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

(1)

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

Adharis, Azis

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from

it. Please check the document version below.

Document Version

Publisher's PDF, also known as Version of record

Publication date:

2019

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):

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

Systems. University of Groningen.

Copyright

Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policy

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.

(2)

531423-L-sub01-bw-Adharis

Processed on: 27-5-2019 Processed on: 27-5-2019 Processed on: 27-5-2019

Processed on: 27-5-2019 PDF page: 9PDF page: 9PDF page: 9PDF page: 9

Chapter

General Introduction

1

(3)

531423-L-sub01-bw-Adharis

10

1.1 Carbohydrates

Carbohydrates are one of the most abundant natural organic substances on Earth and they mainly serve as structural and functional elements of cellular materials. For instance, cellulose is a major component in the cell walls of plants and carbohydrates construct ŶĞāðÖóĴðŋłāŋĕŶĞāłŽóķāĢóÖóĢùŭ̍RłėāłāũÖķ̇óÖũðŋĞƘùũÖŶāŭÖũāùāƩłāùÖŭðĢŋĿŋķāóŽķāŭ consisting of carbon (C), hydrogen (H), and oxygen (O) atoms with the molecular formula

C_m(H₂O)_n also known as hydrates of carbon.1_{Some sweet compounds like xylose C}

5(H2O)5,

glucose C₆(H₂O)_6,and sucrose C₁₂(H₂O)₁₁ follow that formula, but deoxyribose C₅H₁₀O₁₁, a

sugar part of deoxyribonucleic acid, and other particular carbohydrate molecules are łŋŶĢłķĢłāƒĢŶĞŶĞāũŽķā̍¦Ğāũāĕŋũā̇ÖóóŋũùĢłėŶŋŶĞāR!ùāƩłĢŶĢŋł̇ÖłŋŶĞāũŶāũĿĕŋũ carbohydrates is saccharides; which includes monosaccharides, oligosaccharides, and

polysaccharides as well as their derivative components.2

1.1.1 Monosaccharides

Monosaccharides are the simplest carbohydrates and they are the building blocks of other carbohydrates exhibiting more complex structures. Monosaccharides typically contain three to nine carbon atoms with some chiral centers. Glyceraldehyde and neuraminic acid

are two examples of monosaccharides with three and nine carbon atoms, respectively.3

¦ĞũāāóķÖŭŭĢƩóÖŶĢŋłŭÖũāŽŭāùĕŋũĿŋłŋŭÖóóĞÖũĢùāŭ̍DĢũŭŶ̇ðÖŭāùŋłŶĞāŶƘťāŭŋĕĢŶŭóÖũðŋłƘķ group, monosaccharides are aldoses or ketoses if the group present in the structure is an aldehyde or ketone. Second, according to the number of carbon atoms, monosaccharides ÖũāĢùāłŶĢƩāùÖŭŶũĢŋŭāŭƒĞāłŶĞāƘóŋłŶÖĢłŶĞũāāóÖũðŋłÖŶŋĿŭ̇ŶāŶũŋŭāŭĕŋũĕŋŽũóÖũðŋł

ÖŶŋĿŭ̇Öłùŭŋŋł̍¦ĞĢũù̇ŶĞāťũāƩƗD or L is added before the monosaccharide group. This

refers to the chirality of the penultimate carbon atom from the carbonyl group.

Emil Fischer (the 1902 Chemistry Nobel Prize Winner) proposed a so-called Fischer projection that is widely used nowadays to draw the open-chain monosaccharides in planar form. If, in the Fischer projection, the hydroxyl group of the penultimate carbon atom is

located on the left side, then it is an L-monosaccharide. In contrast, it is a D-monosaccharide

if that hydroxyl group is located on the right side. Some examples of monosaccharides drawn in the Fischer projection are displayed in Figure 1.1. The availability of chiral carbons in monosaccharides generates the possibility of these molecules to possess stereoisomers

(4)

531423-L-sub01-bw-Adharis

11

Figure 1.1 Fischer projections of monosaccharides with three to six carbon atoms. (a) and (b) are aldoses

while (c) and (d) are ketoses.

mŋłŋŭÖóóĞÖũĢùāŭƒĢŶĞƩƑāŋũĿŋũāóÖũðŋłÖŶŋĿŭłÖŶŽũÖķķƘĕŋũĿÖóƘóķĢóŭŶũŽóŶŽũāĢł ÖŨŽāŋŽŭŭŋķŽŶĢŋłŭ̍mŋłŋŭÖóóĞÖũĢùāŭƒĢŶĞÖƩƑāŋũÖŭĢƗ̟ĿāĿðāũāùũĢłėŋĕÖŶŋĿŭÖũā called furanoses or pyranoses, respectively, which mimic the molecules furan and pyran. The ring formation, which produces a hemiacetal/hemiketal linkage, is the result of the reaction between the carbonyl and one of the hydroxyl groups of the monosaccharides.

Figure 1.2 shows the ring formation of D-glucose, in which the carbon atom of the carbonyl

ėũŋŽťťũŋùŽóāŭÖłāƒŭŶāũāŋóāłŶāũ̍Nāłóā̇ŶƒŋóŋłƩėŽũÖŶĢŋłŭŋĕŶĞāėķŽóŋŭāÖłŋĿāũ are obtained. The Haworth projection, developed by Walter Norman Haworth (the 1937 !ĞāĿĢŭŶũƘpŋðāķũĢơāÂĢłłāũ̜̇ĢŭŽŭāùŶŋĢķķŽŭŶũÖŶāóƘóķĢóĿŋłŋŭÖóóĞÖũĢùāŭ̵ĢłÖŭĢĿťķā three-dimensional perspective. If the hydroxy group of the anomeric carbon is on the

opposite side of the -CH₂OH group in the Haworth projection, it is called an alpha (Ƚ)

anomer. On the other hand, a beta (Ⱦ) anomer is if that hydroxy group is located on the

same side of the -CH₂OH group. In solution, these Ƚ and Ⱦ-anomers are present in an

equilibrium state by a process called mutarotation. Another common way to draw ring structures of monosaccharides is using a chair conformation that considers the spatial interaction between the functional groups in the most favorable energetic condition. Figure 1.3 shows some examples of monosaccharide structures in the chair conformation.

(5)

531423-L-sub01-bw-Adharis

12

Figure 1.2 Cyclization of D-glucose generates glucopyranoses which are drawn in the Haworth

pro-jection and the chair conformation.

Figure 1.3 Monosaccharide structures in the chair conformation. The numbers within the structures indicate the position of functionalization by vinyl groups as discussed in Section 1.3.

1.1.2 Oligo- and polysaccharides

Carbohydrates with two or more monosaccharide units linked together by glycosidic bonds ÖũāóķÖŭŭĢƩāùÖŭŋķĢėŋ̟ÖłùťŋķƘŭÖóóĞÖũĢùāŭ̍4_{Oligosaccharides consist of a small number}

of repeating monosaccharides, generally between two and ten units. In the case of two ŋũŶĞũāāĿŋłŋŭÖóóĞÖũĢùāŭóŋĿðĢłāù̇ŭťāóĢƩóŶāũĿŭÖũāŽŭāùłÖĿāķƘùĢŭÖóóĞÖũĢùāŭŋũ trisaccharides. Figure 1.4 presents some examples of oligo- and polysaccharides.

(6)

531423-L-sub01-bw-Adharis

13

Figure 1.4 Oligo- and polysaccharide structures in the chair conformation. The numbers within the

structures indicate the position of functionalization by vinyl groups as discussed in Section 1.3.

Glycosidic bonds of oligo- and polysaccharides are formed as the result of condensation reactions between the hydroxyl groups of one monosaccharide and the anomeric carbon of other monosaccharides. The anomeric carbon of monosaccharides possess either Ƚ or Ⱦ-hydroxyl groups which means that the formed glycosidic linkage can either be located on the Ƚ or Ⱦ-position. For example, a glycosidic bond in the dissacharide maltose joins an anomeric carbon (C-1) of glucose on the Ƚ-position and another glucose at the C-4 position. On the other hand, a dissacharide cellobiose is constructed by a glycosidic bond between an anomeric carbon (C-1) of glucose on the Ⱦ-position and another glucose at the C-4 position (see Figure 1.5).

