Examination of the Biological Role of the α(2→6)-Linked Sialic Acid in Gangliosides Binding to...

Examination of the Biological Role of the r(2f6)-Linked Sialic Acid in Gangliosides Binding tothe Myelin-Associated Glycoprotein (MAG)

Oliver Schwardt,† Heiko Gathje,‡ Angelo Vedani,† Stefanie Mesch,† Gan-Pan Gao,† Morena Spreafico,† Johannes von Orelli,†

Sørge Kelm,‡ and Beat Ernst*,†

Institute of Molecular Pharmacy, Pharmacenter, UniVersity of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland, Institute forPhysiological Biochemistry, UniVersity Bremen, D-28334 Bremen, Germany

ReceiVed August 25, 2008

The tetrasaccharide 1, a substructure of ganglioside GQ1bR, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program.In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications ofthe core disaccharide, the R(2f3)- and the R(2f6)-linked sialic acid were synthesized. Biphenylmethyland (S)-lactate were identified as suitable replacements for the R(2f6)-linked sialic acid. Combined witha core modification and the earlier found aryl amide substituent in the 9-position of the R(2f3)-linkedsialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their KDs determined insurface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitativeagreement with molecular modeling studies. This new class of glycomimetics will allow to validate the roleof MAG in the axon regeneration process.

Introduction

Unlike the peripheral nervous system (PNS), the injured adultmammalian central nervous system (CNS) inherently lacks thecapacity for axon regeneration.1 Although neurite outgrowth isin principle possible,2 it is actively blocked by inhibitor proteinsexpressed on residual myelin and on astrocytes recruited to thesite of injury.3 To date, three major inhibitor proteins have beenidentified: Nogo A,4 oligodendrocyte myelin glycoprotein(OMpg),a5 and myelin-associated glycoprotein (MAG).6 Thesethree proteins all bind to the Nogo receptor (NgR)7 located onthe neuron surface. The complex then formed by the activatedNgR with the neurotrophin receptor p75NTR permits the trans-duction of the inhibitory signal into the cytosol of the neuron.8

There, the RhoA-ROCK9 cascade is activated, which finallyleads to growth cone collapse (Figure 1).

It was shown that the RhoA-ROCK inhibitory cascade canalso be triggered by the interaction of a complex formed byMAG and brain gangliosides10 with p75NTR.11 MAG (siglec-4)belongs to the siglec family12 of sialic acid-binding immuno-globulin-like lectins. The biological role of MAG as an inhibitorof axonal regrowth after injury was broadly investigated.13

Schnaar10 reported that a limited set of structurally related

gangliosides like GT1b and GQ1bR (Figure 2), known to beexpressed on myelinated neurons in vivo, are functional ligandsfor MAG. These gangliosides have been synthesized in prepara-tive amounts,14 and were therefore available for a structure-affinity relationship (SAR) study. MAG was found to bindpreferentially to Neu5AcR(2f3)-Gal�(1f3)-GalNAc and Neu5-AcR(2f3)-Gal�(1f3)-[Neu5AcR(2f6)]-GalNAc, the terminaltrisaccharide and tetrasaccharide of the brain gangliosides GT1band GQ1bR, respectively.10,15,16 The SAR profile was thenfurther refined by numerous synthetic contributions based onganglioside fragments17 and neuraminic acid derivatives.18

* To whom correspondence should be addressed. Phone: 0041 267 1551. Fax: 0041 267 15 52. E-mail: [email protected].

† Institute of Molecular Pharmacy, Pharmacenter, University of Basel.‡ Institute for Physiological Biochemistry, University Bremen.a Abbreviations: CHO, Chinese hamster ovary; DCE, 1,2-dichloroethane;

DCM, dichloromethane; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone;DMAP, 4-dimethylamino-pyridine; DME, 1,2-dimethoxyethane; DMF, N,N-dimethylformamide; DMP, 2,2-dimethoxypropane; DMTST, dimethyl(m-ethylthio)-sulfonium triflate; Gal, galactose; GalNAc, N-acetylgalactosamine;IgG, immunoglobulin G; KD, dissociation constant; MAG, myelin-associatedglycoprotein; MPM, p-methoxybenzyl; MS, molecular sieves; Neu5Ac,N-acetylneuraminic acid; NgR, Nogo receptor; NIS, N-iodosuccinimide;NMR, nuclear magnetic resonance; OMgp, oligodendrocyte myelin glyco-protein; OSE, 2-(trimethylsilyl)ethoxy; pyr, pyridine; rIP, relative inhibitorypotency; SAR, structure-affinity relationship; STD, saturation transferdifference; TCA, trichloroacetyl; Tf, trifluoromethanesulfonyl; THF, tet-rahydrofurane; Ts, p-toluylsulfonyl.

