Metabolic Engineering of Glycoproteins

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Metabolic Engineering of Glycoproteins

Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

(Dr. rer. nat.)

vorgelegt von

Anne-Katrin Späte

an der

Mathematisch-naturwissenschaftliche Sektion Fachbereich Chemie

Tag der mündlichen Prüfung: 3. Juni 2016

1. Referent: Prof. Dr. Valentin Wittmann 2. Referentin: Prof. Dr. Elisa May

3. Referent: Prof. Dr. Jörg Hartig

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Ich habe nicht nachgedacht, ich habe experimentiert.

Wilhelm Röntgen

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Diese Arbeit entstand im Zeitraum von Juli 2012 bis Februar 2016 im Fachbereich Chemie der Universität Konstanz, in der Arbeitsgruppe von Herrn Prof. Dr. Valentin Wittmann.

Danksagung

Mein besonderer Dank gilt Prof. Dr. Valentin Wittmann für die Überlassung des Themas dieser Dissertation, sein stetes Interesse am Gelingen dieses Projektes, dem entgegengebrachten Vertrauen und der Möglichkeit, eigene Ideen wissenschaftlich zu verwirklichen.

Für die Übernahme des Zweitgutachtens und des Prüfungsvorsitzes möchte ich mich bei Prof.

Dr. Elisa May und Prof. Dr. Jörg Hartig bedanken. Prof. Dr. Elisa May und Prof. Dr. Thomas Mayer danke ich für die kontinuierliche Betreuung dieser Arbeit im Rahmen des Thesis- Komitees der Konstanzer Graduiertenschule Chemische Biologie.

Dem Bioimaging Center der Universität Konstanz danke ich für die Bereitstellung der Fluoreszenzmikroskope. Für die Unterstützung bei der Mikroskopie danke ich Daniela Hermann und Martin Stöckl. Ein großer Dank gilt Anke Friemel für die Hilfe bei der NMR- Analytik, vor allem bei der Analyse der besonders geliebten Dreiringe.

Bei der gesamten AG Wittmann möchte ich mich für eine schöne Zeit an der Uni und gemeinsamen Aktivitäten bedanken. Ein herzlicher Dank gilt dabei vor allem meinen Laborkollegen Oliver Baudendistel und Andrea Niederwieser, die den Laboralltag um Einiges unterhaltsamer gemacht haben. Oli gilt besonderer Dank für die humorvolle Unterstützung bei technischen Problemen, vor allem im täglichen Kampf mit dem Drucker. Bei Andrea möchte ich mich dafür bedanken, dass sie ihrem Küken nicht nur ihr Projekt überlassen hat, sondern auch für chemischen und nicht-chemischen Rat jederzeit erreichbar war. Verena Schart möchte ich für jahrelanges MOE-Teamwork und gemeinsame, oft diskussionsreiche, Kaffeepausen danken.

Ohne euch wäre das Entstehen dieser Arbeit niemals möglich gewesen.

Für die Bereicherung dieser Arbeit mit neuen Ideen und die Weiterführung der Projekte möchte ich mich bei Franziska Doll, Jeremias Dold und Jessica Pfotzer bedanken.

Der AG Mayer danke ich für die herzliche Aufnahme während der Arbeiten in ihrem Labor.

Besonders bei Julia Häfner möchte ich mich für die vielen Tipps und geduldigen Erklärungen bei den ersten biochemischen Experimenten bedanken.

Allen engagierten Mitarbeiterpraktikanten, Bachelor Studenten (Sophie Schöllkopf, Sven Epple) und Hiwis möchte ich für die Bereicherung dieses Projekts durch Synthesen, Ideen und der nicht zu vernachlässigenden Energie durch Mitarbeiterkuchen danken.

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Für die aufmerksame und kritische Durchsicht dieser Arbeit danke ich Sven Epple, Jessica Pfotzer und Verena Schart.

Ein herzlicher Dank gilt auch meinen Freunden, die neben dem von Zeit zu Zeit nötigen Ansporn auch für einen wertvollen Ausgleich zum täglichen Laboralltag gesorgt haben.

Meiner Familie danke ich für die liebevolle Unterstützung während des Studiums und die Begeisterung für die in bunt gedruckten Ergebnisse dieser Arbeit.

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List of abbreviations

AAC azide-alkyne cycloaddition

AGM1, EC 5.4.3.2 N-acetylglucosamine phosphate mutase AGX1/2, EC 2.7.7.23 UDP-GlcNAc pyrophosphorylase

UDP-GalNAc pyrophosphorylase

AIDS auto immune deficiency syndrome

Asn asparagine

CMP cytidine 5’-monophosphate

CMP-Neu5Ac CMP-N-acetylneuraminic acid CMP-Neu5Gc CMP-N-glycolylneuraminic acid CMPNS, EC 2.7.7.43 CMP-Neu5Ac synthetase

CMP-Sia CMP-sialic acid

CTP cytidine 5’-triphosphate

EC 1.1.1.271 GDP-4-keto-6-desoxy D-mannose-3,5-epimerase-4- reductase

EC 2.3.1.3 glucosamine N-acetyltransferase

EC 2.7.1.7 mannokinase

EC 2.7.1.8 glucosamine kinase

EC 2.7.7.13 mannose-1-phosphate guanylyltransferase EC 2.7.7.9 α-D-glucose-1-phosphate uridyltransferase EC 3.1.3.29 N-acylneuraminic acid 9-phosphatase

EC 5.4.2.10 phosphoglucosamine mutase

EC 5.4.2.8 phosphomannomutase

DAinv Inverse-electron-demand Diels-Alder

ER endoplasmic reticulum

FPGT, EC 2.7.7.30 fucose-1-phosphate guanylyltransferase

Fru-6-P D -fructose-6-phosphate

Fuc L-fucose

Fuc-1-P L -fucose-1-phosphate

FUK , EC 2.7.1.52 L-fucose kinase

Gal D -galactose

Gal-1-P D-galactose-1-phosphate

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GALE, EC 5.1.3.2 UDP-galactose-4-epimerase GALK1/Gk1, Ec 2.7.1.6 galactokinase 1