(7)

531423-L-sub01-bw-Adharis

14

Figure 1.5RķŽŭŶũÖŶĢŋłŋĕŶƒŋėķŽóŋŭāŭķĢłĴāùðƘÖėķƘóŋŭĢùĢóðŋłùÖŶùĢƦāũāłŶťŋŭĢŶĢŋłŭ̇ƒĞĢóĞķāÖùŭ ŶŋŶƒŋùĢƦāũāłŶùĢŭÖóóĞÖũĢùāŭ̍

The glycosidic bond in maltose is called Ƚ-(1,4)-linkage, while in cellobiose it is Ⱦ-(1,4)-linkage. ŽũťũĢŭĢłėķƘ̇ŶĞĢŭŭĢłėķāùĢƦāũāłóāķāÖùŭŶŋŶĞāŭĢėłĢƩóÖłŶùĢŭťÖũĢŶĢāŭŋĕŶĞāťũŋťāũŶĢāŭŋĕ both molecules. For example, cellobiose has a planar structure with a water solubility of 0.12 g mL-1_{. In contrast, the maltose structure is slightly twisted with a water solubility of 1.08 g}

mL-1_{. The twisted structure of maltose results in a helical structure if the number of glucosyl}

units is large enough like in the case of maltooligosaccharide and amylose. On the other hand, the Ⱦ-1,4-linkages between the glucosyl units provide planar and rigid structures of cellooligosaccharide and cellulose.

Amylose and cellulose are linear polysaccharides composed of hundred to thousand glucosyl units.5,6_{Amylose is commonly isolated from starch that contains about 20–30%. The remaining}

polysaccharide in starch is amylopectin, an amylose-like polysaccharide with additional glycosidic bonds at the Ƚ-1,6-position that creates a branched structure.5_{Cellulose is the main}

component of the cell walls of plants, particularly in the woody part of plant tissues.

1.2 Enzymes as biocatalysts in synthetic organic

chemistry

Enzymes are catalysts that perform biosynthetic reactions in the cells at a remarkably high rate under mild reaction conditions (temperature, pH, and pressure).7_{The enzymatic reaction rates}

are about 106_{to 10}12_{-fold of the uncatalyzed reaction, even up to 10}20_{times in some special cases.}8

In general, enzymes are proteins or conjugated proteins. However, some ribonucleic acids, namely ribozymes, are also found to have catalytic properties like protein-based enzymes.9

(8)

531423-L-sub01-bw-Adharis

15

¦ĞāłÖĿāŭŋĕāłơƘĿāŭÖũāėāłāũÖŶāùðƘÖùùĢłėŶĞāŭŽƧƗ̞ase to the substrate of these enzymes (like lactase, cellulase, and amylase) or the type of the enzymatic reaction (such as cellodextrin phosphorylase, DNA polymerase, and alcohol dehydrogenase). Exceptions are made for some enzymes that were named before the accepted convention such as pepsin, thrombin, and trypsin. The International Union of Biochemistry and Molecular ĢŋķŋėƘóķÖŭŭĢƩāùŶĞāāłơƘĿāŭĢłŶŋŭĢƗóÖŶāėŋũĢāŭðÖŭāùŋłŶĞāóÖŶÖķƘơāùũāÖóŶĢŋłŭŶĞÖŶ is oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases which have

an Enzyme Commission (EC) number from one to six, respectively (Table 1.1).10,11_{In August}

2018, a new class of enzymes, namely translocases, with the EC number of 7 were added

into the enzyme nomenclature.12_{According to the ExplorEnz database, about 7500 enzymes}

ƒāũāóķÖŭŭĢƩāùŭŋĕÖũƒĢŶĞĞÖķĕŋĕŶĞāĿðāķŋłėĢłėŶŋŋƗĢùŋũāùŽóŶÖŭāŭÖłùŶũÖłŭĕāũÖŭāŭ̍11

Table 1.1!ķÖŭŭĢƩóÖŶĢŋłŭ̇āƗÖĿťķāŭ̇ÖłùðĢŋóÖŶÖķƘŭĢŭũāÖóŶĢŋłŋĕāłơƘĿāŭ̍

Enzyme class Examples Catalyzed reaction

EC 1. Oxidoreductases Peroxidase, Laccase, Glucose oxidase Oxido-reductions of substrate EC 2. Transferases Glycosyltransferase, Transaldolase,

Transketolase

Group transfer from donor to acceptor

EC 3. Hydrolases Lipase, Glucosidase, Protease Hydrolytic cleavage of chemical bonds

EC 4. Lyases Decarboxylase, Hydratase, Aldolase Cleavage of chemical bonds by elimination

EC 5. Isomerases Cycloisomerase, Racemase, Epimerase

Interconvert isomer changes within one molecule

EC 6. Ligases Carboxylase, Ligase, Chelatase Coupling of two molecules with the hydrolysis of triphosphate EC 7. Translocases Ubiquinol oxidase, Ascorbate

ferrireductase, Protein-secreting ATPase

Movement of ions or molecules across membranes or their separation within membranes

The energy diagram of a typical chemical reaction with enzymes (in vivo) and without enzymes is presented in Figure 1.6. In principle, the role of enzymes as biocatalysts is identical to chemical catalysts in terms of speeding up the reaction rate without changing the total free energy from reactants to products. This is achieved by providing an alternative reaction mechanism, which has a lower activation energy than the non-óÖŶÖķƘơāùũāÖóŶĢŋł̍āŭĢùāŭ̇āłơƘĿāŭŭĞŋƒÖĿÖơĢłėāƧóĢāłóƘÖłùŭťāóĢƩóĢŶƘÖŭƒāķķÖŭ the fact that they are non-toxic, natural, and renewable materials in comparison with

conventional catalysts.13_{As a result, in vitro enzymatic reactions have been developed in}

order to provide chemical synthesis in a green approach.

(9)

531423-L-sub01-bw-Adharis

16

Figure 1.6 Energy diagram of a chemical reaction with and without enzyme.

ũŋðÖðķƘŶĞāƩũŭŶin vitro enzymatic reaction was reported in the 1930s for the synthesis of esters catalyzed by lipase in organic solvents.14,15_{However, it began to gain widespread}

interest after Zaks and Klibanov published their articles in the early 1980s dealing with ùĢƦāũāłŶķĢťÖŭāŭÖŭóÖŶÖķƘŭŶŭĕŋũƑÖũĢŋŽŭŋũėÖłĢóũāÖóŶĢŋłŭĢłÖłĞƘùũŋŽŭŭŋķƑāłŶŭ̍16,17_Since

then, biocatalysts in synthetic organic chemistry have been one of the most studied topics with a constantly growing reputation. Many enzymes are found to be able to work not only with their native substrates but also with unnatural substrates which results in broad variety of materials.18_{Research and development in both chemistry and biology areas}

ĞÖƑāŭĢėłĢƩóÖłŶķƘóŋłŶũĢðŽŶāùŶŋŶĞāŽŭÖėāŋĕāłơƘĿāŭĢłĢłùŽŭŶũĢÖķÖťťķĢóÖŶĢŋłŭÖŭķĢŭŶāù in Table 1.2.

Table 1.2 Some examples of enzyme applications in industry.19

Industry Enzymes Application

Baking Xylanase Lipase Glucose oxidase

Dough conditioning

Dough stability and conditioning Dough strengthening

Dairy food Protease Lipase Lactase

Milk-clotting, infant formula !ĞāāŭāƪÖƑŋũ

Lactose removal Detergent Protease

Lipase Amylase

Protein stain removal Lipid stain removal Starch stain removal Fat and oils Lipase

Phospholipase

¦ũÖłŭāŭŶāũĢƩóÖŶĢŋł

De-gumming, lyso-lechitin production Textile Cellulase Amylase Catalase !ŋŶŶŋłŭŋĕŶāłĢłė̇ùāłĢĿƩłĢŭĞĢłė De-sizing Bleach termination

(10)

531423-L-sub01-bw-Adharis

17

1.3 Enzymatic synthesis of saccharide-vinyl (macro)

monomers

The demand to develop sustainable materials and production methods, especially in the