Figure 1. MAG, Nogo66, and oligodendrocyte myelin glycoprotein(OMgp) all bind to the Nogo-66 receptor (NgR). The inhibitory signalis transduced into the cytosol of the neuron via the coreceptor p75NTR.MAG also binds to GT1b with p75NTR as coreceptor and therebytransduces the inhibitory signal into the cytosol. Intracellularly, the smallGTPase RhoA is activated, which leads to a collapse of the growthcone (adapted from Filbin et al.3b).

J. Med. Chem. 2009, 52, 989–1004 989

10.1021/jm801058n CCC: $40.75 2009 American Chemical SocietyPublished on Web 01/28/2009

Although the exact biological role of the MAG/gangliosideinteraction has not been resolved yet, in some systems, axonregeneration inhibited by MAG could be completely reversedby sialidase treatment, suggesting that MAG indeed usessialidated glycans as its major axonal ligands.19 The excellentcorrelation of the degree of neurite outgrowth with the bindingaffinities of the tested oligosaccharides further contributes tothe validation of MAG as a therapeutic target.20 Overall, thesefindings suggest that potent glycan inhibitors of MAG couldbe valuable therapeutics to enhance axon regeneration.

A comparison of the R-series ganglioside GQ1bR with theclosely related GT1b (Figure 2) shows that an additional sialicacid linked R(2f6) to GalNAc enhances binding affinity toMAG 10-fold.15b,17b A detailed SAR study revealed that thehydroxyl groups of the glycerol side chain of the R(2f6)-linkedsialic acid are not required for binding.17b When the R(2f6)-Neu5Ac was substituted by a sulfate group, MAG-mediatedadhesion was found to be retained, indicating that an importantcontribution of this moiety originates from the acid func-tion.17b-d,21 Finally, gangliosides or therefrom deduced glycans,which are missing the terminal R(2f3)-linked Neu5Ac, showedno or only weak interactions with MAG, indicating theoutstanding relevance of this moiety for binding.15b,17a,b,21

The binding properties of the tri- and tetrasaccharides 1-422,23

(Figure 3) toward a soluble MAG-Fc chimera have beeninvestigated using a hapten inhibition assay.24 The affinities weremeasured relative to the reference compound 1, which has arelative inhibitory potency (rIP) of 1. Interestingly, the replace-ment of the GalNAc moiety present in the natural epitope 1 bya Gal residue (f2) had no significant influence on the rIP. Asimilar trend was observed for the binding affinities of thetrisaccharides 3 and 4. As expected,15b,17b the tetrasaccharides

1 and 2 showed an improved affinity (approximately 4-fold)compared to the corresponding trisaccharides 3 and 4.

Results and Discussion

In this study, we present the synthesis and biologicalevaluation of mimics of tetrasaccharide 1. For that purpose, arylamide modifications in the 9-position of the R(2f3)-linkedNeu5Ac were combined with the replacement of the R(2f6)-linked Neu5Ac with various noncarbohydrate substitutents.

Modification of the r(2f3)-Linked Neu5Ac. Initially, thepreviously identified beneficial effect of aryl amide modificationsin the 9-position of methyl and benzyl sialosides18b,d was appliedto trisaccharide 4 (f 16, Scheme 1).

For the synthesis of 16, the known galactose building block525 was transformed by standard procedures into the p-methoxybenzyl-protected acceptor 7. The galactosylation of 7with donor 826 in the presence of dimethyl(methylthio)-sulfonium triflate (DMTST)27 yielded disaccharide 9 in 76%.After deprotection applying Zemplen conditions (f10), selective6-O-benzoylation (f11) was accomplished with benzoyl cya-nide at -40 °C in 67% yield. Sialidation of 11 with donor 1228

using DMTST as promoter gave 13 in 63% with goodstereoselectivity (R:� ) 3:1). After acetylation, the azidoderivative 14 was converted into benzamide 15 using modifiedStaudinger conditions.18d,29 Finally, deprotection yielded testcompound 16 in 74% yield ready for biological testing in thehapten inhibition assay (Table 1).

Replacement of the r(2f6)-Linked Neu5Ac by Lactates.In a saturation transfer difference (STD) NMR study, the bindingcontribution of the R(2f6)-linked sialic acid moiety has beendetermined.30 In the complex of MAG with 1, only one sizable

Figure 2. The brain gangliosides GT1b and GQ1bR.

Figure 3. Relative inhibitory potencies (rIPs) of oligosaccharides 1-4.22,23 rIPs were calculated by dividing the IC50 of the reference compound1 by the IC50 of the compound of interest. This results in rIPs above 1.00 for compounds that inhibit better than the reference compound and lowerthan 1.00 for substances inhibiting weaker. OSE ) 2-(trimethylsilyl)ethoxy.

