Vesela, Haifei, Mélanie, Stéphanie Steve Stefan

Alena Carlos

Fabien Antoine Marco

Tati, Raph, Yannick Anne Thomas

Michelle Christine Sylvie

Merci Alison

D'

Keywords: Glycolipid-and Lectin-Dependant Endocytosis Studies Glycosphingolipid, metabolic labelling, copper-free click chemistry Glycosphingolipides, marquage métabolique, chimie click sans cuivre Je suis reconnaissante d'

Current biological techniques do not permit the functional reintroduction of long-chain glycosphingolipids (GSLs) into cells. We aim to develop molecular tools permitting cellular reconstitution of functional GSLs in a controlled manner, combining metabolic labelling of sphingolipids with copper-free click chemistry. Molecular tools are validated with the bacterial Shiga toxin, which gains entry to cells independently of the canonical intracellular clathrin machinery on binding to its GSL cell receptor, globotriaosylceramide (Gb3-Cer), through a recently described mechanism of endocytosis, termed GL-Lect (glycolipid-lectin). According to the GL-Lect hypothesis, cellular or pathogenic lectins drive the construction of endocytic pits by reorganising membrane lipids so as to favour the formation of narrow membrane curvature. The GSL reconstitution technique that is described here is applicable to the discovery of GSL binding partners in the study of GL-Lectmediated endocytosis.

Live is if you were to die tomorrow, learn as if you were to live forever Mahatma Gandhi I thank all my friends from CRI, who managed to laugh in the face of PhD stress with me, whilst nourishing overpriced pints, whenever we managed to get out of the lab.

I thank the BIOPOL Innovative Training Network (European H2020 funding) that funded the project and my international training stations, and grouped together 16 PhD students, making us spend quality-science-time together across Europe. We had never felt so different, yet so alike.

After countless hours complaining on the phone, I retain some gems from my mum, who said: "You don't learn unless you're suffering;" and my dad, who said "You're doing a PhD because once you're a Dr., you never have to say if you're a bloody Miss or Mrs., because you'll be a Dr.!" I would be nowhere without you. Carolyn, I know one day your wisdom will come crashing down on me. I love you and M&M to the end and back.

INTRODUCTION

Endocytosis mechanisms

Endocytosis encompasses the cellular uptake of diverse cargo, including macromolecules, membrane components, nutrients and fluids, by their encapsulation in membrane vesicles which bud inwards to be pinched off from the plasma membrane (PM).

From the PM, cargoes are trafficked through endosomes, where they can be (1) recycled back to the PM, [START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF] sorted via the retrograde route to the Golgi apparatus and in some cases to the endoplasmic reticulum (ER), or (3) targeted to the late endosomes and lysosomes for degradation (Figure 1.1.1a). The best characterised mechanism of endocytosis is mediated by clathrin, which coats the PM on binding to its adaptor proteins, forming a concave lattice structure (Figure 1.1.1b) 1 to bend and engulf portions of the membrane containing endocytic cargoes. These clathrin-coated vesicles (70-150 nm) 2 are eventually scissionned from the PM with assistance from intracellular protein machinery including a GTPase, dynamin (Figure 1.1.2), recruited by curvature-inducing BAR domain proteins, which themselves bind to and constrict negatively charged, curved membranes. (1)

(2)

(3)

Clathrin-independent endocytosis (CIE) pathways include macropinocytosis and phagocytosis (Figure 1.1.2), in which large portions of the membrane (>1 μm) are engulfed by membrane structures supported by actin filaments. In a different category of CIE, vesicles of closer size to clathrin-coated vesicles are formed by other cytosolic proteins. Caveolae are membrane invaginations formed from caveolin proteins and cavins (cavin1 -4). They function as mechano-sensing domains which flatten out to buffer membrane tension. Caveolae can bend the membrane to form caveolar carriers (50-80 nm) that undergo endocytosis with the aid of dynamin, ATPase EHD2 and BAR domain protein pacsin2. Which cargoes are transported specifically by caveolae remains unclear, 4-6 and their endocytic uptake is highly dependent on cholesterol and sphingolipid content of the lipid bilayer. 7 Flotillin can localise in caveolae and itself outlines another clathrin-and caveolae-independent process. 4,5 Unlike clathrin, which is located in the cytosol and binds via membrane-bound adaptors, caveolins and flotillins are integral transmembrane proteins implicated in lipid rearrangement. 4 Glycosylphosphatidylinositol-anchored proteins (GPI-ACs) have a high propensity to traffic through clathrin-, caveolin-and dynamin-independent endocytosis, giving the name to the clathrin-independent carrier (CLIC)/GPI-AC-enriched early endosomal compartment (GEEC) pathway. 8 The protein machinery driving endocytic uptake of these high capacity (100-200 nm) 9 carriers is complex, implicating BAR domain proteins and Rho family GTPases. 10 CIE carriers lack regular protein coats, thus are harder to image by electron microscopy compared with clathrin-coated cargos. Several other CIE mechanisms have since been characterised, including those in which lipids are redistributed to bend membranes. 8,11-13 The importance of membrane lipid content is currently studied by indirect, secondary techniques (discussed in section 1. 3-4 and 1.7), since these molecular mechanisms take place below the resolution limit of light microscopy (200 nm).

CIE mechanisms can be classified by the mechanical actors in vesicle formation, such as GTPases (e.g. dynamin, RhoA and CDC42), BAR domain proteins (e.g. endophilin, pacsin2) and actin, or by their cargoes, such as GPI-APs, CD44, major histocompatibility class I (MHCI) molecules and interleukin 2 receptor. 9 Adding to this complexity in characterisation, these categories present significant interchangeability and compensatory roles in both cargo and membrane-bending machinery. Experimentally removing, manipulating or probing one factor can often be compensated by another. 14,15 small-scale circular carriers such as clathrin-or caveolin-mediated pits. 5 1.2 Glycolipid-and Lectin-dependent endocytosis Lectins are proteins that bind to glycan (sugar, carbohydrate, saccharide) moieties of glycoproteins or glycolipids (GL). The GL-Lect hypothesis proposes that certain mammalian or pathological Lectins, once bound to the PM through GLs and/or glycoproteins, utilise GlycoLipids to induce nanodomain formation in the lipid bilayer, membrane curvature, and thereby, the biogenesis of endocytic pits. 16 Clathrin-independent endocytosis (CIE) of the bacterial galactose-binding lectin, ricin, was the first CIE mechanism described. 17,18 Bacterial ricin, Shiga and cholera toxins bind via their respective B-subunits (RTB, STxB, CTxB) to their mammalian glycosyl receptors:

galactosyl lipids and proteins, Gb3-Cer, or respectively (Figure 1.2.1). Once bound to the host cell and endocytosed to early sorting endosomes, the toxins bypass degradation in late endosomes and lysosomes. They are instead transported via the retrograde route (Figure 1.2.2), through the trans-Golgi network (TGN) and the Golgi stack to the endoplasmic reticulum (ER). 19 In the ER, the disulphide bond linking Figure 1.2.1) is cleaved to induce toxicity. 12,20 In the case of ricin and Shiga toxin, the A-subunit, a catalytic RNA N-glycosidase, translocates to the cytosol to induce toxicity by protein synthesis inhibition. The homopentameric B-subunits of AB5 Shiga and cholera toxin, STxB, CTxB, as well as the pentameric VP-1 capsid protein of Simian polyoma virus SV40, have flat, doughnut-like structures (Figure 1.2.3a), where they dock onto the membrane via their glycosphingolipid (GSL) receptors. The lectins' polyvalent binding is only of small individual affinity (dissociation constant Kd ~ 10 -3 M for STxB) for each GSL, but combine to a large avidity (Kd ~ 10 -9 M). 21 STxB contains three globotriose (Gb3) binding pockets per monomer, thus 15 total Gb3 binding sites. CTxB and SV40 protein have one GM1 binding pocket per monomer, totalling five GM1 binding sites. 22 Of note, these three lectins have no sequence similarity, and yet the glycan receptors are all positioned around the edge of the protein in a seemingly overlapping geometry (Figure 1.2.3a), indicating a convergent evolution of structure. The glycan binding sites are of a geometry in which the membrane must bend up around the lectin to fit the glycan part of the GSL receptor molecules into their binding sites, thereby generating negative, inward-oriented curvature. This was recently demonstrated in silico for STxB-Gb3 13-15 in all-atom molecular dynamics studies (Figure 1.2.3b,d). 23 Effects on the membrane underneath the docked STxB, such as suppression of lipid fluctuations, 24 lead to membrane-mediated clustering to induce wider membrane invagination (Figure 1.2.3c). These early endocytic pits are then recognised by cytosolic machinery, dynamin, endophilin and/or actin, 15 for pit elongation and scission, before transport to endosomes. This membrane bending phenomenon is an intrinsic property of GL-Lect complexes and can be reproduced in simple model membrane systems, liposomes. One classical experimental technique applies hydrating with an aqueous solution, a lipid mixture of phospholipid with a small molar percentage of GSL, to obtain spherical giant unilamellar vesicles (GUVs) of homogenous size similar to cells (~10 μM). 25,26 Membrane bending properties of GL-Lect drivers have been widely investigated using GUVs: Gb3 with STxB (Figure 1.2.4a) 27 or bacterial lectin LecA; 28 GM1 with SV40 29 or cholera toxin; 30 and a GSL mixture with a mammalian lectin, galectin-3 (Gal3) (Figure 1.2.4b). 31 Gal3, which undergoes GSL-dependent endocytosis after binding to glycoproteins in cells, spontaneously forms dynamic membrane invaginations when bound to GUVs containing 5% GSL.

GSL function studies

Unpicking the molecular mechanism of membrane curvature formation by the STxB-Gb3 complex can give novel insight on GL-Lect endocytosis. To investigate the relative contribution of driving forces in the clustering of STxB toxins, the system was recently studied in silico and experimentally. 24 There are no attractive interactions between STxB proteins, so how do they cluster? A commonly cited membrane-mediated force, line tension (Gibbs free energy required to maintain domain interfaces) might contribute to clustering due to height or compositional mismatch within the lipid membrane. 32,33 Gb3-Cer and other GSLs often have longer lipid chains than bulk phospholipids, meaning the domain containing STxB-Gb3x≤15 (x indicates numbers of bound Gb3 molecules) should be held higher on the membrane. 34,35 When tested experimentally by varying lipid tail lengths of Gb3-Cer in GUVs, no significant difference in clustering kinetics was observed, 24 demonstrating that height mismatch line tension is not strictly required for clustering. One could also implicate line tension caused by compositional mismatch of the lipids, due to thermodynamically favourable lipid-demixing effects. Pezeshkian et al. suggested that Gb3-Cer domains are not required for clustering to occur, since GUVs formed from 5 or 30 mol% Gb3-Cer showed no b significant difference in binding or invagination; 30 mol% being the Gb3 concentration that one expects to find under STxB molecules. 24 Solevyeva et al. showed that STxB binding induced membrane lipid domain organisation in GUVs, 36 but this reasons for the reverse causality;

that STxB clustering induces compositional mismatch.

To elucidate the mechanism, synthetic were synthesised in the L. Johannes laboratory and tested in GUV experiments. The longer ethylene glycol (EG) spacers between the Gb3 head and the ceramide tail increasingly inhibited STxB clustering and invaginations in GUVs (n=3 or 7, Figure 1.3.1a, b). These experiments gave birth to the fluctuation force hypothesis, proposing that a suppression of fluctuation of the membrane is required for toxins to cluster (Figure 1.3.1c). This effect is commonly alluded to being like drifting boats on the ocean, or cheerios on milk, which aggregate together. This hypothesis was studied by course-grained molecular dynamics simulations, showing that two macromolecules, with dimensions corresponding to the membrane-bound STxB-Gb3x≤15

complex, would randomly drift on a phospholipid bilayer. In the simulation, only the macromolecules tightly bound to the bilayer (Figure 1.3.1d) would be attracted to each other from a distance of several nanometres, whereas loosely bound macromolecules (Figure 1.3.1e) would experience no attraction force. 24 Hence, the tight binding of the toxin to the membrane via Gb3 receptors is sufficient to induce clustering. These results will in turn guide this project, in the constraints on structure of Gb3 analogues proposed and synthesised, which should retain natural lectin binding and membrane bending actively. (d, no spacer) or loosely (e, flexible spacer). 24 Despite the fact that low or high Gb3-Cer concentrations used in GUVs have little effect on STxB clustering, one must still consider that glycolipid organisation may have an effect on lectin binding and endocytosis. 33 The lipid raft hypothesis describes the propensity of cholesterol and sphingolipids, mostly sphingomyelin (SM) and GSLs, to phase separate into denser, more ordered regions in a lipid membrane. 37,38 The theory gained interest because of the dependence of certain biological processes on non-specific cholesterol depletion, or the easy isolation of detergent-resistant membrane domains by treating biological samples with cold detergent, justifying the binominal classification of proteins and processes as "raft" or "non-raft." An enrichment of "raftophile" lipids, cholesterol and sphingolipids, in CIE cargos has been demonstrated in several non-direct methods. 39-41 This phenomenon of lipid organisation is observable with chemically-modified lipids, in liposomes, or using non-specific extraction techniques, but the biological relevance is difficult to evaluate for natural, nonfluorescent lipids in cellulo or in vivo.

Lipid organisation can be driven by a multitude of factors such as actin cytoskeleton organisation, 42 ectodomain association, and membrane pits, ruffles and contact sites. 43 Specific lipid binding pockets in proteins and charge interactions also induce lipid organisation. 20-30% of the PM area is covered by proteins, and lipid solvation rings around proteins could account for the majority of the lipid area remaining. 44 Eggeling et al. recently used super resolution fluorescence scanning microscopy (FSC) to show that fluorescent phospholipids or cholesterol presented no significant lateral heterogeneity, whilst fluorescent SM showed transient trapping in ~80 nm domains, but hardly moved once trapped. This observation does not correspond to lipid raft criteria, but infers specific lipid interactions with membrane components. 45,46 Another recent imaging study by Kraft et al. used secondary ion mass spectrometry (SIMS) with isotopically-labelled sphingolipids and cholesterol reconstituted into cells. 47,48 After fixing and imaging cells, they show 15 N-sphingolipids The high spatiotemporal resolution of fluorescence microscopy has promoted the development of fluorescent GSLs, obtained by some complex chemical syntheses. 51-55 Despite progress in smaller fluorophores and faster imaging techniques, the study of these molecular tools in GUVs and cells has highlighted the precariousness in studying lipid properties with modified lipids. Lipids, including fluorophore-tagged lipids, can separate into ordered (Lo, e.g. Specific study of Gb3-Cer and other long-chain GSLs in cell culture is practically unattainable since they cannot be added directly to cells in culture in a controlled manner. 60 Chinnapen et al., when investigating GM1 and cholera toxin traffic in live cells, reincorporated GM1 species bearing C18 chain length or shorter, despite specifying that the natural profile contains mostly C24 and C16 acyl chains. 30 Pedrosa et al. showed that short C8 acyl chain GlcCer could be taken up with a yield of 20% in cancer cells, and 5% in normal cells, but do not discuss longer chains. 61 Fusogenic liposomes have been used to improve uptake, 62,63 employing neutral and positively charged phospholipids to improve fusion of lipid vesicles with the PM.

Our

GSL function and biosynthesis

The biosynthesis of glycosphingolipids (GSLs) is well documented. However, GSL function and their interactions is still mystifying scientists, apt to the etymology of the word "sphingolipids" -named by J. L. W. Thudichum in 1884 after the enigmatic sphinx. 34 Over 400 unique oligosaccharide head groups make up the enigmatic "glycolipidome" in mammals. 67 GSLs constitute 2-10% of total lipids in membranes, 68 where sphingomyelin (SM) is estimated at 15-20% of the plasma membranes (PM). 69 GSLs are found at higher concentrations at the apical side of epithelial cells, and in certain cell types including oligodendrocytes and neurons.

Their fingerprint expression levels vary throughout development and pathogenesis, amongst cell and tissue type.

Functions of groups of GSLs have been studied by genetic suppressions. Mice deficient in all GSLs, obtained with a glucosylceramide synthase (GCS) gene knockout, do not survive beyond gastrulation, 70 similar to lactosylceramide synthase knockout mice. 71 Genetic suppression of the gala-and ganglio-series GSLs in mice shows phenotypes in nervous system or neuronal functions respectively (Figure 1.4.1). 72,73 GSLs have classically been used as differentiation markers, especially the stage-specific embryonic antigens (SSEA1-4). 74,75 GSLs are implicated in cell migration, adhesion and signal transduction, 76 as well as clathrinindependent endocytosis. 13,31 Many GSL-protein interactions are implicated in signal transduction, 77 such as the activation of epidermal growth factor receptor (EGFR). 78 GLSs undergo individual and polyvalent interactions with proteins, as proposed in the lipid raft hypothesis, where they play a direct role in membrane organisation and activation of raftlocalised proteins. 79 Such polyvalent interactions means that GSLs have low individual affinity for protein interaction partners, making GSL-protein interactions difficult to identify by standard immunopurification techniques. High resolution mass spectrometry (HRMS)

techniques are improving rapidly in GSL analysis, particularly shot-gun lipidomics. 80,81 In spite of this, a topological and temporal interaction map of GSL function and interactions is still far from reach. 1.4.3b). 83 Dihydroceramides ( C16-24) are dehydrogenated by dihydroceramide desaturase (DES) to ceramide (C16 -24). In the ER, ceramide can be galactosylated to galactosylceramide by galactosylceramide synthase (GalCS), going on to form sulfatide GSLs in the Golgi. 69 This represents a small but pathologically important family of GSLs. 67,84 Ceramide is transported from the ER to the Golgi to form the two major families of sphingolipids, SM and GSLs. Sphingomyelin synthase (SMS) transfers the phosphocholine head group from the cell's most abundant glycerophospholipid, phosphatidylcholine (PC), 69 to ceramide, forming SM and diacylglycerol (DAG), in the trans-Golgi and PM. 85,86 Glucosylceramide (GlcCer), the precursor of most GSLs, is synthesised in the cytosolic leaflet of the Golgi by glucosylceramide synthase (GCS), a.k.a. UDP-glucose ceramide glucosyltransferase (UGCG). The stable UGCG gene knockout cell line, GM95, is a useful tool in the study of GSL function. 87 In lesser part, ceramide is phosphorylated to ceramide-1phosphate (C1P) by ceramide kinase (CK). 88,89 The only irreversible degradation route leaving the sphingolipid network is the catabolic pathway. Ceramide is reversibly hydrolysed to sphingosine by ceramidases (CDase), which are organelle specific, pathway specific and substrate specific. 85 Sphingosine is phosphorylated to sphingosine-1-phosphate (S1P) by sphingosine kinase (SK), while the reverse reaction is catalysed by S1P phosphatase (S1PP). S1P can then be irreversibly degraded to ethanolamine-1-phosphate and C16 fatty acid by S1P lyase in the ER.

Ceramide is (re)formed in the salvage pathway, by reacylation of sphingosine, predominantly by the CerS5 enzyme. 83,90 This pool cycles back to form 50-90% of total sphingolipid content. 91 Ceramide is also reformed by the degradation of GSLs by glycosylceramidase (GCS) or galactosylceramidase (Gal-CDase) in lysosomes, the degradation of SM by sphingomyelinase (SMase), and of C1P by ceramide-1-phosphate phosphatase (C1PPase). GSLs are localised between the Golgi, where they are synthesised, and the PM, where they are recognised by antibodies and lectins. 92 Localising GSLs in cellular compartments is challenging since functionalisation with a fluorophore can change considerably their localisation and traffic. Antibodies can be used to recognise GSLs as antigens. However, antibodies cannot cross the PM without disrupting the localisation of the GSL in question. 93,94 Reports differ on GSLs' actual localisation. 34,89,95 1.5 Biomedical relevance

In this project, we develop tools for the investigation of GSLs in GL-Lect endocytosis.

GSLs are of medical interest for the investigation of GSL lysosomal storage diseases, caused by mutations in sphingolipid degradation genes. Major examples include Gaucher disease, (GlcCer accumulation), Fabry (Gb3-Cer), Tay-Sachs and Sandhoff (GM2) diseases. 86,96 These can be treated with substrate reduction therapy, such as the glucosyl-ceramide synthase inhibitor N-butyldeoxyno-jirimycin, 84 and enzyme replacement therapy at high cost.

Moreover, better comprehension of the molecular mechanism of toxins, which hijack mammalian GSLs for cellular uptake via GL-Lect endocytosis, will aid the development of treatment. Shiga toxin, the major virulence factor of Shigella dysenteriae, and Shiga-like toxins, also known as verotoxins, secreted by enterohemorrhagic strains of Escherichia coli bacteria, have a highly conserved structure. 97 All bind specifically to Gb3-Cer, which is expressed on kidneys and brain tissues, 98 and some strains bind to Gb4-Cer with lower affinity. Infection induces diarrhoea, and eventually haemolytic uremic syndrome (HUS) and acute renal failure, with high infant fatality rate across the world, and complex cases can have neurological symptons. 20 Cholera toxin, secreted by Vibrio cholerae, causes cholera, a lethal disease with high fatalities in the developing world, and outbreaks of disease in conflict zones is regularly reported in standard news media around the world. 99 Currently no curative treatment is known for disease caused by the Shiga and cholera toxin-producing bacteria. 20 Gb3 is expressed in several cell types of interest to cancer research: cancer cells and cancer stem cells themselves, 100,101 and antigen-presenting dendritic cells, which stimulate the immune system. The interest in GSLs spans the development of cancer chemotherapeutics, in which it is paramount that effective drug targets are cell-specific to reduce non-specific toxicity. GSLs as cell biomarkers is under-explored territory in the state of chemotherapeutic research, and yet their expression levels are known to change drastically with pathogenesis. 101,102 Furthermore, study of the GL-Lect mechanism will inform the development of lectin-or STxB-conjugated drug-vectors; the rational for which aims at following the non-degradative retrograde pathway, from which STxB benefits, to target and invade host cells (Figure 1.5.1). 98 This route is complementary to the standard pathway followed by large antibody-drug conjugates (ADCs), 103 which are normally taken up by clathrin-mediated endocytosis and sorted to lysosomes to be degraded. STxB-drug conjugates have shown promise in targeting Gb3-overexpressing cancer cells. 104-110 Endogenous lectins and their GL-Lect interactions with GSLs are coming to light. 2,16 For example, galectin-3, which is deregulated in human cancers, is endocytosed with CD44 and β1-integrin in a clathrin-independent mechanism dependent on GSLs and N-glycosylation. 31 Gb3-Cer is the cell receptor for the Shiga toxin, but no endogenous functions are currently known. Although GSLs are not classically "druggable" compounds due to their undesirable pharmacokinetic properties, research into their function and therapeutic role requires methods to manipulate their expression in cells. Hence a robust and versatile reconstitution method and related syntheses of these products are of great interest. project. This rapidly developing technology permits identification and quantification of the lipidome using high resolution mass spectrometry (HRMS) to access exact masses at a partsper-million (ppm, 10 -6 ) mass unit accuracy, coupled with high-throughput data handling.