ƩāķùŋĕťŋķƘĿāũŭ̇ĞÖŭĢłóũāÖŭāùĢłŶĞāķÖŭŶùāóÖùāŭ̍20,21_{This is reasonable considering}

that the main components for polymer production are based on fossil resources which have been predicted to be exhausted in the next several hundred years. In addition, the manufacturing of fossil resources often creates environmental issues like global warming, ťŋķķŽŶĢŋł̇ÖłùùÖłėāũŋŽŭƒÖŭŶā̍RłŋũùāũŶŋŭĢėłĢƩóÖłŶķƘùāóũāÖŭāŶĞāùĢŭÖùƑÖłŶÖėāŭ created by the use of fossil resources, the European Union and the United Nations, through their policies and legislation, support the use of eco-friendly materials and simultaneously

limit the consumption of environmentally hazardous substances in industry.22,23

Sustainable polymers contain some or all portions of building blocks that are derived from

renewable feedstocks.20–22_{One of the most promising examples of renewable feedstocks is}

biomass and its derivatives since these resources are produced by nature via photosynthetic pathways. Especially carbohydrates, one of the most abundant biomass, are interesting materials for the production of durable polymers. However, the chemical industry is still

only using a small percentage of the accessible carbohydrates,24,25_{although carbohydrates}

give rise to a new family of functional water-soluble monomers and polymers. One example for this is glycopolymers, synthetic polymers composed of saccharides as the pendant

group.26

Glycomonomers, the precursors of glycopolymers, contain saccharide units that are linked to some polymerizable groups. Currently, vinyl groups are the most exploited ones. The preparation of glycomonomers from saccharides and vinyl containing molecules can be

carried out using chemical reactions.27–29_{However, this approach is time-consuming given}

that it involves multiple protection steps of hydroxyl groups of saccharides in the course of the reactions. An elegant way to synthesize glycomonomers is by using enzymes as the

biocatalysts.30–33_{As already mentioned in Section 1.2, enzymes exhibit an excellent reaction}

ŭťāóĢƩóĢŶƘóŋĿťÖũāùŶŋŋŶĞāũƒĢŭāŽŭāùóĞāĿĢóÖķóÖŶÖķƘŭŶŭ̍¦Ğāũāĕŋũā̇ĢłŶĞāāłơƘĿÖŶĢó ŭƘłŶĞāŭĢŭŋĕėķƘóŋĿŋłŋĿāũŭ̇ŶĞāƑĢłƘķėũŋŽťóÖłŭťāóĢƩóÖķķƘðāÖŶŶÖóĞāùŶŋÖťÖũŶĢóŽķÖũ position, among multiple possible positions, of saccharides without involving the tedious protection steps. Some vinyl groups of the synthesized glycomonomers are presented in Figure 1.7.

(11)

531423-L-sub01-bw-Adharis

18

Figure 1.7 Some examples of vinyl functionalities of glycomonomers.

¦ŋ ŶĞā ðāŭŶ ŋĕ ŋŽũ Ĵłŋƒķāùėā̇ ŶĞā ƩũŭŶ ƒŋũĴ ŋł ŶĞā ŭƘłŶĞāŭĢŭ ŋĕ ƑĢłƘķ ĿŋłŋĿāũŭ containing saccharide moieties by biocatalytic approaches was published by Klibanov and

coworkers in 1986.34,35_{In this work, lipases were used to catalyze the acylation of various}

monosaccharides. The following sections discuss some of the latest results found in the ķÖŭŶŶƒŋùāóÖùāŭ̍¦ĞāāÖũķƘƩłùĢłėŭĢłŶĞĢŭÖũāÖƒāũāŭŽĿĿÖũĢơāùĢłŭāƑāũÖķāƗóāķķāłŶ

review articles published in the early 2000.29,36–38_{Enzymes from the class of hydrolases (EC}

3) such as lipases, proteases, and glycosidases, were mainly used for the bio-catalytically synthesized glycomonomers since they are able to catalyze the formation of ester-, amide-, and glycosidic bonds as well as reverse hydrolysis reactions.

1.3.1 Lipases

Lipases are a subclass of esterases that, in nature, catalyze the hydrolysis reaction of ester bonds of long-chain triglycerides to diglycerides, monoglycerides, glycerols, and fatty

acids.39_{Lipases can be isolated from various origins such as bacteria, fungi, and mammals.}

In vitro, lipases can perform the reverse reaction of its natural functions, meaning that

lipases create ester bonds from alcohols and carboxylic acids of substrates in dry organic solvents.

Dŋũ ŶĞā óÖŭā ŋĕ ėķƘóŋĿŋłŋĿāũ ŭƘłŶĞāŭĢŭ̇ ķĢťÖŭāŭ óÖŶÖķƘơā ŶĞā ŶũÖłŭāŭŶāũĢƩóÖŶĢŋł ŋĕ ĞƘùũŋƗƘķ ėũŋŽťŭ ŋĕ ŭÖóóĞÖũĢùāŭ ƒĢŶĞ ùĢƑĢłƘķ āŭŶāũŭ̍ ¦Ğā ŶũÖłŭāŭŶāũĢƩóÖŶĢŋł ũāÖóŶĢŋł occurs in an equilibrium state. Therefore, in order to shift the equilibrium reaction to the products side, divinyl esters are favored over carboxylic acids as substrates. The esters generate vinyl alcohol as a side product which immediately converts, via tautomerization, to volatile acetaldehyde. Figure 1.8 exemplarily shows the lipase-catalyzed synthesis of glycomonomers from glucose and divinyl adipate.

(12)

531423-L-sub01-bw-Adharis

19

Figure 1.8 Reaction scheme of the lipase-catalyzed synthesis of glycomonomers.40

Table 1.3 summarizes some examples of glycomonomers that were synthesized by lipases. Lipase from Candida antarctica was the most frequently used biocatalyst, however, other sources of lipases from Alcaligenes sp., Pseudomonas sp., and Thermomyces sp. were also utilized. These lipases show a good activity in commonly available organic solvents like acetonitrile, pyridine, acetone, DMF, t-butanol, and butanone. Nevertheless, working with a particular solvent may be more preferred than the others. For instance, Cameron et al. reported that by replacing the solvent from acetonitrile to butanone for the synthesis of

methyl 6-O-methacryloyl-Ƚ-D-glucoside, the glycomonomer yield was improved from

˖ˏͮŶŋ˗˔ͮÖłùŶĞāũāÖóŶĢŋłŶĢĿāƒÖŭùāóũāÖŭāùĕũŋĿƩƑāŶŋŶƒŋùÖƘŭ̍41_{In general, the}

ķĢťÖŭā̟óÖŶÖķƘơāùŶũÖłŭāŭŶāũĢƩóÖŶĢŋłũāÖóŶĢŋłƒÖŭťāũĕŋũĿāùðāŶƒāāłŋłāÖłùƩƑāùÖƘŭ at 30–60 °C to obtain a product yield up to 85%.

Lipase from Candida antarctica selectively catalyzes the monosubstitution of vinyl groups towards a primary alcohol at the C-6 position of monosaccharides with pyranose

structures,42–46_{at the C-6 position of reduced disaccharides,}47,48_{and at C-6 or C-6’’ positions}

of trisaccharides.49_{Mono- and disubstitution of vinyl groups was observed when fructose,}

sucrose, and trehalose were used as the substrates.50,51_{This happened probably because}

they possess two primary alcohols on the Ⱦ-position allowing the enzymes to perform two substitutions of vinyl groups in a saccharide unit. Also, this enzyme shows a reaction ŭāķāóŶĢƑĢŶƘŶŋƒÖũùŭùĢƑĢłƘķāŭŶāũŭƒĢŶĞùĢƦāũāłŶÖķĴƘķóĞÖĢłķāłėŶĞŭ̍DŋũāƗÖĿťķā̇mĢŽũÖ

et al. reported that divinyl sebacate (Figure 1.7c) yielded a better substrate conversion than

divinyl adipate (Figure 1.7b) during the synthesis of glycomonomers.48_{A similar pattern}

was observed during the enzymatic polymerization of polyesters where this lipase exhibits

a preference towards more hydrophobic diols and diesters.52

A functionalization at the C-4 position of monosaccharide rhamnose with divinyl adipate

was reported by Raku and Tokiwa using lipase from Pseudomonas sp.53_{In that case, a}