990 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 Schwardt et al.

Scheme 1a

a (a) p-methoxybenzyl chloride, NaH, DMF, 0°C, 3 h, 61%. (b) 80% aq AcOH, 90°C, 1.5 h, 50%. (c) DMTST,27 DCM, 0 °C, 16 h, 76%. (d) NaOMe/MeOH, rt, 2 h, 85%. (e) BzCN, MeCN/NEt3 (4:1), -45°C, 1 h, 67%. (f) DMTST,27 MeCN, 0 °C, 16 h, 47% (R-isomer), 16% (�). (g) Ac2O, pyr, DMAP,rt, 16 h, 87%. (h) BzCl, Ph3P, DCE, rt, 16 h, 57%. (i) i. DDQ, DCM/H2O, rt, 3 h; ii. NaOMe/MeOH, rt, 5 h, then aq NaOH, rt, 3 h, 74%.

Scheme 2a

a (a) i. Bu2SnO, benzene, 80°C, 3 h; ii. 18, 19 or 20, CsF, DME, rt, 1 d; 21: 83%; 22: 61%; 23: 83%. (b) Bu3SnH, cat. AIBN, benzene, 80°C, 3 h; 24:72%; 25: 86%; 26: 87%. (c) NaOMe/MeOH, rt, 1 d, then aq NaOH, rt, 7 h; 27: 68%; 28: 87%; 29: 60%.

R(2f6)-Linked Sialic Acid in Gangliosides Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 991

STD effect arising from its N-acetyl group could be detected,thus confirming the reported moderate contribution of thismoiety to the binding to MAG.18c In addition, from affinitystudies with a MAG mutant as well as from docking studies toa homology model of MAG, a salt bridge formed by thecarboxylate of the R(2f6)-linked Neu5Ac and Lys67 waspostulated.22 To mimic this salt bridge, the 6-OH of GalNAcwas alkylated with lactic acid derivatives.

The synthesis of the derivatives 27-29 (Scheme 2) startedfrom trisaccharide 17.22 The regioselective alkylation of the 6-O-position of the GalNAc-moiety was achieved in a two-stepprocedure:31 (i) activation of the 4,6-diol 17 with dibutyltin oxideto yield a 4,6-O-dibutylstannylene acetal intermediate and (ii)in situ treatment under basic conditions with the enantiomericallypure lactic acid triflates (R)-18, (R)-19, or (S)-20.32 Completeinversion at the lactate stereocenter afforded exclusively (S)-configured 21 and 22 and (R)-configured 23. The N-trichloro-acetyl group was then reduced with tributyltin hydride,33

yielding the acetates 24-26. Whereas the 3-phenyllactatederivative 22 exclusively formed the desired product 25, thelactates 24 and 26 were both obtained as inseparable 2:1-mixtures with an unidentified byproduct (presumably an orthoa-mide). However, the pure mimics 28-29 could chromatographi-cally be isolated after the final hydrolytic deprotection (Table1).

Replacement of GalNAc by lyxo-Hexitol. STD NMRinvestigations30 revealed only a marginal contribution of theN-acetate of the GalNAc unit to binding. This observationsuggested the use of the corresponding 1,2-dideoxy-galactosebuilding block leading to less polar derivatives (Scheme 3).

Catalytic hydrogenation of the commercially available galactalderivative 30 afforded peracetylated 1,2-dideoxygalactose 31.Transesterification with sodium methoxide gave 32, whichwas transformed into the corresponding benzylidene 33. TheDMTST27-promoted coupling of 33 with glycosyl donor 3434

Scheme 3a

a (a) H2 (1 bar), 10% Pd-C, MeOH, 3 h, 97%. (b) NaOMe/MeOH, rt, 3 h, 99%. (c) PhCH(OMe)2, cat. p-TsOH, MeCN, rt, 18 h, 84%. (d) DMTST,27

MS 3 Å, DCM, 0°C, 2 d, 79%. (e) 80% aq AcOH, 60°C, 3 h, 89%. (f) NaOMe/MeOH, rt, 18 h, then aq NaOH, rt, 6 h, 73%. (g) i. Bu2SnO, benzene, 80°C, 3 h; ii. 18, 19, or 20, CsF, DME, rt, 1 d; 38: 83%; 39: 72%; 40: 85%. (h) NaOMe/MeOH, rt, 1 d, then aq NaOH, rt, 7 h; 41: 81%; 42: 76%; 43: 96%.


yielded 35 in 79%. Deprotection (f36), followed bysaponification finally gave trisaccharide 37.

