łƑĢũŋłĿāłŶÖķķƘ

friendly pathways towards the synthesis of vinyl-based oligocelluloses”, Carbohydrate Polymers, 2018, 193, 196–204.

3. A. Adharis, T. Ketelaar, A. G. Komarudin and K. Loos, “Synthesis

and Self-Assembly of Double-Hydrophilic and Amphiphilic Block Glycopolymers”, Biomacromolecules, 2019, 20, 1325–1333.

4. A. Adharis and K. Loos, “Synthesis of Glycomonomers via Biocatalytic Methods”,

Methods in Enzymology, 2019, 627, (Accepted).

5. A. Adharis and K. Loos, “Green Synthesis of Glycopolymers Using an Enzymatic Approach”, Submitted upon invitation to Macromolecular Chemistry and Physics, 2019.