GalNAc N-acetyl-D-galactosamine

GALT, EC 2.7.7.10 galactose-1-phosphate uridylyltransferase GAT, EC 2.3.1.4 acetyl-CoA:D-glucosamine-6-phosphate N-

acetyltransferase GCK, EC 2.7.1.2 glucose kinase

GMD, EC 4.2.1.48 GDP-^D-mannose dehydratase

GDP guanidine 5’-diphosphate

GDP-4-keto-6-deoxy-Man GDP-4-keto-6-deoxy-^D-mannose GDP-4-keto-6-deoxy-Gal GDP-4-keto-6-deoxy-L-galactose

GDP-Fuc GDP-^L-fucose

GDP-Man GDP-^D-mannose

GFAT, 2.6.1.16 fructose-6-phosphateamidotransferase GK, EC 2.7.1.6 GalNAc-1-kinase

Glc D-glucose

Glc-1-P D-glucose-1-phosphate

Glc-6-P D-glucose-6-phosphate

GlcN D-glucosamine

GlcN-6-P D-glucosamine-6-phosphate

GlcNAc N-acetyl-D-glucosamine

GlcNAc-1-P N-acetyl-D-glucosamine-1-phosphate GlcNAc-6-P N-acetyl-D-glucosamine-6-phosphate

GNE/MNK UDP-GlcNAc 2-epimerase/ManNAc 6-kinase

GPI glycosylphosphatidylinositol

GPI, EC 5.3.1.9 glucose-6-phosphate isomerase

HIV human immunodeficiency virus

Kdn 3-deoxy-non-2-ulosonic acid

Man D-mannose

Man-1-P D-mannose-1-phosphate

Man-6-P D-mannose-6-phosphate

ManNAc N-acetyl-D-mannosamine

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ManNAc-6-P N-acetyl-D-mannosamine-6-phosphate

MGE metabolic glycoengineering

MPI, EC 5.3.1.8 mannose-6-phoshpate-isomerase NAGK, EC 2.7.1.59 N-acetlyglucosamine kinase

NanE/NanK, EC 5.1.3.9/EC 2.7.7.9 N-acylglucosamine-6-phosphate 2-epimerase

Neu Neuraminic acid

Neu5Ac N-acetylneuraminic acid

Neu5Ac-9-P N-acetylneuraminic acid 9-phosphate

Neu5Gc N-glycolylneuraminic

OGA O-GlcNAcase, nuclear cytoplasmic O-GlcNAcase and acetyltransferase

OGT O-GlcNAc transferase

PEP phosphoenol pyruvate

ppGalNAcT polypeptide-N-acetylgalactosamine transferase RENBP, EC 5.1.3.8 N-acetylglucosamine 2-epimerase

SAS Neu5Ac-9-P synthase

N-acetylneuraminic acid phosphate synthase

Ser serine

ST sialyltransferase

Thr threonine

UDP uridine 5’-diphosphate

UDP-Gal UDP-D-galactose

UDP-GalNAc UDP-N-acetyl-D-galactosamine

UDP-Glc UDP-D-glucose

UDP-GlcNAc UDP-N-acetyl-D-glucosamine

UTP uridine 5’-triphosphate

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1. Introduction and aim of the project

“Sugars are everywhere. They are the foundation of all life on Earth.”^[1]

In fact, their indispensability for energy delivery and structural support has been known for a long time. However, it was only during the past decades that the importance of glycans as modifiers of proteins and lipids and their inherent contribution to a myriad of cellular processes beyond energy delivery was recognized.

For example, glycans are involved in cellular processes like protein folding, solubility or trafficking.^[2-3] Protein-protein interactions, for instance binding of hormones to receptors, can also be mediated by carbohydrates.^[2-3] Further, the development of tumorous tissue can often be correlated to changes in the glycosylation pattern. Glycan alteration correlated to cancer can exemplary occur as loss of expression, appearance of truncated structures as well as overexpression of glycans.^[2,4-5] Glycans are not only important for interactions between biomolecules, they are also essential for cell-cell interactions. Exemplary, the sperm-egg interaction during fertilization is influenced by a carbohydrate-dependent mechanism;

however, the exact mechanism has not been elucidated yet.^[2,6] Another case is the interaction between leucocytes and endothelial cells during inflammation which is mediated by the recognition of Sialyl Lewis X (a tetra-saccharide on the endothelial cells) by L-selectins on leucocytes.^[3,7] Furthermore, glycans can promote binding of pathogens, toxins or symbiotic agents to cells. One bacterial representative is E.coli that can bind to galactose-galactose motives in the urinary tract, resulting in inflammation of the bladder.^[2] Plasmodium falciparum, a protozoan parasite that causes malaria, binds to sialic acid containing glycans on red blood cells.^[2] A viral example is the glycoprotein gp120 of HIV that interacts with T-lymphocytes leading to acquired immune deficiency syndrome (AIDS).^[8-9]

Being involved in many diseases, carbohydrates represent interesting and promising targets for therapies. The development of treatments relying on carbohydrates is highly dependent on the knowledge of the sugars’ function. However, as glycans are secondary gene products and an extremely complex class of biomolecules, studying their function within cells and organisms has been challenging. To further elucidate their structures and functions, the development of methods to visualize them is essential. A powerful visualization tool that has emerged during the past two decades is metabolic glycoengineering (MGE).^[10-12] It allows the introduction of modified unnatural monosaccharides into glycoconjugates where they can be ligated with a probe for visualization or a tag for purification. This strategy relies on the use of bioorthogonal ligation reactions for example the azide-alkyne cycloaddition (AAC)^[13-14] or the inverse-