ŶũÖłŭāŭŶāũĢƩóÖŶĢŋłŋóóŽũũāùÖŶŶĞāŭāóŋłùÖũƘÖķóŋĞŋķŭĢłóāłŋťũĢĿÖũƘÖķóŋĞŋķƒÖŭťũāŭāłŶ in the rhamnose structure. If the disaccharide trehalose - bearing a primary alcohol at the C-6’ position - was used as the substrate for that lipase, monosubstitution of vinyl

groups selectively occurred in the position of that alcohol.54_{Moreover, a monosubstitution}

of trisaccharides with divinyl adipate was observed when lipase from Thermomyces sp.

catalyzed the reaction.49

(13)

531423-L-sub01-bw-Adharis

20 T ab le 1 .3 L ip as e-cat al yz ed s ynt he se s of g ly co m on om er s. The s tr u ct u re of s ac ch ar id es a n d v inyl g ro u p s c or re sp on d t o F ig u re 1 .3, 1 .4, a n d 1.7 . Sacc h ar id es V in yl g rou p V in yl p o si tion E n zy m e s o u rc es Re ac tion c o nd it ion Y ie ld (% ) R ef s. 1b C-6 Can di da an tar ct ica A ce to n it ri le , 5 0 °C, 1 d 50 40 1c C-6 A lc al igene s s p. P yr id in e, 3 0 °C, 7 d 31 55 1e C-6 Ca nd id a a nt arc ti ca t-B ut an ol , 5 0 °C, 1 d 34 50 2d C-6 Can di da an tar ct ica A ce to n e, 6 0 °C, 1 8 h 82 4 2 2e C-6 Can di da an tar ct ica A ce to n it ri le , 5 0 °C, 5 d 70 32, 43 2e C-6 Can di da an tar ct ica But an on e, 6 0 °C, 2 d 85 4 1 3d , e C-6 Ca nd id a a nt arc ti ca t-B ut an ol , 5 0 °C, 1 d 100 v 44 ,4 5 5e C-6 Can di da an tar ct ica A ce to n e, 5 0 °C, 5 d 74 46 6e C-6 Can di da an tar ct ica A ce to n it ri le , 5 0 °C, 4 d 4 8 32 9b C-4 Ps eu do m on as s p. P yr id in e, 3 0 °C, 7 d 11 53 11 e C-1, C-6, C-1, 6 Ca nd id a a nt arc ti ca t-B ut an ol , 5 0 °C, 1 d 9 5 v 50 14 b, c C-6 Can di da an tar ct ica P yr id in e, 5 0 °C, 3 d 70 v, 9 9 v 47 ,4 8 16 b, c C-6 Can di da an tar ct ica P yr id in e, 5 0 °C, 3 d 7 v, 4 4 v 47 ,4 8 17 b C-6, 6’ Can di da an tar ct ica A ce to n e, 5 0 °C, 2 d 21 51 18 b C-6, 6’ Can di da an tar ct ica A ce to n e, 5 0 °C, 2 d 20 51 18 c C-6’ Ps eu do m on as s p. P yr id in e, 6 0 °C, 3 d 19 54 19 c C-6’ Ps eu do m on as s p. P yr id in e, 6 0 °C, 3 d 4 4 54 20 b C-6’ ’ Can di da an tar ct ica P yr id in e, 6 0 °C, 3 d 4 2 49 20 b C-6’ ’ Ther momy ces la nu gi no su s P yr id in e, 6 0 °C, 1 d 75 49 21 b C-6 Can di da an tar ct ica P yr id in e, 6 0 °C, 3 d 38 49 21 b C-6’ ’ Ther momy ces la nu gi no su s P yr id in e, 6 0 °C, 1 d 50 49 Ƒ̵̵͚Ž ðŭ Ŷũ ÖŶ āó ŋł Ƒā ũŭ Ģŋ ł̛ ͮ ̜̇Ƙ Ģā ķùù ÖŶ ÖÖ ũāł ŋŶÖ ƑÖ Ģķ Öð ķā ̍

(14)

531423-L-sub01-bw-Adharis

21

1.3.2 Proteases

Proteases, or proteinases/peptidases, naturally hydrolyze peptide bonds of amino acids

during the metabolism of proteins in all organisms.56_{Depending on the type of proteases,}

ŶĞāĞƘùũŋķƘŭĢŭũāÖóŶĢŋłĿÖƘðāŭťāóĢƩóÖŶťÖũŶĢóŽķÖũŭĢŶāŭŋĕÖťũŋŶāĢłŭŶũŽóŶŽũāŋũłŋł̟ ŭťāóĢƩóũāŭŽķŶĢłėĢłĢłùĢƑĢùŽÖķÖĿĢłŋÖóĢùŭ̍DŋũŶĞāin vitro reaction, proteases work in ÖŭĢĿĢķÖũÖťťũŋÖóĞķĢĴāķĢťÖŭā̒ŶĞÖŶĢŭóÖŶÖķƘơĢłėŶũÖłŭāŭŶāũĢƩóÖŶĢŋłŋĕŭÖóóĞÖũĢùāŭƒĢŶĞ divinyl esters during the synthesis of glycomonomers in organic solvents. For example, Lin and coworkers used galactose and several divinyl esters as the substrate for protease

(see Figure 1.9).57

Figure 1.9 Reaction scheme of the protease-catalyzed synthesis of glycomonomers.

Unlike lipases, proteases are able to produce the highest yield of glycomonomers when

monosaccharides are combined with divinyl esters bearing a few methylene groups,57

meaning that this enzyme has a good selectivity towards more hydrophilic substrates.

Comparable results were reported for dissaccharide57,58_{and trisaccharide}59,60_substrates.

However, an exception was found when glucose reacted with divinyl adipate (bearing four methylene groups). This resulted in an increased yield compared to the reaction with divinyl succinate (bearing two methylene groups) and divinyl sebacate (bearing

eight methylene groups).61_{It is interesting to note that some lipases were successfully}

commercialized in the immobilized state whereas proteases are only available in the liquid or granulate form, which limits their reusability drastically.

Numerous examples of glycomonomer syntheses by proteases are outlined in Table 1.4 in which the enzymes were mainly obtained from Bacillus species, especially Bacillus subtilis. Like lipases, proteases also work well in organic solvents especially pyridine and DMF. Protease-catalyzed synthetic reactions are typically performed in one to seven days with a temperature between 30 and 55 °C. The highest yield of the isolated product was reported to be up to 85%.

(15)

531423-L-sub01-bw-Adharis

22 Tab le 1 .4 P rot ea se -c at al yz ed s ynt he se s of g ly co m on om er s. The s tr u ct u re of s ac ch ar id es a n d v inyl g ro u p s c or re sp on d t o F ig u re 1 .3, 1 .4, a n d 1 .7 . S ac ch ar id es V in yl g rou p V in yl p o si tion E n zy m e s o u rc es Re ac tion c o nd it ion Y ie ld (% ) R ef s. 1 a, b , c C-6 B ac illu s s ub tili s P yr id in e, 5 5 °C, 3 d 30 , 5 3, 3 5 61 1b C-6 B ac illu s s ub tili s D M F, 5 0 °C, 5 d 80 v 62 1e C-6 B ac illu s s ub tili s D M F, 4 5 °C, 1 d 36 50 2e C-6 B ac illu s s ub tili s D M F, 4 5 °C, 7 d 33 63 4 a, b , c C-6 B ac illu s s ub tili s P yr id in e, 5 0 °C, 0 .5 /3 /5 d 70 , 4 7, 3 5 57 5 a, b , c C-6 B ac illu s s ub tili s P yr id in e, 5 0 °C, 0 .5 /4 /5 d 85 , 6 9, 4 3 57 7b C-2 B ac illu s s ub tili s D M F, 3 0 °C, 7 d 55 53 8b C-2 B ac illu s s ub tili s D M F, 3 0 °C, 7 d 32 53 10 b C-4 (1 0 y) , C -5 ( 10z ) B ac illu s s ub tili s D M F, 5 0 °C, 7 d 6 6 6 8 10 b C-5 ( 10 z) St reptomy ces s p. D M F, 3 0 °C, 7 d 46 6 9 11 e C-1, C -6 B ac illu s s ub tili s D M F, 4 5 °C, 1 d 6 0 v 50 12 e C-1, C -6 B ac illu s s ub tili s D M F, 4 5 °C, 3 d 33 63 13 a, b , c C-6’ B ac illu s s ub tili s P yr id in e, 5 0 °C, 5 d 62, 4 6, 31 57 ,5 8 15 a, b , c C-6’ B ac illu s s ub tili s P yr id in e, 5 0 °C, 5 d 53, 4 2, 3 4 57 17 b C-1 B ac illu s s ub tili s P yr id in e, 5 0 °C, 5 d 55 57 ,5 8 17 b C-1 B ac illu s s ub tili s D M F, 5 0 °C, 5 d 9 0 v 65 17 e C-1 B ac illu s s ub tili s D M F, 4 5 °C, 3 d 70 6 4, 6 6 17 b C-1 B ac illu s li ch en ifo rmi s P yr id in e, 4 5 °C, 2 d 13 51 18 e C-6’ B ac illu s s ub tili s D M F, 4 5 °C, 3 d 25 63 18 b C-6’ B ac illu s li ch en ifo rmi s P yr id in e, 4 5 °C, 2 d 22 51 20 a, b , c C-1 B ac illu s s ub tili s P yr id in e, 5 0 °C, 6 d 55 , 48 , 3 0 59 ,6 0 20 b C-1 B ac illu s li ch en ifo rmi s P yr id in e, 4 0 °C, 1 d 6 5 49 21 b C-6’ B ac illu s li ch en ifo rmi s P yr id in e, 4 0 °C, 1 d 55 49 22 a, b , c C-2’ B ac illu s s ub tili s D M F, 5 0 °C, 5 d 5, 1 0, 1 3 6 7 Ƒ̵̵͚Ž ðŭ Ŷũ ÖŶ āó ŋł Ƒā ũŭ Ģŋ ł̛ ͮ ̜̇Ƙ Ģā ķùù ÖŶ ÖÖ ũāł ŋŶÖ ƑÖ Ģķ Öð ķā ̍

