Ulf Ellervik

LLLLuuuunnnndddd 1111999999998888

Akademisk avhandling som fr avlggande av teknisk doktorsexamen vid tekniska fakulteten vid Lunds Universitet kommer offentligen frsvaras

Kemicentrum, sal C, fredagen den 2 oktober 1998, kl. 13.15.

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A doctoral thesis at a university in Sweden is produced as a monograph or as acollection of papers. In the latter case, the introductory part constitutes the formal thesis,which summarizes the accompanying papers. These have either already been publishedor are manuscripts at various stages (in press, submitted, or in manuscript).

© Ulf EllervikDepartment of Organic Chemistry 2Center for Chemistry and Chemical EngineeringLund UniversityP.O. Box 124SE-221 00 LUNDSWEDEN

ISBN 91-628-3105-4

Printed by Frvaltningsavdelningens repro, SLU Alnarp 1998

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I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all referencesources permission to publish and disseminate the abstract of the above-mentioned dissertation.

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Department of Organic Chemistry 2P.O. Box 124SE-221 00 LundSWEDEN

1998-09-08

LUTKDH/(TKOK-1046)/1-110/(1998)

Ulf Ellervik The Swedish Natural Science Research Council

SYNTHETIC ANALOGS OF SIALYL LEWIS X

English

91-628-3105-4

110

1998-09-08

The sialyl Lewis x (SLex) tetrasaccharide is the smallest recognizable ligand for selectins. The selectinsconstitute a vital part of the inflammatory cascade for recruitment of leukocytes to a site of tissue damageor microbial infection. If too many leukocytes are recruited, normal, not injured, cells can be damaged; aprocess known from chronic inflammatory diseases such as reumatoid arthritis, psoriasis, and from septicshock and reperfusion damage.Sialyl Lewis x saccharides were originally identified as human tumor-associated antigens and are foundon all highly malignant types of cancer cells.

The identification of the SLex tetrasaccharide and later synthetic efforts have elucidated thebiologically important features of the molecule. A large number of analogs and mimics have been designedin order to get simpler and more stable compounds for use as anti-inflammatory drugs.

Sialic acid-containing oligosaccharides, such as sialyl Lewis x, can form lactones under slightlyacidic conditions. These lactones are generally more immunogenic due to their increased rigidity and ithas been proposed that the lactones are the actual immunogens. The lactones are however not hydrolyticallystable and are therefore difficult to investigate and to raise antibodies against.

Lactams were introduced as analogs to lactones and were found to be structurally similar and morestable against hydrolysis. Lactam analogs of a number of monosialylated gangliosides (GM2, GM3, GM4)were synthesized and used with success.

This thesis describes the synthesis of the sialyl Lewis x tetrasaccharide, the Lewis x trisaccharide,the 1’’’→2’-, 1’’’→4’-lactam- and 2-acetamido analogs of the sialyl Lewis x tetrasaccharide, the 2- and 4-acetamido- and 2- and 4-lactam analogs of the Lewis x trisaccharide.

The key-steps in the syntheses were regio- and stereoselective galactosylations of one commonmonosaccharide diol acceptor, stereoselective fucosylation and regio- and stereoselective sialylations toyield the oligosaccharidic products in 10-62% over-all yield from monosaccharidic starting materials.

Sialyl Lewis x, SLex, selectin, lactone, lactam analogs, regioselective synthesis, stereoselective synthesis

Department of Organic Chemistry 2P.O. Box 124, SE-221 00 Lund, Sweden

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List of papersThis thesis summarizes the following papers, which are referred to in the text by theroman numerals I-VII. The papers I-III are reprinted with kind permission from thepublishers.

I Ulf Ellervik, Gran MagnussonCalculated Conformations of Sialyl-Lex- and Sialyl-Lea-LactonesBioorganic and Medicinal Chemistry 1994, 2, 1261-1266

II Ulf Ellervik, Gran MagnussonGlycosylation with N-Troc-protected glycosyl donorsCarbohydrate Research 1996, 280, 251-260

III Ulf Ellervik, Gran MagnussonGuanidine/Guanidinium Nitrate; a Mild and Selective O-Deacetylation Reagentthat Leaves the N-Troc group intactTetrahedron Letters 1997, 38, 1627-1628

IV Ulf Ellervik, Gran MagnussonA High Yielding Chemical Synthesis of Sialyl Lewis x Tetrasaccharide and Lewisx Trisaccharide; Examples of Regio- and Stereodifferentiated GlycosylationsSubmitted to Journal of Organic Chemistry

V Ulf Ellervik, Gran MagnussonSynthesis of Sialyl Lewis x-1ÕÕÕ→2Õ-Lactam and the Corresponding AcetamidoAnalogs of Sialyl Lewis x Tetrasaccharide and Lewis x TrisaccharideSubmitted to Journal of Organic Chemistry

VI Ulf Ellervik, Gran MagnussonSynthesis of Sialyl Lewis x-1ÕÕÕ→4Õ-Lactam and the Corresponding AcetamidoAnalog of Lewis x TrisaccharideSubmitted to Journal of Organic Chemistry

VII Ulf Ellervik, Hans Grundberg, Gran MagnussonSynthesis of two Lactam Analogs of the Lewis x TrisaccharideSubmitted to Journal of Organic Chemistry

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AbbreviationsAc AcetylAc2O Acetic anhydrideAcOH Acetic acidAIBN AzoisobutyronitrileAgOTf Silver trifluoromethanesulfonate, silver triflateAW Acid washedBF3Et2O Boron trifluoride etherateBn BenzylBu ButylBz Benzoylcf. CompareCOSY Correlation spectroscopyDMAP 4-DimethylaminopyridineDMF N,N-DimethylformamideDMTST Dimethyl(methylthio) sulfonium triflatee.g. For example (exempli gratia)ELISA Enzyme linked immunosorbent assayeq. Equatorialet al. And others (et alii)Et EthylFuc FucoseGal D-GalactopyranoseGalNAc 2-Acetamido-2-deoxy-D-galactopyranoseGc GlycolylGlc GlucoseGlcNAc 2-Acetamido-2-deoxy-D-glucopyranoseGM Monosialylated gangliosideGSL Glycosphingolipidh HourHetCor Heteronuclear correlation spectroscopyHSEA Hard sphere exoanomerici.e. That is (id est)in vitro ÒIn glassÓ, experiments on cultured cellsin vivo Experiments on intact organismsLea Lewis aLex Lewis xMe MethylMM2, MM3 Force fields for molecular mechanics calculationsMS Molecular sievesMs Methanesulfonyl, MesylMSB Methyl sulfenyl bromideMST Methyl sulfenyl triflateMT MethyltriflateNeuAc 5-N-Acetyl neuraminic acid, sialic acidn.d. Not determinedNFmoc 9-Fluorenylmethoxycarbonylamino

ix

NMR Nuclear magnetic resonancenOe Nuclear Overhauser effectNOESY Nuclear Overhauser effect spectroscopyNPhth N-PhthalimideNTCP N-TetrachlorophthalimideNTeoc TrichloroethoxycarbonylaminoNTroc TrichloroethoxycarbonylaminoPd-C Palladium on activated charcoalPh PhenylPMP p-MethoxyphenylPST Phenyl sulfenyl triflatePy PyridineQBr Tetrabutylammonium bromideRMS Root mean squareSLea Sialyl Lewis aSLex Sialyl Lewis xSuLea Sulfated Lewis aSuLex Sulfated Lewis xTBAF Tetrabutylammonium fluorideTf TrifluoromethanesulfonylTHF TetrahydrofuranTLC Thin layer chromatographyTMS TrimethylsilylTMSEt 2-(Trimethylsilyl)ethylTrocCl 2,2,2-TrichloroethylchloroformateTs Toluenesulfonyl, tosylTsOH p-Toluene sulfonic acidvs Versus

x

Contents

The biological relevance of sialyl Lewis x ...................................................................................................1

1.1 Carbohydrates...........................................................................................................................................................11.2 Sialyl Lewis x Ð Biological background................................................................................................................2

1.2.1 Leukocyte recruitment and inflammation ..........................................................................................................21.2.2 Cancer and metastasis.........................................................................................................................................5

1.3 Structure of sialyl Lewis x.......................................................................................................................................51.3.1 The sialyl Lewis x saccharides ............................................................................................................................51.3.2 The conformation of sialyl Lewis x in solution ..................................................................................................71.3.3 The bioactive conformation of sialyl Lewis x in complex with selectins ...........................................................91.3.4 Sialyl Lewis x conformation, summary............................................................................................................10

Synthetic analogs of sialyl Lewis x ................................................................................................................11

2.1 Biological assays .....................................................................................................................................................112.2 Modification of the glucosamine residue...........................................................................................................12

2.2.1 Deoxy analogs....................................................................................................................................................122.2.2 Modification of the acetamido group ................................................................................................................122.2.3 Deoxynojirimycin derivatives...........................................................................................................................132.2.4 Other analogs ....................................................................................................................................................132.2.5 The importance of the aglycon ..........................................................................................................................142.2.6 Summary ...........................................................................................................................................................14