The lactic acid derivatives 41-43 were obtained by thepreviously applied strategy. Activation of diol 36 with dibutyltinoxide and subsequent treatment with the lactic acid triflates (R)-18, (R)-19, or (S)-20 in the presence of cesium fluoride yieldedthe lactic acid derivatives 38-40 with complete inversion atthe lactate stereocenter. After deprotection, the test compounds41-43 were isolated in good yields ready for evaluation in thehapten inhibition assay (Table 1).

Replacement of the r(2f6)-Linked Neu5Ac by BenzylEthers. To explore the already mentioned lipophilic contact ofthe N-acetate of the R(2f6)-linked Neu5Ac predicted by STDNMR,30 the benzyl ether derivatives 45 and 47 were synthesized(Scheme 4). Starting from 35, reductive opening of thebenzylidene acetal with trimethylaminoborane and aluminumchloride35 (f 44, 74%) followed by saponification gave the6-O-benzyl derivative 45 in 68% yield. For the synthesis of thebiphenylmethyl analogue 47, diol 36 was first treated withbiphenyl-4-carbaldehyde in the presence of cat. amounts ofp-TsOH to yield the corresponding acetal intermediate, whichwas subsequently reduced to ether 46 (51% yield from 36). Finaldeprotection afforded test compound 47 (Table 1).

Combined Modifications of the r(2f3)- and ther(2f6)-Neu5Ac. In the last series of compounds, the two mostsuccessful modifications, namely the introduction of a benza-mide at the 9-position of the R(2f3)-linked Neu5Ac (seecompound 16, Table 1) and the replacement of the R(2f6)-linked Neu5Ac by a biphenylmethyl substituent (see compound47, Table 1) were combined in antagonist 59. Because anadditional p-chloro substituent on the benzamide moiety further

enhances the affinity,18d p-Cl-benzamide 60 was synthesizedas well (Scheme 5).

Starting from acceptor 33, disaccharide 48 was obtained in87% by galactosylation with donor 8 using NIS/TfOH aspromoter. After removal of the benzylidene protection, diol 49was reacted with biphenyl-4-carbaldehyde and the acetalintermediate reduced to ether 50. Subsequent debenzoylationgave 51 in 82% yield. Acetonide formation (f52), peracety-lation (f53), and cleavage of the acetal protection afforded 54.The coupling of 54 and 12 was promoted by NIS/TfOH, yieldingalmost exclusively the R-isomer of 55. After acetylation (f56), the azido group was converted into the benzamides 57 and58 using modified Staudinger conditions. Finally, deprotectionyielded the test compounds 59 and 60 (Table 1).

Biological Evaluation

Hapten Inhibition Assay. For the biological evaluation ofthe synthesized MAG antagonists, the previously reportedinhibition assay24 was applied. Thus, a recombinant proteinconsisting of the three N-terminal domains of MAG and the Fcpart of human IgG (Fc-MAGd1-3) was produced by expressionin CHO cells and affinity purification on protein A-agarose.36

The relative inhibitory potencies (rIP) of the test compoundswere determined in microtiter plates coated with covalentlyattached sialic acids as competitive ligands for Fc-MAGd1-3.By complexing the Fc-part with alkaline phosphatase-labeledanti-Fc antibodies and measuring the initial velocity of fluo-rescein release from fluorescein diphosphate, the amount ofbound MAG could be determined. At least three independenttitrations were performed for each compound. To ensure

Scheme 4a

a (a) Me3N ·BH3 (4 eq), AlCl3 (6 eq), H2O (2 eq), THF, rt, 4 h, 74%. (b) NaOMe/MeOH, rt, 17 h, then aq NaOH, rt, 7 h, 68%. (c) i. biphenyl-4-carbaldehyde, cat. p-TsOH, MeCN, 70 °C, 4 h; ii. Me3N ·BH3 (4 eq), AlCl3 (6 eq), H2O (2 eq), THF, rt, 3 h, 51%. (d) NaOMe/MeOH, rt, 16 h, then aqNaOH, rt, 7 h, 70%.


comparability with earlier results,22 the affinities were measuredrelative to the reference compound 122 (Table 1, entry 1).

The biological evaluation of trisaccharide 16 (Table 1, entry3) revealed an approximately 60-fold improvement in affinity.18b,d

However, the replacement of the R(2f6)-linked sialic acidmoiety in the natural epitope 1 (entry 1) by lactate residues ledto a relevant decrease in affinity for both stereoisomers 27 and29 (entries 4 and 5). The additional introduction of a lipophilicphenyl group at C-3 of the (S)-lactic acid moiety (compound28, entry 6) did not show a measurable effect.