(16)

531423-L-sub01-bw-Adharis

23

Protease from Bacillus subtilis selectively catalyzes the monosubstitution of vinyl groups to primary alcohols of saccharides at various positions. The substitution was located at the

C-6 position of monosaccharides with pyranose structures,50,57,61–63_{at the C-1 or C-6 position}

of monosaccharides with furanose structures,50,63_{at the C-6’ position of the disaccharides}

lactose/maltose/trehalose,57,58,63_{as well as at the C-1 position of the disaccharide sucrose and}

ŶũĢŭÖóóĞÖũĢùāũÖƧłŋŭā̍57,58,64–66_{Besides that, monosubstitution was also found at the secondary}

alcohol (at the C-2 position) when fucose and Ⱦ-cyclodextrin served as the substrates.53,67

ũŋŶāÖŭāŭ ĕũŋĿ Ŷƒŋ ũāŭŋŽũóāŭ óÖł ťũŋƑĢùā ùĢƦāũāłŶ ũāÖóŶĢŋł ŭāķāóŶĢƑĢŶƘ Öłù ŶĞĢŭ ƒÖŭ

observed when L-arabinose was used as the substrate. In solution, L-arabinose possesses two

ĢŭŋĿāũŭŶĞÖŶāƗĞĢðĢŶùĢƦāũāłŶŭŶũŽóŶŽũāŭ̇āĢŶĞāũĕŽũÖł̟ŋũťƘũÖł̟ķĢĴāĿŋķāóŽķāŭ̍ũŋŶāÖŭā

from Bacillus subtilisóÖŶÖķƘơāùŶĞāŶũÖłŭāŭŶāũĢƩóÖŶĢŋłŋĕL-arabinose on both isomers68

while protease from Streptomyces sp. only worked with the furanose isomer69_{under similar}

reaction condition.

1.3.3 Glycosidases

Glycosidases, or glycosyl hydrolases, are carbohydrate-active enzymes that in vivo catalyze

the hydrolytic degradation of glycosidic bonds in carbohydrates.70_{Considering their}

versatility, glycosidases have been utilized extensively in industrial processes. For instance, glycosidases are applied as biocatalysts during the degradation of biomass (cellulose and other plant-derived polysaccharides) yielding mono- and oligosaccharides for biofuels and

biobased chemical manufacturing.71–73

For the synthesis of glycomonomers, glycosidases catalyze the monosubstitution of vinyl groups at the anomeric carbon (the C-1 position) of saccharides. Depending on the enzyme type, the linkage of the vinyl group may be on the Ƚ or Ⱦ-position of the anomeric carbon ƒĞĢóĞũāŭŽķŶŭĢłÖłŋĿāũĢóÖķķƘťŽũāťũŋùŽóŶŭ̍FķƘóŋŭĢùÖŭāŭťũŋƑĢùāÖùĢƦāũāłŶķŋóÖŶĢŋłĕŋũŶĞā attachment of vinyl groups compared to lipases and proteases which usually functionalize the saccharides at their primary alcohols. It was reported that functionalization at the primary alcohol of monosaccharides could be a disadvantage because the resulted

glycopolymers were found to lose their activity with saccharide-binding proteins.46

Harnessing glycosidases for the glycomonomer synthesis is done via two approaches, namely thermodynamically controlled reversed hydrolysis and kinetically controlled transglycosylation. Both methodologies were exploited in the past with their advantages

and limitations summarized elsewhere.74_{¦ĞāĕŋķķŋƒĢłėťÖũÖėũÖťĞŭùĢŭóŽŭŭðũĢāƪƘŶĞā}

ùĢƦāũāłóāŭðāŶƒāāłŶĞāŭāŶƒŋĿāŶĞŋùŭĕŋũŶĞāŭƘłŶĞāŭĢŭŋĕŭÖóóĞÖũĢùā̟ƑĢłƘķĿŋłŋĿāũŭ listed in Table 1.5.

(17)

531423-L-sub01-bw-Adharis

24

1.3.3.1 Thermodynamically controlled reverse hydrolysis

Hydrolysis reactions of saccharides by glycosidases are performed under equilibrium conditions. In principle, reverse hydrolysis reactions are used for the synthesis of glycomonomers by reacting monosaccharides with vinyl-based alcohols. Nevertheless, a small amount of water is still required to maintain the enzyme activity and to increase the solubility of the monosaccharides in the reaction mixture. Consequently, this method is limited to monosaccharide substrates since oligo- and polysaccharides can be hydrolyzed by glycosidases in the presence of water.

Figure 1.10 shows an example of the thermodynamically controlled reverse hydrolysis of glucose and 2-hydroxyethyl acrylate catalyzed by Ⱦ-glucosidase derived from

almonds.75_{Some vinyl-based alcohols (Figure 1.7 f-k) were tested as well, but the obtained}

glycomonomer yields were rather low (up to 21%) although highly concentrated glucose solutions were used to shift the equilibrium reaction to the product side. In order to improve the product yield, Kloosterman et al. optimized the reaction time, the composition

of alcohols and water, as well as adding an organic solvent into the reaction mixtures.31

As a result, the synthesized glycomonomers were obtained in a higher yield (up to 41%) than in the previous report.

In addition, less product yield was generated when 4-hydroxybutyl acrylate was used rather than 2-hydroxyethyl acrylate as the substrate in both studies, revealing the enzyme has preferences towards hydrophilic alcohols. To overcome the issue with hydrophobic alcohols, Gill and Valivety proposed to conduct the enzymatic reaction in plasticized glass

phases using Ⱦ-galactosidase from E. coli, A. niger, and A. oryzae.33_{In their study, the highest}

yield up to 71% was achieved by mixing saccharides/alkyl glycoside/dopant with a small ÖĿŋŽłŶŋĕðŽƦāũ̇ĕŋķķŋƒāùðƘĿāķŶĢłėŶĞāũāÖóŶĢŋłĿĢƗŶŽũāÖłùƩłÖķķƘÖùùĢłėÖķóŋĞŋķŭ and enzymes.