2.3 Modification of the galactose residue.................................................................................................................142.3.1 Deoxy analogs....................................................................................................................................................142.3.2 Fluoro- and acetyl analogs ................................................................................................................................152.3.3 Other analogs ....................................................................................................................................................152.3.4 Summary ...........................................................................................................................................................15

2.4 Modification of the fucose residue ......................................................................................................................162.4.1 Deoxyanalogs.....................................................................................................................................................162.4.2 Epimers ..............................................................................................................................................................162.4.3 Other modifications...........................................................................................................................................172.4.4 Summary ...........................................................................................................................................................17

2.5 Modification of the sialic acid residue................................................................................................................172.5.1 Modification of the glycerol side chain .............................................................................................................172.5.2 Modification of the acetamido group ................................................................................................................182.5.3 Replacement of sialic acid by sulfate.................................................................................................................182.5.4 Replacement of sialic acid with phosphate........................................................................................................192.5.5 Replacement of sialic acid by a carboxylic acid ................................................................................................192.5.6 Summary ...........................................................................................................................................................20

2.6 Sulfated analogs......................................................................................................................................................202.6.1 Sulfated sialyl Lewis x analogs .........................................................................................................................202.6.2 Sulfated Lewis x analogs...................................................................................................................................212.6.3 Summary ...........................................................................................................................................................22

2.7 Other modifications ...............................................................................................................................................222.7.1 Positional isomers of SLex ................................................................................................................................22

xi

2.7.2 Thio-linked SLex................................................................................................................................................232.8 Multivalent sialyl Lewis x analogs......................................................................................................................24

2.8.1 Examples of multivalent analogs......................................................................................................................242.8.2 Liposomes containing SLex...............................................................................................................................262.8.3 Polymers ............................................................................................................................................................272.8.4 Summary ...........................................................................................................................................................27

2.9 Mimics of sialyl Lewis x ........................................................................................................................................272.9.1 Mimics derived from SLex ................................................................................................................................272.9.2 Other mimics .....................................................................................................................................................292.9.3 Miscellaneous ....................................................................................................................................................292.9.4 Summary ...........................................................................................................................................................30

2.10 Summary ................................................................................................................................................................30

Lactones of sialyl Lewis x; introduction to lactams .............................................................................31

3.1 Ganglioside lactones ..............................................................................................................................................313.1.1 Biological importance of ganglioside lactones ..................................................................................................323.1.2 The conformation of ganglioside lactones.........................................................................................................32

3.2 Lactam analogs of gangliosides ...........................................................................................................................333.3 Lactones of sialyl Lewis x......................................................................................................................................33

3.3.1 Reported lactones of sialyl Lewis x ...................................................................................................................343.3.2 Calculated conformations of sialyl Lewis x lactones........................................................................................36

3.4 Lactam analogs of sialyl Lewis x .........................................................................................................................383.5 Objectives of this thesis .........................................................................................................................................38

Synthesis of new analogs of sialyl Lewis x and Lewis x ..................................................................41

4.1 Glycoside synthesis: general concepts................................................................................................................414.2 Syntheses of SLex reported in the literature......................................................................................................424.3 Protection of amino groups ..................................................................................................................................44

4.3.1 The acetamido group, NHAc ............................................................................................................................454.3.2 The N,N-diacetyl group, N(Ac)2......................................................................................................................454.3.3 The phthalimido group, NPhth.........................................................................................................................464.3.4 The tetrachlorophthalimido group, NTCP .......................................................................................................474.3.5 The trichloroethoxycarbonylamino group, NTroc, NTeoc...............................................................................474.3.6 The N-pent-4-enoyl group, NPent ...................................................................................................................484.3.7 The azido group, N3..........................................................................................................................................48

4.4 Synthetic strategy ...................................................................................................................................................494.5 The acceptor.............................................................................................................................................................504.6 The galactosyl donors ............................................................................................................................................52

4.6.1 Synthesis of the donor for SLex. Scheme 4.4....................................................................................................554.6.2 Synthesis of the donor for SLex-2-lactam. Scheme 4.5. ...................................................................................564.6.3 Synthesis of the donor for SLex-4-lactam. Scheme 4.6. ...................................................................................574.6.4 Synthesis of the donor for Lex-2-lactam. Scheme 4.7. .....................................................................................574.6.5 Synthesis of the donor for Lex-4-lactam . Scheme 4.8. ....................................................................................59

4.7 Special methods ......................................................................................................................................................604.7.1 Regioselective openings of 4,6-O-benzylidene acetals .....................................................................................604.7.2 Alkylation via stannylene acetals .....................................................................................................................624.7.3 Inversion of glucos- to galactosamine derivatives............................................................................................62

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4.8 Regio- and stereoselective glycosylations..........................................................................................................634.8.1 A literature survey of regioselective glycosylations.........................................................................................634.8.2 Glycosylation with methylsulfenyl triflate, MST............................................................................................644.8.3 Reaction conditions ...........................................................................................................................................654.8.4 The syntheses.....................................................................................................................................................664.8.5 Molecular mechanics minimization..................................................................................................................684.8.6 Discussion on regioselective glycosylations.....................................................................................................70

4.9 Fucosylation.............................................................................................................................................................714.9.1 Methods for α-fucosylation...............................................................................................................................714.9.2 Attempted fucosylation of acceptors carrying NPhth......................................................................................724.9.3 Removal of the tetrachlorophthaloyl (NTCP) group........................................................................................724.9.4 Fucosylation of N-acetamido acceptors ............................................................................................................74

4.10 Sialylation...............................................................................................................................................................754.10.1 Sialylation, general comments........................................................................................................................754.10.3 De-O-acetylation of the acceptors...................................................................................................................774.10.4 Regioselective sialylation leading to tetrasaccharides....................................................................................79

4.11 Alkylation and lactamization of compound 227 ............................................................................................804.12 Final deprotection.................................................................................................................................................80

4.12.1 Formation of 128 by deprotection of compound 232......................................................................................814.12.2 Formation of 129 by deprotection of compound 218......................................................................................814.12.3 Formation of 130 and 132 by deprotection of compound 233 .......................................................................814.12.4 Formation of 134 by deprotection of compound 220......................................................................................824.12.5 Formation of 131 by deprotection of compound 234......................................................................................834.12.6 Formation of 135 by deprotection of compound 219......................................................................................844.12.7 Formation of 136 by deprotection of compound 235......................................................................................854.12.8 Formation of 137 by deprotection of compound 221......................................................................................854.12.9 Comments about the reagents used for deprotection .....................................................................................86

4.13 Summary of the synthetic sequences................................................................................................................87

NMR Ñ configuration and conformation of sialyl Lewis x related compounds ..................89

5.1 Virtual long-range couplings of the anomeric proton.....................................................................................895.2 Determination of regioselectivity in galactosylations .....................................................................................905.3 Sterical crowding in the protected trisaccharides ............................................................................................905.4 Determination of regio- and stereoselectivity in sialylations.........................................................................915.5 Abnormal chemical shift differences due to proximity to oxygen................................................................92

5.5.1 Chemical shift differences of H-5 in the fucose residue....................................................................................925.5.2 Induced chemical shifts due to lactamization..................................................................................................93

5.6 Summary ..................................................................................................................................................................94

Summary and future perspectives ..................................................................................................................95

Acknowledgements..................................................................................................................................................97

References ......................................................................................................................................................................99

Appendix A .................................................................................................................................................................108

Appendix B..................................................................................................................................................................109

1

1

The biological relevance of sialyl Lewis x

ÒThe boundary between biology and chemistry has eroded in recent years. Theintegration of these disciplines has brought an increasingly molecular perspectiveto bear on biological systems. Once thought of as intractable, biomolecules are nowmanipulated as compounds with defined structures and chemical reactivitiesÓ.2

HE UNDERSTANDING of biomolecules is increasing rapidly. Biomolecules of allkinds are manipulated for investigation and regulation of biological events. Theimportance of molecules such as DNA, RNA, and proteins is since long well

established, while carbohydrates earlier were considered to be of no other use than as asource of energy and as construction material. The discovery of the important role ofcarbohydrates for recognition and signaling in living organisms opened a new era ofsynthetic carbohydrate chemistry3,4.The protein-carbohydrate interactions are often rather weak compared with otherinteractions of biomolecules but the weak interactions can be reinforced by multivalentbinding (i.e. several binding sites).

1.1 Carbohydrates

The importance of carbohydrates in molecular recognition originates from their uniquestructural diversity. The specific recognition of a carbohydrate moiety by a protein (e.g.a lectin or an antibody) is dependant on a number of more or less well defined structuralmotifs in the saccharide (indicated in Figure 1.1):

T

2

O

NHAc

HO

HO

OH

O

OHOMe

OH

OO

OMe

HOOH

OH

Methyl-β-D-glucopyranoside

L-fucopyranoside

2-acetamido-2-deoxy-D-galactopyranoside β-(1-4)-coupling

α-(1-3)-couplingHydroxyl groups for hydrogen bonding

Rigid 4C1-conformation of saccharide ring

Hydrophobic patches for non-polar recognition

Figure 1.1 Structural motifs in a trisaccharide.