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1. Introduction and aim of the project electron-demand Diels-Alder (DAinv) reaction^[15-18]. First experiments exploited the reaction of a ketone with a hydrazide,^[19] whereas nowadays azides are considered valuable chemical reporters. While many groups have focused on the application of the Staudinger Ligation^[20] or the AAC in its copper-catalyzed^[21-23] or strain-promoted^[24-25] version, our lab has recently introduced the DAinv reaction between a terminal alkene and a 1,2,4,5-tetrazine for metabolic glycoengineering.^[26] In addition to being an azide-independent reaction, the DAinv is irreversible,^[27] does not need metal catalysis and can be performed under physiological conditions.^[28] However, the reactivity of terminal alkenes left room for improvement. Thus, this dissertation focused on the further development of dienophiles as reporters aiming to accelerate the reaction for its application in biological issues. To this end, it should first be investigated whether the cellular machinery also tolerates carbamates instead of amide linkages. If accepted, carbamate linkages could be beneficial concerning the reactivity of the derivatives and offer a facilitated synthetic access to modified sugars. Next, acceleration of the reaction ought to be achieved either through manipulation of the electron density of the reaction partners (an electron rich alkene and an electron poor tetrazine) or the exploitation of ring strain of a cyclic alkene. For application of the novel reporter in a dual labeling strategy it is essential to investigate the orthogonality of the new reporter to the compounds of the Click reaction. As sialic acids are an often occurring class of saccharides on cell-surfaces and well accessible for labeling, initial experiments should be performed with mannosamine derivatives, the precursors of sialic acids. Having found a promising new reporter, the expansion to glucosamine and galactosamine derivatives is desirable as it enables the detection of non-sialylated glycoconjugates. Together with our collaboration partners, the sugars developed within this project should be applied to answer biological questions.

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2. State of the art and placement of the work within the field 2.1. Glycoconjugates

Carbohydrates are a class of biomolecules that contributes to essential functions of an organism. Next to energy delivery and structural function, they are also key components for functional regulations such as cell-cell interaction and signal transduction. In these cases the sugars usually occur as glycoconjugates (Figure 1). Especially the cell surface is rich of carbohydrates, but also intracellular glycosylation (mostly O-GlcNAcylation) has a significant impact on the function of a protein. The sugar moiety (then termed glycan) can be found attached to proteins as well as lipids.^[2-4]

Figure 1: ‘Common classes of glycoconjugates in mammalian cells.’ ^[4]

2.1.1. Glycolipids

As the name indicates, a glycolipid is formed by the attachment of mono- or oligosaccharides to a lipid core structure. Cell surfaces are especially rich in glycolipids which can be divided in two main structural groups. Glycosphingolipids can for example be important for cell-cell interactions by presenting recognition markers. Further, they are involved in the organization of membrane domains. The structure of glycosphingolipids is defined by a fatty acid attached to the amino alcohol sphingosine (ceramides). Glycophospholipids on the other hand are built on phosphatidylglycerol. They are also termed glycosyl phosphatidylinositol (GPI-anchors) as the phosphatidylinositol is linked to a glucosamine moiety and they serve as an anchor for proteins on the cell surface.^[2-3]

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2.1. Glycoconjugates

2.1.2. N -Glycoproteins

In N-glycoproteins, the glycan is attached to the amide nitrogen of an asparagine side chain.

N-acetylglucosamine (GlcNAc) is the hexose linked to the asparagine (always beta). As a prerequisite for glycosylation, the asparagine must be part of a consensus sequence: Asn-X-Ser or Asn-X-Thr, with X being any amino acid but proline. The different types of N-glycans share a common core structure but differ in the terminal elaborations, leading to different categories such as for example high-mannose and complex types. Their assembly can be divided in three major steps. First, a lipid-linked precursor oligosaccharide (Glc3Man9GlcNAc2) is formed, which is then transferred en block to the polypeptide. These initial steps occur in the rough endoplasmic reticulum (ER). In a last step, the oligosaccharides are processed; meaning, some sugar residues are cleaved by glycosidases (trimming) and new sugars are added at the non- reducing termini of the glycan. The trimming usually also happens in the ER, while further modifications can be made during the glycoprotein’s migration through the Golgi apparatus.^[2-

3,29]

2.1.3. O -Glycoproteins

Most of the time, O-Glycoproteins are formed through the attachment of a sugar residue to a serine or threonine of a protein. The two occurring main classes of O-glycoproteins are mucins and O-linked N-acetylglucosamine (O-GlcNAc).

Mucins

The main task of mucins is to retain water at surfaces that are exposed to the environment.

Their core structure is formed by an N-acetylgalactosamine (GalNAc) which is α-linked to a serine or threonine and bears a galactose (Gal) residue. An example of a typical further modification would be the disialylation of this core structure. In contrary to the synthesis of N- glycans, the sugars are added one by one and there is no en bloc transfer. Another difference is the absence of a consensus O-glycosylation sequence, as many different transferases can install the GalNAc at a serine or threonine residue, allowing different amino acids to surround the glycosylation site.^[2-3,30]

O-GlcNAcylation

O-GlcNAc is a highly dynamic glycosylation, β-attached to a serine or threonine residue of a protein. It is attached by the O-GlcNAc transferase and removed by O-GlcNAcase. So far, the effect of O-GlcNAcylation on the function of the protein has been difficult to show. It was proposed to act as a modulator of phosphorylation, as the same sites can be glycosylated or phosphorylated. The many functions of O-linked glycans remain to be further explored.^[2-3,31]

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2.2. Biosynthesis of glycans

The function of glycosylation is still not satisfyingly enlightened. To fully elucidate their function, the development of tools to investigate glycans is essential. One of the techniques that nowadays allow visualization of glycosylation patterns in and on cells as well as in small organisms is the previously mentioned metabolic glycoengineering (MGE), which allows the introduction of modifications into glycan structures. As a prerequisite for the understanding and application of MGE it is fundamental to understand the metabolism of monosaccharides within the cells. Thus, the following chapter addresses the metabolic fate of selected sugars.

For better readability sometimes the abbreviated names of sugars, derivatives and enzymes are used without mentioning the full name. The corresponding abbreviations can be found in the list of abbreviations.