(18)

531423-L-sub01-bw-Adharis

25 T ab le 1 .5 Gl yc os id as es -c at al yz ed s ynt he se s of g ly co m on om er s. The s tr u ct u re of s ac ch ar id es a n d v inyl g ro u p s c or re sp on d t o F ig u re 1 .3, 1 .4 , a n d 1 .7 . S ac ch ar id es V in yl g roup V in yl p o si tion E n zy m e Re ac tion c o nd it ion Y ie ld (% ) R ef s. 1 f, g , h , i , j , k C-1ȾȾ -g lu co sid as e f rom a lmond ! ĢŶ ũÖŶ āð Ž Ʀ āũť N˔ ̍ˑ̇˒ ˖΅!̇ː ̟˒ù 16 , 3, 3, 21 , 1 8, 1 4 75 1 f, g , h C-1ȾȾ -g lu co si d as e f ro m a lm on d W at er /d iox an e, 5 0 °C, 1 d 4 1, 4 3, 21 31 1f C-1Ⱦ C el lu la se f ro m Tri ch od erm a r ee se i ! ĢŶ ũÖŶ āð Ž Ʀ āũť N˔ ̇˔ ˏ΅!̇˒ù 57 6 1f C-1Ƚ Am yl og lu co si d as e f ro m A sp erg illu s ni ge r óā ŶÖŶ āð Ž Ʀ āũť N˔ ̇˔ ˔΅!̇˔ù N/ A 77 1 l, m , n C-1ȾȾ -g lu co si d as e f ro m a lm on d W at er , 5 0 °C, 1 h 16 , 1 2, 1 8 30 4g , o C-1ȾȾ -g al ac to si d as e f ro m E scher ich ia col i Ğŋ ŭť Ğ ÖŶ āð Ž Ʀ āũť N˖ ̇˓ ˏ΅!̇ː ̍˔ù 67 , 39 33 4g C-1ȾȾ -g al ac to si d as e f ro m A sp erg illu s ni ge r Ğŋ ŭť Ğ ÖŶ āð Ž Ʀ āũť N˖ ̇˓ ˏ΅!̇ː ̍˔ù 43 33 4o C-1ȾȾ -g al ac to si d as e f ro m A sp erg illu s o ry za e Ğŋ ŭť Ğ ÖŶ āð Ž Ʀ āũť N˖ ̇˓ ˏ΅!̇ː ̍˔ù 64 33 4g C-1ȾȾ -g al ac to si d as e f ro m Sul folobu s sol fatar ic us Ğŋ ŭť Ğ ÖŶ āð Ž Ʀ āũť N ˕ ̍˔ ̇˖ ˔΅!̇ː ̍˔ Ğ 75 – 80 78 15 f, g , h C-1Ƚ A m yl as e f ro m B ac illu s s te aro th er m op hilu s óā ŶÖŶ āð Ž Ʀ āũť N˔ ̇˔ ˔΅!̇˓ù 57 7 p ̵̵͚̓pŋŶÖ ƑÖ Ģķ Öð ķā ̇Ŷ Ğāťũ ŋùŽ óŶƒ Öŭł ŋŶĢ ŭŋ ķÖ Ŷā ù ̍ Fi g u re 1 .1 0 The rm od yn am ic al ly c ont ro lle d r ev er se hyd rol ys is r ea ct io n c at al yz ed b y Ⱦ-g lu co si d as e.

1

(19)

531423-L-sub01-bw-Adharis

26

1.3.3.2 Kinetically controlled transglycosylation

In the synthesis of glycomonomers by kinetically controlled transglycosylation, glycosidases catalyze the reaction between saccharides working as glycosyl donors, and vinyl-based alcohols serving as glycosyl acceptors. The enzymatic reaction is carried out in aqueous solutions. As shown in Figure 1.11, water acts not only as the solvent, but also as a competing reactant to glycosyl acceptors in the transglycosylation reaction. Therefore, it is crucial to optimize the time required for the reaction, as short reaction times may provide a low substrate conversion while hydrolysis of the synthesized glycomonomers is likely to occur during the longer reaction times.

Figure 1.11 Competing transglycosylation and hydrolysis catalyzed by Ⱦ-glucosidase.

In contrast to the previous approach, oligo- and polysaccharides can be used as the substrate of glycosidases in this method. For example, cellulase from Trichoderma reesei

catalyzed the formation of 2-(Ⱦ-glucosyloxy)ethyl acrylate from cellulose.76_{In addition,}

the same glycomonomer with the acrylate unit linked at the Ƚ-position was prepared

from starch using amyloglucosidase from Aspergillus niger.77_{Moreover, maltogenic amylase}

from Bacillus stearothermophilus was able to catalyze the synthesis of maltose-based

glycomonomers from starch.77_{A disadvantage was the low glycomonomers yield of less}

than 5%.

In order to improve the product yield, activated saccharides can be used as the glycosyl donors rather than native saccharides. Activated saccharides usually contain aryl or vinyl groups which are much better leaving groups in transglycosylation reaction than

glycosyl units of natural sugars.79,80_{For instance, Santin et al. reported p-nitrophenyl Ⱦ-}_D

(20)

531423-L-sub01-bw-Adharis

27

isolated glycomonomer yield of about 75%.78_{Another way to improve the product yield is}

by adding cosolvents like organic solvents81–83_{or ionic liquids}84–86_{to increase the}

enzyme-substrate interactions. For example, the yield of glucosyl-(meth)acrylamide monomers

was improved from 16% to 68% upon addition of the ionic liquid BMIMPF₆ and replacing

the native cellobiose with activated glucose.30

Glycomonomers synthesized by both thermodynamically controlled reversed hydrolysis and kinetically controlled transglycosylation possess one vinyl group that was selectively attached either at the Ƚ or Ⱦ-position of the anomeric carbon (the C-1 position) of saccharides. This result supports the excellent selectivity of glycosidases for the ŭƘłŶĞāŭĢŭŋĕÖłŋĿāũĢóÖķķƘťŽũāÖłùĿŋłŋĕŽłóŶĢŋłÖķťũŋùŽóŶŭ̍ƒāķķ̟ùāƩłāùŭŶũŽóŶŽũā of glycomonomers provides a good starting point for the investigation of the interaction

of glycopolymers with sugar-binding proteins.87,88

1.3.4 Glycosyltransferases

Glycosyltransferases are enzymes that catalyze the formation of glycosidic linkages by

transferring glycosyl units from glycosyl donors to glycosyl acceptors.89,90_{In the preparation}

of saccharide-vinyl macromonomers (Table 1.6), various glucose-based monomers were utilized as the glycosyl acceptors, and either natural saccharides or glucosyl phosphates were used as the glycosyl donors. However, the number of glycosyltransferases used for this purpose was limited, probably because the isolation process of these enzymes on the

practical scale is rather challenging.18

Cyclodextrin glycosyltransferase from Bacillus macerans catalyzes the synthesis

of 2-(Ⱦ-maltooligooxy)ethyl (meth)acrylate macromonomers.91_{In that reaction,}

Ƚ-cyclodextrin and 2-(Ⱦ-glucosyloxy)ethyl (meth)acrylate are served as the glycosyl donor ÖłùÖóóāťŶŋũ̇ũāŭťāóŶĢƑāķƘ̍¦ĞāėķƘóŋĿŋłŋĿāũŭóŋłŶÖĢłāùŋłāŶŋƩĕŶāāłėķŽóŋŭƘķŽłĢŶŭ that are linked through Ƚ-(1,4)-glycosidic bonds while the Ⱦ-linkage of the vinyl group remained intact after the coupling reaction.

(21)

531423-L-sub01-bw-Adharis

28 Tab le 1 .6 Gl yc os ylt ra n sf er as e-ca ta ly ze d s ynt he se s of g ly co m on om er s. The s tr u ct u re of t h e s ac ch ar id es a n d v in yl g ro u p s c or re sp on d t o F ig u re 1 .4 a n d 1 .7 . S ac ch ar id es V in yl g rou p V in yl p o si tion E n zy m e Re ac tion c o nd it ion Y ie ld (% ) R ef s. 23 f, g C-1Ⱦ Cy cl od ex trin gl yc os yl tr an sf er as e f ro m B ac illu s m ac era n s ¦ ũĢ ŭð Ž Ʀ āũť N˕ ̇˕ ˏ΅!̇ 1.5 h 38 9 1 24 f, g , h , l , m C-1Ⱦ C el lo de x tr in pho spho ryl as e f rom Clos tr idiu m t her mo ce llu m N 1 1 ð Ž Ʀ āũť N˖ ̍˔ ̇˓ ˔΅!̇˒ù 6 5, 6 7, 5 8, 6 3, 7 0 9 2, 93

(22)

531423-L-sub01-bw-Adharis

29

In another example, a cellooligosaccharide-based glycomonomer was prepared from

2-(Ⱦ-glucosyloxy)ethyl methacrylate and Ƚ-D-glucose 1-phosphate.92 Cellodextrin

phosphorylase from Clostridium thermocellum (CtCdP) was utilized as the biocatalyst. The synthesized macromonomer possesses an average number of nine glucosyl units which are coupled by Ⱦ-(1,4)-glycosidic bonds. A disadvantage is that the solubility issue of the product hinders the enzyme ability to extend the number of glucosyl units further. Recently, the versatility of this enzyme with some unnatural substrates was reported

by us.93_{In this work, several types of vinyl glucosides bearing (meth)acrylate/(meth)}

acrylamide functionalities were applied as the glucosyl acceptors (Figure 1.12) and the isolated yields were about 65%. Interestingly, these vinyl glucosides were synthesized from renewable materials following an enzymatic approach in aqueous solutions, thus, providing an environmentally friendly process.