Highly adapted lipid extraction protocols are employed with direct infusion, without chromatographic separation. Direct infusion allows exact quantification since a constant flow of lipid sample of known concentration is analysed, with the use of appropriate internal standards. 111,112 To investiage biomolecule localisation and function, small chemical reporter groups, such as fluorophores for fluorescence imaging, or affinity tags like biotin for purification, can be incorporated in vitro, before their incorporation and study in cells or organisms. For in vitro tagging, selective or orthogonal chemical conjugations are required for specific tagging in a non-perturbing site on the biomolecule. For small biomolecules such as lipids and carbohydrates, where even the smallest fluorophores can perturb lipid function, 57 the reporter group can be added in cellulo by means of a minute, latently reactive tag or labelling group. After cellular incorporation and biological function, the tag can be specifically conjugated to a reporter group (e.g. fluorophore or biotin) for visualisation or isolation (Figure 1.6.1). 113 Such chemical conjugation (or scission) reactions must occur specifically in a complex biological environment, and are termed bioorthogonal. The bioorthogonal reactant partners must be specifically, mutually reactive whilst being inert to their environment, and as small as possible. Since applications are often in live cell culture, the reaction must occur at a similar timescale to standard cellular processes. 114 Early specific, orthogonal chemical reactions were used to chemically modify proteins in vitro. Reactions include disulfide exchange or maleimides for cysteine residues, and Nhydroxysuccinimides and iso(thio)cyanates for lysine residues (Figure 1.6.2a). 115 However, for complete regiospecificity, these reactions require that only one active thiol or amine is present in the conjugation environment. Ketones and aldehydes can be condensed with aminooxy and hydrazide forming oxime or hydrazine conjugations respectively (Figure 1.6.2b). These were among the first specific chemical conjugations applied to the surface of whole, live cells. Bertozzi et al. described the metabolic labelling of cell surface glycans using a ketone-labelled analogue of N-acetylmannosamine (ManNAc), Figure 1.6.3), which is metabolised into sialic acid-containing glycolipids and glycoproteins and presented at the cell surface, where they selectively conjugated with a hydrazide-biotin reporter. 116-118 These aminooxy and hydrazide conjugations require high concentrations of reactants, and fall prey to high cross-reactivity with natural ketones and aldehydes. The Staudinger ligation uses azide-and arylphosphine-functionalised partners and has been applied to bioorthogonal live cell labelling 119-121 and prodrug release, 122 since bonds are formed and broken in the reaction mechanism (Figure 1.6.3a). Azides are ideally small, nonperturbing and stable in cells and organisms. 123 For example, an azide group is present in the well-known anti-AIDS drug, 3 '-azidothymidine (AZT). 124 The bioorthogonal Staudinger ligation derives from the classic reduction of azides with phosphines, 125 using a functionalised aromatic phosphine which traps the aza-ylide intermediate, forming a stable conjugation. The traceless Staudinger reaction (Figure 1.6.3b) leaves a perfectly small amide bond after the conjugation and intramolecular elimination of the arylphosphine. These variants are less applied in live cell studies due to poor kinetics, selectivity and solubility. 126 Saxon and Bertozzi showed live cell metabolic labelling with an azide-labelled analogue of ManNAc, Ac4ManNAz (Figure 1.6.3c). 119,127 The hydrophobic, peracetylated glycan is used to increase cell permeability, and is deacetylated by non-specific cytosolic esterases to ManNAz in the cell. 128 ManNAz then follows the endogenous anabolic sialic acid pathway to SiaNAz, and is incorporated into sialic acid-containing proteins and glycolipids. 129

The azide-functionalised sialosides were then conjugated specifically with an arylphosphinebiotin for quantification. Unfortunately, Staudinger ligations suffer from impractically slow kinetics (k2≈3×10 -3 M -1 s -1 ), require high concentrations (>250 µM triarylphosphine), 127 and phosphines are prone to enzymatic or atmospheric oxidation. The term click chemistry was coined to describe reactions which are fast, specific, high yielding, occur in mild (aqueous, aerobic, biological) conditions, and produce inert or no byproducts. 131 Reactions coming close to meeting these criteria are now so popular they are used by chemists and non-chemists, in common, commercial laboratory kits. The heat-driven 1,3-cycloaddition between alkyne and azide was described by Huisgen in the the 1960s. 132 The copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) variation (Figure 1.6.4a), first described in 2002, 133,134 is still an extremely useful orthogonal conjugation in organic synthesis, 132,135 allowing complex molecules to be coupled specifically, in the presence of diverse functional groups, without need for protecting groups. CuAAC reactions came to dominate click chemistry for its extremely fast reaction times and specificity (10-200 M -1 s -1 in the presence of mM Cu(I)). Sadly, the copper, solubilised in a ligand complex, is abundantly taken up into cells and causes rapid toxicity by the formation of reactive oxygen species (ROS). 136 This is augmented by ascorbate, the standard reductant used to form Cu(I) from Cu(II), which also promotes ROS formation. 136,137 Copper is also reported to chelate to amino acids, impeding protein structure and function.

To combat this, ligands have been developed promising to reduce toxicity whilst retaining copper catalysis and solubility (Figure 1.6.2b). 138 These have been successfully

applied in cell surface labelling. Cyclooctynes can react with azides without copper catalysis (Figure 1.6.5a). They have higher reactivity due to their strained cyclic structure, in a reaction termed the strainpromoted azide-alkyne cycloaddition (SPAAC). 113 In 1961, Witting and Krebs reported that a stable cyclooctyne reacted "like an explosion" with phenylazide, but it was not until 2004 that Agard, Prescher and Bertozzi synthesised OCT (Figure 1.6.5b) and showed its specific reaction on cells labelled with SiaNAz, without copper catalysis. 142 The standard 1,3-dipolar SPAAC occurs between the LUMOπ , alkyne-HOMOπ,azide (Figure 1.6.5c). 143 The electronic properties of the alkyne have been fine-tuned by adding electronwithdrawing groups (EWGs, Figure 1.6.5c) to lower LUMO energy of the electron-rich alkyne. When choosing a click reagent, one must consider that increasing reactivity is often detrimental to specificity and stability. Lipids with terminal ω-alkyne or ω-azide groups (Figure 1.7.3a,b) have been widely used to study fatty acylated-protein localisation. 157 3). 60-62 The study of GSL function is thus challenging and new molecular tools are highly desirable. We aim to synthesise biomolecule analogues able to reconstitute the biological and mechanochemical properties of GSLs. Tools must be reconstituted in an easy protocol, whilst retaining biological function.

We aim to develop a biorthogonal, clickable kit of GSL precursors from proof-ofconcept studies on GSL globotriaosylceramide (Gb3-Cer) and the Shiga toxin (STxB). To reconstitute a functional Gb3-Cer analogue, we attempt to commandeer the native GSL biosynthesis and traffic; supplementing cells with a clickable Figure 1.8.1), which is natively metabolised to and presented at the plasma membrane (PM), to which we click the functionalised oligosaccharide head group of choice by a highly selective, copper-free bioorthogonal click reaction. The maintaining biological activity, by using the smallest least perturbing chemical groups possible is constantly balanced with having a fast and specific click reaction (Figure 1.8.2a). We base our design on previous studies showing that clustering of STxB was greatly inhibited with long, flexible linkers, but a small linker in the central region (n=1, 10Å) permitted native function (Figure 1.8.2b,Introduction 1.3). 173 We design our final click analogue with dimensions of this size or smaller. Previously a GM1-phospholipid analogue retained its native function in binding and trafficking with the cholera toxin and simian virus 40. 29 We consider the use of a Gb3-phospholipid, proposed to be soluble enough to permit efficient cellular reconstitution (Discussion 3.1).

If these neo-glycolipids are functional and reconstituted with a feasible yield, this strategy could potentially tolerate further chemical modifications, allowing further probing of native GSL interactions. One long-term goal is to reconstitute photoactivatable, traceable GSLs, for example containing a diazirine group (Figure 1.8.2c), to identify GSL interaction partners (cf. Perspectives). Gb3-EG1-Cer is able to induce membrane bending. 24 (c) Potential photoactivatable and reconstituable Gb3.

Specificity

Functionality Reactivity

RESULTS

Synthesis of biomolecule analogues 2.1.1 Reconstitutable globotriose phospholipid

We aim to synthesise a Gb3 with retained native function that can be reintroduced into cells in culture. Gb3-phospholipid analogues, or function-spacer-lipid (FSL) constructs, have shown to retain similar biological activity as the natural Gb3-Cer with known binding lectins: Shiga toxin (verotoxin1) and HIV-1. 29,174,175 Crucially, phospholipids (PLs) are more efficiently incorporated into cells in culture than GSLs (Introduction 1.3). 60,62 Hence, we proposed that a Gb3-PL would be a valuable tool. The spacer between glycan functional head and lipid tail of the FSL must be biostable, meaning it must not be recognised and digested by cellular clearance machinery. We require the spacer between the Gb3 head group and phospholipid tail to be short to retain membrane bending properties (Introduction 1.3). 24 This contributes to the originality of this endeavour, since only long Gb3-phospholipids have been synthesised in the literature. 29,174,175 Synthetic routes to install isothiocyanate and azide functions at the anomeric position of glycans are well documented. 176 Isothiocyanates conjugate specifically with amines to form a thiourea (Scheme 2.1.1.1). The classic bioorthogonal copper-catalysed azide-alkyne cycloaddition (CuAAC, Introduction 1.6) forms a triazole. Both thiourea and triazole are stable in cells.

Our project is applied to the Gb3 triose, synthesised in a four-step synthesis (Section 2.1.3). We began testing syntheses on a lactose model for matters of cost and scale. Lactose (Scheme 2.1.1.2) was peracetylated with acetic anhydride in pyridine, to which the isothiourea (SCN) group was installed with the use of trimethylsilyl thiocyanic acid (TMSSCN)

and SnCl4 as a Lewis acid catalyst to form peracetate lactosyl isothiocyanate 1. 24,177 This was conjugated to 1, [START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF] under basic conditions to form thiourea conjugate 2. Despite an orthogonal deprotection of four acetyl groups being described, 178 this hydrazine-mediated deprotection was unfeasible in our hands without simultaneous breakdown of phospholipid ester groups, and conjugate 3 was not fully characterised. Alternatively, deprotection of isothiocyanate 1 was tested prior to conjugation with DOPE, but isothiocyanate was unstable to methoxide conditions. It proved equally impossible to install the isothiocyanate group without hydroxyl protecting groups. Installation of an azide with sodium azide in DMF via the cyclic intermediate 11 to form azidophopholipid 13, confirmed by MS (Scheme 2.1.1.5). However, constant atmospheric hydrogenation of the unsaturated lipid tails became problematic during the lengthy purification from the ever-present hydrolysis product 12, and full characterisation was not completed. This reaction was initially tested on 1-decaoctanol to obtain alkyne-phosphate 14.

We trialled a peptidic conjugation of an alkyne-carboxlic acid, as described, 180 to form alkyne-phospholipid 16 from DOPE 15. However, the small scale and poor acid reactivity of propiolic acid impeded isolation. In the face of the difficulties of purification of such unstable and amphiphilic phospholipid products, and the small scale nature of synthesis, the strategy was discontinued and we transferred our attention to a sphingolipid-based strategy.

Clickable sphingolipid and globotriose

According to the scheme proposed ( It is worth noting that attempts to form the azide directly from the mesylate 26 resulted in an oxazaline side product 28b (Scheme 2.1.2.2). This oxazoline side product dominated when using a N 2 -tert-butyloxycarbonyl (Boc) protecting group during azide formation. This obliged us to use the electron-withdrawing dichloroacetamide protecting group. 185 From a previous synthesis in the laboratory, we characterised another cyclisation side product, a cyclic carbamate (oxazoline) 29, formed from the iodide 27 with NaN3 and ensuing deprotection with KOH (Scheme 2.1.2.2). This cyclic carbamate was unhelpfully similar to AzSph in Rf, molar mass, 1 H and 13 C NMR, and is likely formed from the hydrolysis of the dichlorooxazoline side product 28b. We confirmed its structure by a weak staining in ninhydrin on TLC, a lack of azide bands in IR, and a strong C 19 -H 1a,b coupling in NOESY NMR. Other azide-functionalised sphingolipids and their cellular metabolism are also of interest. We expected their metabolism profile to be slightly different to that of AzSph due to the high specificity of ceramide synthase (CerS) enzymes (Discussion 3.5). From the biosynthesis described in the Introduction 1.4, sphinganine (Scheme 2.1.2.5) is metabolised to dihydroceramide by all ceramide synthase (CerS) enzymes [1][START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF][3][4][START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF][6], which install C16-24 acyl chains, whereas sphingosine is converted to ceramide by CerS5, which has a specificity for C16 chains. We wished to investigate the metabolism of another azidolipid closer in structure to sphinganine. The phytosphingosine analogue has no double bond, like in sphinganine. Phytosphingosine was converted into [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrinindependent carriers[END_REF] in a threestep synthesis with a 13% overall yield (Scheme 2.1.2.6). The amine is initially protected with the dichloroacetamide protecting group to form 32. Then, unlike the previous synthesis, the azide group was installed with a selective, Mitsunobu-like azidolysis, using NaN3 and PPh3/CBr4, forming 33, along with the cyclisation side product 34. 188,189 N 2 -dichloroacetamide group is then removed under standard basic conditions to yield [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrinindependent carriers[END_REF]. The cyclisation side product 34 presumably forms after the activation of the primary alcohol with PPh3 + /Br -, via an attack of the alcohol in the C-3 position (Scheme 2.1.2.7). These reaction conditions gave varying results of 1:1 (product/side) at a 50 mg scale, but 1:4 at a 200 mg scale. In contrast to AzSph, the synthesis of 1-azidophytosphingosine (AzPhy) was much shorter and thus lower-yielding steps were possible. To confirm the structure of side product 34, the N 2 -dichloroacetamide group was removed in standard deprotection conditions after separation and the resulting spectra corresponded to the described 2-epi-Jaspine B. 190 Scheme 2.1.2.6: Azidophytosphingosine synthesis with major cyclisation side product.

Scheme 2.1.2.7: Proposed selective azidolysis mechanism and cyclisation side reaction.

The N-dichloroacetamide protecting group was installed since cyclisation could hinder the synthesis in an analogous way (Scheme 2. 1.2.8). An N 2 -Boc group was also tested, however no product nor the known cyclised derivatives were observed.

Scheme 2.1.2.8: Selective azidolysis reaction with different N-protecting groups.

Cyclooctyne-functionalised glycans

According to the constraints of our reconstitution strategy (Introduction 1.8), the clickable Gb3 head group must click with AzCer specifically in cell culture to form a Gb3-Cer analogue with a short, straight, biostable spacer. Cyclooctynes react quickly with azides without copper catalysis. The monofluorocyclooctyne (MFCO) was chosen for its small size and high specificity. Its synthesis had already been completed in the laboratory. 148,191,192 In this chapter I will describe the various clickable glycans synthesised. We suspected that the bent angle within the final reconstituted Gb3- , could hinder its function, so we additionally synthesised an analogue with a flexible linker region between the triose and MFCO, which would relieve this angle strain. We tested the syntheses of two flexible linkers, ethanolamine (EA) and glycine (Gly) (Scheme 2. 1.3.7). We worked on the 1.3.3). Although the tert-butyl protecting group should remain stable in basic conditions, approximately half is lost during the base-catalysed coupling reaction. Thus, when the coupling is complete, the remaining O-t Bu-glycine-MFCO is deprotected in situ (one-pot, without purification) with TFA/TIS/CH2Cl2 (5:5:90). By mass spectrometry (MS), we observe trace formation of the doubly conjugated MFCO-Gly-Gly, which is easily separated. Synthesis was complicated by the instability of MFCO, which would break down to various highly polar compounds that were not characterised. In the case of the lactose syntheses, the α/β isomers are not separated, since the applications of these model molecules are to test the cellular kinetics of an MFCO-glycoside, followed by MS, for which the stereochemistry has no affect. Gb3 analogues must be of βstereochemistry for their biological interaction with STxB. We tested the DCC coupling reaction in CH2Cl2 (condition

Glycan click

To test the click reaction in cellulo, we incubated cells with AzSph in cell medium, in conditions optimised to have stable, non-toxic levels of AzCer (0.5 μM, 6-16h) not occur at all, or the product is formed on a small scale and is not extracted for analysis. We considered that the bioavailability of the reagent, as well as the reactivity of the cyclooctyne, To improve deacetylation we carried out fluid-phase uptake of a commercial esterase, pig liver esterase, which has been shown to increase drug metabolism. 195,196 We incubated cells with AzSph (0.5 μM, 8h), followed by either sequential treatment with Ac-Lac-MFCO (15 μM, 16h), then esterase (50 or 500 mU, 8h), or co-treatment with Ac-Lac-MFCO (15 μM) and esterase (50 or 500 mU) for 24h. No significant difference in the acetylation profile of esterase-treated cells was observed (Graph 2.2.3.10). When peracetylated glycan- cyclooctynes where tested in vitro with commercial pig liver esterase, only minor deacetylation of 1 or 2 groups was observed, even at high enzyme concentrations in PBS buffer. Deacetylation could not be improved by first incubating cells with the peractylated glycan, followed by AzSph. In fact, no click product was formed (data not shown). Sarkar et al. described a bioactive disaccharide must be used at concentrations exponentially higher than the concentration of the same peracetylated disaccharide to show activity. 128 We must avoid using cytotoxic levels of our clickable glycan, since cytotoxicity levels of lactose-MFCO (tested by MTT assay as before (section 2.2.2)) were non-negligible.

EC90 concentration was 10 μM, compared to lactose (EC90=27 μM). Over 50% of cells were viable in conditions tested (EC50>100 μM) over 24h (Graph 2.2.3.11).

Graph 2.2.3.11: Lactose-MFCO cytotoxicity on GM95 cells over 24h.

Lactose-MFCO Lactose

We showed that incubating cells with higher concentrations of lactose To circumvent the necessity of using peracetylated clickable glycans, we would ideally have our azide group present in high amounts at the plasma membrane (PM). The current dogma suggests that the PM houses longer lipids, and transmembrane proteins with longer transmembrane domains, whereas the Golgi and ER membranes are thinner. 197 To increase presence of a clickable azidolipid at the PM, we would install a longer lipid chain.

According to the natural biosynthesis (Introduction 1.5, Figure 1.4.3b), sphinganine

(Scheme 2.2.4.1
) is acylated to dihydroceramide by all ceramide synthase (CerS) enzymes [1][START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF][3][4][START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF][6] with various acyl chains C16-24. Whereas, sphingosine is acylated to ceramide by CerS5, which has a specificity for C16 chains. Like plant-derived phytosphingosine is accepted in mammalian cells for cosmetic and biomedical studies, [START_REF] Lakshminarayan | Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrinindependent carriers[END_REF] showed similar cytotoxicity to that of ; EC50 = 2 μM and 3 μM respectively, in GM95 cells.

DISCUSSION

Phospholipid strategy

The lack of methods for efficient reconstitution of natural, long-chain GSLs has thus far hindered their study. Phospholipids are more easily taken up into cell culture, to the extent that they are used to make liposomes for transfection and drug delivery agents. 63,198 Synthetic glyco-phospholipids, with enhanced cellular uptake, could constitute a valuable tool in studying the "glycocode" represented by GSL head groups. 199,200 Previously, phospholipid analogues of GSLs, or function-spacer-lipid (FSL) constructs, have been used to study GSL binding and antigen properties. 175 The Gb3-phospholipid analogue (Scheme 3.1.1), showed verotoxin (STxB) binding on TLC and could protect cells in culture against HIV-1 virus infection, similar to natural Gb3-Cer. 174 Ewers et al. showed that a GM1 analogue with a phosphatidylethanolamine (PE) lipid tail could be recognised by Simian virus 40 (SV40) which induced membrane curvature on giant unilamellar vesicles (GUVs), and allowed for SV40 trafficking into cells. 29 We set out to form a FSL construct of Gb3 with a short spacer region between the phospholipid tail and triose head group. The length of the spacer is imperative 3). 24 The lipid raft theory proposes that sphingolipids and cholesterol separate into ordered domains in a lipid membrane, 38,201 whereas common phospholipids such as 1,2-dioleoyl-sn- GM1-phospholipid analogues demonstrates otherwise. 29 Furthermore, Shiga toxin binding on Gb3-Cer-presenting liposomes can induce lipid organisation in basic membrane models, 36,202 indicating that initial Gb3-Cer domain organisation is not necessary for toxin binding and clustering. STxB-induced membrane invagination is not affected when liposome models are constituted of 5 or 30mol% Gb3-Cer, indicating that dense clusters of GSL are not necessary for membrane bending (Introduction 1.2). 24 We can thus propose that preliminary sphingolipid organisation is not necessary for STxB binding, clustering and membrane bending. In order to avoid using protecting groups, we sought to use specific conjugation conditions between small chemical moieties that are easily installed onto the two starting materials: triose and phospholipid. Because of the instability of ester and phosphoester bonds in phospholipids, this specific conjugation must take place in mild conditions. Click chemistry reactions became popular for their fast kinetics, high yields and mild conditions. The coppercatalysed alkyne-azide cycloaddition (CuAAC) is perhaps the most well-known and presents the smallest spacer group, a triazole (Scheme 2.1.1.1). The triazole is also considered to be highly biostable. 204 The attempted syntheses of alkyne-or azide-functionalised clickable phospholipids are described (Results 2.1.1). However, a clickable phospholipid was not isolated to click with the Gb3 head group due to the small scale, lack of sufficiently anhydrous conditions, and the instability of the cyclic phosphate intermediate 11 central to the synthesis. Reactions were successful on 1-octanol and 1-decaoctanol models, forming phosphates 9 and 14 (Scheme 3.1.4). If the synthesis were to be revisited, one must take all precautions possible to obtain the driest possible conditions and timely purify products. Kolb, Finn and Sharpless coined the term click chemistry in a review published in 2001, 131 in which they describe the need for protecting groups as "the most insidious problem" of synthesis in its growing complexity, and hail chemistry's liberation from organic solvents. Using protecting groups innately requires two extra steps: protection and deprotection, and an overall poorer atom economy. 205 As hydrophobic groups, they simultaneously necessitate the use of unecological organic solvents over aqueous solvents.

The draw of protecting group-free chemistry is manifold, reducing costs in time and resources. They were probably not considering phospholipid synthesis when they imagined a straightforward water-soluble synthesis. However, one can still try to apply these principles to lipids.

Clickable sphingolipid synthetic strategy

We diverted our efforts to another click-based strategy: to reconstitute Gb3 by metabolically labelling sphingolipids with an azide moiety, onto which we could click the functionalised oligosaccharide head group of choice. The choice of chemical click groups, azide and cyclooctyne, was primarily based on their small size and biocompatibility, since the length and flexibility of the final Gb3 analogue was of highest importance. Simultaneously, the click partners must be selective enough to react on the complex cellular environment, on live cells.

We sought the smallest possible spacer region between lipid and glycan, which conjugates specifically and quickly, ideally within 24h. 192,206,207 The toolbox of azide click partners includes the Staudinger phosphine reagents, alkynes for copper-catalysed cycloaddition, and cyclooctynes for strain-promoted copper-free alkyne-azide click. A cyclooctyne appeared to be most adapted to our system due to the range of size and reactivity available, relatively simple formation, and their stability in storage and in cellulo compared with phosphines, which are highly sensitive to oxidation. Amongst the variety of cyclooctynes, monofluorocyclooctyne (MFCO) was chosen for its ideal balance between small size and fast reactivity with azides (Introduction 1.6). 114,145 The reactivity is not as high as some popular dibenzocyclooctyne derivatives (DIBO, DIBAC, etc, Figure 3.2.2) but the small size of MFCO is highly desirable in our project. It is easily accessible via a four step synthesis (Scheme 3.2.3), 148 and had been previously used in the laboratory. 191 The major drawback we found was that the free carboxylic acid MFCO 22 was unstable and could not be stored for more than 6 months. Azides are ideally small and stable in cells (Introduction 1.6). 147 An azide was installed at the head position (C-1) in the place of the natural hydroxyl group of sphingosine, since this would form the most linear GSL analogue. The azide group is less hydrophilic than the hydroxyl group, and may or may not be held within the hydrophobic layer of the lipid bilayer when in a biological environment. Despite this, we were confident it would retain its reactivity with a hydrophobic cyclcooctyne partner, as many literature reports suggest that click reaction in cellulo and in liposomes can occur with lipids functionalised with a hydrophobic moiety held within the membrane. Even before the rise in click chemistry, the azide was a common functional group on the synthetic route to amines, to which azides are easily reduced. 210 The azide group is so stable and convenient it is used as an amine protecting group, 181 the latter being sensitive to oxidation and carboxylation in long term storage. Phytosphingosine (Scheme 2.1.2.4) is an ideal inexpensive starting material, with stereochemistry in the 2,3-positions identical to that of sphingosine, and multiple syntheses to sphingosine have been reported. 182,211,212 Synthesis of sphingosine was mastered in the laboratory, to obtain natural Gb3-Cer for various structure-function studies. 24,27,35,54 We follow the synthesis described by Kim et al. We chose the N-dichloroacetamide protecting group following the investigations by Haifei Gao, a previous PhD student, who tested first the N-Boc group and characterised the oxazoline cyclisation product (Results 2.1.2, Scheme 2.1.2.2), and showed its formation was less important when an electron-withdrawing protecting group was used. This cyclisation continued to hamper the synthesis during the installation of a leaving group at the C-1 position even with the dichloroacetamide group. We observed that a stepwise transformation from mesylate (Ms), to iodide, to azide gave the highest yield with less cyclisation product.