In a second approach, the contribution of the N-acetate ofthe GalNAc moiety was explored. Its replacement by 1,2-dideoxy-Gal in trisaccharide 37 (entry 7) raised the affinity closeto the one of the parent tetrasaccharide 1 (rIP 0.76 vs 1.0). Whenthe similar modification was applied to the pseudo-tetrasaccha-rides 27-29, improved affinities for the lactic acid derivatives41 (entry 8) and 43 (entry 9), but not for the phenyllactic acidderivative 42 (entry 10), were obtained.

For further investigating the lipophilic contact emanating fromthe R(2f6)-linked sialic acid moiety,30 benzyl ether 45 (entry11) and biphenylmethyl ether 47 (entry 12) were synthesized.Whereas 45 (entry 11) turned out to be less active than the parenttetrasaccharide 1 (rIP 0.59 vs 1.0), 47 (entry 12) proved to besuperior (rIP 1.25).

In a last step, the affinity enhancing modifications at the siteof the R(2f3)- as well as at the R(2f6)-linked sialic acid werecombined in one molecule leading to mimic 59 (entry 13), whichbinding affinity is improved by more than 300-fold comparedto the parent tetrasaccharide 1 (entry 1). Similar to previousfindings,18d the p-chloro substituent on the benzamide in 60(entry 14) led to a further significant enhancement of the affinity(rIP: 377 vs 314), making oligosaccharide mimic 60 one of themost active synthetic MAG ligands described to date.

Surface Plasmon Resonance. The interaction between MAGand the MAG-antagonists 16, 59, and 60 has been analyzed bya surface plasmon resonance based biosensor (Biacore) at 25°C.37 Figure 4 shows the sensorgram for 59, which was usedfor the kinetics analysis of the binding process. The curve wasglobally fitted to a Langmuir 1:1 binding model employingstandard Biacore software.

Association (kon) and dissociation rate constants (koff) as wellas affinities (KD) were determined (Table 2). The KDs correspondwell with the results obtained in the hapten inhibition assay24

(Table 1). Whereas for trisaccharide 16 a KD of 32 µM wasmeasured, the introduction of the biphenylmethyl moiety in 59or 60 improved affinities to the low micromolar level. Thebeneficial effect18d of an additional p-chloro substituent on thebenzamide as realized in 60 could also be confirmed.

By resolution of the affinities into the individual associationand dissociation rate constants, it was possible to reveal thebinding kinetics of the three antagonists 16, 59, and 60. Theincrease in lipophilicity originating from the introduction ofthe biphenylmethyl moiety (59 and 60) led to a substantialimprovement of the association rate constants by a factor of 20and 30, respectively. Because the three investigated antagonistsshow rather similar koffs (0.3-1.05 s-1), the improved affinityof 59 and 60 results predominantly from enhanced associationrate constants.

Molecular Modeling

The homology model for the ligand-binding domain of MAG(mouse; UniProt ) P20917) was based on the three-dimensionalstructure of sialoadhesin (mouse, SIGLEC-1, PDB code )

Table 1. Relative Inhibitory Potencies (rIPs) of OligosaccharideMimicsa

a The rIP of each substance was calculated by dividing the IC50 of thereference compound 122 by the IC50 of the compound of interest. This resultsin rIPs above 1.00 for derivatives binding better than 1 and rIPs below1.00 for compounds with a lower affinity than 1. The previously usedreference compound 422,23 with a 3.3-fold lower affinity, reflecting thecontribution of the R(2f6)-linked sialic acid moiety, is also included inTable 1 (entry 2).


1QFP, 2.8 Å resolution) and generated using the Fugue38 andOrchestrar39,40 concepts as implemented in Sybyl 7.341 followinga standard protocol. The identity in primary sequence betweenthe two species is 29.9%. After generation, the homology modelwas subjected to a full refinement in aqueous solution usingthe AMBER 7.0 software package.42

The three-dimensional structures of compounds 1, 59, and60 were generated using the MacroModel software43 and

optimized in aqueous solution by means of the AMBER* forcefield.44 Atomic partial charges (MNDO/ESP) were then gener-ated using MOPAC.45 The ligands were first manually dockedto the binding pocket of the MAG model using the salt-bridgeto Arg118 and the hydrogen bonds to Asn125, Gln126, Thr127,and Tyr128 as anchor points. Next, the protein-ligand complexwas minimized in aqueous solution and then subjected to amolecular dynamics equilibration protocol (10 ps at 100 K,

Scheme 5a

a (a) NIS/TfOH, DCM, -20 °C, 87%. (b) 80% AcOH, 60°C, 83%. (c) i. biphenyl-4-carbaldehyde, cat. p-TsOH, MeCN; ii. Me3N ·BH3 (4 eq), AlCl3 (6eq), THF, rt, 55%. (d) NaOMe/MeOH, 82%. (e) DMP, p-TsOH, MeCN, 78%. (f) Ac2O, pyr, 94%. (g) 80% AcOH, 80°C, 81%. (h) NIS/TfOH, MeCN,-40°C, 61%. (i) Ac2O, pyr, 80%. (j) BzCl or p-Cl-BzCl, PPh3, DCE, rt, 16 h; 57: 65%; 58: 75%. (k) NaOMe/MeOH, rt, 3 h, then aq NaOH, rt, 16 h; 59:79%; 60: 75%.