Epimers (e.g. glc vs gal, D vs L)Ring size (e.g. pyranosides, furanosides)Anomeric configuration (i.e. α vs β)Intersaccharidic linkages (e.g. 1→2, 1→3)Sequence and branching of oligosaccharide chainOther substitution (e.g. COO-, SO3

-, acyl groups, NH3+)

Variation of these factors results in a tremendous number of different oligosaccharidesout of a rather small number of building blocks5.

1.2 Sialyl Lewis x Ð Biological background

The surface of a living cell is densely covered with carbohydrates bound to proteins(glycoproteins), lipids (glycolipids, glycosphingolipids) or polysaccharidicproteoglycans. This is called the cell coat, or glycocalyx. The carbohydrates serve asprotection against mechanical and chemical damage and as receptors in specific cellrecognition processes6. Such diverse processes as sperm-egg interaction, blood clotting,bacterial adhesion and the inflammatory cascade have been proved to depend oncarbohydrate-protein interactions. The carbohydrate chains can be more or lessbranched and usually contain less than 15 monosaccharidic units. Gangliosides arecomplex glycolipids containing at least one sialic acid (NeuAc) residue which gives thecarbohydrate chain specific characteristics. One of the best known gangliosides is thesialyl Lewis x tetrasaccharide (SLex), that can be expressed as glycoprotein orglycosphingolipid, and is important in a number of interactions7.

1.2.1 Leukocyte recruitment and inflammation

In the case of a tissue damage or a microbial infection, white blood cells, leukocytes, arerecruited to the site of injury. When the site is encountered, the leukocytes migrate intothe tissue to repair and protect. The recruitment of leukocytes, referred to as theinflammatory cascade, is a highly complicated process with several steps and signalsystems included8-13 (cf. Figure 1.2).

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Leukocyte

Endothelial cells

Injury

Selectincarbohydrate ligand

Blood vessel

I II III IV II

Figure 1.2 The mechanism of leukocyte recruitment.

The leukocytes circulate in the bloodstream (I). In the case of an injury, certainsignal substances, cytokines, are released as signals for the endothelial cells to displaycarbohydrate binding proteins, selectins. Ligands on the leukocyte can then interactwith the selectins on the endothelial cells. The interaction is weak but the leukocyteslows down and starts to roll, propelled by the blood stream, along the wall of the vessel(II). Another kind of proteins, the integrins, create a stronger interaction which stops theleukocyte (III) and it can then migrate into the surrounding tissue (IV).

If too many white blood cells are recruited to a site of injury, normal (i.e. notinjured) cells can be damaged. This process is known from chronic inflammatorydiseases such as rheumatoid arthritis and psoriasis, in septic shock and in reperfusioninjury following a heart attack, stroke or organ transplant. It is of the highest importanceto understand the inflammatory cascade in order to develop new and more efficientanti-inflammatory drugs.

The selectins is a small group in the carbohydrate-binding lectin family. Sinceselectins are dependant on calcium, they are referred to as C-type lectins. There are threedifferent selectins, E-, P- and L-selectin, according to the cell type on which each wasinitially found (i.e. endothelium, platelets and lymphocytes). They consist of an amino-terminal C-type lectin domain that is highly conserved between different selectins, anepidermal growth factor (EGF)-like domain and a varying number of short consensusrepeat (SCR) sequences (cf. Figure 1.3). A number of different ligands that arerecognized by the selectins have been found.

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Lectin domain

EGF-like domain

Short Concensus Repeat Domain

Cell surface

L-Selectin E-Selectin P-Selectin

Figure 1.3 The different selectins.

P-selectin

The P-selectin (also known as CD62, PADGEM, LECAM-3 and GMP-140) is kept inintracellular stores in blood platelets and endothelial cells. It can be expressed on the cellsurface within a few minutes after exposure to histamine, substance P, peroxide radicalsor thrombin. The only identified ligand for P-selectin is PSGL-1, a mucin-like homo-dimer with O-linked (serine-threonine) oligosaccharides.

E-selectin

The E-selectin (also known as ELAM-1, LECAM-2) is activated by interleukin-1 (IL-1) ortumor necrosis factor α and is expressed on the cell surface a few hours after activation.The ligand for E-selectin is ESL-1. ESL-1 differs from other selectin ligands since it is aglycoprotein with only five potential oligosaccharide sites in the polypeptide chain. Thismakes multivalency binding more unlikely. The oligosaccharides are primarily N-linked(asparagine).

L-selectin

The L-selectin (also known as LAM-1, LECAM-1, Leu-8) is, in contrast to E- and P-selectins, expressed on the surface of leukocytes. The ligands, GlyCAM-1, CD34 andMAdCAM-1, are expressed on the endothelial cells and are all mucin-like and denselyclustered with O-linked oligosaccharides. The L-selectin is involved in leukocyterecruitment but also in the recirculation of lymphocytes (homing).

5

C-type lectins are known to recognize carbohydrate ligands, and E-selectin was found torecognize a carbohydrate ligand with a terminal tetrasaccharide known as sialyl Lewisx14-16 (SLex, section 1.3). It was later found that L- and P-selectins also recognized SLex,although with lower affinity. L- and P-selectin also bind sulfated molecules such asheparin and sulfatides. The binding affinities of SLex to the selectins are rather low, andtypical KD values are in the millimolar range. There are still a lot of questions regardingthe true ligands for selectins.

The discovery of the tetrasaccharide ligand SLex caused tremendous efforts in thesearch for analogs with high binding affinity to the selectins, for use as new potentialanti-inflammatory drugs (chapter 2).

1.2.2 Cancer and metastasis

There is a dramatic change in the composition of the glycocalyx of malignant cancercells compared with normal cells17. Sialyl Lewis x (SLex)18,19 was together with sialyldimeric Lewis x (SLex-Lex)20 and sialyl Lewis a (SLea)21,22 in fact originally identifiedas human tumor-associated antigens. These oligosaccharides are found, asglycosphingolipids (GSL) or glycoproteins, on all highly malignant types of cancer cells.The survival time for patients with SLex-expressing tumors is significantly shorter thanfor patients with other kinds of tumors. SLex and SLea have thereby been used as serummarkers for the diagnosis of cancer23. SLex is preferentially expressed on cancer cellsfrom the lung, ovary, liver, kidney and breast, while SLea have been found on cancercells from colon, pancreas and the biliary tract.

The role of SLex for cancer cells is probably related to metastasis. The cancer cellscan induce the expression of E-selectin on endothelial cells and thereby bind to the cellwall to invade the tissue and form a new tumor. The mechanism is similar to theinflammatory cascade. Apart from the use as tumor markers, antibodies raised towardsSLex may be used as a passive cancer vaccine. Some recent reviews have appeared onthe role of SLex and its analogs in cancer24,25.

1.3 Structure of sialyl Lewis x

The design of new and more efficient ligands for biological systems, i.e. drug design,relies on two different approaches. The traditional way is to synthesize a small numberof carefully chosen analogs while combinatorial methods produce a large number ofmore randomized analogs, a molecular library. Regardless which approach is chosen,knowledge of structure and conformation of the ligand is of the highest importance for asuccessful result.

1.3.1 The sialyl Lewis x saccharides

The sialyl Lewis x ganglioside 1 was first isolated and characterized from humankidney18. The hexasaccharide consists of a Lewis x trisaccharide, Galβ1-4-(Fucα1-3)-GlcNAc, a well known blood group antigen in the Lewis series, that is sialylated andbound to a lactose disaccharide unit (Figure 1.4). The lactose residue is in turn bound toa ceramide.

6

O

OH

HO

O

OH

O

NHAc

R

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

OH

HO

O

OHOMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

NHAc

R

OH

OO

O

OH

HO

O

OH

O

NHAc

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

OH

HO

O

OH

O

NHAc

OH

OO

OMe

HOOH

OH

O

OH

HO

O

OH

O

OH

OH

HOO

O

OH

HO

O

OH

O

AcHN

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

OH

HO

O

OH

O

OH

O

OH

HOO

Sialyl Lewis x hexasaccharide, (SLex-6),

NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1'Cer

C13H27

NHCOC17H35

OH

Sialyl dimeric Lewis x nonasaccharide, (SLex-Lex),

NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-1'Cer

O C13H27NHCOC17H35

OH

Sialyl Lewis x tetrasaccharide, (SLex),

NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-RSialyl Lewis a tetrasaccharide, (SLea),

NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc-R

O

OH

HO OH

O

NHAc

R

OH

OO

OMe

HOOH

OH

Lewis x trisaccharide, (Lex),

Galβ1-4(Fucα1-3)GlcNAc-R

HOO

OH

HO

HO

OHOMe

HOOH

OH

O

NHAc

R

OH

OO

Lewis a trisaccharide, (Lea),

Galβ1-3(Fucα1-4)GlcNAc-R

Sialic Acid Lewis x trisaccharide Lactose disaccharide Ceramide

2

3 4

5 6

1

Figure 1.4 Representative oligosaccharides in the Lewis series.

The smallest ligand recognized by the selectins is the terminal sialyl Lewis xtetrasaccharide26 (3) that is, in the following, referred to as sialyl Lewis x or SLex.Another ligand that is recognized by the selectins is the sialyl Lewis a tetrasaccharide (4)that is isomeric to SLex with inverted connectivity of the fucose- and galactose-residues(Fucα1-4- and Galβ1-3-).