Figure 1.12 Enzymatic synthesis of cellooligosaccharide-based glycomonomers catalyzed by CtCdP

(m = 1 or 2, A = O or NH, and R = H or CH3).

1.4 Glycopolymers

The polymerization of glycomonomers forms a new type of polymers called glycopolymers. The potential applications of glycopolymers have attracted much research in the last decades starting in the 1970s when it was proven that synthetic glycopolymers with polyacrylamide backbones could interact with carbohydrate-binding proteins called

lectins.94,95_{FķƘóŋťŋķƘĿāũŭāƗĞĢðĢŶÖĞĢėĞÖƧłĢŶƘÖłùŭťāóĢƩóĢŶƘŶŋķāóŶĢłŭ̇ŶĞŽŭĿĢĿĢóĴĢłė}

glycolipids and glycoproteins at the cell surface.88,96_{Moreover, glycopolymers have been}

used as inhibitors that prevent the adhesion of pathogens to the cell surface during an

infection.97_{Other applications of glycopolymers cover, for example, drug delivery,}98–100

biosensors,101,102_{and diseases inhibitors.}97,103

Glycopolymers were developed in various structures like linear homo- and block (co)

polymers, dendrimers, and star polymers as shown in Figure 1.13.104–110_{In the initial work,}

glycopolymers were prepared by free radical polymerization (FRP) which results in high

molecular weight polymers with a broad dispersity.111–113_{After the birth of living/controlled}

ũÖùĢóÖķťŋķƘĿāũĢơÖŶĢŋł̇ƒāķķ̟ùāƩłāùŭŶũŽóŶŽũāŭŋĕėķƘóŋťŋķƘĿāũŭƒāũāŭƘłŶĞāŭĢơāùŽŭĢłė

techniques like atom–transfer radical polymerization (ATRP),114–116_{reversible addition–}

(23)

531423-L-sub01-bw-Adharis

30

fragmentation chain transfer (RAFT) polymerization,117–119_{nitroxide–mediated radical}

polymerization (NMP),120–122_{and cyanoxyl–mediated radical polymerization (CMRP).}123–125

The discussion in the following section will be limited to FRP and RAFT polymerization, two techniques that are utilized in the experimental chapters of this thesis.

Figure 1.13óĞāĿÖŶĢóŭŶũŽóŶŽũāŭŋĕėķƘóŋťŋķƘĿāũŭƒĢŶĞùĢƦāũāłŶÖũóĞĢŶāóŶŽũāŭ̍

1.4.1 Free radical polymerization (FRP)

FRP is a very well-established, robust, and simple method for the synthesis of polymers from wide varieties of vinyl monomers including (meth)acrylates, (meth)acrylamides, and styrenes. Polymerization by FRP method involves three fundamental steps namely initiation, propagation, and termination.

The inititation step comprises the generation of radicals from an initiator followed by the reaction of these radicals with a monomer. Depending on the initiator molecules, ŶĞāĕŋĿÖŶĢŋłŋĕũÖùĢóÖķŭóÖłðāĢłùŽóāùðƘùĢƦāũāłŶāƗŶāũłÖķŶũĢėėāũŭ̇ŭŽóĞÖŭŶĞāũĿÖķ or photochemical treatments and reduction–oxidation reactions. The commonly used initiators are based on azo-, peroxide, and persulphate compounds.

The propagation step is characterized by a continuous addition of monomers that react ƒĢŶĞũÖùĢóÖķŭ̵ũāŭŽķŶĢłėĢłũÖùĢóÖķťŋķƘĿāũĢóóĞÖĢłŭ̍¦ĞāķÖŭŶŭŶāťĢŭŶĞāŶāũĿĢłÖŶĢŋłĢł which the formed radicals react with each other, thus, terminating the growing chain. The termination step occurs in two modes namely recombination or disproportionation reaction. In the case of recombination, radical polymeric chains can react either with each other or with the initiator radicals. On the other hand, disproportionation happens when a Ⱦ-hydrogen atom is abstracted from one radical and transferred to another radical. As a result, two polymer chains are formed with a saturated and an unsaturated chain end, respectively. Figure 1.14 ilustrates the overall steps involved in the FRP of a saccharide-vinyl monomer with azobisisobutyronitrile (AIBN) used as the thermal initiator.

(24)

531423-L-sub01-bw-Adharis

31

Figure 1.14 Reaction scheme of the initiation, propagation, and termination during a FRP of a

sac-charide-vinyl monomer.

¦ĞāƩũŭŶŭŶŽùƘŋłťŋķƘĿāũĢơÖŶĢŋłŋĕŭÖóóĞÖũĢùā̟ƑĢłƘķĿŋłŋĿāũŭðƘDƒÖŭũāťŋũŶāùĢłŶĞā

early 1960s,126–128_{in which methacrylate or (meth)acrylamide-containing glycomonomers}

were successfully homo- and copolymerized. FRP is still widely used in the last few years although this method shows a drawback considering the control of the microstructure of the synthesized glycopolymers. However, regarding an important property of the ėķƘóŋťŋķƘĿāũŭ̇ŶĞāðĢŋÖóŶĢƑĢŶƘ̇ŶĞāĿĢóũŋÖũóĞĢŶāóŶŽũāāƗĞĢðĢŶŭÖũāķÖŶĢƑāķƘķŋƒĢłƪŽāłóā

in comparison to the carbohydrate type in the glycopolymer.129_{Furthermore, the dispersity}

of glycopolymers is often not the main concern especially when they are used for hydrogel

materials.130–132_{For instance, a glycomonomer called N-acryloyl-}_D_{-glucosamine was}

copolymerized with 2-hydroxyethyl methacrylate and N,N’-methylenebisacrylamide

which results in novel hydrogels to be applied for mucosa-mimetic materials.130_In

another example, Deng et al. prepared interpenetrating polymer network hydrogels

(25)

531423-L-sub01-bw-Adharis

32

which were composed of a glycopolymer and collagen.132_{The synthesized copolymers}

were biocompatible and showed a promising application as a corneal substitute material.

1.4.2 Reversible addition–fragmentation chain transfer (RAFT) polymerization

RAFT polymerization was discovered in 1998133_{and since then, it became one of the most}

famous living/controlled radical polymerization techniques for the synthesis of well-ùāƩłāùŭŶũŽóŶŽũāŭŋĕťŋķƘĿāũŭ̍RłťũĢłóĢťķā̇D¦ťŋķƘĿāũĢơÖŶĢŋłĕŋķķŋƒŭŶĞāĿāóĞÖłĢŭĿ of FRP including an initiation, propagation, and termination step. In order to control the amount of radicals and consequently also the microstructure and dispersity, RAFT polymerization uses chain transfer agents (CTAs) in addition to the initiator. As can be seen in Figure 1.15, this CTA is involved in the equilibrium reaction between the active and ùŋũĿÖłŶťŋķƘĿāũóĞÖĢłŭùŽũĢłėŶĞāťŋķƘĿāũĢơÖŶĢŋł̍RłÖłāƧóĢāłŶŭƘŭŶāĿ̇ŶĞāÖùùĢŶĢŋł̓ fragmentation equilibrium rate is higher than the propagation rate to ensure a similar chain length of the synthesized polymers. Therefore, the CTA has a crucial role and must be carefully selected to achieve a successful RAFT polymerization. An additional advantage of this polymerization technique is that the end groups are retained and can be precisely ùāƩłāùðƘŶĞā!¦̍

Equation 1.1 can be used to calculate the theoretical molecular weight of the polymers

prepared by the RAFT method where [M], MW_M, and conv are the initial concentrations, the

mass, and the conversion of the monomers, respectively. [CTA] and MW_CTA are the initial

concentrations and the mass of the CTA, respectively.