Figure 4. Biacore sensorgram (A) and fitting curve (B) of compound 59 binding to MAG (for the immobilization see Experimental Section) forwhich a kinetics analysis was performed. The Sensorgram was fitted to a Langmuir 1:1 binding model utilizing standard Biacore software in theglobal fitting mode. Eight concentrations for 59 of a 2-fold dilution series starting from 50 µM (top) were used. The global fit yielded the followingparameters: KD ) 5.00 × 10-6 M, RUmax ) 19.4, koff ) 1.05 s-1, kon ) 2.1 × 105 M-1s-1. From koff, a half-life of 0.66 s is calculated.


heating to 310 K during 50 ps, 10 ps at 310 K, cooling during10 ps to 293 K; 1 ps ) 10-12 s), followed by structure sampling

at 293 K during 100 ps (at 0.2 ps intervals). The lowest-energybinding modes of tetrasaccharide 1 and mimic 60 are comparedin Figure 5.

Both compounds, 1 and 60, form an important salt bridge(carboxylate of the R(2f3)-linked Neu5Ac with Arg118), arelevant hydrogen bridge (backbone carbonyl of Phe 129 with9-OH and 9-NH, respectively), and a crucial hydrophobiccontact (between the acetamido group of the R(2f3)-linkedNeu5Ac and the side chains of Trp22 and Tyr124). Whereas 1establishes a second salt bridge between the carboxylate of theR(2f6)-linked Neu5Ac and Lys67, prominent interactions withthe two hydrophobic pockets near the surface of MAG are

Table 2. Dissociation Constants KD from Surface Plasmon ResonanceExperiments (Equilibrium Analysis) Performed at 298 K withFc-MAGd1-3 Immobilized on a CM5 Chip

ligand

16 59 60

rIP 61.5 314 377KD, µM 32.6 5.00 2.83kon, M-1 s-1 1.1 × 104 2.2 × 105 3.1 × 105

koff, s-1 0.3 1.05 0.8t1/2, s 2.3 0.67 0.87

Figure 5. A homology model of MAG, based on a 3D-structure of sialoadhesin (siglec-1),46 complexed with tetrasaccharide 1 (left) and glycomimetic60 (right). The images have been generated using the VMD software.47 In both compounds, the carboxylate of the R(2f3)-linked Neu5Ac formsa salt bridge with Arg118 and hydrophobic interactions are established between its acetamido group and the side chains of Trp22 and Tyr124,which are part of a small hydrophobic pocket. Both compounds form an important hydrogen bridge with the backbone carbonyl of Phe129; in 1,with OH in the 9-position of the R(2f3)-linked Neu5Ac and in 60, with the corresponding amide NH. Tetrasaccharide 1 establishes a second saltbridge between the carboxylate of the R(2f6)-linked Neu5Ac and Lys67. In contrast, antagonist 60 seeks for additional hydrophobic interactions:(i) the p-chloro benzamide substituent and the side chains of Tyr127 and Glu131 defining a secondary hydrophobic pocket, and (ii) the biphenylmethylmoiety and the side chains of Tyr60, Tyr69, and Tyr116 lining the main hydrophobic pocket.

Table 3. Semiquantitative Estimation of the Binding Affinitiesa

ligand KD (exp.) rIP Eligand-receptor Edesolvation T∆S Einternal strain Ebinding KD (calcd)

1 >1.0 × 10-3 1.0 -21.4 +10.0 +6.3 +3.6 -1.5 7.6 × 10-2

59 5.0 × 10-6 314 -23.2 +6.9 +4.3 +4.8 -7.2 4.3 × 10-6

60 2.8 × 10-6 377 -23.2 +6.8 +4.3 +3.5 -8.6 0.4 × 10-6

a All energies are given in kcal/mol, KD in M. The desolvation energies reflect the partial desolvation of the ligands at the protein surface. Proteinsligandand internal energies were calculated using the AMBER force field44 as implemented in MacroModel43 and appropriately scaled.