Converting directly from mesylate to azide, for example, produced the cyclisation product in majority. Although lengthy, one must accept in multistep synthesis that the highest overall yield may be obtained from a strategy with more steps. 212 As is often the case in chemical biology project, we did not further optimise the synthesis, but continued to cellular testing of AzSph after the final deprotection step.

The synthesis of 1-azidosphingosine (AzSph) has been reported in the thesis of M.

Garrido Martínez (Scheme 3.2.4). 184 The synthesis begins with an amino acid starting block and uses a bridging protecting group on the C2 and C3 position. Thus they could not observe the same cyclisation as ours. However, they were confronted with the formation of a pyrroline side product and an extremely low-yielding deprotection step. TLC indicated approximately a 50:50 ratio. We did not attempt to purify the R/S isomers since we considered that the right-left tilt in the plane of the length of the Gb3 analogue would not have much effect on Shiga toxin binding and membrane bending.

Model membrane testing

Giant unilamellar vesicles (GUVs) provide a single bilayer of homogeneous size similar to that of a cell (typically diameters of 10-30 µm). GUV testing is the classical method for investigating membrane bending properties, as they can be observed under the microscope.

We followed the protocol of the pioneering report of membrane tubulation induced by Shiga toxin on GUVs containing Gb3-Cer. 27 Although membrane invaginations can differ with the tension and osmolarity of GUVs, the results are a robust indication of basic mechanochemical properties of the glycolipid-lectin interaction.

The two clickable glycan head groups, Gb3-MFCO 37 and Gb3-Gly-MFCO 38, were tested in order to compare the importance of the strained angle at which the AzCer is held once clicked to the Gb3-MFCO 37. The flexible glycine linker would alleviate the angle constraint. We remained within the constraints set out in the structure-activity study of Gb3-EGn-Cer analogues (Introduction 1.3), which concluded that 10Å constituted an important guideline for the maximum length spacer region, as in Gb3-EG1-Cer. Gb3-Cer analogues 35

and 36 were within this constraint, and both GSL analogues retained membrane bending properties in complexation with STxB in GUVs.

Scheme 3.3.1: Final Gb3 analogues spacer regions compared with confirmed functional Gb3 analogue. 24 GUVs are extremely simplified systems compared to the cell membrane, which contains much more heterogeneity in lipid and protein composition. We considered this indication of membrane curvature on GUVs sufficient to test the molecules in cells, despite the fact that the cellular system is so chemically different.

We tested the click reaction on LUVs constituting 5% AzCer, since LUVs are simpler to prepare and have similar chemical reactivity to GUVs, and are often used in the literature. 216,217 GUVs are typically used for microscopy observations, while LUVs can be obtained in high quantities for biochemical studies. We observed very slow click kinetics. This could be explained by the concentrations used, since click cycloadditions are extremely dependent on concentration. Salomé et al. use similar LUV concentration (100 nM of lipid)

and observed 37% conversion after 24h with the solution phase reagent in large excess, whereas we used 1 equivalent. 218 Since this experiment required a large quantity of click reagents, we did not optimise the experiment, but preferred to use the material for cellular studies.

Sphingolipid metabolism

We began cellular testing on GM95 cells, a stable mutant cell line derived from mouse melanoma MEB-4 cells, which are deficient in GSLs due to a lack of glucosylceramide synthase (GCS) activity. Since we can envisage our reconstitution technique being applied to any glycoside head group, for the functional reconstitution of any GSL analogue, not only Gb3, we chose a system in which we could validate efficient reconstitution of any GSL. This cell line has been used in many previous studies on GSL function. 11,31,82 Chinese hamster ovary (CHO) cells were used to validate our results further in a second cell line, since they express some GSLs but lack the Gb3 synthase enzyme.

We followed the metabolism of Most reports describing the cellular incorporation of modified sphingolipids use a low concentration 165,208,220 compared with concentrations of modified fatty acids or phospholipids described, and for short incubation times. 140,155,159,161,221 Sphingolipids, particularly ceramide, are well-known modulators in apoptosis. Under stress, ceramide is produced from SM and GSLs, inducing apoptosis signalling. 88,222-224 Ceramide analogues which inhibit CerS, have been shown to inhibit proliferation or induce apoptosis. 225-227 Standard lipid membranes have a low concentration of ceramide (Cer <1%) compared with sphingomyelin (SM, 5-8%) and GSLs (2-8%), and even lower sphingosine (Figure 3.4.3). 80 Our sphingolipid analogue may affect such signalling pathways. We tested the cytotoxicity of AzSph using the MTT assay, measuring EC50 and EC90 values (concentrations at which 50% and 90% of cells are viable) for two cell lines (Table 3.4.1).

We observe a higher cytotoxicity of AzSph (and sphingosine) in GM95 than CHO cells.

GM95 cells are deficient in the GlcCer synthase (GCS) gene (UGCG) which catalyses the first step on the pathway to all major GSLs (Scheme 3.4.5), and thus GM95 cells have a 1.5 times higher SM content compared to the parent cell line. 87 Thus, one of the two pathways to reduce sphingolipid stress signalling, via conversion of ceramide to GSLs/SM, is absent in GM95 cells. This ill-balanced sphingolipid equilibrium may explain why they may be more sensitive to AzSph cytotoxicity. Cellular uptake of AzSph is fast due to its small size and hydrophobicity. We observe no significant signal of AzSph in cells, meaning it is immediately acylated. This corresponds with the above-described report of photoSph (Figure 3.4.2), which describes photoCer formation within 5 minutes. 220 CerS enzymes are described to be highly selective enzymes (Introduction 1.5), and it appears that one CerS acts on AzSph preferentially, resulting in a more abundant C16:0 fatty acid chain in AzCer. CerS5 is specific for the palmitoyl (C16) chain, and CerS6 for the palmitoyl (C16) and myristyl (C14) chain. 83 Furthermore, CerS5 has highest preference for the acylation of sphingosine via the salvage pathway, whereas all other CerSs act on dihydrosphingosine to form dihydroceramide in the de novo pathway. 90,91 Since our AzSph more closely resembles sphingosine in its double bond, it is likely that AzSph is acylated by CerS5.

This in turn could justify the induced cytotoxicity, since C16:0 chains have been implicated in apoptosis signalling. C16 ceramide has indeed been described as proapoptotic in different published reports. 229-233 This may depend on cell and tissue type, and will be involved in a complex signalling cascade. The cause of cytotoxicity and the identity of acylation enzymes could be studied further with small molecule inhibitors, e.g. CerS inhibitor fumonisin B, manipulation of CerS enzyme expression, interference with apoptotic cascades such as those depending on caspases, or broadening experiments to a further cell types.

Since each GSL is often present at a very small molar percentage of the PM (Figure 3.4.3), we considered that even a low concentration of our modified lipid at the PM should be sufficient to observe lectin binding, this being our final validation. To avoid cytotoxic effects of ceramide analogues, we incubated cells with a low AzSph concentration (0.5 μM) to obtain a stable ratio of approximately [START_REF] Mcmahon | Molecular mechanism and physiological functions of clathrin-mediated endocytosis[END_REF][3] The lateral localisation of Gb3-Cer on the cell surface appears to be important to its receptor activity (Introduction 1.3). In the natural system, Gb3-Cer is transported from the Golgi to its functional position as a lectin receptor on the cell surface. Gb3-Cer negative (Gb3 -

) cells incubated with external Gb3-Cer/BSA can permit low levels of binding of STxB at the cell surface. However, the lectin's traffic is always dysregulated from the natural retrograde route (Figure 1.3.4). Externally supplemented Gb3-Cer with short acyl chains (natural long chain species fail to be reconstituted) may function non-canonically because it does not organise correctly within biological membranes. We hypothesised that AzCer with the natural complement of acyl chains would be incorporated into native lipid transport of the cell, allowing it to be recycled through a native pathway, to the PM, although temporarily, to undergo click reaction and present Gb3-Cer at the surface.

We studied the intracellular localisation of AzCer by immunofluoresence (IF) experiments. When cells were treated with AzSph, then stained with alkyne or cyclooctyne fluorophores, AzSph metabolites (AzCer) appear to be localised in intracellular membranes, possibly the ER, where CerS enzymes are known to be localised. 83 One must consider a margin of error in lipid localisation, since performing click reactions on AzSph with a fluorophore may change its localisation. In general, fixation is poorly effective on lipids, which contain few nucleophilic, fixable groups, even after the most robust fixation techniques. 93 A permeabilisation step with Saponin was the cleanest method to wash cells of nonspecific unclicked fluorophore. However, this treatment destabilises the membrane and adds even greater possible error. Despite this, we see repeatedly specific staining compared to negative controls, and similar staining in live cell imaging. AzCer localisation in all intracellular membranes is rational since the substrate closely resembles ceramide, which is synthesised in the ER and should have little intracellular specificity. Furthermore, short chain lipids are considered to preferentially localise in the Golgi and ER, whereas long chain lipids would preferentially localise in the PM, observed by differences in the thickness of transmembrane domains of PM/Golgi/ER proteins. 197 GM95 cells were too poorly adhesive to undertake lengthy IF experiments to study the intracellular localisation of AzCer metabolites. GSLs have long been known as modulators of cell adhesion. 87,234,235 Jwa Hidari et al. showed that GM95 cells lost the ability to adhere to standard extra-cellular matrix proteins when treated with sphingomyelinase, which removes SM from the cell surface, whereas GSL-expressing MEB-4 cells, from which GM95 cells were mutated, showed no loss in adhesion. 87 An inhibitor GCS and major GSL synthesis, PDMP, blocked adherence of macrophages during differentiation, further indicating GSLs are implicated in cell adhesion. 225 In the same way, PPMP treatment of naïve E14 mouse stem cells sporadically reduced adhesion during differentiation (unpublished). Click protocols for imaging the localisation of clickable lipids were optimised on CHO and HeLa cells (Experimental 6.2.6).

In cellulo click

We synthesised Gb3-MFCO We observed that the reaction with lactose-MFCO 49 was poorly efficient, since there is no significant drop in cellular AzCer levels even when using an excess of cyclooctyne Alexa488-DIBO is a commonly-used reagent for live-cell staining 62,162,163 , and is able GSLs can be analysed (Figure 3.4.3), their surface presentation is difficult to quantify.

To increase the permeability of the glycan-cyclooctynes, which would allow them to react with intracellular AzCer, we incubated AzCer-labelled cells with the peracetylated substrates, obtained from the second-to-last step of the glycan-cyclooctyne synthesis (Results 2.1.3). The acetyl group is used in chemistry to protect chemically reactive functional groups, but is also a common signalling adduct in biology, regulating the genetic expression in the cell nucleus, for example. Non-specific carboxylesterases (CES) are known to remove acetyl groups from small molecules in the cytosol. 236 Acetyl groups (CH3CO-) are hydrophobic groups that have historically been used to mask more polar, hydrophilic hydroxyl groups, augmenting the permeability of drugs in prodrugs. A drug is a substance with a non-nutritive effect on the body. A prodrug is a compound that, after administration, is metabolised in the body into a pharmacologically active drug. Prodrugs are designed to aid delivery or bioavailability. Examples include aspirin (acetylsalicylic acid) which is metabolised to active salicylic acid (Fig 3.5.1a), and heroin (diacetylmorphine), metabolised to morphine after permeating the blood brain barrier. The technique has been widely used in for the metabolic labelling of cells with azidoglycans (Introduction 1.7). 127 The concept was first proved for disaccharide uptake by Sarkar et al., 128 and made famous by Bertozzi et al. for metabolic labelling of clickable glycans (Figure 3.5.1b). 237,238 All chemical biology papers cite the action of non-specific, intracellular (carboxy)esterases. 127 Such esterases acting on glycans are likely to be the CES enzymes, CES1 and CES2, responsible for the metabolism of common prodrugs, localised in the cytosol and endoplasmic reticulum (ER). 236 Disappointingly, the deacetylation of functionalised di-and trisaccharides was poor and incomplete after 48h. This project aims to reconstitute functional GSL analogues with which one can further test their biological activity in cells. Therefore, reconstitution protocols must be practical for standard laboratory research work, where >48h incubation times are unfeasible. We are currently pursuing the upregulation of one of these genes, Ces2, to increase deacetylation of our incorporated product. Ces2 is reported to preferentially mediate the metabolism of permeating drugs corresponding to our substrate's size -a large hydroxyl and small acetyl group. 236,239 One recent paper has shown that such an upregulation can increase esterase activity by three times, 240 which -in our case -may be sufficient to catalyse the full metabolism. Prescher et al. suspected that plasma CESs could reduce the bioavailability of ManNAz in mice treated with Ac4ManNAz, with the objective of labelling all mouse tissues. 127 They trialled the use of plasma Ces1-deficient mice to reduce deacetylation in the plasma, but saw no difference in labelling of splenocyte (cells rich in sialiosides) (Figure 3.5.1c) and tissues compared with control mice. This indicates some compensatory effect of multiple CES enzymes.

Optimisation of lipid localisation

To obtain an azide labelling at the plasma membrane (PM), we envisaged different azide-lipid precursors, which might prove to have a different metabolite profile, and thus a different cellular localisation. Longer chain lipids are thought to be preferentially housed in the PM, whereas shorter lipids localise to the Golgi or ER. Although this theory is difficult to verify, a study of protein transmembrane domains showed that longer hydrophobic domains exist in PM-localised proteins than the ER-and Golgi-localised proteins. 197 AzSph is acylated to AzCer (C16:0) by the CerS5 enzyme, which has high specificity for sphingosine formed in the salvage pathway. 91 Sphinganine, formed in the de novo synthesis pathway, is metabolised by other CerSs, which have individual specificities for longer fatty acid chains (Graph 3.6.1, Introduction 1.5). An azide analogue of sphinganine holds promise as it may be metabolised like sphinganine, via the de novo pathway to long chain ceramides, which should have a higher propensity to localise to the PM. Phytosphingosine is structurally more similar to sphinganine than sphingosine as it has no double bond in the C4 position.

Phytosphingosine is a plant-derived starting material from which we begin the AzSph synthesis. was easily obtained synthetically (Results 2.1.2). Phytosphingosine is a popular cosmetic product and literature suggests that it is widely accepted by mammalian cells. 241 Indeed, AzPhy showed cytotoxicity similar to that of AzSph; EC50 = 2 μM and 10 μM in GM95 and CHO respectively, compared with 3 μM and 2 μM for AzSph. As hoped, the profile of AzPhy metabolites is closer to the profile of native ceramide than that of AzSph, comprising a higher percentage of long chain lipids. We suggest that this is due to the metabolism of AzPhy by some other CerS enzyme(s), which have other fatty acid chain specificities (Graph 3.6.1). There were no traces of dehydroxylation of azidophytoceramides into mammalian azidoceramides.

In immunofluorescence experiments, no difference in localisation was observable compared with AzSph. Furthermore, AzPhyCer did not show improved reactivity in cellular click experiments. HRMS-detectable levels of click product are formed from surface click with Lac-MFCO 49, yet their levels are not significantly different from that observed with AzSph (Graph 2.2.4.6). One can suggest that the change in lipid length of AzPhyCer does not significantly change its localisation.

PERSPECTIVES

Our primary aim to reconstitute functional Gb3 analogues in cells using click chemistry could be attained by optimising our protocol or our clickable reagents. The upregulation of a major non-specific esterase, Ces2, is envisaged to increase deacetylation (Discussion 3.5).

Alternatively, because permeable, acetylated glycosides are much better clicked than free glycosides, one could consider using partially acetylated glycosides. These products could be taken up by cells and more easily deacetylated. Sarkar et al. describe that mono-, di-and trimethylation of disaccharides (Figure 4.1, lanes [3][4][START_REF] Mayor | Clathrin-independent pathways of endocytosis[END_REF] greatly increases their cellular uptake compared with hydrophilic glycan (lane 2). We can envisage the partial acetylation of Gb3-MFCO 37 or partial deprotection of peracetyl Gb3MFCO 47. Enzymatic deacetylation of 47 using pig liver esterase was very poor. Chemical acetylation of 37 with few equivalents of acetylating reagent is low yielding and non-specific, but a viable possibility. The uptake of hydrophilic glycans could also be increased by electroporation or transfection reagents. The suite of this project could go to investigating biological interactions of other GSLs, the strategy of the clickable Gb3 head group would in principle be applicable to any glycan head group. The synthetic steps required to functionalise the glycan with MFCO are as follows: hydroxyl protection, anomeric deprotection, activation, azidolysis, reduction to amine and peptide coupling with the cyclooctyne. Using this many steps is difficult to envisage for commercial oligosaccharides bought at milligram scale. Enzymatic methods, such as the use of glycosyltransferases, are ideal for direct synthesis, with retention of stereochemistry.

Such methods have been applied for the synthesis of Gb3 243 and GM3. 244 We could envisage the chemoenzymatic synthesis of other glycosides from simpler functionalised starting materials, such as GM3-MFCO from lactose-MFCO with the α-2,3-sialyltransferase. If the glycoside head group can be efficiently reconstituted, one can envisage the synthetic incorporation of a crosslinking, photoactivatable group. One easy route would be to incorporate a diazirine-functionalised amino acid in the place of glycine in compound 36 (Figure 4.4). Since compound 36 has shown acceptable membrane bending properties in GUVs in complexation with STxB, we could imagine that its biological function would not be hindered, and the crosslinking group could be used to isolate interaction partners with the glycan head group of choice. To purify the lipid-protein bound complex, an additional alkyne would be required in the lipid tail to permit affinity purification. Basic principles of organic chemistry suggest that every synthesis is feasible with the right budget and time. Now, with the developments of chemical biology and the surprising plasticity of the natural system, we can begin to believe that we can manipulate and understand each part of the cellular system. All that's needed is the right controls: molecules which control cellular function, and positive/negative control experiments to verify the results!

CONCLUSIONS

We plates (2×20 μl).

2. Plates dried at 65°C for 5 min then vacuum dried for 30 min.

3. Plates separated with plastic shoots and placed inside Teflon cases. Covered with parafilm leaving a small gap. 6ml sucrose (300 mOs) added and the capsule sealed over.

4. Electrodes connected to AC (10 Hz, 1.1 V (AC) at 65°C for 2 h.

Cytotoxicity assay

The day before, 50,000 cells/well seeded (200 μl in DMEM++) in a 96-well 9.7 Hz,1H),5.36 (d,J 3.2 Hz,1H,5.13 (dd,J 10.3,7.9 Hz,1H),5.01 (dd,J 10.3,3.7 Hz,1H),4.96 (dd,J 10.4,3.4 Hz,1H) J 6a-6b 13.8, J 6a-5 2. 2 Hz, 6a-H), 4.14 (1H, dd, J 6'a-6'b 11.1, J 6'a-5' 6.5 Hz, 6'a-H), 4.11-4.07 (2H, m, 6b-H, 6'b-H), 3.91-3.85 (1H, m, 5'-H), 3.82 (1H, dd, J 4-5 9.6 Hz, J 4-3 9.4 Hz, 4-H), 3.66 (1H, m, 5-H), 2.16, 2.14, 2.11, 2.07, 2.06, 2.05, 1.97 (21H, 7s, COCH3). Compound 1 (10 mg, 14.8 μmol, 1 eq) and 1, 11 mg,14.8 μmol, 1 eq) were mixed in dimethylformamide (DMF, 1 ml), to which N, N-diisopropylethylamine (DIPEA, 0.1 ml, 0.57 mmol, 40 eq) was added. The solution was stirred overnight. The reaction mixture was evaporated and purified by silica column chromatography (CH2Cl2/MeOH [9:1]) to afford 2 (14 mg, 67 %). Rf (CH2Cl2/MeOH [9:1]=0.3;

N-1,2-dioleoyl

1 H NMR (300 MHz, CDCl3) δ 7. 1H,NH), 5.89 (s, 1H, NH), 5. 4H,HC=CH), 5. 40 -5.28 (m, 3H), 5. 2H),2H),4.47 (d,J 28.1 Hz,3H),4.14 (s,5H),5H), 3. 84 -3.69 (m, 2H) 31 P NMR (121 MHz, MeOD) δ 1.17; 13 C NMR (75 MHz, MeOD) δ 83.21, 72.38, 66.81, 65.24, 60.00, 56.28, 51.85, 32.98, 31.78, 30.47, 26.90, 23.70, 14.28; ESI-MS 320.4 1, 2-dioleoyl-sn-glycerol (12 mg, 20 μmol, 1 eq), COP (11 μl, 60 μmol, 3 eq) and 4-DMAP (0.25 mg, 2 μmol, cat.) were dissolved in dry toluene and cooled to 0°C. EtN3 (8 μl, 60 μmol, 3 eq) was added dropwise and the reaction mixture stirred at 0°C for 15 minutes then at rt for 4h.

The reaction mixture was filtered through Celite and washed with dry toluene. Solvent was removed under reduced pressure to afford the cyclic phosphate 11 ( 31 P NMR δ 18.92 (s)). The product was redissolved with NaN3 (7 mg, 110 μmol, 5 eq) in DMF (1.5 ml) and heated to 80°C for 6h. The reaction mixture was dried and purified by silica column chromatography (CH2Cl2/MeOH [95:5 to 85:15]) to afford the compound 13 (10 mg, 65 % over two steps). Rf (CH2Cl2/MeOH [85:15])=0.5; 1 H NMR (300 MHz, CDCl3) δ 6. 75 -5.21 (m, 5H, H h , H c ), 4. 44 -azide hydrochloride (0.87 g, 4.15 mmol, 1.2 eq) was added and the solution was stirred for 24 h. The solution was filtered and the filter cake washed with CH2Cl2/MeOH (10:1). The mother liquor was evaporated and purified by silica column chromatography [START_REF] Howes | Molecules, mechanisms, and cellular roles of clathrinindependent endocytosis[END_REF][START_REF] Hinze | Local actin polymerization during endocytic carrier formation[END_REF](11)[START_REF] Johannes | Shiga toxins -from cell biology to biomedical applications[END_REF](13)[START_REF] Ferreira | Mechanisms of Carrier Formation during Clathrin-Independent Endocytosis[END_REF][START_REF] Renard | Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis[END_REF][START_REF] Johannes | Glycolipids and Lectins in Endocytic Uptake Processes[END_REF][START_REF] Moya | Inhibition of coated pit formation in hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin[END_REF][START_REF] Schmid | Endocytosis: Past, Present, and Future[END_REF](19)[START_REF] Bergan | Shiga toxins[END_REF] to afford the described compound 18 as a white solid (0.85 g, 2.46 mmol, 75 %). Rf (CH2Cl2/MeOH [10:1])=0.5; 1 H NMR (300 MHz, MeOD) δ 3.92 (dd, J 11.5, 3.4 Hz, 1H, H 1b ), 3.75 (dd, J 11.6, 8.0 Hz, 1H, H 1a ), 3. 63 -3.55 (m, 1H, H 2 ), 3. 55 -3.48 (m, J 4.4 Hz, 1H, H 3 , H 4 ), 1. 2H,CH2), 1.29 (s, 24H), 0.90 (t,J 6.7 Hz,3H).