7

1.3.2 The conformation of sialyl Lewis x in solution

The conformation of an oligosaccharide is usually defined by the angles of the glycosidicbond. The glycosidic dihedral angles are defined as φ = τ(H1-C1-Ox-Cx) and ψ = τ(C1-Ox-Cx-Hx) for linkages to the GlcNAc moiety (Figure 1.5) while the NeuAc-Gal angles aredefined as φ = τ(C1-C2-Ox-Cx) and ψ = τ(C2-Ox-Cx-Hx)27. Other definitions have alsoappeared28,29.

O

C1HO

HO OH

Cx O

NHAc

R

OH

HOOxHO

H1 Hxφ ψ

Figure 1.5 Definition of dihedral glycosidic angles.

The dihedral glycosidic angles have been determined using quantitative analysis ofNMR data in connection with computer calculations. The NMR techniques usually relyon the measurement of nuclear Overhauser effects, nOe, which give a mean value ofseveral possible conformations according to a Boltzmann distribution. Essentially threedifferent computational methods have been used with carbohydrates:• Molecular dynamics (MD): shows how the carbohydrate moves during a short

period of time, usually less than 5 ns.• Molecular mechanics: a fast and reliable method to calculate low energy

conformations. The most used force fields are MM2 and the newer MM3.• Hard Sphere Exoanomeric (HSEA) calculations: a very fast method to give

preliminary conformational data. The GESA (geometry of saccharide) program is anextension of HSEA

A combination of several techniques is often used to calculate the conformation.

Conformation of the Lewis x trisaccharide

The Lewis x trisaccharide part of sialyl Lewis x has been shown to have very lowconformational flexibility. An MM3 calculation showed that more than 99.5 % of theconformers belonged to the same conformational family with only minor (±5°) variationof the angles28. The RMS (root mean square) differences for the time-averagedglycosidic torsion angles were 50% lower for the Lewis x part of the tetrasaccharidecompared with the NeuAc-Gal linkage in a molecular dynamics calculation30. The φ,ψ-angles obtained in different studies are given in Table 1.1. A few older conformationalstudies of Lex are mainly consistent with these data31,32.

Although there are some small differences especially between the fucose φ angles,the conformational space for the Lewis x trisaccharide is well defined with the fucose-and the galactose-moieties stacked in a very tight conformation (cf. section 5.3 and 5.5).The Lex-conformation is conserved in the sialyl Lewis x tetrasaccharide (see below).

8

Table 1.1 Conformational studies on the Lewis x trisaccharide part of the sialyl Lewis x tetrasaccharide.

Reference Galβ-(1-4)GlcNAc Fucα(1-3)GlcNAc Methodφa ψa φa ψa

Poppe (1997)33 46 18 48 24 NMRImberty (1995)28 44 18 35 31 MM3Paper I (1994) 43 11 28 31 MM2Rutherford(1994)30

50 15 48 22 NMR, MD

Ichikawa(1992)34,35

48 15 22 30 NMR, GESA, MM2

aSome original φ and ψ angles were recalculated to fit the φ/ψ-definition given above. φ = τ(H1-C1-Ox-Cx)and ψ = τ(C1-Ox-Cx-Hx)

Conformation of the NeuAc-Gal residue

A conformational search of the NeuAc-Gal disaccharide results in a very flexiblestructure with several minima36. However, when the NeuAc-Gal is present in the SLextetrasaccharide, it is more restricted due to steric hindrance and intramolecularhydrogen bonding. The NeuAc-Gal bond is anyway much more flexible than the otherintersaccharidic bonds in the tetrasaccharide. Several investigators have used NMR andcomputational methods for determination of conformations of SLex in solution assummarized in Table 1.2.

Table 1.2 Different conformations for the NeuAc-Gal moiety of the sialyl Lewis x tetrasaccharide.

Reference Minimaa NeuAcα-(1-4)Gal

Method

φa ψaPoppe (1997)33 A

BC

-60-100180

0-50

0

NMR

Paper I (1994) Global -36 35 MM2Rutherford(1994)30

AB

-70-160

5-20

NMR, MD

Mukopadhyay(1994)29

ABC

-146-73

-127

-211140

Ichikawa (1992)34 ABCD

163-170-7968

-57-87

-20

NMR, GESA,MM2

Wormald (1993)37 -130 -95 NMRaThe notation A-D is referred to the notations in the original publications.bSome original φ and ψ angles were recalculated to fit the φ/ψ-definition givenabove. φ = τ(C1-C2-Ox-Cx) and ψ = τ(C2-Ox-Cx-Hx)

9

We performed a molecular mechanics calculation (MM2) on the SLex tetrasaccharideusing a dihedral driver around the NeuAc-Gal coupling with 30° increments (paper I).Figure 1.6 shows an energy map of the resulting conformational space (lowest 3 kcal areshown) together with energy minima conformations from Table 1.2.

-180 -90 0 90 180

ψ

-180

-90

0

90

180

φ

Conformations according to Poppe

Conformations according to Ichikawa

Conformations according to Rutherford

Conformation according to Wormald

II

III

I

I

Conformations according to Mukopadhyay

Global minimum according to the MM2 calculation

Figure 1.6 Conformational space of SLex, obtained by molecular mechanics (MM2) calculation (Paper I).

Three different energy minima were obtained from the MM2 calculation (I-III). The datafrom Table 1.2 fit very well into the energy map. The conformation determined byWormald37 is an exception, probably being a virtual conformation. Virtualconformations can be seen when there is a fast (on the NMR time scale) exchangebetween several conformers. Both NMR and computational methods indicates severalconformations of the NeuAc-Gal residue with low energy barriers. The MM2-calculatedenergy minimum I is consistent with minima for the NeuAcα2-3Gal segment in mostgangliosides38.

1.3.3 The bioactive conformation of sialyl Lewis x in complex with selectins

The bioactive conformation of a carbohydrate must not necessarily be the same as theconformation in solution. The three-dimensional structure of E-selectin has beendetermined at 2.0 resolution using X-ray crystallography39 but the SLex-selectincomplex has not yet been crystallized. Determinations of the bioactive conformationshave been done using NMR techniques instead. Essentially four different investigationshave been reported.• In the first investigation (Cooke40, 1994), the conformation of SLex in complex with

E-selectin was determined by transfer nOe experiments. The conformation of boundSLex differed from the virtual solution conformation, thus indicating that onedistinct conformation is recognized by the protein. The preferred conformation isclose to IchikawaÕs34 conformation C (NeuAc-Gal φ = -79°, ψ = 7°; cf. Table 1.2).

• In the second investigation (Hensley41, 1994), the conformation of SLex in complexwith a soluble E-selectin, was determined with NMR techniques. The authorsdescribe the conformation of bound SLex as similar to the solution conformation,without mentioning which solution conformation they refer to.

10

• In the third and more thorough investigation (Peters42, 1995) of the SLex-E-selectincomplex, one rigid structure was indicated, in contrast to the multiple flexiblesolution conformations discussed above. The φ/ψ -angles for the Lewis xtrisaccharide part were essentially the same as for the solution conformation.Furthermore only one conformation was adopted for the NeuAc-Gal coupling,analogous to the Ichikawa34 C or Rutherford30 A conformations.

• In the fourth and most extensive study so far (Poppe33, 1997) the conformations ofSLex bound to E-, P- and L-selectin were determined. The E-selectin-bound SLextetrasaccharide adopted the Poppe A conformation (φ/ψ = -60, 0°), which is veryclose to the Rutherford30 A and Ichikawa34 C conformations (Table 1.2). Anintramolecular hydrogen bond between the NeuAc carboxyl group and Gal-OH2was of great importance to the E-selectin bound conformation. The (-60, 0°)conformation was also adopted by SLex when bound to P-selectin, while L-selectinrecognized the Poppe B conformation (-100, -50°).

The crystal structure of E-selectin has been used for determination of bioactiveconformations of SLex; however the investigations are at best approximate39,43. Despitethe drawbacks, these investigations show the importance of the hydroxyl groups of thefucose moiety for interaction with calcium ion.

1.3.4 Sialyl Lewis x conformation, summary

The Lewis x trisaccharide part of the sialyl Lewis x tetrasaccharide is rigid with very lowconformational flexibility, whereas the NeuAc-Gal intersaccharidic bonds show a highdegree of flexibility with several energy minima in solution. Three minima have beenshown to be most important for the solution conformation. One conformation (close to IIin Figure 1.6) has been shown to be most important for the SLex complexed with E- andP-selectin while another conformation (close to I in Figure 1.6) is preferred by the L-selectin. The conformational flexibility of SLex is probably essential for the function ofthe SLex-selectin interactions. Figure 1.7 shows the most important conformation (II) ofSLex.

NeuAc

Gal

Fuc

GlcNAc

Figure 1.7 Stereoview of the most important conformation of SLex, (methyl glycoside).

11

2Synthetic analogs of sialyl Lewis x

N ORDER TO examine the bioactive conformation of SLex and to pinpoint thegroups that are important for the binding of different selectins, a large number ofSLex analogs have been synthesized as described in a recent review by Wong44. The

objective of this chapter is to summarize important modifications of the saccharides andtheir effects on the biological activity.