2.2.1. Sialic acid biosynthesis

Sialic acids, also referred to as N-acylneuraminic acids, occur predominantly at the termini of N-glycans, O-glycans and glycosphingolipids. They contribute to cellular processes like signal transduction and cell-cell interaction. The family of sialic acids comprises more than 50 members, among which N-acetylneuraminic acid (Neu5Ac), neuraminic acid (Neu), 3-deoxy- non-2-ulosonic acid (Kdn) and N-glycolylneuraminic acid (Neu5Gc) are considered the four core structures (Figure 2). The further level of diversity arises from modifications like O-acetyl or O-methyl at their hydroxyl groups.^[2-3]

Figure 2: Structures of neuraminic acid and related compounds: Neu5Ac, Neu, Kdn and Neu5Gc.

The most common representative is N-acetylneuraminic acid (Neu5Ac) whose precursor is N-acetylmannosamine (ManNAc). First, ManNAc is phosphorylated to ManNAc-6-P by the GNE/MNK (UDP-GlcNAc 2-epimerase/ManNAc 6-kinase), a bifunctional enzyme that can also convert UDP-GlcNAc to ManNAc-6-P and thus introduce GlcNAc derivatives into the pathway (Scheme 1). The condensation of ManNAc-6-P with phosphoenolpyruvate (PEP) catalyzed by the Neu5Ac-9-P synthase (SAS), forms Neu5Ac-9-P, which is then dephosphorylated by the N-acylneuraminic acid 9-phosphate phosphatase to yield the free sialic acid Neu5Ac. Upon activation into the nucleotide donor CMP-Neu5Ac by the CMPNS (a process that occurs in the

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2.2. Biosynthesis of glycans nucleus), the activated CMP-Neu5Ac is transported to the Golgi and transferred to glycoconjugates by sialyltransferases (ST). It is noteworthy that the GNE-domain of the GNE/MNK can be inhibited by a feedback inhibition wit CMP-Neu5Ac. Thus, the introduction of ManNAc derivatives derived from GlcNAc is hampered if an excess of (unnatural) CMP-Neu5Ac is present.^[32-36]

O COOH

O HO HO

OH OH

NH O

O HO HO HN HO

OH O

O HO

HN

2-O₃PO HO

OH O ManNAc

ManNAc-6-P GNE/MNK

SAS

O COOH

OH HO

HO

2-O₃OP OH NH O

Neu5Ac-9-P

O COOH

OH HO

HO OH OH

NH O

O CMP

COOH HO

HO OH OH

NH O

CMP-Neu5Ac

RENBP O

HO NH OH HO

OH

GlcNAc

phosphatase sialyltransferase

(Golgi)

Neu5Ac CMPNS

Nucleus O

PEP

Scheme 1: Biosynthesis of N-acetylneuraminic acid and its incorporation into sialoglycans.

2.2.2. Mucin-type O -glycosylation

The core structure of mucin type O-glycosylation is a GalNAc residue α-linked to the hydroxyl group of a serine or threonine side chain (Scheme 2). To this end, GalNAc is first phosphorylated by the GalNAc-1-kinase (GK) to form GalNAc-1-P which is then activated by UDP-GalNAc-pyrophosphorylase (AGX1/2) to form UDP-GalNAc. After being transported from the cytosol to the Golgi, the GalNAc residue is transferred to the protein by a polypeptide-N- acetylgalactosamine transferase (ppGalNAcT). This Tn-Antigen can then be further modified by glycosyltranferases yielding several mucin-type O-glycoproteins.^[2-3,30]

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Scheme 2: Biosynthesis of mucin-type O-glycoproteins.

2.2.3. O -GlcNAcylated proteins

The de novo synthesis of O-GlcNAcylated proteins (Scheme 3) starts with the conversion of glucose to Fru-6-P which is then converted to Glucosamine-6-phosphate (GlcN-6-P) by the fructose-6-phosphateamidotransferase (GFAT). GlcN-6-P is then acetylated by the acetyl- CoA:D-glucosamine-6-phosphate N-acetyltransferase (GAT) yielding GlcNAc-6-P. GlcNAc-6-P can also be introduced through the salvage pathway by phosphorylation of GlcNAc at the 6-OH group by the N-acetylglucosamine kinase (NAGK). Next, the N-acetylglucosamine phosphate mutase (AGM1) catalyzes the shift of the phosphorylation from GlcNAc-6-P to GlcNAc-1-P. The sugar is then activated to form UDP-GlcNAc by the UDP-GlcNAc pyrophosphorylase (AGX1 or AGX2) and then transferred from the nucleotide sugar donor to a serine or threonine of the protein by the O-GlcNAc transferase (OGT). O-GlcNAcylation is a reversible modification; the O-GlcNAcase (nuclear cytoplasmic O-GlcNAcase and acetyltransferase; OGA; NCOAT) can hydrolytically remove GlcNAc residues from modified proteins.[2-3,31,37-39]

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Scheme 3: Metabolism of GlcNAc.

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The cleaved GlcNAc can be reused through the salvage pathway. BOYCE et.al. could show that UDP-GalNAc can be converted to UDP-GlcNAc and by the UDP-galactose 4-epimerase (GALE), thereby allowing GalNAc derivates to enter the pathway to O-GlcNAcylation.^[37] Thus, a cross talk between galactosamine and glucosamine derivatives is possible. This is not the only option that allows interconversion from one monosaccharide to another. Possible epimerization points for natural sugars as well as unnatural sugars should be considered when performing MGE experiments and can be found in the next section.

2.2.4. Interconversion between monosaccharides

The natural sugars can be interconverted into each other, rendering the understanding of the diverse glycosylation pathways a challenge.

Scheme 4: Selected interconversions between monosaccharides.