(2S,3S,4R)- 4H),6H),4.03 (dd,J 10.9,4.2 Hz,1H),3.91 (dd,J 10.9,5.7 Hz,1H), 3. 2H),3.56 (dd,J 9.9,5.5 Hz,1H),2.51 (d,J 3.4 Hz,1H),1.99 (d,J 3.6 Hz,1H), 1. 63 -1.46 (m, 2H), 1.26 (s, 24H), 1.08 (s, 9H), 0.88 (t,J 6.6 Hz,3H); 13 C NMR (75 MHz, CDCl3) δ 136.03, 136.00, 132.93, 132.85, 130.44, 74.52, 72.74, 64.55, 63.76, 32.34, 32.23, 30.09, 29.78, 27.15, 26.07, 23.11, 19.50, 14.55. Compound 19 (0.84 g, 1.45 mmol, 1 eq) was dissolved in dry CH2Cl2 (8 ml), to which dry Et3N (0.60 ml, 4.34 mmol, 3 eq) and SOCl2 (0.127 ml, 1.735 mmol, 1.2 eq) were added and stirred at 0 °C. After 30 min the reaction mixture was washed with brine (2×50 ml) and extracted with EtOAc (2×50 ml), dried (MgSO4) and evaporated. The intermediate cyclic sulfite was dissolved in CCl4/CH3CN/H2O (12 ml, 1:1: 1) and to the solution was added Ru(III)Cl3 (18 mg, 0.07 mmol, 0.05 eq) and NaIO4 (0.90 g, 4.22 mmol, 3 eq). The reaction mixture was stirred for 2 h, diluted with EtOAc (30 ml), washed with sat. NaHCO3 solution (2×20 ml), extracted with EtOAc, dried (MgSO4), evaporated and purified by silica column chromatography (10% EtOAc in cyclohexane) to afford 20 (0.83 g, 89 %). Rf (cyclohex:EtOAc [6:1])=0.8; 1 H NMR (300 MHz, CDCl3) δ 7. 74 -7.64 (m, 4H), 7. 6H),4.98 (ddd,J 10.2,5.2,2.6 Hz,1H),4.92 (dd,J 9.9,5.2 Hz,1H),4.04 (dd,J 11.3,2.4 Hz,1H),3.89 (dd,J 11.3,5.2 Hz,1H),3.69 (ddd,J 9.7,5.1,2.4 Hz,1H), 2.00 -1.66 (m, 2H), 1. 41 -1.30 (m, 2H), 1.27 (s, 22H), 1.09 (s, 9H), 0.88 (t, J 6.7 Hz, 3H); 13 C NMR (75 MHz, CDCl3) δ 135. 98, 132.60, 132.36, 130.56, 128.39, 86.85, 80.26, 63.97, 59.62, 32.33, 30.06, 29.86, 29.76, 29.70, 29.36, 28.53, 27.33, 27.14, 25.59, 23.10, 19.57, 14.52. Compound 20 (772 mg, 1.20 mmol, 1 eq) was dissolved in dry toluene (8 ml), to which Bu4NI (487 mg, 1.32 mmol, 1.1 eq) and 1, 8-diazabicyclo(5.4.0)undec-7-ene (DBU, 274 mg, 1.80 mmol, 1.5 eq) was added. The reaction mixture was refluxed for 2 h, the solvent evaporated and redissolved in a solution of concentrated H2SO4 (0.1 ml), H2O (0.8 ml) and tetrahydrofuran (THF, 10 ml) and stirred for a further 1 h. Purification by silica column chromatography (10% EtOAc in cyclohexane) afforded 21 (0.72 g, 60 %). Rf (cyclohex:EtOAc [10:1])=0.4; 1 H NMR (300 MHz, CDCl3) δ 7. 74 -7.65 (m, 4H), 7. 6H),5.74 (dtd,J 15.5,6.5,0.5 Hz,1H),5.43 (ddt,J 15.4,7.1,0.5 Hz,1H),4.22 (dd,J 11.8,5.4 Hz,1H),3.82 (dd,J 10.8,6.3 Hz,1H),3.77 (dd,J 10.6,4.6 Hz,1H),3.51 (dd,J 11.2,4.9 Hz,1H), 1. 98 -1.59 (m, 2H), 1. 36 -1.28 (m, 2H), 1.26 (s, 20H), 1.07 (s, 9H), 0.88 (t, J 6.7 Hz, 3H); 13 C NMR (75 MHz, CDCl3) δ 135.98, 135.85, 133.15, 130.31, 128.22, 73.23, 67.28, 64.53, 32.69, 32.33, 30.08, 29.88, 29.77, 29.59, 29.35, 27.12, 23.10, 19.51, 14.53. Compound 21 (1.95 g, 3.48 mmol, 1 eq) was dissolved in dry pyridine (12 ml) at 0 °C, to which BzCl (960 µl, 8.29 mmol, 2.4 eq) was added dropwise and the reaction mixture was stirred for 7.5 Hz, 2H, Si(C6H5)2), 7. m,Ph),m,Si(C6H5)2), 5.91 (1H, ddd, J 5-6 6.8 Hz, J 5-6' 6.8 Hz, J 5-4 15.2 Hz, H 5 ), 5.69 (1H, dd, J 3-4 7.9, J 3-2 4.7 Hz, H 3 ), 5.52 (1H, dd, J 4-5 15.4, J 4-3 5H,TBDPS),6H,aromatic),2H,Bz), 5. 90 -5.77 (m, 1H, H 5 ), 5. 48 -5.34 (m, 2H, H 3 , H 4 ), 3. 72 -3.59 (m, 2H, H 1a , H 1b ), 3.10 (q, J 5.1 Hz, 1H, H 2 ), 1.97 (q, J 6.9 Hz, 2H, H 6 ), 1. 22H,11CH2), 0.99 (s, 9H, t Bu), 0.80 (t, J 6.6 Hz, 3H, CH3); 13 C NMR (75 MHz, CDCl3) δ 165.51 (CO benzyl), 137.91 (C 5 ), 135.75, 135.59, 135.59, 133.38, 133.22, 133.00), 130.71, 130.27, 130.23, 129.87, 129.81, 129.75, 128.79, 128.49, 128.08, 127.90, 127.82, 127.13, 125.05 (C 4 ), 76.56 123 (C 3 ), 64.93 (C 1 ), 55.56 (C 2 ), 32.61 (C 6 ), 32.11 (C 16 ), 29.85, 29.77, 29.63, 29.55, 29.39, 29. 2H,Bz), 7. 60 -7.45 (m, 5H, arom), 7.41 -7.20 (m, 6H, arom), 7.12 (t, J 7.3 Hz, 2H, Bz), 5. 94 -5.81 (m, 1H, H 5 ), 5.84 (s, 1H, CHCl2), 5.66 (t, J 3-2, 3-4 7.5 Hz, 1H, H 3 ) 5.43 (dd, J 4-5 15.4, J 4-3 7.5 Hz, 1H, H 4 ), 4.27 (ddd, J 2-3 7.2, J 2-1a 3.5, J 2-1b 4.0 Hz, 1H, H 2 ), 3.84 (dd, J 1a-b 10.6, J 1a-2 3.0

(
Hz, 1H, H 1a ), 3.65 (dd, J 1a-b 10.6, J 1b-2 4.0 Hz, 1H, H 1b ), 1.95 (q, J 6-5 6.9 Hz, 2H, H 6 ), 1. 32 -1.09 (m, 22H), 0.99 (s, 9H), 0.81 (t, J 6.7 Hz, 3H); 13 C NMR (75 MHz, CDCl3) δ 165.17 (COCHCl2), 163.57 (CO benzyl), 138.01 (C 5 ), 135.53, 135.45, 133.10, 132.34, 132.18, 130.06, 130.00, 129.88, 129.71, 128.42, 127.91, 127.79, 124.13 (C 4 ), 73.59 (C 3 ), 66.57 (CHCl2), 61.72 (C 1 ), 53.13 (C 2 ), 32.37 (C 6 ), 31.94 (C 16 ), 29.68, 29.59, 29.47, 29.38, 29.25, 28.82, 26.77 ( t To a solution of compound 24 (888 mg, 1.18 mmol, 1 eq) in THF (20 ml), benzoic acid (187 mg, 1.53 mmol, 1.3 eq) was added and the solution cooled to 0°C. To this, tetra-n-butylammonium fluoride (TBAF, 1M solution in THF, 1.53 ml, 1.3 eq) was added dropwise over 3 minutes. The reaction was warmed to rt over 10 minutes and stirred for 4 h before complete conversion.

The reaction was quenched with water (10 ml) and extracted with CH2Cl2 (2×10 ml), dried (MgSO4), evaporated and purified by silica column chromatography (0. (C 16 ), 29.67, 29.59, 29.45, 29.37, 29.24, 28.77, 22.71 (C 17 ), 14.15 To a solution of compound 25 (510 mg, 0.99 mmol, 1 eq) in pyridine (15 ml) at -10°C, methanesulfonyl chloride (MsCl, 109 μl, 1.27 mmol, 1.4 eq) was added. The solution was warmed to rt after 5 min and stirred for 3 h before co-evaporation with toluene. (C 6 ), 31.93 (C 16 ), 29.66, 29.57, 29.43, 29.37, 29.21, 28.71, 22.70 (C 17 ), 14.15 (C 18 ); ESI-MS 590. (C 6 ), 31.94 (C 16 ), 29.67, 29.58, 29.43, 29.38, 29.21, 28.73, 22.72 (C 17 ), 14.16 (C 18 ), 5.17 To a solution of compound 27 (380 mg, 0.61 mmol, 1 eq) in DMF (2.5 ml), sodium azide (396 mg, 6.1 mmol, 10 eq) was added and the mixture stirred for 14 h. N-2 9.0 Hz, 1H, NH), 6. 04 -5.92 (m, 1H, H 5 ), 5.61 (t, J 3-4, 3-2 6.8 Hz, 1H, H 3 ), 5.49 (dd, J 4-5 15.3, J 4-3 7.4 Hz, 1H, H 4 ), 4.41 (m, 1H, H 2 ), 3. 70 -3.54 (m, 2H, H 1a,b ), 2.06 (dd, J 6-5 6.9 Hz, J 6-6' 14 Hz, 2H, H 6 ), 1. 12H,CH2), 0.87 (t,J 6.6 Hz,3H,CH3). 13 (C 16 ), 29.67, 29.59, 29.44, 29.38, 29.21, 28.76, 22.72 (C 17 ), 14.16 (C 18 300MHz, CDCl3) δ 5. 87 -5.68 (m, 1H, H 5 ), 5.43 (dd, J 4-5 15.4, J4-3 7.2 Hz, 1H, H 4 ), 4.00 (t, J 3-4, 3-2 6.1 Hz, 1H, H 3 ), 3.48 (dd, J 1a-b 12.2 Hz, J 1-2 (C 16 ), 29.68, 29.62, 29.48, 29.37, 29.23, 29.11, 22.71 (C 17 ), 14.15 (C 18 (C 6 ), 29.69, 29.59, 29.56, 29.52, 29.37, 25.68, 22.70, 14.14 48, 128.93, 128.81, 128.72, 128.70, 128.67, 128.64, 128.60, 128.58, 128.57, 128.54, 128.28, 128.08, 128.03, 127.98, 127.93, 127.87, 127.84, 127.80, 127.60, 127.55, 125.74, 118.85, 114.87, 103.41 (α), 103.13 (β), 101.24 (β), 83.08, 82.10, 81.90, 79.85, 77.67, 75.80, 75.70, 75.66, 75.62, 75.43, 75.32, 75.15, 74.16, 73.71, 73.61, 73.46, 72.83, 72.50, 69.85, 68.20, 68.08, 56.05; ESI-HRMS:

Calcd for [MNa] + 1533.7702; found 1533.6707.

( 300MHz, CDCl3) δ 5.57 (d, J 4-3 2.3 Hz, 1H, H 4'' ), 5.37 (dd, J 3-2 11.0, J 3-4 3.2 Hz, 1H, H 3'' ), 5.20 (t, J 3-2, 3-4 7.6 Hz, 1H, H 3 ), 5.17 (dd, J 2-3 11.0, J 2-1 3.6 Hz, 1H, H 2'' ), 5.09 (dd, J 2-3 10.7, J 2-1 7.8 Hz, 1H, H 2' ), 4.97 (d, J 1-2 3.4 Hz, 1H, H 1'' ), 4.84 (t, J 2-3, 2-1 9.0 Hz, 1H, H 2 ), 4.72 (dd, J 3-2 10.8, J 3-4 2.1 Hz, 1H, H 3' ), 4.63 (d, J 1-2 8.7 Hz, 1H, H 1 ), 4. 57 -4.33 (m, 4H, H 1' , H 5'' , H 6a , H 6'a ), 4. 21 -4.03 (m, 4H, H 6 , H 6''a,b , H 5' ), 4.00 (s, 1H, H 4' ), 3. 92 -3.62 (m, 3H, H 4 , H 5 , H 6' ), 2.11 (s, 6H), 2.07 (s, 6H), 2.05 (s, 9H), 2.04 (s, 3H), 1.99 (s, 3H), 1.97 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 170. 62, 170.44, 170.41, 170.39, 170.33, 170.04, 169.53, 169.49, 168.84, 101.10 (C 1' ), 99.64 (C 6'' ), 20.89, 20.76, 20.68, 20.63, 20.60, 20.56, 20.47 Compound 43 (63 mg, 66.3 μmol) was dissolved in EtOAc (1 ml), to which Pd/C (10%, 7 mg) was added and dihydrogen gas was bubbled through the solution for 10 min. The reaction was stirred in H2 atmosphere for 3h, filtered through Celite and evaporated to afford amine Compound 47 (24 mg, 22.3 47 -4.37 (m, 1H), 4.27 (t, J 5-6 6.4 Hz, 1H), 3.99 (s, 1H), 3. 5H),3.81 (d,J 3.6 Hz,1H),3.79 (d,J 3.0 Hz,1H), 3. 77 -3.63 (m, 3H), 3. 63 -3.51 (m, 4H), 3. 51 -3.41 (m, 2H), 2. 43 -2.20 (m, 4H), 2. 19 -1.78 (m, 4H), 1. 75 -1.58 (m, 1H), 1. 58 -1.37 (m, 1H). 13 80.42, 79.71, 78.36, 77.27, 76.55, 74.61, 73.20, 72.80, 72.65, 71.28, 71.03, 70.54, 62.65, 61.58, 61.49, 47.59 (d, J 24.8 Hz, C 17 ), 34.96 (C 16 ), 30.11 (C 15 ), 26.72 (C 14 ), 21.01 (C 13 ). 19

N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-Dglucopyranosyl) 1-fluoro-cyclooct-2-yne-1-carboxamide (53)

To a solution of MFCO 45 (18 mg, 106 μmol, 1 eq), the heptaacetyl-lactose-β-NH2 52 (85 mg, 120 μmol, 1.1 eq) and hydroxybenzotriazole (HOBt, 19 mg, 127 μmol, 1.2 eq) in CH2Cl2 (1 ml) at 0°C, N,N 44 mg,212 μmol,2 eq) was added at the reaction mixture was warmed to rt after 10 min, and stirred overnight. The crude reaction mixture (α/β≈1:11) was evaporated and purified by silica column chromatography (40 to 90 % EtOAc in cyclohexane) containing some dicyclohexylurea impurity. This was removed by precipitation in Et2O to afford 53 (52 mg (β), 62 %). Rf (EtOAc/cyclcohexane [1:1])=0.4. 1 H NMR (500 MHz, CDCl3) δ 6. 94 (ddd, J 26.6, 9.4, 4.8 Hz, 1H, NH), 5.35 (d, J 3.1 Hz, 1H, H 4' ), 5.31 (td, J 9.2 Hz, 3.9 Hz, H 3 ), 5.17 (qd, J 9.5 Hz, 1.2 Hz, 1H, H 1 ), 5.11 (dd, J 10.4, 7.9 Hz, 1H, H 2' ), 4.94 (dd, J 10.4, 3.5 Hz, 1H, H 3' ), 4.88 (td, J 9.5, 6.9 Hz, 1H, H 2 ), 4.46 (dd, J 7.8, 1.1 Hz, 1H, H 1' ), 4. 41-4.45 (m, 1H, H 6 ), 4. 10-4.17 (m, 2H, H 6 , H 6' ), 4.08 (dd, J 11.1, 7.2 Hz, 1H, H 6' ), 3.87 (t, J 6.8 Hz, 1H, H 5' ), 3.78 (td, J 8.8 Hz, 2.3 Hz, 1H, H 4 ), 3.73 (dt, J 10.0, 2.2 Hz, 1H, H 5 ), 2. 47 -2.16 (m, 4H, H 11 96 -1.79 (m, 4H, H 12 , H 14 ), 1. 74 -1.56 (m, 1H, H 13 ),

1. 48 -1.34 (m, 1H, H 13 ). 13 C NMR (126 MHz, CDCl3) δ 170. 95, 170.76, 170.38, 170.33, 170.16, 170.09, 169.37, 168.98, 110.12 (dd, J 48.1, 10.4 Hz, C 10 ), 100.90 (C 1' ), 94.04 (dd, J 187.4, 34.4 Hz, C 8 ), 86.51 (d, J 45.5 Hz, C 9 ), 78.16 (d, J 20.1 Hz, C 1 ), 75.91 (C 4 ), 74.74 (C 5 ), 72.17 (C 3 ), 71.01 (C 3' ), 70.94 (C 2 ), 70.78 (C 5' ), 69.00 (C 2' ), 66.63 (C 4' ), 61.87 (d, J 8.2 Hz, C 6 ), 60.90 (C 6' ), 46.06 (dd, J 75.5, 24.7 Hz, C 15 ), 33.78 (d, J 6.5 Hz, C 14 ), 28.83 (C 13 ), 25.43 (C 12 ), 20.90, 20.75, 20.67, 20.64, 20.56, 20.53. 19

N-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl) 1-fluoro-cyclooct-2-yne-1carboxamide (49)

To a solution of the protected lactose 53 (48 mg, 63 μmol, 1 eq) in dry MeOH (1.5 ml), CH3ONa

(1.27 ml, 0.63 mmol, 10 eq, 0.5 M solution in MeOH) was added. The reaction was stirred for 30 minutes and filtered through a pad of Dowex 50WX8 (previously protonated HCl, 0.1M).

The crude mixture was evaporated and purified by reverse phase C18 silica column chromatography (10-20% CH3CN/H2O) to obtain clickable lactose 49 (21 mg, 71 %).

Rf ( t BuOH/EtOH/H2O [2:2:1])=0.7. 1 H NMR (500 MHz, MeOD) δ 4.96 (dd, J 1-2 9.0, J 1-N 3.0 Hz, 1H, H 1 ), 4.39 (d, J 7.6 Hz, 1H, H 1' ), 3. 91 -3.86 (m, J 2.6 Hz, 3H), 3.91 -3.81 (m, 3H), 3.81 -3.75 (m, 1H), 3.72 (dd, J 11.5, 4.3 Hz, 1H, H 6' ), 3. 68 -3.54 (m, 4H), 3.54 -3.44 (m, 3H), 2. 44 -2.26 (m, 4H), 2. 16 -1.79 (m, 4H), 1. 77 -1.59 (m, 1H), 1. 54 -1.39 (m, 1H). 13 C NMR (126 MHz, MeOD) δ 170.62 (dd, J C-F 25.8, 5.8 Hz, C7), 108.82 (C10), 103.67 (C1'), 93.84 (dd, J C-F 187.6, 23.7 Hz, C8), 86.75 (d, J 29.7 Hz, C9), 79.78 (d, J 7.3 Hz, C1), 78.65, 76.98 (d, J 4.0 Hz, C2), 75.89, 75.71, 73.38, 71.72, 71.15, 68.91, 61.14, 60.21, 46.21 (d, J 24.8 Hz, C15), 33.57 (C14), 28.73 (C13), 25.33 (C12), 19.63 (C11). 19 F NMR (471 MHz, MeOD) δ -148.56 (d, J 63.4 Hz). To a solution of MFCO 45 (43 mg, 0.25 mmol, 1 eq) and DIPEA (66 μl, 0.38 mmol, 1.5 eq) in CH2Cl2 (2 ml) at 0°C, PFP-TFA (52 μl, 0.30 mmol, 1.2 eq) was added dropwise. The reaction mixture was stirred for 3h until complete activation of the carboxylic acid. The solvent was evaporated and DMF (1 ml) was added, followed by DIPEA (66 μl, 0.38 mmol, 1.5 eq), then O- (bs, 1H, COOH), 6.90 (d, J 3.9 Hz, 1H, NH), 4.12 (qd, J 18.6, 5.3 Hz, 2H, H 10 ), 2. 46 -2.24 (m, 4H, H 2 , H 6 ), 2. 10 -1.79 (m, 4H, H 3 , H 5 ), 1. 74 -1.61 (m, 1H, H 4 ), 1.44 (m, 1H, H 4 ). 13 C NMR ( 126MHz, CDCl3) δ 173.32 (C11), 169.29 (d, J C-F 25.1 Hz, C 9 ), 110.15 (d, J C-F 10.4 Hz, C 3 ), 94.35 (d, J C- F 186.7 Hz, C 1 ), 86.69 (d, J C-F 31.8 Hz, C 2 ), 46.46 (d, J C-F 24.5 Hz, C 8 ), 41.17 (C 10 ), 33.92 (C 7 ), 28.95 (C 6 ), 25.64 (C 5 ), 20.67 (C 4 ). 19 J 10.3, 7.9 Hz, 1H), 4.94 (dd, J 10.4, 3.4 Hz, 1H, H 1 ), 4.84 (t, J 9.6 Hz, 1H), 4.46 (d, J 7.9 Hz, 1H, H 1' ), 4.43 (d, J 11.5 Hz, 1H), 4.18 -3.98 (m, 4H), 3.98 -3.82 (m, 3H), 3.81 -3.69 (m, 2H), 2.49 -2.24 (m, 4H, H 10 , H 14 ), 2.15 (s, 3H), 2.12 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 1.96 (s, 3H), 1. 93 -1.63 (m, 4H, H 11 , H 13 ), 1. 21 -1.02 (m, 2H, H 12 ). 13 C NMR (101 MHz, CDCl3) δ 171. 47, 171.42, 170.43, 170.40, 170.22, 170.15, 169.39, 169.05, 168.56, 109.94 (d, J C-F 10.5 Hz, C 9 ), 101.01 (C 1' ), 94.36 (d, J C-F 186.2 Hz, C 7 ), 86.88 (d, J 31.6 Hz, C 1 ), 78.25, 75.9, 74.64, 72.25 (d, J 4.3 Hz), 70.96 (d, J 23.0 Hz, C 8 ), 69.09, 66.71, 61.95, 60.96, 46.37 (d, J C-F 20.2 Hz, C 15 ), 43.13 (C 17 ), 33.98 (d, J 12.3 Hz, C 13 ), 28.98 (C 12 ), 25.66 (C 11 ), 20.96 (C 10 ), 20.82, 20.73, 20.59. 19 F NMR (282 MHz, CDCl3) δ -146.42 (d, J 16.2 Hz). UPLC-MS (gradient 80-90% CH3CN /H2O, 4 min, ESI) rt=1.14 min; 843 (M -), 846 (MH + ).

N-β-D-galactopyranosyl-(1→4)-1-azido-β-D-glucopyranosyl-N'-glycine-1-fluoro-cyclooct-2yne-1-carboxamide (50)

To a solution of the protected lactose 54 (5 mg, 6 μmol, 1 eq) in dry MeOH (470 μl), CH3ONa

(118 μl, 0.63 mmol, 10 eq, 0.5M solution in MeOH) was added. The reaction was stirred for 1h and filtered through a pad of Dowex 50WX8 (previously protonated HCl, 0.1M). The crude mixture was evaporated and purified by reverse phase C18 silica column chromatography (0 to 50% CH3CN/H2O) to obtain compound 50 (2 mg, 62 %). Rf( t BuOH/EtOH/H2O [2:2:1])=0.8.