2.1 Biological assays

The biological activity, referred to as ÒactivityÓ, ÒinteractionÓ, or ÒrecognitionÓ, isusually measured in some kind of assay. Essentially two different types of assays havebeen used for measuring the activity of SLex analogs; ELISA assays or Cell adhesionassays26,45,46.

The ELISA assay employs a ligand (i.e. SLex or GlyCAM-1 with high affinity toall three selectins) immobilized in microtiter wells. The inhibitor (i.e. a SLex analog)together with an enzyme-tagged anti-IgG-antibody are added to a solution of a selectin-IgG fusion protein (E-, P- or L-selectin), incubated for a certain time, and then added tothe microtiter wells. The wells are washed to remove any unbound selectin-IgG. Theantibodies are tagged with an enzyme that can transform a detection reagent from anuncolored to a colored form. The color is then measured to quantitate the result. Theresult is usually given as the IC50 value, i.e. the concentration of the inhibitor that givesa 50% inhibition of the selectin-SLex complex.

The cell adhesion assay differs from the ELISA in that the analogs binding toselectins are measured in competition with human HL60 cells (i.e. tumor cells with ahigh concentration of SLex ligands on the surface). The HL60 cells are lysed and assayedfor myeloperoxidase activity to quantitate the result.

The results are usually compared to the binding capacity of unmodified SLex.Due to variations in the assays, the results are difficult to compare with each other. Theactivities of the analogs below are therefore given as very crude measures. The poor

I

12

inhibitors are referred to as ÒinactiveÓ or Ònot recognizedÓ. Inhibitors with the sameactivity (i.e. up to 2-3 times better or worse than SLex) are referred to Òas good asÓ theSLex saccharides. The really good inhibitors are specifically mentioned.

2.2 Modification of the glucosamine residue

The GlcNAc residue in SLex is mostly regarded as a rigid backbone of theoligosaccharides. The connectivity between the fucose and the galactose residue (i.e. 1-3or 1-4, SLex and SLea respectively) is not important since both tetrasaccharides show thesame activity against selectins.

2.2.1 Deoxy analogs

The deoxyanalogs 747, 848, and 948 were synthesized and tested against selectins49(Figure 2.1).

O

NHAc

OH

OH

OO O

NHAc

OH

OO O

OH

OH

OO O

OH

OO

SLex 7 (1-deoxy) 8 (1-deoxy-2-hydroxi) 9 (1,2-dideoxy)

O

OH

OO

10 (glycal)

Figure 2.1 Deoxy analogs of the GlcNAc residue.

The 1-deoxy analog 7 was less active than SLex against E-selectin but up to 20times more potent towards P- and L-selectins. Compounds 8 and 9 showed similaractivity as the parent tetrasaccharide.

The glycal analog 1034,50 was as active as SLex tetrasaccharide against E-selectin51.

2.2.2 Modification of the acetamido group

A number of analogs with different nitrogen groups (11-16) were synthesized andtested52,53. The azido- and amino analogs of SLex and SLea enhances the interactionwith E-selectin while replacement by the more sterically demanding N-propionyl group(14) had little or no effect for the SLex analog but decreased the activity of the SLeaanalog at least 4 times.

Hayashi reported the synthesis of the butyryl and naphtoyl amide analogs (15and 16, Figure 2.2)54. These analogs were as active as the SLex tetrasaccharide towardsE-selectin.

Another investigation used a SLex analog with acetamides replaced by amines inboth the GlcNAc and the NeuAc residues55. This analog was 5-10 times as active as theSLex tetrasaccharide in E- and P-selectin assays.

13

O

OH

OR

OH

OO

17

O

R1OR

OH

OO

11 R1 = N312 R1 = NH2 13 R1 = NHAc 14 R1 = NHCOEt 15 R1 = NHCOPr

16 R1 =O

NH

Figure 2.2 Modification of the 2-acetamido group

An early investigation showed that the glucosamine residue could be replaced bya glucose moiety (17) without decreasing the activity of the saccharide against E-selectin26. The SLex(Glc) compound is much easier to synthesize than SLex since lactosecan be used instead of lactosamine. It has been synthesized by a number of groups56-58and used in several analogs48. A later investigation59 indicated that the trisaccharideLex(Glc) was much more flexible than Lex but this does not seem to influence theactivity.

2.2.3 Deoxynojirimycin derivatives

The 1-deoxynojirimycin derivatives 1860 and 1961 of SLex and SLea were synthesized byHasegawaÕs group (Figure 2.3).

N

OH

OH

OO

18 (1-deoxynojirimycin)

MeN

NHAc

OH

OO

19 (2-NHAc-1-deoxynojirimycin)

Me

Figure 2.3 Deoxynojirimycin derivatives.

All compounds Òexhibited potential inhibitory activity against selectin binding in vitroÓ.

2.2.4 Other analogs

The glucosamine residue was replaced by a cyclohexadiol to give compound 20 (Figure2.4)62. This analog was as active as the SLex tetrasaccharide in E- and P-selectin assays.

OO

20

Figure 2.4 Replacement of the GlcNAc residue by a cyclohexadiol

14

2.2.5 The importance of the aglycon

Hasegawa showed in an investigation using different lipid aglycons that the binding ofthe two sulfated Lex-trisaccharide (SuLex) analogs 21 and 22 differed63,64 (Figure 2.5).Compound 22 with a dialkyl aglycon had a stronger binding than the ceramidederivative 21 or the reducing sugar 23 probably due to a positive interaction with twohydrophobic regions on the surface of the E-selectin65. Another investigation alsorevealed that oligosaccharides with fatty acid spacer groups were more active than thereducing saccharides53.

O

OH

OR

OH

OO

21 R =

22 R =

C13H27

NHCOC17H35

OH

23 R = H

Figure 2.5 Spacer groups

2.2.6 Summary

The GlcNAc moiety of SLex functions mainly as a scaffold for the Gal and the Fucmoieties. Position 1 and 2 can be deoxygenated without loss of activity. SLea does nottolerate sterically demanding groups in position 2. The aglycon has been shown to beimportant in some assays.

2.3 Modification of the galactose residue

2.3.1 Deoxy analogs

A number of deoxy analogs of the galactose residue of the SLex tetrasaccharide havebeen synthesized (Figure 2.6). The 4- and 6-deoxy analogs (24 and 25) were prepared byStahl66. Later, Hasegawa67 synthesized the 4- and 6-deoxy SLex-pentasaccharidestogether with the 4,6-dideoxy analog 26.

O

OH

O

OHO

OH

HO

OO

OH

OO

OH

HO

O

OH

SLex 24 (4-deoxy) 25 (6-deoxy) 26 (4,6-dideoxy)

Figure 2.6 Deoxy analogs of SLex.

The 6-deoxy analogs were not recognized by any of the three selectins whereasHasegawaÕs 4-deoxy analog was recognized by P-selectin but not by E- or L-selectins.

15

2.3.2 Fluoro- and acetyl analogs

The 6-deoxy-6-fluoro- (27)66 and the 2-O-acetylated (28)33 analogs shown in Figure 2.7were synthesized.

O

OH

HO

O

F

27 (6-fluoro)

O

OAc

HO

O

OH

28 (2-acetyl)

Figure 2.7 Fluoro- and acetyl analogs

The 6-deoxy-6-fluoro analog showed a significantly lower binding to E-selectin than theparent tetrasaccharide verifying the importance of the 6-hydroxy group66,68. The 2-O-acetylated compound showed the same binding affinity to E-selectin as SLex.

2.3.3 Other analogs

Two other analogs have been synthesized (Figure 2.8). The galactose moiety has beenreplaced by a six-atom bridge to give compound 29. Compound 30 69is an epimer ofSLex with the galactose α(1-4)-coupled to the GlcNAc moiety. Unfortunately no datahave been published concerning the biological activity of these two compounds.

OO

O

NHAc

R

OH

OO

OMe

HOOH

OH

O

COOHHO

OH

AcHN

HO

OH

O

HO

HO

O

OH

O

HOOC

HOOH

AcHNHO

OHO

OH

O

NHAc

O

OH

OHO

HO

OH

29 30

Figure 2.8 Other analogs.

2.3.4 Summary

The galactose residue is important, not only as a linker between the sialic acid and theGlcNAc residue, but also for providing hydroxyl groups for selectin binding. The HO-6group is important for the binding to all three selectins whereas HO-4 group isnecessary for binding to E- and L- but probably not to P-selectin. The 2-hydroxyl groupis not necessary for the binding to E-selectin.

16

2.4 Modification of the fucose residue

The fucose residue is very important for the binding of SLex to selectins. Non-fucosylated analogs showed no activity in an E-selectin assay53. It has been suggestedthat the hydroxyl groups of the fucose residue are involved in the binding to Ca2+ in theSLex-selectin complex39. Several analogs have been synthesized in order to evaluate theimportance of the hydroxyl groups in the fucose residue.

2.4.1 Deoxyanalogs

All three monodeoxy analogs of the SLex-pentasaccharide (Figure 2.9) have beensynthesized as the corresponding ceramide70,71 and ethylglycoside72(Figure 2.9).