Concerning the hexosamines important for the present thesis, it was for example shown that GALE can not only convert UDP-Glc to UDP-Gal, but also UDP-GlcNAc to UDP-GalNAc and vice versa (Scheme 4a).^[37,40] GlcNAc can be epimerized to ManNAc catalyzed by the RENBP, a

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GlcNAc 2 epimerase (Scheme 4b).^[41] Alternatively, GlcNAc can be metabolized to UDP-GlcNAc which yields ManNAc if metabolized by the GNE (Scheme 4c).^[42] Both ways allow GlcNAc to enter the sialic acid biosynthesis. These are only the most prominent examples, more conversions have been summarized before and are depicted in Scheme 5.^[35-36]

O HOHO

OH HN O

OH O HOHO

OH H2NOH O HOHO

OH OHOH

O HOHO

OPO32- HN GlcNAc-6-PO

OH O HOHO

OPO32- H2N GlcN-6-P

OH O HOHO

OPO32- OH Glc-6-P

OH O HOHO

OH OH Glc-1-POPO32-

O HO

HOOH HN GalNAcO

OH O HOHOOH H2N GalN

OH

O HO

HOOH OH Gal

OH O HOHO

OH OH UDP

O HO

HOOH OH UDP-GalUDP

O HO

HOOH OH Gal-1-POPO32-

O HO

HNHO HO OH

O O HO

HN-2O3PO HO OH

O

ManNAc ManNAc-6-P O

COOH OH HO

HO

-2O3POOH NH ONeu5Ac-9-P O

COOH OH HO

HO

OH OH NH O O

CMP COOH HO

HO

OH OH NH O CMP-Neu5Ac

O HO

HOOH HN GalNAc-1-PO

OPO32-

O HOHO

OH HN GlcNAc-1-PO

OPO32-

O HOHO

OH HN O

UDPNAGKAGM1AGX1/2GNE/MNK GAT

RENBP EC 5.1.3.8 EC 2.7.1.8 Fru-6-P EC. 5.4.2.10 EC 2.7.7.9

EC 5.1.3.2

GALK1/GK1 EC 2.7.7.10

or GNE/MNK SAS EC 2.5.1.57 EC. 3.1.3.29 phosphatase CMPNS EC 2.7.7.43

GALE EC 5.1.3.2 O HOHOOH HN UDP-GalNAcO

UDP AGX1/2 EC 2.7.7.23 EC 2.7.1.6

GFAT

GlcNAc GlcN

UDP-GlcNAc Glc UDP-Glc

Neu5Ac

OOH CH2OH

2-O3PO OHHOO HO

OHHO HO OH Man O HO

OH2-O3PO HO OH Man-6-P O HO

OHHO HO OPO32-Man-1-P

O HO

OHHO HO GDP-ManGDP

Fuc-1-P GDP-Fuc

OOH

HO OH GDP O GDP

OH OH

O

EC 1.1.1.271 4-reductase E.C. 1.1.1.271 3,5-epimerase

GDP-4-keto- 6-deoxy L-Gal O GDP

OHO HO GDP-4-keto- 6-deoxy Man

OOH

HO OH OPO32-

OOH

HO OH OH Fuc EC. 2.3.1.3

NanE/NanK (EC 5.1.3.9) NanE/NanK GMD

FUK FPGT EC. 2.7.1.7 mannokinase MPIGPI EC. 5.4.2.8

GCK EC. 2.7.7.13

EC 2.7.1.52 EC 2.7.7.30

both EC 2.7.1.60

EC 5.1.3.14EC 2.7.7.23 GK GALE

GALT

EC 2.7.1.6

EC 2.7.7.23EC 2.7.1.59 EC 2.3.1.4 EC 2.6.1.16 EC 5.3.1.9 EC 5.3.1.8 EC 2.7.1.2

Scheme 5: Enzymatic pathways of selected sugars. Activated sugars that can be transferred to proteins, lipids and saccharide cores are highlighted with a square.

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Anticipatory to the next chapter (dealing with modified sugars), this obviously raises the discussion, if unnatural modified monosaccharides can be used to target a specific class of glycans. As the promiscuity of the enzymes varies tremendously and already a small modification might not allow epimerization anymore, the question of interconversion has to be addressed for each monosaccharide separately. In the following paragraph, the metabolic fate of certain modified sugars - which will be introduced in the next chapter - will be discussed.

GlcNLev for example could not be used to monitor surface glycans, allowing the conclusion that UDP-GlcNLev is probably not converted to ManNLev or epimerized to UDP-GalNLev.^[34,43-

44] It should be kept in mind that since the publication of these findings in 1998, the sensitivity of the detection methods could be improved significantly. Furthermore, it has since been shown, that peracetylated sugars are more efficiently taken up into cells.^[34] Thus double- checking these results might reveal that conversion is taking place at least in a small percentage.

It was reported that azide modified Ac4GlcNAz does not lead to a GlcNAz modified cell- surface.^[39] Nonetheless cell-surface labeling occurred when Ac4GlcNAz was fed to the cells.

This is most likely due to an epimerization of GlcNAz to ManNAz which is further metabolized to an azide-modified sialic acid.^[44] Our results also support that Ac4GlcNAz can be used to label cell surface glycoconjugates.^[45] While an interconversion between GlcNAz and GalNAz is possible, GlcNAlk was shown to be less prone for conversions and thus more suitable to target O-GlcNacylation.^[37,46] However Ac4GlcNAlk was used to label N-glycans, which was shown through a signal decrease after treatment with PNGase F.^[46] Further, it was shown that cross- talk between GlcNAlk and non-carbohydrate protein modifications is possible by a transfer of the N-acyl side chain.^[47]Efforts to tune the selectivity of the sugars by attaching the azide in the 6-position rather than the N-acyl side chain, led to the development of Ac36AzGlcNAc which was shown to be incorporated into O-GlcNAc bypassing NAGK, the enzyme that usually phosphorylates in the 6 position.^[48] An alternative approach was to vary the attachment of the side chain and attach the residue as a carbamate instead of an amide (Ac4GlcPoc, Ac4GalPoc and Ac4ManPoc). Ac4GlcPoc und Ac4GalPoc were shown to lead to the same labeling pattern, whereas the labeling pattern for Ac4ManPoc differs slightly from the two.^[49] It could be speculated that GALE allows the Poc modification at the N-acyl side chain, while the conversion to ManNAc by the RENBP is hampered. However, all three Poc derivates can be used to label NEDD4 (Ac4GlcPoc and Ac4ManPoc well suited, Ac4GalPoc moderate signal) which is known to be O-GlcNAcylated.^[49]

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2.3. Metabolic glycoengineering Concluding, the question about the precise metabolic fate of modified sugars can at this point not satisfyingly be answered. Postulations should be taken with caution and efforts to elucidate the metabolism of unnatural sugars should be undertaken.