1 H NMR (300 MHz, D2O) δ 5.09 (d, J 9.0 Hz, 1H), 4.52 (d,J 7.7 Hz,1H),4.11 (s,2H),2H), 3. 93 -3.67 (m, 8H), 3.66 -3.46 (m, 2H), 2. 56 -2.20 (m, 4H, H10, H 14 ), 2. 20 -1.82 (m, 4H, H 11 , H 13 ), 1. 82 -1.58 (m, 1H, H 12 ), 1. 59 -1.38 (m, 1H, H 12 ). 19 To a solution of amine 44 (21 mg, 23 μmol, 1 eq), carboxylic acid 46 (8 mg, 34 μmol, 1.5 eq) and HOBt (4 mg, 27 μmol, 1.2 eq) in CH2Cl2 (1 ml) at 0°C, DCC (9.5 mg, 46 μmol, 2 eq) was added at the reaction mixture was warmed to rt after 10 min, and stirred for 2h before complete formation of crude product (α/β≈1:11). The reaction mixture was evaporated and purified by silica column chromatography (50 to 90 % EtOAc in cyclohexane) to afford 48 (19 mg (β) 4.85 (t, J 9.5 Hz, 1H), 4.71 (dd, J 10.8, 1.9 Hz, 1H), 4.48 (d, J 7.7 Hz, 1H, H 1' ), 4. 47 -4.38 (m, 3H), 4. 5H,incl. CH2 gly),2H), 3. 93 -3.84 (m, 1H), 3. 82 -3.70 (m, 3H), 2. 49 -2.24 (m, 4H, H 13 , H 17 ), 2.12 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.07 (m, 3H), 2.06 (s, 3H, Ac), 2.05 (s, 6H, Ac), 2.04 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1. 95 -1.76 (m, 4H, H14, H 16 ), 1. 63 -1.55 (m, 1H, H 15 ), 1. 53 -1.44 (m, 1H, H 15 ). 13 C NMR (126 MHz, CDCl3) δ 171. 49, 171.43, 170.80, 170.63, 170.57, 170.53, 170.51, 170.16, 169.59, 169.32, 169.12, 168.87, 168.60, 109.92 (d, J 12-F 10.4 Hz, C 12 ), 100.97 (C 1' ), 99.68 (C 1'' ), 94.36 (d, J 10-F 186. 6 Hz, C 10 ), 86.90 (d, J 11-F 31.5 Hz, C 11 ), 78.20 (C 1 ), 76.07, 74.63, 72.94, 72.54, 72.49, 72.01, 70.88, 69.06, 68.99, 68.02, 67.30, 67.10, 62.12, 61.54, 60.37, 46.39 (d, J 17-F 18.8 Hz, C 17 ), 43.10 (C 8 ), 33.92 (C 16 ), 28.98 (C 15 ), 25.02 (C 14 ), 21.03, 20.98, 20.83, 20.76, 20.74, 20.69, 20.60

N-(α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl)-N'glycine-1-fluoro-cyclooct-2-yne-1-carboxamide (38)

To a solution of compound 48 (18.5 mg, 16.3 (m, 3H, incl. CH2 gly), 3. 89 -3.82 (m, 4H), 3. 3H),3.69 (dd,J 10.7,5.3 Hz,2H), 3. 62 -3.51 (m, 4H), 3.47 (dd, J 3-4 6.1, J 3-2 2.9 Hz, 1H, H 3 ), 3.34 (dd, J 2-1 8.9, J 2-3 2.6 Hz, 1H, H 2 ), 2. 40 -2.26 (m, 4H), 2. 13 -1.83 (m, 4H), 1. 74 -1.62 (m, 1H), 1. 53 -1.42 (m, 1H). 13 C NMR (126 MHz, MeOD) δ 170.34 (C 7 ), 170.11 (d, J 9-F 25.5 Hz, C 9 ), 109.00 (d, J 12-F 10.2 Hz, C 12 ), 104.06 (C 1' ), 101.36 (C 1'' ), 93.83 (d, J 10-F 189.7 Hz, C 10 ), 86.80 (d, J 11-F 31.8 Hz, C 11 ), 79.66 (C 1 ), 79.38, 78.41, 76.83, 75.98, 75.25, 73.32, 72.40, 71.50, 71.35, 69.97, 69.72, 69.24, 61.35, 60.41, 60.19, 46.41 (d, J 17-F 24.8 Hz, C 17 ), 41.92 (C 8 ), 33.67 (C 16 ), 28.84 (C 15 ), 25.42 (C 14 ), 19.72 (C 13 ). 3.48 (dd,J 10.1,5.2 Hz,2H,H10), 2. 52 -2.18 (m, 4H, H 6 , H 2 ), 2. 18 -1.77 (m, 4H, H 3 , H 5 ), 1. 78 -1.57 (m, 1H, H 4 ), 1. 56 -1.37 (m, 1H, H 4 ). 13 C NMR (101 MHz, CDCl3) δ 169. 56 (d, J 24.2 Hz, C 9 ), 109. 68 (d, J 10.5 Hz, C 3 ), 94.57 (d, J 186.6 Hz, C 1 ), 87.11 (d, J 31.8 Hz, C 2 ), 62.05 (C 11 ), 46.48 (d, J 24.5 Hz, C 8 ), 42.39 (C 10 ), 33.93 (C 7 ), 28.95 (C 6 ), 25.71 (C 5 ), 20.67 (C 4 ). 19 To a solution of peracetyl lactose (2×100 mg, 0.157 mmol, 1 eq) in CH2Cl2 (1 ml) was added trichloroacetonitrile (Cl3CCN, 258 μl, 2.56 mmol, 20 eq) and DBU (65 μl, 0.384 mmol, 2.5 eq).

The reaction was stirred overnight, evaporated and by silica column chromatography (12%-14% acetone in toluene + 0.5% Et3N) to afford 65 (100 mg (1:3 α/β), 41% %). Rf (acetone/toluene/Et3N [20:80:0.5])=0.4. 1 H NMR (300 MHz, CDCl3) δ 8.65 (s, 1H, NH), 6.48 (d,J 3.7 Hz,1H, H 1 ), 5.55 (t, J 3-2, 3-4 9.7 Hz, 1H, H 3 ), 5.35 (d, J 4-3 3.0 Hz, 1H, H 4' ), 5.12 (dd, J 2-3 10.3, J 2-1 7.9 Hz, 1H, H 2' ), 5.05 (dd, J 2-3 10.1, J 2-1 3.8 Hz, 1H, H 2 ), 4.95 (dd, J 3-2 10.3, J 3-4 3.3 Hz, 2H, H 3' ), 4. 57 -4.40 (m, 2H, H 1' , H 6 ), 4. 20 -4.02 (m, 4H, H 5 , H 6 , 2H 6' ), 3. 92 -3.77 (m, 2H, H 4 , H 5' ),

2.15 (s, 3H), 2.10 (s, 3H), 2.06 (s, 6H), 2.03 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H). 13 C NMR (75 MHz, CDCl3) δ 170. [START_REF] Römer | Shiga toxin induces tubular membrane invaginations for its uptake into cells[END_REF]170.18,170.08,170.01,169.29,169.01,160.98 (C=NH), 101.20 (C 1' ), 92.91 (C 1 ), 90.70 (CCl3), 75.89 (C 4 ), 71.12 (C 3' ), 70.93 (C 5 ), 70.73 (C 5' ), 69.97 (C 2 ), 69.58 (C 3 ), 69.16 (C 2' ), 66.61 (C 4' ), 61.51 (C 6 ), 60.78 (C 6' ), 20.83, 20.76, 20.60, 20.46, 20.42. ν/cm -1 3311 (NH), To a solution of trichloroacetamidate 65 (95 mg, 0.122 mmol, 1 eq) and (1R,8S,9s)bicyclo[6.1.0]non- 22 mg,[START_REF]2-azidoethyl octadecyl phosphate (14) 210 1-Octadecanol (100 mg, 370 μmol, 1 eq) was dissolved in dry toluene (3 ml), and cooled to 0°C. To this solution, COP (82 μl, 481[END_REF]146 mmol, 1.2 eq) with activated molecular sieves (4Å) in freshly distilled CH2Cl2 (2 ml), after cooling to -25°C, TMSOTf (11 μl, 61 μmol, 0.5 eq) was added. The reaction was stirred at for 4h and quenched with Et3N, evaporated and purified by silica column chromatography (10 % acetone in toluene) to afford 66 (32 mg (β pure), 34 %). Rf (acetone/toluene [3:7]) =0.6. 1 H NMR (500 MHz, CDCl3) δ 5.34 (d, J 4-3 3.3 Hz, 1H, H 4' ), 5.19 (t, J 3-4,3-2 9.3 Hz, 1H, H 3 ), 5.10 (dd, J 2-3 10.3 Hz, J 2-1 7.9 Hz, 1H, H 2' ), 4.95 (dd, J 3-2 10.4 Hz, J 3-4 3.4 Hz, 1H, H 3' ), 4.88 (dd, J 2-3 9.5 Hz, J 2-1 8.1 Hz, 1H, H 2 ), 4. 53 -4.45 (m, 3H, H 1 , H 1' , H 6 ), 4. 17 -4.02 (m, 3H, H 6 , H 6' ), 3.92 (dd, J 7a-7b 10.6, J 7-8 7.3 Hz, 1H, H 7a ), 3.86 (t, J 6.9 Hz, 1H, H 5' ), 3.78 (t, J 9.5 Hz, 1H, H 4 ), 3. 60 -3.57 (m, 1H, H 5 ), 3.54 (dd, J 7a-7b 10.6, J 7-8 8.6 Hz, 1H, H 7b ), 2. 35 -2.15 (m, 6H, H 10 , H 11 ), 2.14 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 2.04 (s, 6H), 2.02 (s, 3H), 1.96 (s, 3H), 1. 57 -1.39 (m, 2H, H 10 ), 1. 35 -1.21 (m, 1H, H 8 ), 0. 96 -0.83 (m, 2H, H 9 ). 13 C NMR (126 MHz, CDCl3) δ 170.37, 170.16, 170.09, 169.84, 169.59, 169.11, 101.13 (C 1β ), 99.90 (C 1'β ), 98.92 (C 12a ), 98.71 (C 12b ), 76.37 (C 4 ), 72.93 (C 3 ), 72.58 (C 5 ), 71.78 (C 2 ), 70.99 (C 3' ), 70.68 (C 5' ), 69.13 (C 2' ), 66.97 (C 7 ), 66.59 (C 4' ), 61.99 (C 6 ), 60.77 (C 6' ), 29.17 (C 10a ), 29.10 (C 10b ), 21.42 (C 11 ), 20.87, 20.83, 20.75, 20.65, 20.52, 20.01, 19.93 (C 9 ), 18.06 (C 8 ). ESI-HRMS:

Calcd for [MNH4 + ] 786.3179. Found: m/z 768.3173. 6.48 (d, J 1-2 3.7 Hz, 1H, H 1 ), 5.56 (t, J 3-2, 3-4 9.8 Hz, 1H, H 3 ), 5.27 (d, J 4-3 2.7 Hz, 1H, H 4' ), 5.05 (dd, J 2-3 10.4, J 2-1 7.9 Hz, 1H, H 2' ), 5.00 (dd, J 2-3 10.3, J 2-1 3.8 Hz, 1H, H 2 ), 4.88 (dd, J 10.4, 3.4 Hz, 1H, H 3' ), 4.40 (d, J 2-1 7.9 Hz, 2H, H 1' ), 4.36 (m, 1H, H 6 ), 4. 12 -4.03 (m, 3H, H 6 , 2H 6' , H 5 ), 4. 03 -3.92 (m, 2H, H 4 , H 5' ), 3. 84 -3.73 (m, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.95 (s, 3H), 1.88 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 170. 43, 170.38, 170.27, 170.17, 169.21, 169.15, 168.85, 101.26 (C 1' ), 90.54 (CCl3), 87.61 (C 1 ), 75.88, 73.85, 71.12, 70.80, 70.78, 69.35, 69.21, 66.66, 61.63, 60.88, 20.96, 20.77, 20.61 Peracteyl lactose- 1-O-bicyclo[6.1.0]nonyne 66 (29 mg, 38 μmol) was solubilised in dry MeOH (1 ml) and CH3ONa (530 μl, 0.5M in MeOH) was added dropwise. The reaction was stirred for 1h30 before Dowex H+ was added, the reaction mixture filtered, concentrated and purified

Figure 1

 1 Figure 1.1.1: (a) Intracellular vesicular trafficking pathways 3 (b) Electron microscopy image of clathrin-coated pits in hypertonic fibroblasts, scale bar 100 nm 1

Figure 1 . 1 . 2 :

 112 Figure 1.1.2: Mechanisms of endocytosis. Dynamin-dependent pathways (+) are typically associated with

Figure 1 . 2 . 1 :

 121 Figure 1.2.1: Crystal structures of ricin, Shiga and cholera toxins 19 with their respective receptors: galactose (presented on GLs and glycoproteins), globotriaosylceramide (Gb3-Cer), or GM1-ceramide.

Figure 1 . 2 . 2 :

 122 Figure 1.2.2: Retrograde trafficking of the Shiga toxin 12

Figure 1 . 2 . 3 :

 123 Figure 1.2.3: (a) Crystal structure overlay of STxB, (green Protein Data Bank (PDB) 1BOS), CTxB (red; PDB 1FGB), VP1 protein (blue, PDB 3BWR) bound to GSL receptors (corresponding colour) 8 (b, d) Local curvature of STxB binding to 13-15 Gb3-Cer in phospholipid bilayer 23 (c) STxB binding, inducing local curvature, clustering and membrane invagination 8

Figure 1 . 2 . 4 :

 124 Figure 1.2.4: Giant unilamellar vesicles (GUVs) showing membrane bending induced by lectins and GSLs. (a) DOPC/cholesterol/porcine Gb3-Cer/BodipyFL-C5-HPC (green) [64:30:5:1] incubated with STxB-Cy3 (red); (b) PC/cholesterol/sphingomyelin/PS/GSL-mix/Nickel-NTA-DOGS/lissamine-rhodamine-PE (0.1mol%) [56.9:20:12:3:5:3:0.1] incubated with His-tagged galectin Gal3-His 31

Figure 1 . 3 . 1 :

 131 Figure 1.3.1: (a) Structures of Gb3-ethyleneglycol (EG)n-Cer analogues, n=1,3,7 (b) quantification of GUV experiments showing that long, flexible spacer region inhibits tabulation (c) Suppression of membrane fluctuation induces clustering of macromolecules bound to lipid bilayer. (d,e) Course-grain simulations of pentameric toxin-like nanoparticles bound either tightly (d, no spacer) or loosely (e, flexible spacer).24

  concentrated into microscale domains (Figure 1.3.2b), and 18 O-cholesterol evenly dispersed in the PM (Figure 1.3.2c), contesting the existence of nanoscale lipid rafts. These findings correlate with recent superresolution fluorescence microscopy performed with fluorescent analogues.49,50

Figure 1 . 3 . 2 :

 132 Figure 1.3.2: Scanning electron microscopy (a) and highresolution SIMS images (b,c) of a representative clone 15 fibroblast cell. (b) 15 N-sphingolipids are enriched in micron scale domains on the PM, (c) whereas 18 Ocholesterol showed no enrichment.

  Figure 1.3.3: GUVs with 5mol% Gb3 analogue 1 (a,b) or 2 59 (c,d) or wild type Gb3-Cer (Figure 1.2.1) 58 (e) (DOPC/SM/Chol/Gb3-Cer [40:35:20:5] with <1% fluorescent phospholipid (green, Ld), incubated with STxB-Cy3 (red).

Figure 1 . 3 . 4 :

 134 Figure 1.3.4: GM95 cells incubated with Gb3 commercial mix (Matreya) with bovine serum albumin (BSA) (20 μM) (a) or without (b), then incubated with STxB-Alexa488 to observe surface binding (4°C, 30 min). Short chain Gb3 C18:1 shows binding at 4°C (c) and uptake but misdirected traffic at 37°C (d). (e) When internalised on natural Gb3, STxB is transported to the Golgi and colocalises with CTR433, a Golgi marker.

Figure 1

 1 Figure 1.4.1: Stepwise synthesis of GSLs from ceramide (Cer). Knockout of pathways shows phenotypes in early development, myelin (nervous system) or neuronal function. Modified from D'Angelo et al. 82

Figure 1 . 4 . 2 : 86 BFigure 1

 142861 Figure 1.4.2: Sphingolipid biosynthesis, modified from S Merscher et al.86

Figure 1

 1 Figure 1.5.1: Cellular incorporation and traffic of antibody-drug conjugates (ADCs) and STx and its conjugates

Figure 1 . 6 . 1 :

 161 Figure 1.6.1: Tagging a biomolecule and analysing its cellular localisation or metabolism products using a reporter molecule, often fluorophore or biotin affinity tag.

Figure 1 . 6 . 2 :

 162 Figure 1.6.2: (Bio)orthogonal chemical conjugations. (a) Disulfide exchange and maleimide conjugations for cysteine residues and N-hydroxysuccinimides and iso(thio)cyanate and iso(thio)cyanates for lysine residues. (b) Oxime/hydrazine with aldehyde/ketones.

Figure 1 . 6 . 3 :

 163 Figure 1.6.3: Bioorthogonal chemical conjugations (a) Staudinger and (b) traceless Staudinger ligations. (c) ManNAc, Natural sialic acid precursor. Synthetic analogues ManLev and Ac4ManNAc, used for metabolic labelling of sialic acid-containing glycoproteins and glycolipids. 117,130

Figure 1

 1 Figure 1.6.4: (a) Copper-catalysed azide-alkyne cycloaddition (CuAAC). (b) Cu-chelating ligands for CuAAC; TBTA: R, R'=CH2Ph; THPTA: R, R'=(CH2)3OH; BTTAA: R= t Bu; R'=CH2CO2H.

  to the cyclooctyne.144 Chemical evolution of click reagents has led to studies on nitrones, nitrile oxides and sydnones, which react quicker than azides with cyclooctynes (Figure1.6.6 a-c). For example, strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions demonstrate rate constants up to 60 M -1 s -1 . 147 However, their syntheses are not commonly mastered and their use as tags in biomolecules can prove more cumbersome than the classic azide. Nitrones were stable enough to efficiently label large proteins in live cells without degradation. 149 However, nitrone-labelled glycans fared less well, as the nitrone modification was not accepted into the biosynthetic pathway.150 Inverse electron demand Figure 1.6.6d, e) conjugations come out ahead as the quickest bioorthogonal reactions, but leave a remarkably large scar in the structures of modified biomolecules.

  Ac4GalNAz for spatiotemporal labelling of zebrafish embryo morphology (Figure1.7.1), displaying early (red) and late (green) gestational post-translational glycosylation, using a fluorophore functionalised with difluorocyclooctyne (DIFO, Figure 1.6.5b). Wang et al.showed that Ac3ManNAz could be coupled with a tumour-selective self-immolative linker in the anomeric (C1) position to specifically label tumour tissue in mice.154

Figure 1

 1 Figure 1.7.1: Zebrafish embryo labeled with Ac4GalNAc. DIFO-647 (red) click 60 hours postfertilisation (hpf), then DIFO-488 click 62 hpf after an additional 1h pulse of Ac4GalNAc labelling. Scale bars 100 μm (A), 10 μm (B).

Figure 1 . 7 . 2 :

 172 Figure 1.7.2: Clickable choline analogues, (a) propargylcholine 155 and (b) 1-azidoethylcholine, 156 are metabolised into phosphatidylcholine (PC), ether phospholipids (ePC) and sphingomyelin (SM). (c) Propargylcholine treatment (100 μM) shows similar profile of choline-containing lipids by ESI-MS/MS analysis. (d) Alkyne-lipids undergo CuAAC click reaction with alkyne-fluorophore (fluorescence microscopy image).

Figure 1 . 7 . 3 :

 173 Figure 1.7.3: Clickable lipids. (a,b) Fatty acids, 158,159 (c) alkyne-oleoyl-lysophosphatidic acid (d) ω-alkynesphinganine (e) palmitoyl-lyso-propargyl-phosphatidyl-choline. 161 (f) Golgi-specific transcyclooctene ceramide. 165

Figure 1 . 7 . 4 :

 174 Figure 1.7.4: Clickable glycolipids localise in intracellular cellular membranes (a, analogue 1) and PM (b, analogue 2). Short chain LacCer analogues 47 and 48 localised to the PM. Analogues 3 and 49 were incorporated using fusogenic liposomes, localising in the PM.

Figure 1 . 7 . 5 :

 175 Figure 1.7.5: Photocrosslinkable groups, benzophenone (a), 3-phenyl-3-(trifluoromethyl)diazirine (b), and aryl azide (c) with their reactive radical forms. 167 (d) Confirmed photoactivatable sphingosine probe; protocol involves live cell incubation, UV light activation, extraction and click with a fluorophore for visualisation, or biotin-streptavidin beads for purification and mass spectrometry characterisation. 170

Figure 1 . 8 . 1 :

 181 Figure 1.8.1: GSL click reconstitution. Sphingolipid precursor, 1-azidosphingosine (AzSph) is natively metabolised to 1-azidoceramide (AzCer), ideally presented at the PM to be clicked with the prepared clickable oligosaccharide (Gb3-MFCO) to form the final Gb3 analogue. (Lipid membrane not to scale)

Figure 1 . 8 . 2 :

 182 Figure 1.8.2: (a) Balance between reactivity and specificity of click partners, with the biological functionality of final clicked product. (b) Gb3-EGn-Cer analogues able to bind Shiga toxin in liposome models, whilst only

Scheme 2 . 1 . 1 . 1 : 6 .

 21116 Scheme 2.1.1.1: Biostable chemical conjugations

Figure 1 . 8 . 1 ,

 181 Scheme 2.1.2.1: 1-Azidosphingosine (AzSph) synthesis.

Scheme 2 . 1 . 2 . 5 :

 2125 Scheme 2.1.2.5: Ceramide precursors: sphingosine (CerS5 substrate) and sphinganine (CerS1-6 substrate); plant-derived phytosphingosine, and its azide-functionalised analogue.

38 .

 38 Scheme 2.1.3.1: Final clicked Gb3-AzCer analogues.

Scheme 2 .

 2 Scheme 2.1.3.3: One-pot peptidic coupling and deprotection to formMFCO-Gly.

Scheme 2 . 1 . 3 . 4 :

 2134 Scheme 2.1.3.4: Clickable lactose model synthesis.

2. 2

 2 Biological validation of analogues 2.2.1 Model membrane validation We conducted preliminary validation of the biological function of final clicked Gb3 analogues by testing their binding and membrane bending properties with the B-subunit of the Shiga toxin (STxB) (Introduction 1.2). These mechanochemical properties of the GSL analogues are traditionally tested in model membrane systems called giant unilamellar vesicles (GUVs), or liposomes. This robust technique shows spontaneous, dynamic tubule formation of STxB bound to the natural Gb3 in the liposome bilayer, indicating that GL-Lect dependant endocytic interactions could occur in a cellular environment. We use this technique to test each final synthetic product. To follow the process by fluorescence microscopy, we use STxB-Alexa488, where the green fluorophore (Alexa488) is conjugated via a lysine-specific sulfodicholorphenol (SDP) functionality. A red fluorescent phospholipid, Rhod-SDP-PE, is used to stain the liposome (Figure 2.2.1.1).

Figure 2 .

 2 Figure 2.2.1.1: Canonical STxB-Cy3(wild type, red) binding and invagination of membrane in GUVs (64 mol% DOPC; 30mol% chol; 5mol% porcine Gb3; 1mol% BodipyFL-C5-HPC (green)). 27 Schematic structures of STxB bound with 15 Gb3, 193 STxB-Alexa488 with Gb3 binding sites, and Lissamine Rhodamine phosphoethanolamine (Rhod-SDP-PE).

   Chinese hamster ovary (CHO) cells were incubated with AzSph to study its cellular metabolism. Cells were incubated with AzSph (10 μM) in media for various lengths of time. The cells were then lysed, the lipids extracted, and the azide-metabolites clicked with a fluorophore (Alexa488-alkyne and copper catalyst, or tetramethyl-rhodamine (TAMRA)-dibenzocyclooctyne (DIBO), Experimental 6.2.3). This reaction mixture was spotted and migrated on high performance thin layer chromatography (HPTLC) plates, and visualised with a fluorescent plate reader. When possible, samples were compared with pure reference AzSph and AzCer previously clicked in vitro with the fluorophore (Alexa488-AzSph and -AzCer, or TAMRA-AzSph and -AzCer). AzCer from cell lysate samples was often below detection limit on the fluorescent plate reader after HPTLC migration. We obtained better results with the more lipophilic (fast migrating) clickable fluorophore TAMRA-DIBO. In one experiment (Figure 2.2.2.1)fluorescent band of reference compounds AzSph and AzCer are visible. However, no band is observable in the treated cells that could correspond to clicked AzCer. Figure 2.2.2.2 shows bands which would specifically correspond to TAMRA-AzCer in treated cells. After scanning the plate, the fluorescent bands were scratched off to isolate silica. Lipid species were extracted from the silica in a dichloromethane/methanol mixture and analysed by MALDI-MS. By this method, exact masses of the AzSph and AzCer synthetic reference compounds were observed, thus confirming their migration properties. In MALDI-MS, we were able to see the TAMRA-AzCer (C16:0) formed (Figure 2.2.2.3), showing that the C16:0 chain is enzymatically added to AzSph in cells with high preference (Discussion 3.4).