O

OMe

HOOH

OH

O

OMe

HOOH

O

OMe

HO

OH

O

OMe

OHOH

31 (2-deoxy-fucose) 32 (3-deoxy-fucose) 33 (4-deoxy-fucose)SLex

Figure 2.9 Deoxyanalogs of the fucose residue

None of these compounds were recognized by E- or L-selectin, while both the 2- and 4-deoxy-analogs bound to P-selectin with approximately the same binding strength asSLex.

2.4.2 Epimers

The 2-epi-, 4-epi- and 2,4-di-epifucopyranosyl analogs of the SLex pentasaccharideganglioside have been synthesized by Hasegawa73,74 (Figure 2.10).

O

OMe

HOOH

OH

O

OMeHO

OHOH

O

OMeHO

OHOH

34(2-epi-fucose)

35(4-epi-fucose, quinovose)

36(2,4-di-epi-fucose, rhamnose)

Figure 2.10 Epimers of the fucose residue.

None of these analogs were recognized by any of the selectins.

17

2.4.3 Other modifications

A few other modifications have been done on the fucose residue as depicted in Figure2.11.The arabino analog 3772 of the SLex pentasaccharide showed about 20% of theactivity of SLex against E-selectin while the 2-O-methyl analog 38 was not at allrecognized by any of the selectins.

A 5-thio-fucose analog of the Lewis x trisaccharide (39) has been synthesized75but this interesting compound has not yet been tested against selectins.

O

O

HOOH

OH

O

OMe

HOOH

OMe

37(arabinose) 38(2-O-methylfucose)

O

SMe

HOOH

OH

39

Figure 2.11 Other modifications of the fucose moiety2.4.4 Summary

The presence and configuration of all hydroxyl groups in the fucose moiety seem to beimportant for the binding of E- and L-selectins, while only HO-3 is crucial for thebinding of P-selectin. Removal of the 6-methyl group of fucose decreases the activityagainst E-selectin slightly.

2.5 Modification of the sialic acid residue

The sialic acid residue is included in the SLex epitope recognized by the selectins butseveral investigations have concluded that only the anionic moiety is essential forbinding. Non-sialylated oligosaccharides without an anionic residue had little or noactivity53.

2.5.1 Modification of the glycerol side chain

Pentasaccharide analogs with a truncated or epimerized glycerol side (Figure 2.12) weresynthesized by Hasegawa46,76.

O

COOHHO

OH

AcHNHO

OH

O

COOHOH

AcHNOH

O

COOHHO

AcHNHO

OH

O

COOHHO OH

AcHNHO

OH

SLex 40(C7-NeuAc)

41(C8-NeuAc)

42(8-epi-NeuAc)

Figure 2.12 Analogs with modified glycerol side chain.

18

All analogs bound to E-, P-and L-selectin to the same extent as SLex indicating that theglycerol side chain plays no important role for the selectin recognition.

2.5.2 Modification of the acetamido group

A pentasaccharide analog of SLex without the 5-acetamido group (i.e. aketodeoxynonanoic acid, KDN) was synthesized by Hasegawa46 (43). A similartetrasaccharide analog was synthesized together with the 5-amino analog 44 (Figure2.13) by Unverzagt55. The N-glycolyl analog 45 was synthesized by Hasegawa77.

O

COOHHO

OH

AcHNHO

OH

SLex

O

COOHHO

OH

HO

OH

45(NeuGc)

O

COOHHO

OH

H2NHO

OH

44(Neu)

43(KDN)

O

COOHHO

OH

HNHO

OHO

HO

Figure 2.13 Modification of the 5-acetamido group.

All three analogs recognized E-, P- and L-selectin with approximately the same efficacyas the natural compound indicating that the 5-acetamido group is not important for thebinding to the selectins. However another investigation using the 5-deoxy analog 45showed a significant decrease in activity in an E-selectin assay53.

2.5.3 Replacement of sialic acid by sulfate

In 1993, an equimolar mixture of sulfated Lex and Lea tetrasaccharides was isolatedfrom an ovarian cystodenoma glycoprotein. The mixture exhibited E-78 and L-selectin79binding properties. The two tetrasaccharides (SuLex and SuLea) were sulfated inposition 3 of the galactose residue (Figure 2.14) and thereby being sulfated analogs ofSLex and SLea.

O

OH

HO

HO3SO

OH

O

AcHN

OH

OO

OMe

HOOH

OH

O

OH

HO

O

OH

OH

46 SuLex-tetrasaccharide

O

OH

HO

HO3SO

OHOMe

HOOH

OH

O

NHAc

O

OH

OO O

OH

HO OH

OH

47 SuLea-tetrasaccharide

Figure 2.14 Sulfated Lex and Lea tetrasaccharides.

This discovery initiated several synthetic programs aiming at sulfated analogs.The SuLex tri-80-82, tetra-81, and pentasaccharides83 were synthesized, as well as thetri-81,82,84, tetra-81 and pentasaccharides85 of SuLea. These saccharides were testedagainst E-selectin and were all active86. The Lea analogs were slightly more active thanthe Lex compounds and the pentasaccharides were more active than the smaller analogs.

19

The SuLea pentasaccharide proved to be at least 15 times more potent than the SLextetrasaccharide.

An investigation using SuLex-trisaccharide analogs, with the GlcNAc residuechanged to a Glc, showed that the sulfated analogs are recognized not only by E- butalso by P- and L-selectins. However, binding to the latter showed characteristics distinctfrom SLex recognition46,63. It was suggested that SLex and SuLex are recognized by E-selectin in the same way, but differently by P- and L-selectin.

A molecular dynamics simulation of the interaction between E-selectin and asulfated Lewis x saccharide has been reported87. This investigation implies that thecarbohydrate portion of the SuLex interacts in essentially the same way as that of SLex.

2.5.4 Replacement of sialic acid with phosphate

After the discovery of the importance of sulfated Lex and Lea structures, a variety ofanalogs with other anionic substituents have been synthesized.

Hasegawa reported a series of Lex analogs where the GlcNAc moiety wasreplaced by Glc or 1-deoxy-Glc and the sialic acid residue by a phosphate, as depicted inFigure 2.15 (48)49,88. The phospho analogs were recognized by all selectins, although toa lower degree than the parent SLex compound.

O

OH

HO

Na2O3PO

OH

O

OH

OH

OO

OMe

HOOH

OH

ORR = H or alkyl

48

Figure 2.15 Phosphate analogs of Lex.

A similar phospho Lewis a trisaccharide was synthesized by Kiessling89 and wasfound to bind as strongly as the sulfated analog to E- and L selectins.

2.5.5 Replacement of sialic acid by a carboxylic acid

The sialic acid residue has been replaced by an acetic acid moiety(49)88. This analogshowed Òsignificant competitive inhibition activityÓ against all three selectins whereasthe analog 5090 with a sterically fixed carboxylic acid group was found to be inactiveagainst E-selectin (Figure 2.16).

The 3Õ-C-carboxymethyl derivative 51 was much more active than thecorresponding SLex tetrasaccharide91.

20

HO

OO

HOOCHO OH

AcHN

HO

OH

HO

OHOOC

HO

OHOOC

O

HO

OHOOC

HO

OHOOC

HR H R R = H (54)R = Ph (55)

R = H (52)R = Ph (53)

49 50

HO

51

HOOC

SLex

Figure 2.16 Replacement of the sialic acid by a carboxylic acid.

A number of lactic acid derivatives were synthesized92 to investigate the role ofthe absolute configuration of the carboxylic acid moiety. The (R)-series (52 and 53) wereinactive whereas the (S)-series (54 and 55) proved to be only 2-3 times less active thanSLex.

2.5.6 Summary

The side chain and the acetamido group of the sialic acid residue of sialyl Lewis x arenot essential for the binding to selectins. The sialic acid moiety can be replaced by avariety of other anionic groups with retained or even increased binding to the selectins.The mechanism of binding is however different for the three selectins and only E-selectin seems to bind SuLex in the same way as SLex.

2.6 Sulfated analogs

The SLex and SLea tetrasaccharides were early found to be the smallest carbohydrateligands recognized by selectins (section 1.2.1). Later it was found that 3Õ-O-sulfo-Lex andLea also were natural ligands (section 2.5.3). Rosen reported in 199493 the presence of 6Õ-O-sulfated SLex in GlyCAM-1, the natural ligand for L-selectin. This resulted in thesynthesis of a variety of sulfated SLex and Lex analogs for testing in selectin assays.

2.6.1 Sulfated sialyl Lewis x analogs

Hasegawa synthesized three different sulfated sialyl Lewis x hexasaccharides accordingto Figure 2.1794-96.

21

O

OH

HO

O

OR2

O

AcHN

OR1

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

OH

HO

O

OH

O

OHO

OH

HOO

C13H27NHCOC17H35

OH

R1 R2HSO3NaSO3Na

SO3NaHSO3Na

565758

Figure 2.17 Sulfated SLex analogs

All three analogs, 56, 57 and 58 were recognized by L- and P-selectins with thesame efficacy as the SLex hexasaccharide. Compounds 56 and 58, sulfated on HO-6 ofthe galactose residue, were not recognized at all by E-selectin. This is in full agreementwith other investigations (section 2.3.1) and indicates the importance of HO-6 forbinding. Compound 57 was recognized with only slightly lower activity.