These modified sugars are the basis for metabolic glycoengineering, where they are applied to modify the structure of glycans.

2.3. Metabolic glycoengineering

A technique that can be applied to modify glycan structures is the above mentioned metabolic glycoengineering (MGE).^[10-12] This tool exploits the promiscuity of cellular enzymes allowing the incorporation of modified unnatural sugars into glycoconjugates (Scheme 6).

Scheme 6: Scheme of metabolic glycoengineering with a mannosamine derivative which is incorporated into a sialoglycoconjugate, followed by a labeling reaction.

In more detail, a modified monosaccharide is synthesized and fed to cells or even introduced into an organism. For in cellulo experiments, usually the peracetlyated form of a monosaccharide is utilized, as the acetylation facilitates diffusion over the membrane.

Unspecific esterases cleave the acetyl groups leading to the free sugar which can then be metabolized according to the above described pathways and incorporated into glycans.^[34]

This technology was applied to alter glycosylation patterns especially on cell surfaces. This might be of interest for therapeutical applications, as modified glycan structures can for example influence virus binding to cell surfaces. In this context, REUTTER et al. could show that the N-acyl group can be elongated and the sugars (Figure 3) are still accepted by the cellular machinery.^[11,50-51]

Figure 3: N-acyl modifications that are accepted by the cellular machinery.

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2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions

This technology was further advanced to label glycans. If a functional group is chosen as modification, it can - after incorporation into a glycan - be reacted in a ligation reaction with a probe of choice. The probe can for example be a biotin tag for enrichment of glycoconjugates or visualization via a streptavidin dye conjugate. Alternatively, the probe can be a fluorophore and thus enable direct visualization of the modified glycan.^[19-20]

If the ligation reaction is chosen wisely, MGE can be an extremely powerful tool for the detection and visualization of a myriad of glycans. In the following part, bioorthogonal ligation reactions that were applied for MGE will be introduced and correlated to the work performed within this thesis.

2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions

For the labeling of biomolecules, bioorthogonal ligation reactions have proven themselves highly valuable. Chemical reactions, whose components do not interfere with unmodified biomolecules and proceed without disturbance of the cellular/biological environment, are considered to be bioorthogonal. The two reacting chemical functions should have a high selectivity for each other and interact rapidly under physiological conditions, while being inert in the biological environment. Ideal properties include that the ligation product is stable and that both product and starting material are nontoxic for the biological environment.^[52-53]

2.4.1. Keto-hydrazide reaction

Scheme 7: The reaction of a ketone with a hydrazide forming a hydrazone can be used as ligation reaction (left).

Stuctures of GlcNLev and ManNLev that were employed for MGE (right).

The first ligation reaction that was employed in combination with metabolic glycoengineering was the reaction of a ketone with a hydrazide-based probe.^[19,54] Experiments with a ketone- modified glucosamine derivative (GlcNLev) revealed that GlcNLev is not or only neglectably incorporated into cell-surface glycoconjugates.^[43] In contrast, a ketone-modified mannosamine derivative (ManNLev, Scheme 7) was fed to cells and incorporated as sialic acids into glycoconjugates. The ketone was reacted with a hydrazide functionalized biotin and visualized by treatment with FITC-labeled avidin via flow cytometry. However, the reaction proceeds

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2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions slowly under physiological pH, warranting the demand for new ligation reactions. Additionally, ketones can occur in biological systems thus the reporters are not truly bioorthogonal.

2.4.2. Azide-based ligation reactions

As an alternative, azides have been shown to be especially valuable reporters for bioorthogonal chemistry. In contrary to aldehydes, ketones or thiols, azides do not occur in biological systems. Due to their small size, azides only cause minimal changes in a molecule which is advantageous to create biomolecules that differ merely a little in structure. They can readily be introduced via nucleophilic replacement of a leaving group with an azide ion or via diazo transfer. Being only weak electrophiles, azides are inert to nucleophilic attacks and thus suited for biological applications. Nevertheless, they react readily with phosphines as well as alkynes and are thus suitable reporters for the Staudinger reaction and the azide-alkyne cycloaddition (AAC).

Staudinger ligation

Scheme 8: Non-traceless version of the Staudinger Ligation.

In 1919 STAUDINGER and MEYER reported the reaction of an azide and triphenylphospine forming an iminophosphorane intermediate under the release of nitrogen.^[55] In aqueous media, the reaction is followed by hydrolysis to an amine and triphenylphosphine oxide; the covalent bond between the two molecules is cleaved, thus the reaction is not suitable as a ligation reaction. A modification of this classical Staudinger reaction, developed by BERTOZZI

and SAXON in 2000 allowed the use of this chemistry as a ligation reaction (Scheme 8).^[20,44] By introducing a phosphine bearing an electrophilic trap, the negatively charged nitrogen of the aza-yilide intermediate is trapped by the ester group. The amide bond formed in this reaction is stable in aqueous environment and links the two molecules even after hydrolysis of the P-N bond. This non-traceless Staudinger Ligation fulfills all necessary criteria for the application as a chemoselective, bioorthogonal ligation reaction. With both moieties being abiotic reporters, the method can even be applied in living systems. Furthermore, the reaction can proceed quantitatively, at room temperature and at reasonable speed (up to k=3.8 x 10^-3 M^-1s^-1 CH3CN/KH2PO4: 1/1).^[56] As kinetics can depend on the solvent, it is difficult to compare reaction rates; thus one should pay close attention to the solvent when comparing values