Figure 6. Comparison (in stereo) of the bound conformation and the nearest low-energy conformation in aqueous solution of tetrasaccharide 1 (left) andthe glycomimetic 60 (right). The bound conformation is colored by atoms, the low-energy conformation in aqueous solution is depicted in blue.


formed by 60: Glu131 and Tyr127 are homing the p-chlorobenzamide substituent and the side chains of Trp59, Tyr60,Tyr69, and Tyr116 lining the main hydrophobic pocket accom-modate the biphenylmethyl moiety.

To assess the feasibility of the bound conformation (asidentified by molecular mechanics and molecular dynamicssimulations), we conducted a conformational search of com-pounds 1, 59, and 60 in aqueous solution and identified thelowest conformer featuring a similar folding as the bound state.For the tetrasaccharide 1, the closest identified conformation islocated only 1.9 kcal/mol above the global minimum; for theglycomimetics 59 and 60, the corresponding energies are 2.8and 1.3 kcal/mol, respectively. This suggests that all threeligands can adopt the bound conformation at very moderatecosts. Comparison of the calculated binding energies with theexperimental binding affinity (Table 3) support the bindinghypothesis identified in silico: the low binding affinity oftetrasaccharide 1 can be explained by its high desolvation energycombined with entropic costs; in contrast hereto, the glycomi-metics 59 and 60 display a higher protein-ligand interactioncombined with lower entropic and desolvation energies whichexplains their higher binding affinity (Table 3).

Conclusions

To investigate the biological role of the sialic acid moi-eties in the lead tetrasaccharide Neu5AcR(2f3)Gal�(1f3)-[Neu5AcR(2f6)]GalNAc (1), a number of oligosaccharidemimics were prepared. On the basis of STD NMR data30 andprevious results,18b,d,22 the terminal R(2f3)-linked Neu5Ac wasmodified by benzamides at the nonreducing end, theGal�(1f3)GalNAc core was simplified, and the R(2f6)-linkedsialic acid was replaced by hydrophilic lactate residues orlipophilic benzyl ethers. The biological evaluation of the mimicswas performed in a competitive target-based assay and bysurface plasmon resonance.

According to docking studies to a homology model of MAG,the R(2f6)-linked sialic acid moiety is forming a salt bridgewith Lys67. When Lys67 was mutated, a substantial loss in theaffinity of tetrasaccharide 1 resulted.22 The loss in affinity causedby the replacement of the sialic acid moiety by lactic acid (f27and 29, entries 4 and 5, Table 1) or phenyllactic acid (f28,entry 6, Table 1) is probably the result of an insufficientpreorganization of the carboxylate in the bioactive conformation.Similar modifications for ligands of E-selectin48 and choleratoxin49 were successful and rationalized by preorganization ofthe bound conformation.

The replacement of the GalNAc moiety in trisaccharide 4 bylyxo-hexitol resulted in a modestly improved affinity (f37, entry7). On the basis of this new scaffold, the (R)- and (S)-lacticacid derivatives 41 and 43 (entries 8 and 9) showed affinitiesin the range of the parent tetrasaccharide 1. Interestingly, bothstereoisomers, the (S)- and the (R)-lactic acid derivatives 41and 43, show comparable affinities.

Because STD-NMR investigations indicated a lipophiliccontact for the N-acetate of the R(2f6)-linked sialic acid in1,30 lipophilic substitutents in the 6-position of the lyxo-hexitolmoiety in 37 were successfully explored (f45, entry 11 and47, entry 12).

Finally, concomitant modifications of the reducing andnonreducing sugars, each with the best so far identifiedsubstituent, proved the additivity of the beneficial effects. Thus,both 59 and 60 (entries 13 and 14) showed a more than 300-fold enhancement in MAG-binding affinity.

The dissociation constants KD obtained by Biacore experi-ments (Table 2) support the results of the inhibition assay.24

The introduction of the biphenylmethyl substituent in the6-position of the lyxo-hexitol moiety caused a 5- to 6-foldimprovement of affinity. Interestingly, the major contributionresults from an enhanced association rate. Thus, the kineticanalysis for 16, 59, and 60 (Figure 4) reveals that kon is changingfrom 104 to 3 × 105 M-1 s-1 and koff is rather stable (0.3-1s-1). For all three antagonists, the corresponding half-lives arein the range of approximately 1 s. In general, high efficacy ofa ligand in vivo correlates with fast association and slowdissociation rates.50 The association rate kon is concentrationdependent, therefore slow association rates can be compensatedby high intracellular concentrations. Slow dissociation rates koff,however, must be achieved by optimizing the contact of ligandand target. It was shown for HIV-1 protease inhibitors that it isbeneficial to reduce the dissociation rate to a point where thecompound acts close to an irreversible binder (koff 10-4s10-3

s-1), resulting in half-lives ranging from minutes to hours.50

Taking this into account, the structure of the ligands presentedin this study have to be further optimized in order to achievemore druglike properties.