Matta synthesized the 6Õ-O -sulfated analogs of the SLex and SLeatetrasaccharides97,98. The binding results99 were in agreement with the results ofHasegawa.

2.6.2 Sulfated Lewis x analogs

Kiessling reported a number of synthetic sulfated Lewis x and Lewis a trisaccharideanalogs100-102 according to Figure 2.18.

O

OH

HO

R2O

OR3

O

OH

OR1

OO

OMe

HOOH

OH

O O

OH

HO

R2O

OR3

OMe

HOOH

OH

O

OH

O

OR1

OO

R1 R2 R3

596061626364

SO3NaHHSO3NaSO3NaSO3Na

HSO3NaSO3NaHSO3NaSO3Na

HHSO3NaSO3NaHSO3Na

R1 R2 R3

65666768

HHHSO3Na

HSO3NaSO3NaSO3Na

SO3NaHSO3NaH

Figure 2.18 Sulfated Lewis x and Lewis a analogs.

To summarize, these analogs bound as good as, or better than the sialyl Lewis xtetrasaccharide to L- and P-selectins but only the 3Õ-O-sulfated analogs (SuLex, 60 and

22

SuLea, 66) bound to E-selectin, which is in full agreement with earlier data (section 2.3.1and 2.5.3).

Kondo prepared a number of Lewis x analogs sulfated in 3- and/or 4-position ofthe galactose103 (Figure 2.19) but no biological data has been published yet.

O

OH

R2O

R1O

OH

O

OH

OH

OO

OMe

HOOH

OH

OC14H29

R1 R2

697071

SO3NaHSO3Na

HSO3NaSO3Na

C14H29

Figure 2.19 Sulfated Lewis x analogs.

2.6.3 Summary

Sulfation in position 6 and 6Õ of SLex and Lex derivatives enhances the binding to L- andP-selectin but decreases the activity against E-selectin.

2.7 Other modifications

2.7.1 Positional isomers of SLex

SLex and SLea are positional isomers and are equally active. Other positional isomershave been synthesized according to Figure 2.20.

Compounds 72, 73 and 74 were synthesized by Hasegawa104 and tested againstE-, P- and L-selectins while compounds 75 and 76 were prepared by Bevilacqua53 andtested in an E-selectin assay. None of these analogs showed any binding to the selectinstested indicating that the over-all structure of the SLex saccharide is of highestimportance.

Another investigation concerning the lectin domain of E- and P-selectins foundthe structure to be incompatible with a 2-6-linked sialic acid105.

23

O

OH

HO

O

OH

O

NHAc

R

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

OH

HO

O

OHOMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

O

NHAc

R

OH

OO

1 (SLex),

NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc2 (SLea),

NeuAcα2-3Galβ1-3(Fucα1-4)GlcNAc

O

OH

HO

O

OH

O

HOOCHO OH

AcHNHO

OH

O

O

OH

OHO

O

HOHO

OH

O

O

OH

OHO

OMe

HOOH

OH OHO

OHO

OH

OCOOH

OH

OH

AcHNHO

HO

72NeuAcα2-3Galβ1-3(Fucα1-2)Glc

73NeuAcα2-3Galβ1-2(Fucα1-3)Glc

OMe

HOOH

OH

OO

HOO

OHO

OHO

OH

OCOOH

OH

OH

AcHNHO

HO

74NeuAcα2-3Galβ1-6(Fucα1-4)Glc

O

OH

HO

O

OH

O

NHAcR

O

HOO

O

HOOCHO OH

AcHNHO

OH O

HOHO

OH

76NeuAcα2-3Galβ1-4(Fucα1-6)GlcNAc

O

OH

HO

HO

O

O

NHAcR

OH

OO

O

HOHO

OH

OCOOH

OH

OH

AcHNHO

HO

75NeuAcα2-6Galβ1-3(Fucα1-4)GlcNAc

Figure 2.20 Positional isomers of sialyl Lewis x.

2.7.2 Thio-linked SLex

A thio-linked analog of the sialyl Lewis x tetrasaccharide (77, Figure 2.21) wassynthesized by Schmidt in 1996106,107. Thioglycosides are much more stable againstenzymatic hydrolysis than normal O-linked glycosides108. Conformational analysis of athiogalabioside showed that the thio analog had a slightly altered conformationcompared to the parent compound109. The C-S bond is about 0.4 longer than the C-Obond and the C-S-C angle differs from the C-O-C angle by about 16°. These differencesresulted in the loss of one important hydrogen bond and this analog was biologicallyinactive. A conformational analysis110 of compound 77 showed that this interestinganalog also were more flexible than the parent tetrasaccharide. The biological activityhas not been reported yet.

24

O

OH

HO

S

OH

O

OH

S

OH

SS

OMe

HOOH

OH

O

HOOCHO OH

AcHN

HO

OH

77

Figure 2.21 Thiolinked SLex.

2.8 Multivalent sialyl Lewis x analogs

The natural ligands to the selectins are multivalent with respect to the oligosaccharides(cf. section 1.2.1). It has been proposed that recognition by a selectin is multivalent andseveral analogs with two or more SLex residues have been prepared. Multivalentanalogs of oligosaccharides have been used with success in a number of other cases111-115. The activity of multivalent analogs are based upon the molarity of SLex units.

2.8.1 Examples of multivalent analogs

Wong synthesized a number of bivalent sialyl Lewis x analogs (Figure 2.22)51,116 whichwere tested in an E-selectin assay. The galactose-based dimers (78-82) were all moreactive than the monomeric tetrasaccharide with the general activity trend being 3,6-linked > 2,3 ≥ 4,6 ≥ 2,6 > monomer. The 3,6-linked dimer was 5 times more active thanthe monomer. The dimers linked to butanediol and pentanediol showed no increasedactivity.

O

OH

HO

SLex-β-O

O-β-SLex

OEt

81 (3,6)

O

SLex-β-O

HO

HO

O-β-SLex

O(CH2)5COOMe

80 (2,6)

O

OH

SLex-β-O

SLex-β-O

OH

O(CH2)5COOMe

79 (3,4)

O

OH

SLex-β-O

HO

O-β-SLex

O(CH2)5COOMe

82 (4,6)

O

SLex-β-O

HO

SLex-β-O

OH

O(CH2)5COOMe

78 (2,3)

SLex-β-OO-β-SLex

SLex-β-O O-β-SLex

83 (C4) 84 (C5)

Figure 2.22 Bivalent analogs of SLex.

25

Kretzschmar synthesized a number of trivalent SLex analogs117. Compounds 85, 86 and87 are based on nitromethane-trispropionic acid (Figure 2.23). They were tested againstE- and P-selectins and were found to have higher activity than the monomerictetrasaccharide. Compound 86, with a medium spacer length, proved to be the bestfollowed by 85 and 87.

SLex-β-HN

SLex-β-HN

SLex-β-HN

O

OO

NO2 NH

NH

NH

O

OO

NO2SLex-β-O

SLex-β-O

SLex-β-O

NH

NH

NH

O

OO

NO2HN

HN

HN

O

O

OSLex-β-O

SLex-β-O

SLex-β-O

85 86

87

Figure 2.23 Trivalent analogs of SLex based on nitromethane-trispropionic acid.

Later, Kunz prepared two trivalent SLex analogs based on cyclic peptides (Figure2.24)118. The two analogs 88 and 89 were 2-3 times as active as the monomeric SLextetrasaccharide.

NH

HNHN

NH

NH

NH

HN

O

OO

O

O

OO

Et

NH-β-SLex

SLex-β-HNPh

O

O

89

O

NH-β-SLex

NH

HNHN

NH

NH

NH

HN

O

OO

O

O

OO

SLex-β-HN NH-β-SLex

OO

ONH-β-SLex

88

HO

Figure 2.24 Trivalent analogs of SLex based on cyclic peptides

26

Roy reported three dendritic structures containing 2, 4 or 8 SLex tetrasaccharides1998119 (Figure 2.25). The biological activity of these interesting structures is now underinvestigation.

SLex-β-SO

Gly-Gly Lys-β-Ala-OHSLex-β-S

O

Gly-Gly Lys2-Lys-β-Ala-OH

SLex-β-SO

Gly-Gly Lys4-Lys2-Lys-β-Ala-OH

2 4

8

90 91

92

Figure 2.25 Dendritic SLex analogs.

2.8.2 Liposomes containing SLex

DeFrees reported the synthesis of 93 (Figure 2.26) and the preparation of liposomesthereof120. The liposomes contained 0.5-5 mol% of 93 and had an average diameter of100 nm. These liposomes were presented to E-selectin and were more than 5000 times asactive as monomeric SLex. The liposomes were also tested in vivo in a cat myocardialinfarction model and found to be 40 times more active than the monomer121.

HO

O

AcHN

O

O

HO

OH

OH

OH

HO

OHNaOOC

OO

OH

NHR

O

OMe

HOOH

OHHO

OO

OH

OHOEt

R =NH

SHN

OO

HN

O

O

O

OPO

ONaO C17H35

O

O

O

C17H35O

n

n = 42-48

93

Figure 2.26 Compound 93 for liposomes.