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resulting from different studies. An even more elegant version is the traceless Staudinger ligation (simultaneously published by the groups of BERTOZZI and RAINES) in which the phosphine oxide is eliminated from the final product through hydrolysis.^[57-58] This is an advantage if the ligation is used for a synthetic application, in which a residual phosphine oxide is not desirable. Limitations of the Staudinger Ligation are its rather slow reaction kinetics and the resulting high concentrations of phosphine that are needed. Furthermore, the Staudinger Ligation is limited by the stability of the phosphines. By increasing its nucelophilicity, in order to accelerate the reaction, one also increases the probability for oxidation by air, thus acceleration of the reaction remains challenging.^[59] Despite these limitations, the Staudinger Ligation has successfully been applied for MGE for mammalian as well as bacterial glycans.^[20,60]

Azide-alkyne cycloaddition (click reaction)

Another ligation reaction relying on azides is the 1,3-dipolar azide-alkyne cycloaddition (AAC).

The cycloaddition of azides and alkynes was reported by HUISGEN^[61] long before its potential as ligation reaction was recognized by SHARPLESS and MELDAL^[62], who could achieve the necessary acceleration of the reaction by catalysis with Cu(I).

Scheme 9: Copper-catalyzed (left) and strain-promoted (right) azide-alkyne cycloaddition.

Using alkynes (AAC) rather than phosphines (Staudinger Ligation) as ligation partner for the azide, the reaction rate of the ligation was significantly improved. While the addition of copper accelerated the reaction and enabled the performance at room temperature, copper is toxic for living systems (due to the production of reactive oxygen species and inhibition of enzymes), thus application in living cells is limited. To avoid copper toxicity, strained alkynes can be applied for an accelerated Cu-free click reaction. Building on findings that showed that cyclooctynes react explosively with azides^[63] a cyclooctyne derivative was developed to label azides in cellular glycans. However, the first generation of cyclooctynes (OCT) react relatively slow (k=0.0012M^-1s^-1, CD3CN).^[24] To improve the reactivity, several substituted cyclooctynes (Figure 4) were synthesized and evaluated.

Figure 4: Selected strained alkyne reagents.

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2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions The introduction of two fluorine atoms was synthetically challenging, but resulted in a difluorocyclooctyne (DIFO) which has accelerated kinetics (k=0.076 M^-1s^-1, CD3CN)^[64]. Further improvements in the cyclooctyne development led to dibenzocyclooctyne DIBO (k=0.17 M^-1s^-1, MeOH; k=2.3 M^-1s^-1, H2O/CH3CN)^[25] and aza-dibenzocyclooctyne DIBAC (k=0.31 M^-1s^-1, CD3OD)^[65], which are easier to synthesize and react quite well. Another strained alkyne reagent is the 3,3,6,6-tetramethyl-thiacycloalkyne TMTH that is highly reactive (k=4.0 M^-1s^-1, CD3CN) which has so far not been conjugated to a dye.^[66] Both versions of the AAC have successfully been employed for metabolic glycoengineering with azide as well as alkyne modified monosaccharides (Figure 5). The azide reporter has been attached to peracetylated mannosamine (Ac4ManNAz)^[20], galactosamine (Ac4GalNAz)^[44], glucosamine (Ac4GlcNAz^[44] and Ac36GlcNAc^[48]) and fucose (Ac4Fuc6Az)^[22-23]. Recently, inositol derivatives carrying an azido group were applied to label glycosylphosphatidylinositol anchored proteins.^[67] As ligation reactions, the Staudinger ligation or the AAC can be applied in combination with the azide modified sugars. The azide modification is superior to a terminal alkyne, as it allows the performance of the ligation reaction with a strained alkyne (attached to a probe) to avoid the toxicity of the copper. A cyclooctyne attached to a hexosamine is expected to be too bulky to be accepted by the bioenzymatic machinery. Consequently, the ligation of these alkynes has to be copper catalyzed. A way to circumvent the limitation of size is to attach the residue directly to the sialic acid rather than its precursor mannosamine. Next to small terminal alkynes (SiaNAl and Neu5Proc)^[68-70] also the sterically demanding BCN (BCNSia)^[71] was thus successfully used to modify sugars. Alternatively, 9-Az-NeuAc was used to perform strain-promoted azide-alkyne cycloaddition (SPAAC).^[72] So far only terminal alkynes attached as amides (Ac4ManNAlk, Ac4GlcNAlk, Ac4GalNAlk)^[21,73] or carbamates (Ac4ManPoc, Ac4GlcPoc, Ac4GalPoc)^[49] have been attached to hexosamines or fucose (Ac4Fuc6Alk)^[23,73] and were incorporated into glycoconjugates.

In cooperation with Prof. SCHERER we were able to apply GalNAz for the visualization of cupulin in zebrafish. By injecting the modified sugar and monitoring it via the copper catalyzed AAC we could show that the cupula, that is important for the function of the ear, is renewed regularly.

(manuscript in preparation and ^[74])

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Figure 5: Unnatural sugar derivatives modified with reporter groups for click chemistry. The depicted abbreviations correspond to the names used in the original publications and are thus not uniform.

The development and application of azide based ligation reactions has greatly advanced the ligation reaction field. To further expand the panel of ligation reactions, an azide-independent ligation reaction would be advantageous, as it allows the combination of two orthogonal ligation reactions.

2.4.3. Tetrazine ligation with isonitriles

Our lab focusses on the DAinv reaction between an alkene and a tetrazine as an alternative ligation reaction to the AAC for metabolic glycoengineering. In parallel to our work, another tetrazine-based ligation reaction was developed by the scientists around LEEPER.^[75-77]

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Scheme 10: Tetrazin ligation of isonitriles with 1,2,4,5-tetrazines.