Figure 2 .

 2 Figure 2.2.2.1. Incubation of AzSph (10 μM, 3h) in GM95 cells, allowing cellular AzCer synthesis. Reference compounds indicate migration properties. Cannot observe AzCer in treated cell lysate.

Figure 2 .Figure 2

 22 Figure 2.2.2.2: Time course of AzSph treated cells. We observe TAMRA-AzCer in a specific manner in AzSph treated cells.

Figure 2 .

 2 Figure 2.2.2.4: MTT reaction occurring in living cells producing strong purple formazan.

2 %

 2 :0 C16:0 C16:1 C17:0 C18:0 C20:0 C22:0 C22:1 C23:0 C23:1 C24:0 C24:1 C24:

  (IF) experiments were also performed on HeLa cells(Figure 2.2.2.5) and CHO cells, and all three cell types showed a similar staining in all intracellular membranes, in the ER and Golgi. Live cell imaging showed similar co-localisation withER-and Golgi-labelling by transfection of CHO cells with Golgi and ER markers, galactosyltransferase-GFP and RDEL-HALO. The staining was relatively weak at the plasma membrane (PM) where the click reaction would ideally occur, but we considered that constant recycling of membranes by vesicular transport would allow temporary presentation of AzCer at the PM, which could undergo click reaction.

Figure 2 .

 2 Figure 2.2.2.5: AzSph treated HeLa cells (5 μM, 1h), fixation, Alexa488-alkyne (2.5 μM) and copper (0.4 μM), imaging by confocal microscopy.

Figure 2 .

 2 Figure 2.2.2.6: GM95 cells, AzSph (0.5 μM, 8h), click with TAMRA-DIBO (0.5 μM), fixation, confocal imaging.

49 Figure 2 . 2 . 3 . 1 :MFCO 49 , 2 66 Figure 2 . 2 . 3 . 2 : 4 ,

 4922314926622324 Scheme 2.2.3.1: In cellulo click protocol. Cells incubated with AzSph, followed by cyclooctyne, lysed, extracted and analysed by HRMS (Experimental 6.2.5).

Figure 2 8 :

 28 Figure 2.2.3.3: Ac-Lac-MFCO-AzCer 68 and Ac-Gb3-MFCO-AzCer 69.

Figure 2 . 2 . 3 . 4 :

 2234 Figure 2.2.3.4: Ac-Lac-

Graph 2 . 2 . 3 . 10 :

 22310 Ac-Lac-MFCO-AzCer lipid profile of cells treated with AzSph (0.5 μM, 8h), then sequential or co-treatment with Ac-Lac-MFCO (15 μM) and pig liver esterase.

Graph 2 . 2 . 3 . 13 :

 22313 Sphingolipids and GSLs present, as a molar % of all lipids in cells treated with AzSph (

Scheme 2 . 2 . 4 . 1 :

 2241 Scheme 2.2.4.1: Ceramide precursors: sphingosine (CerS5 substrate in salvage pathway) and sphinganine (CerS1-6 substrate in de novo pathway), with possible azidolipid analogues.

3 :Figure 2 . 2 . 4 . 1 :

 32241 Figure 2.2.4.1: CHO cells treated +/-AzPhy or AzSph (1 μM, 6h), followed by Alexa488-DIBO (1 μM, 16h, green) or TAMRA-DIBO (1 μM, 16h, red), fixed, permeabilised and imaged by epifluorescence.

  to the receptor function, as shown by Pezeshkian et al., where Gb3-spacer-Cer analogues with spacer lengths >1 nm inhibited STxB clustering, curvature generation and trafficking into cells (Introduction 1.

glycero- 3

 3 Scheme 3.1.1) occupy less ordered regions of the lipid bilayer. Despite theoretical reasoning that phase separation of phospholipids and sphingolipids in the biological membrane may inhibit receptor function of an FSL construct, the above-mentioned study by Ewers et al. on a

Scheme 3 . 1 . 1 :

 311 Scheme 3.1.1: Natural Gb3-Cer; synthetic Gb3-phospholipid analogue, FSL construct; common phospholipids DOPC and DOPE.

Scheme 3 . 1 . 4 :

 314 Scheme 3.1.4: Clickable phospholipid analogue models successfully isolated and characterised.

Figure 3 . 2 . 2 :

 322 Figure 3.2.2: Evolution of alkyne reagents for strain-promoted azide-alkyne cycloaddition. 150

181 to sphingosine 6 ( 6 (

 66 Scheme 3.2.3: Envisageable alternatives to our AzSph synthesis.

Scheme 3 . 2 . 4 :

 324 Scheme 3.2.4: Garrido Martinez synthesis to1-azidosphingosine. 184

  into 1-azidoceramide (AzCer) in cells via acylation by ceramide synthase (CesS) enzymes. We first analysed azide metabolites by clicking with an alkyne-functionalised fluorophore in vitro, after cell treatment and lysis. This method, pioneered by Thiele et al., involves separating the clicked biological sample, based on their polarity, on high performance thin layer chromatography (HPTLC) plates. This normally involves a purification step on the cell lysate; either protein precipitation 159 or lipid extraction, 219 to purify lipid components from aqueous metabolites, proteins and genetic molecules present in cells. We observe a specific band for AzCer clicked with a fluorophore (Fig 3.4.1) without carrying out an extraction or purification step, but by simply resuspending cell lysate in methanol. Only once we began performing a lipid extraction step (Experimental 6.2.3), were we able to observe cellular AzCer by mass spectrometry (MS) from extracted silica of HPTLC plates or directly after extraction. Testing began with the hydrophilic fluorophore Alexa488-alkyne, and a specific, fast-migrating band which corresponded to AzCer-Alexa488 click product were observed by HPTLC (Figure 3.4.1). The migration properties of click product AzSph-Alexa488 was very similar to unclicked fluorophore and difficult to distinguish. With hydrophobic, copper-free clickable TAMRA-DIBO, we could distinguish AzCer and AzSph metabolites (Figure 2.2.2.1). Many reports use a hydrophobic clickable coumarin. We performed all HPTLC experiments with high concentrations of AzSph (10 μM) due to the extremely poor resolution of HPTLC plates. To analyse the molar mass of azide-lipid species, silica from HPTLC plates was scratched off in specific regions, extracted in chloroform/methanol and analysed by MALDI-MS. Large quantities were required likely due to the poor ionisability of the amphiphilic, zwitterionic fluorophore-clicked lipid. Cellular testing at 10 μM of AzSph induced remarkable cell death after 6h (Fig 3.4.1).

  However, Collenburg et al. showed that ceramides modified in the lipid chains were non-toxic in T cells, up to 25 μM for 2h. 162 Haberkant et al. tested the bifunctional sphingolipid, photoSph (Fig 3.4.2), at 0.1 μM, mentioning that no loss in cell viability is observed from 0.1 to 5 μM. 220 They cite the role of ceramide in apoptosis signalling as a reason for this toxicity.

Figure 3 . 4 . 1 :

 341 Figure 3.4.1: Incubation of AzSph (10 μM) on GM95 cells shows formation of AzCer. Cytotoxicity beyond 6 h. HPTLC separation of azido-metabolites clicked with alkyne-fluorophore (Cu(I), 3 mM) following lipid extraction.

Figure 3 . 4 . 2 .

 342 Figure 3.4.2. Photoactivatable sphingosine, photoSph, investigated by P. Haberkant et al., is converted into photoCeramide within 5 min, and photoSphingomyelin (SM) and trisaccharide glycosphingolipid GM3 at longer incubation times. 220 Visualised by spraying with 3 H-specific Amplify Fluorographic Reagent.

Figure 3 . 4 . 3 :

 343 Figure 3.4.3: Lipidome of MDCK cells in the endothelial (red) and mesenchymal (blue) states.80

  reagent. In comparison, click reaction was efficient with hydrophobic clickable fluorophore TAMRA-DIBO. This was observed by a major consumption of cellular AzCer and appearance of the clicked product TAMRA-DIBO-AzCer. To confirm the lipid extraction of the clicked glycolipid, we synthesised in vitro purified Gb3-and lactose-. These were used as internal standards, spiked into biological samples before the lipid extraction, to ensure that the click product is extracted and analysed by HRMS by our protocol. Since we observe the Gb3-and lactose-MFCO-AzCer (C22:1) internal standards after extraction, we can confirm that our extraction protocol is adapted to the desired glycolipid products, and that cellular click reaction forming lactose-MFCO-AzCer is inefficient since no cellular product is observed.We had hypothesised that, although AzCer may be predominantly present in intracellular membranes, it would cycle to the plasma membrane (PM) by vesicular trafficking in order to undergo click reaction at the cell surface(Fig 3.5.1a). Since we observed no click reaction with hydrophilic, non-permeating cyclooctynes (lactose-MFCO, Alexa488-DIBO), we suggested that very little AzCer is present at the PM. This corresponds with the commonly accepted localisation of natural ceramide, synthesised in the ER, glycosylated in the Golgi and thus depleted at the PM.

  to specifically stain AzSph and AzPhy in IF experiments on live CHO cells(Fig 3.5.1b). Such experiments were performed with overnight incubation of fluorophore, indicating that the AzCer recycles to the PM overnight to click with extracellular fluorophore. Alternatively the fluorophore could penetrate to the intracellular AzCer. Alexa488-DIBO click product was not observed in HRMS experiments because it is too hydrophilic to be extracted in our lipid extraction protocol, not because it was not taken up or clicked. After click at the surface, the click product is endocytosed to intracellular membranes, as likely occurs to the small percentage of the Gb3-MFCO-AzCer product. This would explain why no STxB binding is observed.

Figure 3 . 5 . 1 :

 351 Figure 3.5.1: (a) Recycling of azide-lipid to the PM (b) IF experiment of ±AzSph (6h), Alexa488-DIBO (16h) (Results Figure 2.2.4.1).

Figure 3 .

 3 Figure 3.5.1: (a) Conversion of aspirin to salicylic acid. (b) Metabolic labelling of clickable peracetylated alkyne-derivatived glycan, incorporated into the native glycoprotein biosynthesis and clicked to be isolated via biotinylated azide. 238 (c) Quantification of surface azides by mean fluorescence intensity (MFI) of splenocytes from mice deficient in plasma Ces1 (triangles) or wild-type B6D2F1 mice (circles, males; squares, females) treated with Ac4ManNAz. 127

1 :

 1 Native ceramide chain length and the specificity of ceramide synthase (CerS) enzymes. Garrido et al. studied the metabolism of three ω-alkyne sphingolipids 1-3 (Figure 3.6.2), 208 analogues of sphingosine, sphinganine and ketosphinganine. In the natural biosysnthesis, ketosphinganine is reduced to sphinganine by 3-ketosphinganine reductase (3-KSR) (Introduction 1.4). ω-Azidosphinganine 2 and ω-azidoketosphinganine 3 show a slighter higher percentage of long C24:1 chains compared with ω-azidosphingosine 1, indicating they are better substrates for CerS enzymes using longer CoA-acyl chains. Likewise, Gaebler et al. described major transformation of ω-alkyne-sphinganine (Figure 1.7.3) to C24:1 dihydroceramide in liver microsomes, highly expressing CerS2, and moderate to weak acylation with other acyl chains in CerS2-suppressed tissue samples. 161

Figure 3 .

 3 Figure 3.6.2: ω-Azidosphingolipid probes and their acyl chain profiles (6h, 5 μM), later converted to 1phosphated and SM analogues.208

Figure 4 . 1 :

 41 Figure 4.1: Mono-and disaccharide uptake into CHO cells depending on level of hydrophobicity. Secondary readout for glycoside uptake based on inhibition effect of naphthol-glycosides.

Figure 4 . 2 :

 42 Figure 4.2: Other potential azidosphingolipids could produce different localisation of azide labelling. Neutral fluorophore FR4 localises intracellularly, whereas analogous zwitterionic FR5 localises to the PM. 242

Figure 4 . 3 :

 43 Figure 4.3: Potential enzymatic synthesis of lactose-MFCO 49 into GM3-MFCO.

Figure 4 . 4 :

 44 Figure 4.4: Gb3 analogue 36, synthesised in 15 convergent steps (27 steps), and the envisageable photocrosslinkable Gb3 analogue to study native Gb3 interaction partners.

1 .

 1 have synthesised novel clickable biomolecule analogues and tested their validity in model membranes and cell culture. Two different glycan-cyclooctyne head groups, Gb3-MFCO and Gb3-Gly-MFCO, when clicked with 1-azidoceramide, show promising model membrane activity when bound to fluorescent STxB. This indicates that the proposed structural modification in the spacer region between glycan head and lipid tail is tolerated in biological function. The synthesis of glycan-cyclooctynes can be applied to other glycans and bioorthogonoal click reagents for the study of other GSLs. Similarly, the synthesis of 1azidosphingosine and 1-azidoceramide can be extended to other azido-sphingolipid analogues for a varied metabolism profile. We have shown efficient metabolic labelling of sphingolipids using clickable 1azidosphingosine, which is rapidly incorporated into cells and metabolised into 1azidoceramide. 1-azidoceramide localises in intracellular membranes, thus click reaction with non-cell-permeating reagents such as glycan-cyclooctynes is poorly efficient. 1-Azidoceramide undergoes specific and efficient bioorthogonal click reaction with permeable, peracetylated glycan-cyclooctynes. We have quantified this in cellulo click reaction by HRMS lipidomics. Endogenous deacetylation of clicked glycolipid analogues is inefficient in two cells lines tested. Several pathways to change the localisation of lipid or glycan parts are available, and should permit the reconstitution of a biologically functional glycolipid analogue into cells. With this technique in hand, one can envisage a precise and efficient reconstitution of long chain glycolipid analogues for the study of GL-Lect endocytosis. Lipid mixture made up in prewashed glass vial and spread onto indium tin oxide (ITO)

 1 -

 1 Wash 1x PBS and 2x ammonium bicarbonate solution (150 mM) at rt  Scratch cells in 1 mL ammonium bicarbonate solution at 4 °C (6 wells per condition)  Centrifuge 5 min at 4700 RPM at 4 °C  Remove supernatant  Lipid extraction  Resuspend pellet in 200 µL H2O  Add 1000 µL CHCl3/MeOH (10 :1) Vortex 75 min  Centrifuge quickly in small benchtop centrifuge  Keep supernatant  Dry the solvent  Re-suspend in 100 μL of CHCl3/MeOH/H2O (50:40:5) Click reaction (2h in Eppendorf shaker at 800 RPM, 25 °C) Cell lysate/reference (150 µL; references at 50 μM, 300 µL)  2 -Alexa/TAMRA (100 μM final, stock 1.25 mM)  3 -Cu(I) (2 mM) (Cu(BF4)) (100mM)  4 -45 μL EtOH (volume total=100ul)  Spot supernatant mix (20 μL) on HPTLC plate (10 cm×10 cm) and dry 10 min under vacuum or with heat gun  Elute TLC in cuvette (CHCl3/MeOH/H2O 50:40:10) and scan

  -sn-glycero-3-phosphoethyl-N'-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl)thiourea (2) 177

  2S,3R)-(E)-2-azido-3-O-benzoyl-1-O-tert-butyldiphenylsilyl-4-octadecene-1,3-diol (22) 248

3 h at 0

 0 °C to rt, quenched with H2O(40 ml) and stirred for a further 20 min. The reaction mixture was extracted with CH2Cl2 (3×50 ml), washed with HCl (1 M, 100 ml), saturated NaHCO3 solution (100 ml) and brine (100 ml), dried (MgSO4), evaporated and purified by silica column chromatography (20 -10% EtOAc in cyclohexane) to afford 22 (2.01 g, 87 %). Rf (cyclohex:EtOAc [10:1])=0.8; [α] 22 D=-9.0° (c 7.0, CHCl3); 1 H NMR (300 MHz, CDCl3) δ 8.03 (d, J

(C 18 )

 18 ; ESI-MS 512.4 (M-H -); ESI-HRMS: Calcd for [MH + ] 514.2490. Found: m/z 514.2485.

5 (

 5 (C 1 ); ESI-MS 641.MNH4 + ); ESI-HRMS: Calcd for [MNa + ] 646.1327. Found: m/z 646.1322.

35 Compound 41 ( 1 .

 35411 02 g, 0.673 mmol) was dissolved in acetic acid(20 ml) and Pd/C (10wt%, 400 mg) was added. Dihydrogen gas was bubbled through the solution then the reaction mixture was stirred for 1 hour in a dihydrogen atmosphere at rt for 1 h, then at 80°C for 2 h.The reaction mixture was filtered through Celite and washed with methanol and evaporated to obtain a white powder. The crude debenzylated triose-MP was solubilised in acetic anhydride/pyridine (1:2) and stirred overnight. The reaction mixture was diluted with CH2Cl2, washed with HCl (1M, 3×150ml), dried (MgSO4), evaporated and purified by silica columnThe peracetylated globoside 42c (400 mg, 0.41 mmol) and SnCl4 (1.0 ml, 1M in CH2Cl2) were dissolved in anhydrous CH2Cl2 (5 ml) and stirred at RT. After 5 min, TMSN3(50 μl, 0.50 mmol) was added, and the reaction mixture was stirred at rt for 48h. The resulting solution was washed with NaHCO3 and extracted with CH2Cl2. The organic phase was washed with water, dried over MgSO4, and concentrated in vacuum. The crude product was purified by flash chromatography on silica gel (cyclohexane/EtOAc, 85/15) to afford 43 (217 mg, 56%) as colourless foam. Rf (EtOAc/cyclohexane[75:25])=0.7; [α] 22 D=+38.0 (c 3.0, CHCl3); 1 H NMR (

  ESI-HRMS: Calcd for [MH + ] 494.2038. Found: m/z 494.2032 N-Glycine-1-fluoro-cyclooct-2-yne-1-carboxamide (46) 148

t

   0.38 mmol, 1.2 eq). The reaction was stirred overnight, diluted with CH2Cl2, evaporated and extracted in EtOAc, washed with water. The partially protected material is fully deprotected in a solution of trifluoroacetic acid (TFA)/triisopropylsilane (TIS)/CH2Cl2 (5:5:90) for 4h, then dried and purified by silica column chromatography (EtOAc/cyclohexane/formic acid[25:75:0.5 to 30:70:0.5] to afford 46 as a racemic mixture (34 mg, 59 %). Rf (EtOAc/cyclohexane/AcOH [50:50:0.5])=0.2. 1 H NMR (500 MHz, CDCl3) δ 8.84

  F NMR (282 MHz, CDCl3) δ -146.70 (s). ESI-HRMS: Calcd for [MH + ] 228.1036. Found: m/z 228.1030.Compound 46(6 mg, 26 μmol, 1 eq), heptaacetyl-lactose-β-NH2 52(17 mg, 31 μmol, 1 eq), HOBt(6 mg, 40 μmol, 1.3 eq) and DCC(19 mg, 77 μmol, 3 eq) were stirred in dry DMF(1 ml) for 3h at 30°C. The crude reaction mixture was evaporated and purified by silica column chromatography (40 to 80 % EtOAc in cyclohexane) containing some dicyclohexylurea impurity. This was removed by precipitation in Et2O to afford 54(4.5 mg, 50 %). Rf (EtOAc/cyclcohexane[3:1])=0.4. 1 H NMR (400 MHz, CDCl3) δ 6.91 (s, 1H, NH),6.59 (dd, J 9.0, 4.4 Hz, 1H, NH), 5.35 (d, J 3.0 Hz, 1H, H 4 ), 5.30(t, J 9.0 Hz, 1H), 5.18 (t, J 9.3 Hz, 1H), 5.10 (dd,

19 F

 19 NMR (471 MHz, MeOD) δ -148.36. ESI-HRMS: Calcd for [MH + ] 713.2781. Found: MFCO 47 (0.8 mg, 0.7 μmol) and 1-azidoceramide 30 (0.5 mg, 0.78 μmol) were stirred in CH2Cl2 for 48h. The solvent was evaporated and the racemic product (R/S) was purified by silica column chromatography (0 to 2.5% MeOH in CH2Cl2) to obtain the isomeric clicked 69 (1 mg, 94%) and 1-N-MFCO-Gb3 (0.1 mg) recuperated. Rf (2.5% MeOH/CH2Cl2)=0.3. ESI-HRMS: Calcd for [M+H + ] + 1720.9585, [M+H + +NH4 + ] +2 869.4962. Found: m/z 1720.9589 and 869.4965.N-2-hydroxyethyl-1-fluoro-cyclooct-2-yne-1-carboxamide (59)Stable intermediate 58 (Scheme 2.1.3.7) (9 mg, 49 μmol, 1 eq) was dissolved in THF (240 μl) and cooled to 0°C, to which freshly distilled ethanolamine in solution with THF (50 μl, 1:3) was added dropwise. The reaction mixture was stirred for 4h, evaporated and purified by silica column chromatography (EtOAc/cyclohexane[1:1]) to afford 59 as a racemic mixture (3 mg, 29%) Rf (EtOAc)=0.5. 1 H NMR (300 MHz, CDCl3) δ 6.80 (s, 1H, OH), 3.77(t, J 4.9 Hz, 2H, H 11 ),

  1744 (C=O), 1676 (C=N), 1368 (C-H), 1213 (C-O), 1044 (C-O), 732 (C-Cl). ESI-HRMS: Calcd for [MNH4 + ] 797.1336. Found 797.1334.

  Amide decomposition impurity 67 (15 mg, 16%) closely followed 66 in chromatography purification. (Rf (acetone/toluene[3:7]) =0.5. 1 H NMR (500 MHz, CDCl3) δ 8.44 (s, 1H, NH),

  . UPLC-MS (gradient 90-100% CH3CN/H2O, 4 min, ESI) rt=1.14; 780 [MH + ].

Table of contents

 of Abbreviations……………………………………………………………………………………………………………..……………. 1 INTRODUCTION ................................................................................................................................ 1.1 Endocytosis mechanisms ........................................................................................................

	1.2

  1.6 Bioorthogonal conjugations and click chemistryChemical biology uses chemical techniques to effect or probe biological systems at the molecular level. This includes the development of fluorophores, small-molecule protein

	inhibitors/activators, non-natural amino acids and any other synthetically modified
	biomolecule. Genetic tagging of proteins, for example, with green fluorescent protein (GFP),
	has significantly promoted research in molecular biology. However, smaller chemical
	modifications are needed for smaller metabolites, lipids, carbohydrates and post-
	translational modifications. Although chemical synthesis may never attain the complexity of
	nature's highly regulated protein factory, progress is constantly being made in establishing
	better, subtler and more specific manipulation and higher-resolution study of the cellular
	system.