27

Another liposome preparation was made by Rice 1998122. Biological data of theseliposomes (Figure 2.27) have not yet been presented.

SLex-β1-2Manα1

SLex-β1-2Manα1

63

Manβ1-4GlcNacβ1-4GlcNAcβ1-Tyr-NHCO-PEG-SO2(CH2)2-S-(CH2)2-O-PO(OH)-O-Liposome

94

Figure 2.27 Compound 94.

2.8.3 Polymers

Biotinylated polyacrylamide-type glycoconjugates containing SLex or SLea wereprepared by NifantÕev for use in cell free assays for E-selectin ligands123. A SLexpolymer has also been published by Thoma124 but without further data.

2.8.4 Summary

A number of polyvalent SLex analogs have been reported in the literature. Theoligomeric analogs (2-8 SLex units) showed slightly enhanced activity (2-5 times moreactive) while liposomes containing SLex showed very high activity (5000 times moreactive). Investigations using linkers of different length showed the best result with aÒmedium sizedÓ linker.

2.9 Mimics of sialyl Lewis x

The boundary between analogs and mimics (or mimetics) is difficult to define. Somecompounds are easily labeled as analogs while others are mimics. In this thesis I havechosen to define analogs as compounds with only minor modifications of the parentcarbohydrate (i.e. modification in only one monosaccharide unit) and mimics as moredrastically modified carbohydrates (i.e. more than one monosaccharide unit altered) ornoncarbohydrate compounds.

An large number of analogs has been reported in the literature and it is out of thescope of this thesis to give a complete summary. Instead a very brief description ofdifferent types of mimics is given. A thorough review was recently published byWong44 but it should be noted that some of the references are displaced.

2.9.1 Mimics derived from SLex

Most of the reported mimics are derived from the SLex tetrasaccharide by replacingparts of the molecule by other groups. These modifications strive for i)simplifiedmolecules that are easy to synthesize and ii)molecules that are more stable againsthydrolysis and therefore potentially useful as drugs. A few examples of mimics derivedfrom the SLex tetrasaccharide are shown in Figure 2.28.

28

O

OH

HO

O

OH

O

NHAc

R

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

- NeuAc O

OH

HO

O

OH

O

NHAc

R

OH

OO

OMe

HOOH

OH

HOOC

- GlcNAc

O

OH

HO

O

OH

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

- GlcNAc

O

OH

HO

O

OH

OO

OMe

HOOH

OH

HOOC

- Gal

- GalO

OO

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH

OH

- NeuAc

OO

OMe

HOOH

OH

HOOC

COOH

SLex49

2095

99

102

OO

O

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH100

HOHO

OO

O

OMe

HOOH

OH

O

HOOCHO OH

AcHNHO

OH101

O

OH

HO

O

OH

OO

OMe

HOOH

OH

HOOC

96

O

OH

HO

O

OH

OO

OMe

HOOH

OH

HOOC

97

O

OH

HO

O

OHO

O

OMe

HOOH

OH

HOOC

98

OHN

OMe

HOOH

OH

105N

HO OH

HOOCO

O

OHN

OMe

HOOH

OH

103N

HO OH

HOOCO

O

COOEt

OOH

OHHO

OH

O

O

HOOC

104

Figure 2.28 Mimics derived from SLex.

The fucose moiety was found to be very important for the selectin recognitionand most reported mimics contain a fucose or a mannose moiety. Exchange of NeuAc orGlcNAc groups gives analogs described in section 2.2.4 and 2.5.5 and further removal ofthe galactose moiety gives a series of mimics.

Compounds 99, 100 and 10162 are examples of fucose bound to NeuAc via alinker. The mimics with hydroxyl groups in the linker show better activity than thosewithout, indicating the importance of the hydroxyl groups in the galactose residue.None of these compounds were more active than the parent tetrasaccharide.

Compounds 95125, 96126-128, 97129 and 9892,130,131 are examples of mimicswhere the NeuAc and GlcNAc residues are removed. Mimic 96 is as active as 95indicating that the fucose and galactose moieties are well presented despite theincreased flexibility. Compounds 97 and 98 are about 10 times as active as the parenttetrasaccharide.

29

Compounds 102132, 10344,133, 104134 and 10544, are examples of highlysimplified mimics where only the fucose residue is left from the parent saccharide. Thedifficulties in designing mimics are exemplified with compounds 103 and 105 with thelatter being about 100 times as active as the second44.

Armstrong reported the synthesis of a chemical library of C-fucopeptides asglycomimetics for selectin ligands135. No binding constants were however reported.Wong presented another combinatorial approach with C-fucopeptides to mimic selectinligands44. Some of the products showed positive results.

2.9.2 Other mimics

A number of other mimics have been reported. The three major types are polyanions(106)136, polypeptides (107)137, and natural products such as glycyrrhizin (108)138, asexemplified in Figure 2.29.

O32-POO3

2-POOPO3

2-

OPO32-

O32-PO

OPO32-

106 (polyanion)

107 (polypeptide)

HOOC

H

O

H

O

OH

HO

O

OHOH

OH 108 (glycyrrhizin)

H2N-Asp-Ile-Thr-Trp-Asp-Gln-Leu-Trp-Asp-Leu-Met-Lys-COOH

Figure 2.29 Examples of other types of mimics.

Compound 106 is very active against L-selectin indicating the preference of negativelycharged groups in ligands for L-selectins (i.e. sulfated SLex saccharides). Polypeptidessuch as 107 was found to be up to 1000 times more active than SLex. Compound 108 wasidentified by Brandley138 from a computational search of a 3-D database, together withseveral other compounds of different types. Compound 108 was found to be about asactive as sialyl Lewis x.

2.9.3 Miscellaneous

In an investigation by Kretzschmar139, a SLex mimic was found to be very activeagainst P-selectin. The activity was however batch-dependent and the actual ligandproved to be traces of polyanions released from the anionic exchange resin used. Furtherinvestigations showed very high activities of a number of anionic exchange resins. Thehigh activity probably stems from non-carbohydrate binding sites.

30

2.9.4 Summary

A large number of compounds have been synthesized to mimic the ligands for selectinsand the information has been used to further refine the compounds. The search for theultimate selectin ligand continues.

2.10 Summary

The information from the different analogs was used to make a Òfunctional group mapÓof the important features of sialyl Lewis x (Figure 2.30).

O

OH

HO

O

OH

O

NHAc

R

OH

O

O

OMe

HOOH

OH

O

-OOC

HO OH

AcHNHO

OH Important for conformation

Figure 2.30 Important functional groups for selectin binding of the SLex tetrasaccharide.

All three hydroxyl groups of the fucose moiety, the 4- and 6-hydroxyl groups of thegalactose moiety and the carboxylic acid group of sialic acid are important for thebinding to E- and L-selectin. The 2- and 4-hydroxyl groups of fucose are not importantfor P-selectin. Sulfation of the 6-position of GlcNAc and/or Gal enhances the activitytowards L-selectin. Only the O3-C3-C4-O4 conformation is important in the GlcNAcmoiety. Some investigators have found that nonpolar anomeric groups enhances theactivity.

31

3Lactones of sialyl Lewis x; introduction to lactams

HE CARBOXYLIC acid residue in sialyl Lewis x, can participate in the formationof two different intramolecular esters groups, lactones, which gives the sacchariderigid and well defined conformations. The lactones of a number of biological

important saccharides are suspected to be naturally occurring compounds withimmunogenic properties.

3.1 Ganglioside lactones

Gangliosides are glycolipids that have at least one sialic acid unit. The sialic acid is arather strong carboxylic acid (pKa 2-3) and lactones140 (internal esters) can be formedwith nearby hydroxyl groups as exemplified in Figure 3.1.

O

OH

HO

O

OH

OR

O

HOOC

HOOH

AcHNHO

OH

O

O

HO

O

OH

OR

O

O

AcHN OH

HO

HOHO

O

OH

O

O

OH

ORO

OHOH

AcHN

HO

HO

OH H

Figure 3.1 Examples of lactone formation in a disaccharide.

Gangliosides have been shown to lactonize in vitro at low pH. A method for thepreparation of ganglioside lactones is to treat the ganglioside with HOAc141,142 orHCl143 for several days. The ganglioside lactones are usually stable if stored at pH 6 orlower but are rapidly hydrolyzed in Na2CO3 (aq)142 and slowly (3 weeks) in NaHCO3(aq)144.

T

32

3.1.1 Biological importance of ganglioside lactones

Ganglioside lactones were found in extracts from bovine adrenal glands141. However,since the lactones may be formed during the purification process, the existence oflactones in vivo has been debated for a long time. Positive indications have beenobtained from NaB3H4 (tritium) reduction of ganglioside-containing cells, wherereduced sialic acid residues could be detected 145. The corresponding free acids werepractically unreactive to the reducing agent. Other investigations also point in the samedirection146,147.

Monoclonal antibodies raised against mouse melanoma cells recognized not onlythe GM3 ganglioside 109 (Figure 3.2) but also the lactone 110148. The lactone proved tobe an even stronger immunogen than the open form while the corresponding ethyl esterof GM3 was not immunogenic.

O

OH