Isonitriles were shown to react with tetrazines forming tetraazanorbornadienimines that after a [4+2]-cyclorevision with nitrogen release lead to 4H-pyrazol-4-imine derivatives. However, these can after tautomerisation hydrolyse to aminopyrazoles (not shown).^[78] Nevertheless, LEEPER and his coworkers tackled this challenge by usage of tert-butylisonitrile that cannot tautomerise and consequently not hydrolyze, or by usage of a primary 3-isocyanopropionyl ester that tautomerises to a conjugated enamine which is less prone to hydrolysis.^[76] Attached to hexosamines (Figure 6) these two residues could be employed for MGE in a ligation reaction with a tetrazine derivative.^[75] Reaction rates with the symmetric tetrazine and the primary (3-isocynopropionylester) and the tertiary isonitrile were determined to be 0.12 and 0.57 M^-1s^-1(THF/H2O) respectively; requiring labeling with 100 µM Tz-biotin 1 for 30 min to label the glycoconjugates.^[75-76]

Figure 6: Isonitrile-modified hexosamines.

Later it was demonstrated, that these isonitrile modified hexosamines can be used in combination with azido sugars, for example Ac4GlcN-n-Iso and Ac4GalNAz were visualized in parallel on cell surfaces.^[77]

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2.5. Inverse-electron-demand Diels-Alder reaction for MGE

Over the past decades, the inverse-electron-demand Diels-Alder (DAinv) reaction has gained increasing importance as a ligation reaction.^[15-17] This [4+2] cycloaddition was initially developed by CARBONI and LINDSEY as a synthetic route to pyridazines.^[79]

Scheme 11: Diels-Alder reaction, a [4+2] cycloaddition.

The name results from the fact that the electron demand of the DAinv reaction is inverse to the classical Diels-Alder reaction which was developed by DIELS and ALDER and later awarded with a Nobel prize in chemistry.^[80] Whether the reaction (Scheme 11) occurs with classical or inverse electron demand depends on the residues of the diene and the dienophile. In the DAinv reaction, the LUMO of the diene overlaps with the HOMO of the dienophile, while the classical Diels-Alder reaction is characterized by an overlap between the HOMO of the diene and the LUMO of the dienophile (Figure 7).^[81] An inverse-electron-demand reaction occurs if an electron poor diene reacts with an electron rich dienophile. The more electron-withdrawing the substituent of the diene is and the more electron pushing the residue of the dienophile is, the faster the reaction proceeds.

Figure 7: Occurring molecule orbitals for Diels-Alder reaction with normal (left) and inverse (right) electron demand.

EWG: electron withdrawing group, EDG: electron donating group.

It was shown that tetrazines react well with alkenes in a DAinv reaction, followed by a retro- Diels-Alder reaction in which nitrogen is released marking the reaction irreversible

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2.5. Inverse-electron-demand Diels-Alder reaction for MGE (Scheme 12).^[15] The resulting dihydropyridazines occur as several tautomeric and isomeric forms and can in a subsequent reaction be oxidized to pyridazines.

Scheme 12: Inverse-electron-demand Diels-Alder reaction between an alkene and a 1,2,4,5-tetrazine.

Its irreversibility is just one of the advantages that make the DAinv reaction an excellent ligation reaction. Moreover, the reaction proceeds without a catalyst, at physiological pH and 37°C.^[28,82] Despite all these benefits, at the starting point of this dissertation, the DAinv reaction had not been published as a ligation reaction in combination with metabolic glycoengineering. The application of the DAinv reaction requires a dienophile that, in addition to possessing the characteristics for bioorthogonality, can be easily attached to a monosaccharide without interfering too much with the structure of the sugar, so that it will still be accepted by the enzymatic machinery.

2.5.1. MGE with terminal alkenes as reporter groups

Looking for small dienophiles, our lab identified terminal alkenes as a class of chemical reporters that react in the DAinv reaction.^[26] The group around LIU also recognized the potential of terminal alkenes and applied them to modify amino acids.^[83] In contrast to intra- chain alkenes that occur in fatty acids, terminal alkenes are advantageous as they are hardly found in biological systems and completely absent in proteins. Terminal, monosubstituted rather than doubly substituted alkenes were chosen, because even though for example a methyl substituent would theoretically accelerate the reaction, its sterical boldness overbalances this effect and thus makes the reaction slower.^[84] Previous to this dissertation, our lab¹ could show that ManNAc derivatives bearing a terminal alkene (Ac4ManNPtl and Ac4ManNHxl, Figure 8) in the side chain are indeed incorporated into cell-surface glycoconjugates and can be labeled through a DAinv reaction with a tetrazine derivative as for example Tz-biotin 2 (Figure 8).^[26]

1 PhD Thesis ANDREA NIEDERWIESER

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2.5. Inverse-electron-demand Diels-Alder reaction for MGE

Figure 8: Mannosamine-derivatives bearing terminal alkenes and Tz-biotin 2.

Combining an alkene modified (Ac4ManNPtl) with an azido modified derivative (Ac4GalNAz), it was further shown that the DAinv reaction and the SPAAC can be performed in the same experiment (Scheme 13).^[26]

Scheme 13: Dual sugar labeling by combination of DAinv and click chemistry.

Depending on the residue, we realized that it could be advantageous to attach the functional group via a carbamate rather than an amide group. Next to steric and kinetic influences, which will be discussed later, they provide an easy synthetic access starting from the corresponding alcohols. Thus, this dissertation started with the investigation whether carbamate-linked terminal alkenes are accepted by the cellular enzymatic machinery. Indeed, we^[26,85] and the group around PRATT^[49] could show that also sugars that feature a carbamate instead of an amide linkage are also accepted by the cellular machinery (Figure 9).

Figure 9: Structures of amide (yellow) and carbamate (blue) bound terminal alkenes and alkynes.

Ac4ManNPoc was developed by the PRATT group and successfully visualized by copper- catalyzed click chemistry.^[49] The synthesis, kinetic and biological evaluation of Ac4ManNPeoc that are part of the present thesis, were published together with the terminal alkenes developed by ANDREA NIEDERWIESER in “Two-Color Glycan Labeling of Live cells by a Combination of Diels-Alder and Click Chemistry”(Chapter 6.1).^[26]

Metabolic Engineering of Glycoproteins