Current techniques for the study of lipids and GSLs are limited. Stable isotope labelling should not disrupt the biochemical interactions of the molecule under study, but incorporation is often low yielding, requiring technically complicated protocols for low sensitivity and high costs, and topological imaging techniques able to localise stable isotopes are still developmental (secondary ion mass spectrometry, SIMS, section 1.3). Radioisotope labelling can be localised but is innately hazardous. Mass spectrometry (MS) coupled to liquid chromatography (LC-MS) has traditionally been used for exact identification for the brute structure of the lipidome, all of the lipids present in a biological sample. Liquid chromatography separation of analyte species allows higher signal to noise ratio, but is not quantifiable. Now, shotgun lipidomics is coming to dominate the field, and is applied in this

  139 For example, Cairo et al. showed that they could induce cell adhesion-like properties in alkyne-lipid labelled Jurkat (a human T lymphocyte line) cells when clicked with azide-coated beads and L-histidine ligand. 140Besanceney-Webley et al.

	developed BTTAA (Figure 1.6.4b), which they showed chelates copper to catalyse the cell
	surface click reaction with azide-labelled sialic acids (SiaNAz) in 3 minutes. This is fast enough
	that copper could be used without significant cell death in Jurkat and HEK cells. They cite the
	use of 30 µM Cu(I), [ligand]/[Cu]=6:1, where 30 µM Cu(I) and no ligand shows no significant
	cell death, and yet 50 µM killed 90% of cells in the same time. 141

  Hang et al. first investigated the metabolism of clickable fatty acids in proteins by Western blotting, 158 whereas Thiele et al.

	developed a lipid extraction technique which would permit the analysis of clickable lipid
	metabolism by thin layer chromatography (TLC). 159,160 Clickable ω-alkyne-oleoyl-
	lysophosphatidic acid, ω-alkynesphinganine and palmitoyl-lyso-propargyl-phosphatidyl-
	choline (Figure 1.7.3c-e) displayed canonical metabolism in cells. 161 Enzyme affinity (Km)
	measurements of such substrates showed no significant difference in binding affinities with
	native biosynthesis enzymes compared with natural lipids. 161 Localisation of clickable lipids,
	clicked to a fluorophore with copper-catalysed or copper-free 162,163 protocols, seem to reliably
	demonstrate, with negative controls, that fluorescent labelling is specific to the lipid
	analogue. 164 Most often the clicked lipid is lodged non-specifically in plasma membrane and
	intracellular membranes, as is conceptualised for natural lipids. One less subtle modification
	in a ceramide analogue with trans-cyclooctene in the lipid tail (Figure 1.7.3f) provides the lipid
	strong Golgi specificity. 165

  We investigated the unusually slow Staudinger reduction (deprotection) of azide 22 to amine 23 (Scheme 2.1.2.4) via the stable phosphazene intermediate, (MH + 902, δ NMR (H 2 )=3.54 ppm), which breaks down overnight to 23 (δ NMR (H 2 )=3.16 ppm) and the NHBz

Scheme 2.1.2.2: Side reactions observed in synthesis of AzSph. Chemical and enzymatic acylation of AzSph 17 with erucic acid (C22:1) to form AzCer 30 (Scheme 2.1.2.3) were trialled. Standard peptidic coupling conditions: EDC and pentafluorophenyl-trifluoroacetic acid (PFP-TFA) with DIPEA in CH2Cl2 and DMF respectively proved effective but lengthier to purify from hydrophobic EDC-urea and PFP side products. The most efficient acylation proved to be the enzymatic activity of SCDase, a commercial transacylation enzyme which can be tuned to acylate or deacylate sphingosine analogues based on the reaction buffer pH. 186,187 SCDase and 0.4% TritonX detergent are mixed in phosphate buffer with the sphingolipid and fatty acid, shaken in an Eppendorf overnight, lyophilised, and purified by silica column with no highly lipophilic impurities. AzCer 30 was used to form final clicked GSL analogues (Scheme 2.1.3.1) for model membrane testing, and as a mass spectrometry (MS) internal standard, permitting precise quantification of the cellular metabolism of AzSph. The 12-step synthesis from phytosphingosine to AzCer 30 was completed with a 3% overall yield. Scheme 2.1.2.3: 1-Azidoceramide (AzCer) chemoenzymatic synthesis.

side product. This phosphazene intermediate may be stabilised by the neighbouring Obenzoyl group, and is stable for over 24h in THF/H2O. We simultaneously observe epimerisation at the C-2 position, which becomes separable after the following protection with dichloroacetyl chloride to form 24 (Scheme 2.1.2.1), confirmed by 1 H-NMR and MS. Scheme 2.1.2.4: Staudinger reduction (N 2 -azide deprotection).

  parallel to the glycine linker, we tested the synthesis of the ethanolamine (EA) linked Gb3 analogue(Scheme 2.1.3.7) which could be used for the same purposes as the Gly analogue. We isolated MFCO-EA 59 by aminolysis of MFCO intermediate 58 with freshly distilled ethanolamine (EA) in THF. The glycosylation of protected Gb3 60 with MFCO-EA was undertaken, via the activated trichloroacetimidate glycoside 61 in the presence of a Lewis acid. Low yield was due to poorly anhydrous conditions, often inevitable on small scale, reforming 60 from 61. 62 formation was confirmed by MS but was isolated on a scale too small to continue to the deprotection step.

	Scheme 2.1.3.9: Clicked Gb3-Cer analogues, from Gb3 head groups and AzCer 30.

C, Scheme 2.1.3.6)

and obtained greater β-stereoselectivity; 1:20 α/β ratio, rather than 1:2 with DMF (condition B). This higher selectivity was reproducible in the synthesis of heptaacetyl-lactose-β-MFCO 53 from corresponding starting materials. When first forming 53, we tested the coupling with PFP-TFA (condition A), expecting these products to have a similar reactivity to the reaction forming peracetyl-Gb3-MFCO 47, however no reaction was observed. Scheme 2.1.3.6: MFCO-Gly coupling reaction conditions. In Scheme 2.1.3.7: Synthesis of ethanolamine-MFCO and glycosylation.

We briefly visited the synthesis of cyclooctyne-glycoside 63 with the commercial (1R,8S,9S)-bicyclo[6.1.0]non-

. As for the previous glycosylation reaction (Scheme 2.1.3.7), we activate the anomeric position of the protected lactose 64 to form the trichloroacatamidate 65, which is glycosylated with the BCN alcohol (Scheme 2.

1.3.8)

. Poor yields were due to hydrolysis of 65 back to 64, and formation of the stable trichloroacetamide 67, as before (Scheme 2.1.3.5). 176 Scheme 2.1.3.8: Synthesis of glycoside-conjugated bicyclo

[6.1.0]

non-

.

Functionalised trioses, Gb3-MFCO 37 and Gb3-Gly-MFCO 38, were clicked in vitro with AzCer, and the products (Scheme 2.1.3.9), a mixture of regio-and stereoisomers (Discussion 3.2), were purified for use as internal mass spectrometry standards, and in model membrane experiments to test their recognition by and membrane-bending with STxB.

  GUVs are formed by mixing the cocktail of desired lipids in chloroform, allowing a lipid film to dry under vacuum and rehydrating in a sucrose solution of known osmolarity under a constant alternating current(10 Hz, 1.1 V, Experimental 6.2.1). Each experiment included a negative control (no GSL) and a positive control (natural. Molar ratio of lipids inTo test the click reaction on a liposome model, we formed large unilamellar vesicles (LUVs, 200 nm) containing AzCer and incubated with equimolar amounts of Gb3-MFCO (5mol% AzCer, 65mol% DOPC, 30mol% cholesterol in liposomes; 4 nmol cyclooctyne, 1 eq; Experimental 6.2.2). LUVs are simpler to form and are less uniformly aesthetic than GUVs, so not ideal for microscopy experiments, but have the same chemical properties for the scale of our interest.

	Rhod-PE	STxB-A488	Merge
	Gb3-Cer (wt)
	GUVs was used as described previously by Römer et al.: 64.9% DOPC, 30% cholesterol, 5%
	Gb3 species, 0.1% Rhodamine-PE. 27 The mass spectrometry (MS) results showed product forming with time, but only a very small
	Highly dynamic invaginations of GUVs are observed after 5-10 min of incubation with proportion of AzCer reacted.
	STxB-Alexa488 with the two Gb3 analogues: Gb3-MFCO-AzCer 35 and Gb3-Gly-MFCO-AzCer
	36, similar to that observed on wild type Gb3 (Figure 2.2.1.2-3). We were thus happy to use
	Gb3-MFCO-AzCer
	35
			C16
			4% C18
			4%
			C22
			24%
	Gb3-Gly-MFCO-Cer 36	C24 68%
	GSL negative
	Figure 2.2.1.2: Natural porcine Gb3-Cer with acyl chain profile, 64 with Gb3 analogues tested in GUVs

these substrates in ensuing cellular experiments. As mentioned in Results section 2.1.3, the cis/trans regioisomers formed from the click cycloaddition were not separated since this will not be controllable in cells, and this test is to evaluate the function of our synthetic analogues in cells. Figure 2.2.1.3. Dynamic membrane invaginations observed on GUVs containing 5% Gb3 species or w/o, and incubated with STxB (green). (64.9mol% DOPC, 30mol% cholesterol, 5mol% Gb3 species, 0.1mol% Rhodamine-PE (red)) Images taken on a Leica SP8 confocal microscope. Graph 2.2.1.1: Formation of Gb3-MFCO-Cer and consumption of AzCer on LUV click reaction.

2.2.2

Cellular metabolism and localisation of AzSph

  -MFCO 49 and Gb3-MFCO 37 (20 μM) permits cellular click reaction with AzCer detectable by lipidomics. Approximately 2% of AzCer is clicked with lactose-MFCO 49 after 24h, and 4% after 48h (Graph2.2.3.12). 10% of AzCer appears to be clicked after 48h with no serum, where cell proliferation is halted. However, over 50% cell death is observed under serum starvation conditions after 48h for CHO cells. Gb3-MFCO 37 showed similar extent of click, forming up to 0.1mol% of all lipids extracted(Graph 2.2.3.13). In spite of these encouraging results, the levels of surface Gb3 is not high enough to observe STxB binding in immunofluorescence (IF) experiments.

Graph 2.2.3.12: AzCer formed from AzSph (0.5 μM, 6h) in CHO cells and clicked product with Lac-MFCO 49 or Gb3-MFCO 37 (20 μM, 24/48h) w/o serum, normalise to ceramide and AzCer.

  AzPhy 31 metabolism with that of AzSph 17 (Results 2.2.2), we incubated cells with AzPhy (0.5 μM) for various time points, lysed the cells, extracted lipid membranes as before (Experimental 6.2.5), and analysed their metabolism profile by HRMS. As expected, we observe acylation to 1-azidophytoceramide (AzPhyCer) (Graph 2.2.4.2) with an acyl chain profile comprising a higher percentage of long acyl chains (C18, C22, C24) than short C16 chains. In GM95 cells, the acyl chain length profile is intermediate to that of AzCer formed from AzSph and native ceramide (Cer). In CHO cells, the profile is almost identical to that of native ceramide, which has fewer long chain ceramides compared to GM95 cells anyway(Graph 2.2.4.3). In both cell types, at 0.5 μM of AzPhy we observe steady acyl chain profiles of natural ceramide over 72h, with approximately 2:1 AzPhyCer/Cer, as observed for AzSph.The quantity of AzPhyCer stabilises in GM95 cells throughout 72h incubation (Graph 2.2.4.4), whereas cellular levels increase continuously in CHO cells(Graph 2.2.4.5).

	(I MTT /II MTT,0 ) To compare Graph 2.2.4.2: Acylation profile of sphingolipid analogues AzSph (forming AzCer, blue) and AzPhy (forming AzPhy AzSph survival AzPhyCer, grey), compared with native ceramide (Cer, green), in GM95 cells after 24h incubation.
	Cell
	-0,3	-0,2	-0,1	0	0,1	0,2	0,3	0,4	0,5	0,6	0,7	0,8	0,9	1
							log(conc)

  click studies apply a standard format: cellular incorporation and native metabolism of a biomolecule analogue labelled with a small clickable moiety, followed by click with a reporter (fluorophore or biotin)(Introduction 1.6, Figure 1.6.1). In this project, we attempt to introduce biological activity using a click conjugation. Few reports attempt to recreate lost or supressed native downstream signalling interactions, despite most papers agreeing that lipids and glycans are important signalling molecules.Prescher et al. even suggest that azide-labelled sialic acids should not interfere with signalling, since they are present at approximately 3% of the sialic acids.127 Our conjugation is performed before the desired biological activity, thus we must have the smallest possible click groups.

Scheme 3.2.1: GSL reconstitution via click chemistry. Most

  170,208 Garrido et al. showed that 1-azidoceramide incorporated into liposomes could undergo efficient copper-catalysed click reaction with an alkyne-fluorophore.209

  Scheme 3.2.5: Standard non-enantioselective MFCO synthesis 148 and formation of diasteriomeric mixture of MFCO-functionalised triose. Click reaction with AzCer gives cis/trans regioisomers. linkers could have been envisaged, for example an ethylenediamine linker (Scheme 3.2.7), which can easily be installed on MFCO 45 and coupled with the reducing sugar 42b. However, this strategy requires an excess of amine-MFCO reagent, and, for the amine linkage to be stable to hydrolysis, it must be "capped" with an acetate group.215 This would add extra bulk to our linker, which may hinder biological activity.

	Scheme 3.2.7: Potential amine functionalisation of reducing sugar.

We synthesised two clickable glycan head groups,

and

, in consideration of the strained angle at which the AzCer is held once clicked to the Gb3-MFCO 37, where the flexible glycine linker would alleviate the angle constraint. Two flexible linkers were considered, glycine and ethanolamine (EA).

Following the principles of convergent synthesis, we decided to conjugate the linker first to MFCO 45, a product formed in a four step synthesis from cheap starting materials, rather than Ac-Gb3-NH2 44 or Ac-Gb3-OH 42b, synthesised from seven or four steps from expensive glycoside starting materials. During the syntheses, the glycine route drew ahead as the most feasible, and will be applicable in later projects incorporating a photoactivable group (Perspectives 4). Scheme 3.2.6: Retrosynthetic analysis of Gb3-linker-MFCO.

Other

Unlike the copper-catalysed click cycloaddition, the copper-free click is not regioselective, thus the cis and trans regioisomers are typically formed in an approximately 50:50 ratio (Scheme 3.2.8), described in the supporting information of first literature report by Pigge et al., and observed by TLC.

148

NMR and UPLC-MS analyses are insufficiently resolved to quantify further. We did not attempt to purify regioisomers to test their activity. In cells, we cannot prevent the two isomers from forming, so the molecule of interest is the cis/trans mixture. Commercially available bicyclo

[6.1.0]

non-

could also hold potential in this project. Is it slightly longer than MFCO, but its functional groups, alcohol and alkyne, are at opposite ends of the cyclooctyne, creating a straighter molecule when clicked (Scheme 3.2.6), in which the cis/trans diastereoisomers are more symmetrical. Scheme 3.2.8: Final clicked Gb3 analogues.

Table 3 .

 3

	4.1: EC50 and EC90 values of AzSph in two cell lines
		GM95 cells		CHO cells
		AzSph (n=9) Sphingosine (n=6) AzSph (n=3) Sphingosine (n=3)
	EC50/μM	3.5	6.2	1.8	>>10
	EC90/μM	0.4	1.8	0.34	2.7

  molecules of total AzCer to total ceramide (Graph 2.2.2.6), and a stable amount of ceramide over 24h, indicating that cells are not under ceramide-signalling stress.Attempts to halt cell proliferation, to thus reduce the dilution of AzCer labelling, serum starvation conditions were tested. The presence of natural sphingolipids in serum should also be noted. No serum conditions induced a higher AzCer/Cer ratio over 48h compared to serum conditions. Eto et al. described an increase in cellular C16 ceramide in serum-free culture conditions due to an onset of apoptosis.

230

Despite this, we do not observe reduction in cell viability at low AzSph concentration.

37 ,

 37 , lactose-MFCO 49, lactose-Gly-MFCO 50 and lactose-BCN 63(Scheme 2.1.3.4, Results 2.1.3). Our final validation of a functional GSL analogue would be via the recognition of the Gb3 head group with its specific lectin binder, the Shiga toxin. However, to first study the click reaction via high resolution mass spectrometry (HRMS) we tested the protocol with lactose-MFCO 49,

obtained on larger scale.

3 Cellular metabolism of AzSph and click with fluorophore 

  Extrude lipid solution by passing it through extruder 15 times, remembering to take final solution from opposite end to which it was injected. The day before, 500,000 GM95 cells/well seeded in

	6.2.2 Large unilamellar vesicle (LUV) click reaction  Lipid mixture made up with 65% DOPC, 30% cholesterol and 5% AzCer in a new glass vial 6.2. Wash 2x DMEM(++)
	 Dry lipid solvent under stream of nitrogen  Add AzSph (10 µM, 1 ml) in DMEM(++)(Penicillin/streptomycin 5%, foetal bovine serum
	 Dry under vacuum for 1h 10%)
	 Resuspend in water at 1mg/ml total lipid  Incubate 37 °C for various times
	 3 vortex/freeze/thaw cycles, freezing in a EtOH/dry ice bath
	 Wash extruder with water (200 nm polycarbonate filter)
	 Click reaction
	5. 8-well coverslip for imaging coated with casein (4mg/ml, 30 min)  50 μL of LUV solution in each reaction vial (0.049 mg/ml AzCer, 0.15 mM, 7.5 nmol per
	6. GUVs cooled to rt 30 mins before use vial)
	7. 8-well coverslip washed 2x PBS  19 μl of 200 nM solution of Gb3-MFCO added (3.8 nmol per vial, equimolar since 50%
	8. Wells filled with 150 ul PBS (300 mOs) and 100 ul GUV solution and mixed with pipette of AzCer faces inside) and mixed with pipette tip
	tip  Reaction left at rt for varying time points (24/6/4/3/2/1h/30min/15min/ctrl (no Gb3))
	9. Visualise GUVs  Reaction stopped by extracting lipids with 70ul CHCl3/MeOH (10:1), vortexed for 60
	10. STxB-Alexa488 (20 or 200nM) added and mixed with pipette tip seconds and sample taken from aq and org layer for TLC and MS.
	11. Visualise tubules mol% μmol		μg		stock conc	vol
	Total		Gly (1) 1,2		Gb3-MFCO (2) Gb3wt (3) 791,0946	GSL neg
	[Stock / AzCer 5	Molar 0,06	Volume 38,7	Molar	Volume 1	Molar	Volume	Molar	Volume
	mg/ml] DOPC 65	% 0,78	added 613,197 %	added 10	%	added 61,3	%	added
	Cholesterol 6 Chol 30	30 0,36	5,6	30 139,1976	5,6 6	30	5,6 23,2	30	5,6
	DOPC	10	64.9 14,9	64.9 14,9	64.9 14,9	69.9 16
	Gb3 variant 1	5	19,8	5	19	5		16,6	0	0
	Liss Rhod PE 1	0.1	0,4		0.1	0,4	0.1		0,4	0.1	0,4

5 Cell treatment of click reagents for lipidomics testing, optimised protocol

	6.2.1. The day before, seed 250,000 GM95 cells/well in 6-well plate (2 ml) 6.2.	plate
	Wash 2x DMEM(++) 2. Wash 2x DMEM (++)
	Add compound (AzSph/AzPhy/cyclooctyne, 100 µL at descending concentrations) in 3. Add AzSph/AzPhy (0.5 µM, 1 ml) in DMEM(++)
	DMEM(++), 3 wells per condition. 4. Incubate at 37 °C (6h to overnight)
	Incubate at 37 °C, 24 h 5. Wash 2x DMEM(++)
	Remove medium 6. Incubate w/o cyclooctyne reagent in DMEM(++) (varying time points)
	Wash cells with 200 µL virgin DMEM at 4 °C 7. Wash 2x with ammonium bicarbonate (150 mM) at 4°C
	Add 150 µL virgin DMEM and 20 µL MTT solution (5 mg.mL -1 in H2O, protected from the 8. Add 1 ml ammonium bicarbonate and scratch cells at 4 °C
	light) 9. Centrifuge 5 min à 13.3K RPM à 4 °C
	Incubate for at least 3h at 37°C in the dark 10. Throw away supernatant
	Gently remove medium 11. Resuspend pellet in 300 µL ammonium bicarb
	(At this step, the plate can be conserved at 4°C to read at a later date) 12. Homogenise with needle and syringe (18G, then 25G needle)
	Add 100 µL DMSO 13. Take 50 μL for protein quantification (MicroBCA kit)
	Shake for 20 min 14. Snap freeze in N2(l)
	Read plate on spectrophotometer at 570 nm 15. Extract cell lysate solution (200 μl, quantity adjusted with water based on protein
	quantification), spiked with internal standards (non-natural AzCer C22:1 and Cer C12:0)
	 Add CHCl3/MeOH (2.75:1, 1 ml)
	 Shake vigorously in Eppendorf shaker for 1h
	 Centrifuge briefly and collect lower organic layer
	 Dry solvent (rotary evaporator or SpeedVac)
	 For Gb3-MFCO-AzCer extraction, a two-step extraction is used: Extract cell lysate
	solution (200 μl), spiked with internal standards (non-natural Gb3-MFCO-AzCer C22:1,
	AzCer C22:1 and Cer C12:0) first with in CHCl3/MeOH (10:1, 1 ml), shaken for 2h. Organic
	layer collected and remaining aqueous layer extracted with CHCl3/MeOH (2:1, 1 ml) as
	before
	16. Resuspend in 100 µL of MS mix (Propanol/MeOH/ CHCl3 [4:2:1] with ammonium acetate
	7.5mM)
	17. Direct infusion HRMS

6 Immunofluorescence test to localise clickable lipid

	6.2.
	1 The previous day, spread 200,000 GM95 cells or 50,000 CHO cells/well+coverslip (in 1 ml
	DMEM++) in a 4-well plate with glass coverslips
	2 Wash 2x with DMEM++
	3 Add 500 μl AzSph in DMEM++ medium or DMEM++ medium only (control)
	4 Incubate, 37°C
	5 Wash 3x with DMEM++
	6 Add clickable fluorophore (TAMRA-or Alexa-DIBO, Alexa488 alkyne) in DMEM++ and
	incubate 1-2h, 37°C
	7 Wash 3x with PBS++ and incubate 30 min, rt
	8 Wash 1x with PBS++
	9 Add 500 μl PFA (4%) and incubate 15 min, rt
	10 Wash 3x with PBS++
	11 Add NH4Cl (50mM) and incubate 30 min, rt
	12 Wash 1x with PBS/BSA/Saponine 1X
	13 Permeabilisation with PBS/BSA/Saponine 1X, rt, 30 minutes
	14 Wash 1x with H2O then add H2O
	15 Wash 3x with PBS++/1%FCS
	16 Mount on a slide with Mowiol
	Copper click solution (1 ml total, 0.4 mM Cu):
	o 860 μL PBS
	o 40 μL CuSO4 10 mM (0.4 mM final)
	o 2 μL alkyne-A488 1.25 mM (2.5 μM final)
	o 66 μL sodium ascorbate 150mM (10 mM final, dark -20°C)