(1.1)

In the area of glycopolymer synthesis, the number of publications on RAFT polymerizations of glycomonomers is about twice the number of reports on ATRP/transition

metal-mediated polymerization.105_{¦ŭĞŋƒŭÖŭĢėłĢƩóÖłŶùũÖƒðÖóĴĢłŶĞāťŽũĢƩóÖŶĢŋłŋĕ}

glycopolymers since the remaining metal traces are limiting the use of these glycopolymers for biomedical applications. Moreover, the RAFT polymerization is applicable with a broad ũÖłėāŋĕĕŽłóŶĢŋłÖķĿŋłŋĿāũŭÖłùāƗťāũĢĿāłŶÖķóŋłùĢŶĢŋłŭŶĞÖŶĿÖƘðāùĢƧóŽķŶŶŋũāÖķĢơā with other methods. For example, in 2003, Lowe et al. reported a RAFT polymerization of unprotected 2-methacryloxyethyl glucoside in an aqueous solution, whereas the ATRP of

(26)

531423-L-sub01-bw-Adharis

33

Figure 1.15 Proposed mechanism of the RAFT polymerization.

RAFT polymerization is generally performed at 60–90 °C using a thermal initiator such as AIBN or 4,4-azobis(cyanopentanoic acid) for the reactions in organic and aqueous solvents, respectively. Due to the natural good solubility of saccharides in water, in recent years, RAFT polymerization of unprotected glycomonomers was mostly performed in aqueous media with the addition of a small amount of organic solvents for dissolution of the CTA. The availability of the reactive thiocarbonylthio-end groups at the glycopolymers allows for the syntheses of not only linear homopolymers but also multiblock copolymers and hyperbranced polymers.

1.5 Aim and outline of the thesis

The main objective of this research is to study the synthesis of various saccharide-vinyl (macro)monomers and their polymerization via eco-friendly routes. In this approach, carbohydrates and enzymes derived from renewable resources serve as starting materials and biocatalysts, respectively. In order to ensure an environmentally friendly process, the monomer synthesis or the polymerization is performed in aqueous media.

Chapter 2 presents the synthesis of glucosyl-(meth)acrylamide monomers catalyzed by Ⱦ-glucosidase from almonds. The kinetically controlled transglycosylation reactions are performed in green solvents – water or water-ionic liquid mixtures. The glycomonomers are then successfully polymerized in aqueous systems by FRP and RAFT polymerization.

(27)

531423-L-sub01-bw-Adharis

34

The prepared glucosyl-(meth)acrylamide monomers in Chapter 2 and the synthesized

glucosyl-(meth)acrylate monomers from the previous study31_{are utilized as glucosyl}

acceptors in the synthesis of cellooligosaccharide-vinyl macromonomers, as described in Chapter 3̍¦ĞāũāƑāũŭāťĞŋŭťĞŋũŋķƘŭĢŭũāÖóŶĢŋłĢŭóÖũũĢāùŋŽŶĢłÖðŽƦāũĿāùĢÖƒĢŶĞ cellodextrin phosphorylase from Clostridium thermocellum as the biocatalyst.

Chapter 4 investigates the green synthesis of glycopolymers via an enzymatic method. The enzymatically synthesized glycomonomers are polymerized by peroxidase from horseradish in an enzyme/hydrogen peroxide/acetylacetone ternary initiating system. The properties of the prepared glycopolymers by this technique are compared with the glycopolymers synthesized by a conventional FRP.

Ŷ ķÖŭŶ̇ ŶĞā ƒāķķ̟ùāƩłāù ŭŶũŽóŶŽũā ŋĕ ðķŋóĴ ėķƘóŋťŋķƘĿāũŭ ŭƘłŶĞāŭĢơāù ðƘ Ö D¦ polymerization is addressed in Chapter 5. Double-hydrophilic and amphiphilic block glycopolymers are prepared from enzymatically synthesized glucosyl-methacrylate monomer. The spontaneous self-assembly of both block glycopolymers in aqueous solutions is explored.

(28)

531423-L-sub01-bw-Adharis

35

1.6 References

1 R. Alén, Carbohydrate Chemistry — Fundamentals and ApplicationṡÂŋũķùóĢāłŶĢƩó̇ĢłėÖťŋũā̇ 1st edn., 2018.

2 G. P. Moss, P. A. S. Smith and D. Tavernier, Pure Appl. Chem., 1995, 67, 1307–1375.

3 A. Blanco and G. Blanco, in Medical Biochemistry, eds. A. Blanco and G. Blanco, Academic Press, London, 1st edn., 2017, pp. 73–97.

4 A. D. Mcnaught, Pure Appl. Chem., 1996, 68, 1919–2008.

5 T. Luallen, in Starch in Food, eds. M. Sjöö and L. Nilsson, Woodhead Publishing, Duxford, 2nd edn.,

2018, pp. 545–579.

6 Suhas, V. K. Gupta, P. J. M. Carrott, R. Singh, M. Chaudhary and S. Kushwaha, Bioresour. Technol.,

2016, 216, 1066–1076.

7 A. Blanco and G. Blanco, in Medical Biochemistry, eds. A. Blanco and G. Blanco, Academic Press, London, 1st edn., 2017, pp. 153–175.

8 S. Borman, Chem. Eng. News, 2004, 82, 35–39.

9 T. M. Picknett and S. Brenner, Encycl. Genet., 2001, 1730.

10 International Union of Biochemistry and Molecular Biology, Biochem. Educ., 1993, 21, 102.

11 A. G. McDonald, S. Boyce and K. F. Tipton, Nucleic Acids Res., 2009, 37, 593–597.

12 K. Tipton, Explor. Prim. source IUBMB Enzym. List, 2018.

13 K. Loos, Ed., Biocatalysis in Polymer Chemistry, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 1st edn., 2010.

14 E. A. Sym, Enzymologia, 1936, 1, 156– 160.

15 E. A. Sym, Biochem. Z., 1933, 258, 304– 324.

16 A. Zaks and A. M. Klibanov, Proc. Natl. Acad. Sci. U. S. A., 1985, 82, 3192–3196.

17 A. Zaks and A. M. Klibanov, Science (80-. )., 1984, 224, 1249–1251.

18 S. Shoda, H. Uyama, J. Kadokawa, S. Kimura and S. Kobayashi, Chem. Rev., 2016, 116, 2307–2413.

19 O. Kirk, T. V. Borchert and C. C. Fuglsang, Curr. Opin. Biotechnol., 2002, 13, 345–351.

20 Y. Jiang and K. Loos, Polymers (Basel)., 2016, 8, 243.

21 A. Gandini and T. M. Lacerda, Prog. Polym. Sci., 2015, 48, 1–39.

22 Y. Zhu, C. Romain and C. K. Williams, Nature, 2016, 540, 354–362.

23 K. G. Satyanarayana, G. G. C. Arizaga and F. Wypych, Prog. Polym. Sci., 2009, 34, 982–1021.

24 F. W. Lichtenthaler and S. Peters, Comptes Rendus Chim., 2004, 7, 65–90.

25 H. Nakajima, P. Dijkstra and K. Loos, Polymers (Basel)., 2017, 9, 523.

26 C. R. Becer and L. Hartmann, Eds., Glycopolymer Code: Synthesis of Glycopolymers and their

Applications, The Royal Society of Chemistry, 1st edn., 2015.

27 M. Ambrosi, N. R. Cameron, B. G. Davis and S. Stolnik, Org. Biomol. Chem., 2005, 3, 1476–1480.

28 M. C. Schuster, K. H. Mortell, A. D. Hegeman and L. L. Kiessling, J. Mol. Catal. A Chem., 1997, 116, 209–216.

29 F̍ÂŽķƦ̇`̍óĞĿĢùÖłù¦̍ÁāłĞŋƦ̇Macromol. Chem. Phys., 1996, 197, 259–274.

30 A. Adharis, D. Vesper, N. Koning and K. Loos, Green Chem., 2018, 20, 476–484.

31 W. M. J. Kloosterman, S. Roest, S. R. Priatna, E. Stavila and K. Loos, Green Chem., 2014, 16, 1837–1846.