7 Immunofluorescence test for clicked glycolipid with fluorescent STxB

	6.2 α-Lactose monohydrate (1.09 g, 3.19 mmol, 1 eq) was dissolved in a solution of acetic Chemical synthesis
	1 The previous day, spread 50,000 CHO cells/well+coverslip (in 1 ml DMEM++) in a 4-well General methods anhydride/pyridine (1:1) and stirred in air overnight at room temperature. The reaction
	plate with glass coverslips All reactions were performed under argon atmosphere unless specified. Commercially mixture was extracted with CH2Cl2 (2×100 ml), washed with brine (200 ml), water (200 ml)
	2 Wash 2x with DMEM++ available chemicals were used without further purification. Anhydrous solvents were freshly and HCl (1M, 200 ml), dried (MgSO4), evaporated and purified by silica column
	3 Add AzSph/AzPhy (0.5 μM, 500 μl) in DMEM++ medium or DMEM++ medium only distilled before use or obtained from solvent purifier Pure SolvTM. 3Å and 4Å molecular sieves chromatography (toluene/acetone, 6:4 to 4:6) to afford peracetyl lactose (2.06 g, 95 %). Rf
	4 Incubate, 37°C were activated immediately before reaction by a heat gun in vacuo. Glycosides and (toluene/acetone [1:1])=0.7; 1 H NMR (300 MHz, CDCl3) δ 6.26 (d, J 3.7 Hz, 1H, 1-Hα), 5.47 (t, J
	5 Wash 3x with DMEM++ phytosphingosine were purchased from TCI. Other lipid starting materials were purchased
	6 Add Gb3-MFCO (20 μM, 500 μl), incubate 37°C from Avanti Lipids. Column chromatography (CC): silica gel 60 (0.040-0.063 mm) Merck.
	7 Wash 3x with DMEM++ at 4°C Analytical thin layer chromatography (TLC): Merck silica gel 60 F-254 pre-coated plates;
	8 Incubate at 4°C for 10 min detection by UV light (254 nm), ceric ammonium molybdate, ninhydrin, orcinol or potassium
	9 Add STxB-Cy3 in DMEM++, 4°C permanganate. 1 H NMR spectra were recorded on Bruker instruments at 300, 400 or 500
	10 Binding 30 min on ice, 4°C (Skip to step 19 to visualise STxB surface binding) MHz. 13 C NMR spectra were recorded at 75, 101 or 126 MHz with complete proton
	11 Add DMEM++ preheated to 37°C decoupling. 31 P NMR spectra were recorded 121.4 MHz with respect to an external standard
	12 Internalisation 45 min, 37°C of 85% H3PO4, w/o proton decoupling. Chemical shifts (δ) in ppm related to protonated
	13 Wash 3x PBS++ (at 4°C for surface binding) solvent as internal reference ( 1 H: CHCl3 in CDCl3, 7.26 ppm; 13C: 13 CDCl3 in CDCl3, 77.0 ppm;
	14 Fix with PFA 4% in PBS, 15 min, rt 1 H: CH3OH in CD3OD; 13 C: 13 CD3OD in CD3OD; 1 H: H2O in D2O). Coupling constants J in Hz.
	15 Wash 1x NH4Cl 50 mM Current notations are used for multiplicity (s: singlet; bs: broad singlet; d: doublet; dd: double
	16 Quench with NH4Cl 50 mM, 30 min doublet; t: triplet; q: quadruplet; m: multiplet). The assignment of 1 H NMR spectra was based
	17 Wash 2x PBS++ on chemical shift correlation (DQF-COSY), whereas the assignment of 13 C NMR spectra were
	18 Wash 1x H2O then add H2O based on Carbon-Proton Heteronuclear Multiple Bond Correlation Coherence (HMBC) and
	19 Mount on a slide with Mowiol+DAPI (Hoechst/Fluoromount 1:20000) Hetero Nuclear Single Quantum Coherence (HSQC). UPLC-MS analyses were obtained on an
	Acquity UPLC BEH C18 pre-column 2.1×5 mm and column 2.1×50 mm, 1.7 μm (Waters) coupled to a
	SQ Detector 2 (Waters) at a flow rate of 0.6 ml/min. High resolution mass spectra (HRMS) were
	obtained in-house on a Q-Exactive Orbitrap (ThermoFisher Scientific) and on a LTQ-Orbitrap
	(Thermo) at the Laboratoire de Spectrométrie de Masse of ICSN CNRS (Gif-sur-Yvette).
	α-1-O-acetyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-D-
	glucopyranoside

,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl isothiocyanate (1) 177

	, 4.49 (d, J 7.8 Hz, 1H), 4.45 (d, J 12.0 Hz, 1H), 4.16 (dd, J 11.0,
	6.1 Hz, 2H), 4.12 -4.07 (m, 1H), 4.07 -3.95 (m, 1H), 3.89 (t, J 6.8 Hz, 1H), 3.80 (d, J 9.7 Hz,
	1H), 2.18 (s, 3H), 2.16 (s, 3H), 2.13 (s, 3H), 2.06 (s, 6H), 2.06 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H).
	2,3Peracetyl lactose (1.87 g, 2.56 mmol, 1 eq) and SnCl4 solution (2.8 ml, 2.78 mmol, 1 eq, 1 M
	solution in CH2Cl2) were mixed in CH2Cl2 (17 ml), to which trimethylsilylisothiocyanate
	(TMSSCN, 0.43 ml, 3.06 mmol, 1.1 eq) was added dropwise at rt. The solution was stirred for
	24 h, then an additional 0.25 equivalents of TMSSCN (0.1 ml, 0.69 mmol). The reaction
	mixture stirred for another 24h, then washed with NaHCO3 (200 ml) and water (2×200 ml)
	and extracted with CH2Cl2, evaporated and purified by silica column chromatography
	(EtOAc/cyclohexane/CH2Cl2, 1:1:1) to afford 1 (0.62 g, 0.91 mmol, 33 %). Rf
	(EtOAc/cyclohexane [1:1])=0.4; 1 H NMR (300 MHz, CDCl3) δ 5.35 (1H, d, J 4'-3' 3.1 Hz, 4'-H), 5.20
	(1H, t, J 3-4 9.2 Hz, 3-H), 5.11 (1H, dd, J 2'-1' 10.3 Hz, J 2'-3' 7.9 Hz, 2'-H), 5.02 (1H, m, 3'-H), 5.00
	(1H, m, 2-H), 4.95 (1H, dd, J 1-2 10.4 Hz, J 3.3 Hz, 1-H), 4.48 (1H, d, J 1'-2' 10.4, 1'-H), 4.48 (1H, dd,

-3-((2-oxido-1,3,2-dioxaphospholan-2-yl)oxy)propane-1,2-diyl dioleate (11) 179

	(MH + ), 342.4
	(MNH4 + ); with the major impurity formed from hydrolysis of 8 (178 mg, 58 %).
	(Z)-(R)1,2-dioleoyl-sn-glycerol 10 (80 mg, 129 μmol, 1 eq), COP (36 μl, 386 μmol, 3 eq) and 4-
	dimethylaminopyridine (4-DMAP, 1.6 mg, 13 μmol, 0.1 eq) were dissolved in dry toluene and
	cooled to 0°C. Et3N (540 μl, 10v% in toluene, 386 μmol, 3 eq) was added dropwise and the
	reaction mixture stirred at 0°C for 15 minutes then at rt for 4h. The reaction mixture was
	filtered through Celite and washed with dry toluene. Solvent was removed under reduced
	pressure to afford the crude compound 11. Rf (cyclohexane/EtOAc [3:1])=0.1; 1 H NMR (300
	MHz, CDCl3) δ 5.41 -5.29 (m, 4H, HC=CH), 5.29 -5.18 (m, 1H, CH(CH2)2O), 4.42 (d, J 10.7 Hz,
	4H, P(OCH2)2), 4.35 -4.24 (m, 2H, CH2OPO3), 4.16 (dd, J 11.9, 5.7 Hz, 2H, CH2OCO), 2.34 (dt, J
	11.4, 7.5 Hz, 4H, COCH2), 2.02 (s, J 5.3 Hz, 8H, HC=CHCH2), 1.61 (s, 4H, COCH2CH2), 1.28 (d, J
	9.7 Hz, 40H, CH2), 0.88 (t, J 6.4 Hz, 6H, CH3); 31 P NMR (121 MHz) δ 18.94 (s). ESI-MS 749.9
	(MNa + ), 764.5 (MK + ).
	1,2

-dioleate-sn-glyceryl-3 (2-hydroxyethyl) phosphate (5), and 1,2-dioleate-sn-glyceryl-3 (2-(dimethyl(prop-2-yn-1-yl) ammonio)ethyl) phosphate (12) 245

	With the intention of forming compound 5, compound 11 (284 mg, 1.2 mmol, 1 eq) was
	dissolved in CH2Cl2, to which N,N-dimethylpropargylamine (0.445 ml, 2.46 mmol, 2 eq) was
	added, then TMSOTf (265 μl, 2.46 mmol, 2 eq) was added dropwise. The reaction was stirred
	for 15 min at 0°C then at rt for 24 h. The reaction mixture was evaporated and purified by
	silica column chromatography (CHCl3/MeOH/NH4OH [9:1:0 to 70:30:3]) to afford the
	hydrolysis impurity 12 (87 mg, 91 % over two steps). Rf (CHCl3/MeOH/NH4OH [70:30:3])=0.5;
	1 H NMR (300 MHz, MeOD) δ 5.40 -5.28 (m, 4H, HC=CH), 5.27 -5.19 (m, 1H, CH(CH2)O), 4.31
	(ddd, J 18.8, 12.0, 4.9 Hz, 2H, CH2OCO), 3.99 (t, J 5.5 Hz, 2H, CH2OPO3), 3.90 (dd, J 11.1, 5.7
	Hz, 2H, HOCH2CH2OPO3), 3.69 (t, J 4.9 Hz, 2H, HOCH2CH2OPO3), 2.32 (q, J 7.1 Hz, 4H, OCOCH2),
	2.03 (d, J 5.4 Hz, 8H, HC=CHCH2), 1.61 (s, 4H, OCOCH2CH2), 1.46 -1.21 (m, 40H, CH2), 0.90 (t,
	J 6.6 Hz, 6H, CH3); 32 P NMR (121 MHz, MeOD) δ 1.45; ESI-MS 744.0 (M -).

2-azido-1-(tert-butyldiphenylsilyoxy)octadecane-3,4-triol (19) 247

	Compound 18 (0.85 g, 2.46 mmol, 1 eq), tert-butyldiphenylsilyl chloride (TBDPSCl, 0.81 g, 2.95
	mmol, 1.2 eq), Et3N (0.86 ml, 6.15 mmol, 2.5 eq) and 4-DMAP (0.015 g, 0.123 mmol, 0.05 eq)
	were dissolved in dry CH2Cl2/DMF (15/3 ml) at 0°C. The reaction mixture was stirred
	overnight, then diluted with EtOAc, washed with brine (2×20 ml), dried with MgSO4 and
	purified by silica column chromatography (10-20% EtOAc in cyclohexane) to afford 19 (0.84
	g, 59 %). Rf (cyclohex/EtOAc [5:1])=0.4, 1 H NMR (300 MHz, CDCl3) δ 7.

-2-amino-3-O-benzoyl-1-O-tert-butyldiphenylsilyl-4-octadecene-1,3-diol

	8.0 Hz, H 4 ), 3.85 (1H, m, H 2 ), 3.75 (2H, m, H 1a , H 1b ), 2.06 -1.97 (2H, m, H 6 , H 6' ), 1.40 -1.18
	(22H, m, 11CH2), 1.09 (9H, s, t Bu), 0.89 (3H, t, J 6.7 Hz, CH3); ESI-HRMS: Calcd for [MNa + ]
	690.4066. Found: m/z 690.4061.
	(2S,3R)-(E)(23) 248,249
	Compound 22 (685 mg, 1.03 mmol, 1 eq) was dissolved in toluene/water (20:1, 10 ml), to
	which PPh3 (538 mg, 2.05 mmol, 2 eq) was added. The reaction mixture was stirred overnight,
	heated to 60°C for 2h until all starting material and phosphorus intermediate had disappeared
	from TLC. Reaction mixture was evaporated and purified by silica column chromatography
	(0.5 to 10% EtOAc in cyclohexane) to afford 23 (494 mg, 78 %) as a mixture of inseparable
	stereoisomers (2S,3R) and (2R,3R) (98:2). Rf (cyclohex:EtOAc [3:1]+1% Et3N)=0.1; 1 H NMR
	(300 MHz, CDCl3) δ 7.88 (d, J 7.2 Hz, 2H, Bz), 7.

  09, 26.99 ( t Bu), 22.88 ( t Bu), 19.40 (C 17 ), 14.32 (C 18 ); ESI-MS 642.8 (MH + ), 664.8 (MNa + ), 1284.3 (2MH 2+ ); ESI-HRMS: Calcd for [MNa + ] 664.4161. Found: m/z 664.4156.

	(2S,3R)-(E)-1-O-tert-butyldiphenylsilyl-2-(2,2-dichloroacetamido)-3-O-benzoyl-4-
	octadecene-1,3-diol (24) 250
	Compound 23 (970 mg, 1.51 mmol, 1 eq) was dissolved in dry CH2Cl2 (20 ml) and cooled to -
	15°C. DIPEA (385 μl, 2.26 mmol, 1.5 eq) was added dropwise, followed by dichloroacetyl
	chloride (182 μl, 1.89 mmol, 1.25 eq) dropwise. After 15 minutes at -10°C, the reaction was
	stirred 30 minutes at rt until complete conversion by TLC. The reaction was quenched with

water, extracted with CH2Cl2, washed with brine, dried (MgSO4), evaporated and purified by silica column chromatography (1 -10% EtOAc in cyclohexane) to afford 24 (918 mg, 81 %). Rf (cyclohex:EtOAc [20:1])=0.3; [α] 22 D=+20.8° (c 5.0, CHCl3); 1 H NMR (300 MHz, CDCl3) δ 7.

-2-(2,2-dichloroacetamido)-1-iodo-3-O-benzoyl-4-octadecene-3-ol (27)

  5 (M-H -), 626.8 (MCl -); ESI-HRMS: Calcd for [MNa + ] 614.2085. Found: m/z 614.2080.

	(2S,3R)-(E)To a solution of compound 26 (62 mg, 0.11 mmol, 1 eq) in distilled acetone (1 ml), sodium
	iodide (146 mg, 0.30 mmol, 10 eq) was added and mixture was refluxed at 65°C for 5h. The
	reaction was quenched with H2O, extracted with CH2Cl2, dried (MgSO4) and evaporated. The
	product was purified by silica column chromatography (10 -50 % EtOAc in cyclohexane) to
	afford 27 (48 mg, 76 %). Rf (EtOAc/cyclohexane [9:1])=0.7; 1 H NMR (300 MHz, CDCl3) δ 7.97
	(d, J 7.3 Hz, 2H), 7.53 (m, 1H), 7.39 (t, J 7.6 Hz, 2H), 6.60 (d, J N-2 9.1 Hz, 1H, NH 2 ), 6.02 -5.88
	NMR (75 MHz, CDCl3) δ 165.07 (COCHCl2), 163.70 (COPh), 139.19 (C 5 ), 133.42 (Bz), 129.75
	(Bz), 129.59 (Bz), 128.55 (Bz),122.82 (C 4 ), 75.43 (C 3 ), 66.29 (CHCl2), 52.36 (C 2 ), 32.40

(m, 1H, H1), 5.55 (t, J 6.9 Hz, 1H), 5.43 (dd, J 15.2, 7.5 Hz, 1H), 4.19 (td, J 11.1, 6.1 Hz, 1H), 3.35 (qd, J 10.8, 5.4 Hz, 1H)

, 1.99 (q, J 6.9 Hz, 1H), 1.

40 -1.04 (m, 12H)

, 0.81 (t, J 6.6 Hz, 2H);

13 C

  C NMR(75 MHz, CDCl3) δ 165.37 (COPh), 163.97 (COCHCl2), 138.70(C 5 ), 133.48 (Bz), 129.76 (Bz), 129.55 (Bz), 128.57 (Bz), 123.22 (C 4 ), 73.93(C 3 ), 66.25 (CHCl2), 51.92 (C 2 ), 50.58(C 1 ), 32.37(C 6 ), 31.94

-1-azido-2-(N-2,2-dichloroacetamido)-3,4,-octadecanediol (3)

  Phytosphingosine(1.1 g, 3.46 mmol, 1 eq) was suspended in THF(40 ml), to which DIPEA(0.8 ml, 4.34 mmol, 1.3 eq) was added and the mixture was cooled to -10°C. Dichloroacetyl chloride (367 μl, 3.82 mmol, 1.1 eq) was added dropwise and the reaction was warmed to rt after 15 min, then stirred at rt for 3h, evaporated and purified by silica column

	chromatography (2.5 to 7% MeOH in CH2Cl2) to afford 32 as a white crystalline solid (1.41 g,
	81 %). Rf (MeOH/CH2Cl2 [5:95]) =0.3; [α] 22 D=+7.9 (c 3.3, CHCl3/MeOH [1:1]); 1 H NMR (500 MHz,
	CDCl3) δ 7.60 (d, J 8.4 Hz, 1H, NH), 5.97 (s, 1H, CHCl2), 4.07 (dt, J 5.0, 3.2 Hz, 1H, H 2 ), 3.84 (dd,
	J 11.6, 3.4 Hz, 1H, H 1a ), 3.70 (dd, J 11.6, 5.4 Hz, 1H, H 1b ), 3.64 -3.55 (m, 2H, H 4 , H 3 ), 1.68 -
	1.35 (m, 4H, H 5 , H 6 ), 1.35 -1.13 (m, 22H), 0.84 (t, J 7.0 Hz, 3H); 13 C NMR (126 MHz, CDCl3) δ
	164.56, 75.08 (C 3 ), 72.59 (C 4 ), 66.33 (CHCl2), 60.57 (C 1 ), 52.30 (C 2 ), 32.86 (C 5 ), 31.89 (C 6 ), 29.66,
	29.62, 29.33, 25.80, 22.66, 14.07; ESI-HRMS: Calcd for [MH + ] 428.2329. Found: m/z 428.2332.
	(2S,3S,4R)N-Protected phytosphingosine 32 (50 mg, 0.117 mmol, 1 eq) and NaN3 (76 mg, 1.17 mmol,
	10 eq) were suspended in DMF (1 ml), to which PPh3 (61 mg, 0.23 mmol, 2 eq) and then CBr4
	(77 mg, 0.23 mmol, 2 eq) in DMF (0.5 ml) was added and the mixture stirred at rt overnight.
	The reaction was quenched with MeOH, evaporated, diluted with CH2Cl2, washed with H2O,
	); IR ν 3354 (NH), 3287 (NH), 3154 (OH, C=C-H), 2916 (C-H), 2848 and purified by silica column chromatography (10 to 25% EtOAc in n-hexane) to afford 33 as
	(C-H), 2133 (N3); ESI-MS 5325.5 (MH + ), 307.4 (MH-H2O + ); ESI-HRMS: Calcd for [MH + ] 325.2967. a crystalline white solid (16 mg, 31 %). Rf (EtOAc/n-hexane [4:6]) =0.7; [α] 22 D=+14.3 (c 0.7,
	Found: m/z 325.2962. CHCl3/MeOH [9:1]); 1 H NMR (500 MHz, CDCl3) δ 7.10 (d, J 8.5 Hz, 1H, NH), 5.97 (s, 1H, CHCl2),
	4.23 (td, J 8.9, 4.6 Hz, 1H, H 2 ), 3.84 (dd, J 12.7, 4.8 Hz, 1H, H 1a ), 3.74 -3.66 (m, 3H, H 1b , H 3 , H 4 ),
	1-azidoceramide (C22:1) (30) 209 1.75 -1.42 (m, 4H, H 5 , H 6 ), 1.42 -1.19 (m, 22H), 0.90 (t, J 6.9 Hz, 3H); 13 C NMR (101 MHz,
	CDCl3) δ 164.22, 74.35 (C 3 or C 4 ), 72.82 (C 3 or C 4 ), 66.27 (CHCl2), 51.01 (C 1 ), 50.65 (C 2 ), 32.74
	(C 5 ), 31.93

Compound 17 (3×1.67 mg, 15.5 μmol, 1 eq) was mixed in 3 separate Eppendorf tubes with erucic acid (3×2 mg, 5.9 μmol, 1.2 eq) in a phosphate buffer solution (pH 7.4, NaH2PO4 (0.1M),

  sieves in a dry, inert atmosphere at -55°C. To the solution,N-iodosuccinimide (NIS, 425 mg, 1.89 mmol, 1 eq) was added, followed by TMSOTf(68 µl, 0.38 mmol, 0.2 eq) dropwise and the reaction mixture was stirred for 2 h. The reaction mixture was neutralised with Et3N (0.2 ml) and stirred for a further 1 h, then diluted with CH2Cl2, washed with aqueous

	4 Å molecular sodium thiosulfate solution (10 %, 2×50 ml) and brine (2×50 ml), dried (MgSO4), evaporated
	to a crude mixture of isomers (6:1 α/β), which is purified by silica column chromatography
	(1.25 to 2% acetone in toluene) to afford triose 6 (1.62 g, 75 %). Rf (toluene/acetone
	[19:1])=0.2; [α] 22 D=+20.0 (c 13.0, CHCl3); 1 H NMR (300 MHz, CDCl3) δ 7.47 -7.12 (m, 50H),
	6.92 (dd, J 69.7, 9.1 Hz, 4H), 5.17 -5.05 (m, 2H), 5.03 -4.89 (m, 2H), 4.89 -4.85 (m, 2H), 4.84
	-4.74 (m, 6H), 4.58 -4.45 (m, 6H), 4.42 -4.17 (m, 6H), 4.14 -4.09 (m, 2H), 4.07 (s, 2H), 4.04
	-3.94 (m, 2H), 3.83 (s, 2H), 3.79 (s, 3H, OCH3), 3.72 -3.62 (m, 3H), 3.52 (t, J 8.7 Hz, 3H), 3.37
	(t, J 8.3 Hz, 2H), 3.18 (dd, J 8.3, 4.7 Hz, 1H); 13 C NMR (75 MHz, CDCl3) δ 129.
	; ESI-HRMS: Calcd for
	[MH + ] 453.2394. Found: m/z 453.2395.

  μmol, 1 eq) was dissolved in dry MeOH (1.6 ml) and CH3ONa (0.5 M solution in MeOH, 0.45 ml, 0.223 mmol, 10 eq) was added. The reaction was stirred overnight, quenched with Dowex50WX8-200, filtered, evaporated and purified by reverse phase C18 silica column chromatography(10 to 20% MeOH in H2O) to obtain the clickable

	trisaccharide 37 (8 mg, 56 %) as a pure crystalline solid. Rf ( n BuOH/EtOH/H2O [2:2:1])=0.5; 1 H
	NMR (400 MHz, MeOD) δ 4.96 -4.91 (m, 2H, H 1 , H 1' ), 4.59 (bs, 1H, NH), 4.

  C NMR (101 MHz, MeOD) δ 172.04 (d,J 25.8 Hz, C 9 ), 110.20 (d, J 10.3 Hz, C 12 ), 105.32 (C 1'' ), 102.65 (C 1' ),95.23 (dd, J 187.6, 18.6 Hz, C 10 ),88.13 (d, J 31.7 Hz, C 11 ), 81.15 (d, J 5.6 Hz, C 1 ),

  , H15 ),2.16 (s, 3H), 2.13 (d, J 5.9 Hz, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.05 (d, J 1.6 Hz, 3H), 2.05 (d, J 2.4 Hz, 3H), 1.96 (s, 3H), 1.96 (s, 3H), 1.

  F NMR (471 MHz, CDCl3) δ -146.44, -146.95. ESI-HRMS: Calcd for [MH + ] 788.2777. Found: m/z 788.2772.

  , 74 %). Rf (EtOAc/cyclcohexane[3:1]) =0.3. 1 H NMR (500 MHz, CDCl3) δ 6.95 (bs, 1H, NH 18 ), 6.67 (d, J 6.1 Hz, 1H, NH19 ), 5.58 (d, J 2.0 Hz, 1H), 5.39 (dd, J 11.0, 3.1 Hz, 1H), 5.28(t, J 8.8 Hz, 1H), 5.18 (m, 2H, incl. H 1 ), 5.09 (dd, J 10.5, 7.9 Hz, 1H), 4.98 (d, J 3.3 Hz, 1H, H 1'' ),

  . UPLC-MS (gradient 5-100% CH3CN/H2O, 4 min, ESI) rt=1.13; 1132 (M -), 1134 (MH + ). ESI-HRMS: Calcd for [MH + ] 1133.3837. Found: m/z 1133.3831.

  μmol, 1 eq) in MeOH (400 μl), CH3ONa (436 μl, 0.5M solution in MeOH, 13 eq) was added dropwise. The reaction was stirred for 45 min at rt, before filtering through acidified Dowex 50WX-800 and washing with MeOH at 4°C to reduce side reactions. The crude mixture was purified by reverse-phase silica column chromatography (H2O/MeOH [100:0 to 0:100) to afford 38(11 mg, 95 %). Rf ( n BuOH/EtOH/H2O [2:2:1])=0.5. 1 H NMR (500 MHz, MeOD) δ 4.95 (d, J 3.7 Hz, 1H, H 1'' ), 4.93 (d, J 9.2 Hz, 1H, H 1 ), 4.42 (d, J 6.1 Hz, 1H, H 1' ), 4.26(t, J 6.2 Hz, 1H), 3.98 (s, 1H),

Scheme 2.1.1.3: Proposed globotriose-phospholipid formed from click conjugation.

Scheme 2.1.1.5: Further syntheses of alkyne and azide phospholipids.

Acknowledgements

Abbreviations

A.U.