With docking studies of the most active ligand 60 to ahomology model of MAG, the beneficial contribution of thetwo lipophilic substituents could be rationalized (Figure 5). Both,the p-chloro group at the 9-position of R(2f3)-linked sialic acidand the biphenylmethyl substitutent in the 6-position of the lyxo-hexitol contribute to binding by hydrophobic contacts. Becauseboth hydrophobic pockets, especially the one formed by Trp59,Tyr60, Tyr69, and Tyr116, are quite spacious, improved affinitymay be realized by further modifications of these substitutents.

Another important issue to be addressed is the metabolicstability of the presented oligosaccharide mimics. In general,the substrate specificity of mammalian sialidases is determinedby the linkage type of the terminal sialic acid residue (2f3,2f6, or 2f8) and does not depend on the structure of theunderlying oligosaccharide.51 Therefore, it cannot be excludedthat the presented mimics are metabolically cleaved by siali-dases. Nevertheless, the new class of MAG blockers presentedin this study constitute an important step toward the developmentof potent oligosaccharide mimics, although pharmacokineticaspects have to be further optimized.

Experimental Section

General Methods. NMR spectra were recorded on a BrukerAvance DMX-500 (500 MHz) spectrometer. Assignment of 1H and13C NMR spectra was achieved using 2D methods (COSY, HSQC,TOCSY). Chemical shifts are expressed in ppm using residualCHCl3, CHD2OD, and HDO as references. Optical rotations weremeasured using Perkin-Elmer polarimeters 241 and 341. Electronspray ionization mass spectra (ESI-MS) were obtained on a Watersmicromass ZQ. The LC/HRMS analysis were carried out using aAgilent 1100 LC equipped with a photodiode array detector and aMicromass QTOF I equipped with a 4 GHz digital-time converter.Reactions were monitored by TLC using glass plates coated withsilica gel 60 F254 (Merck) and visualized by using UV light and/orby charring with a molybdate solution (a 0.02 M solution ofammonium cerium sulfate dihydrate and ammonium molybdatetetrahydrate in aq 10% H2SO4). Column chromatography wasperformed on silica gel (Fluka, 40-60 mesh). Methanol was driedby refluxing with sodium methoxide and distilled immediatelybefore use. Pyridine was freshly distilled under argon over CaH2.Dichloromethane (DCM), dichloroethane (DCE), 1,2-dimethoxy-ethane (DME), acetonitrile (MeCN), toluene, and benzene weredried by filtration over Al2O3 (Fluka, type 5016 A basic). Molecularsieves (3 and 4 Å) were activated in vacuo at 500 °C for 2 himmediately before use.


2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopy-ranosynate)-(2f3)-(6-O-benzoyl-�-D-galactopyranosyl)-(1f3)-2,6-di-O-(4-methoxybenzyl)-�-D-galactopyranoside (13). A mixtureof 11 (77.0 mg, 97.8 µmol), donor 1228 (98.0 mg, 196 µmol), andactivated powdered MS 3 Å (1.00 g) was stirred in MeCN (5 mL)at rt for 6 h under argon. Then DMTST (202 mg, 0.783 mmol)was added at 0 °C. The reaction mixture was stirred at 0 °C for16 h, diluted with DCM (5 mL), and filtered through a pad of celite.The celite was washed with DCM (3 × 5 mL), and the combinedfiltrates were washed with saturated aq NaHCO3 (2 × 10 mL) andH2O (10 mL). The organic layer was dried (Na2SO4), filtered, andconcentrated under reduced pressure. The residue was purified bysilica gel chromatography (0.25% gradient MeOH in DCM) toafford 13 (90.5 mg, 47%) as a white foam (�-isomer: 30.0 mg,16%).

[R]D -3.3, (c 0.91, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.01(s, 9H, SiMe3), 1.01-1.04 (m, 2H, SiCH2), 1.90, 2.04, 2.10, 2.13(4s, 12H, 4 COCH3), 2.11 (m, 1H, Sia-H3a), 2.69 (dd, J ) 4.6,13.1 Hz, 1H, Sia-H3b), 3.11 (dd, J ) 5.4, 13.6 Hz, 1H, Sia-H9a),3.44 (dd, J ) 2.6, 13.6 Hz, 1H, Sia-H9b), 3.49-3.59 (m, 3H, Gal-H5, Gal-H6a, OCH2-Ha), 3.61-3.71 (m, 4H, Gal-H2, Gal-H3, Gal-H6b, Gal′-H4), 3.75-3.81 (m, 1H, Gal′-H2), 3.77, 3.78, 3.80 (3s,9H, 3 OCH3), 3.84 (m, 1H, Gal′-H5), 4.00 (d, J ) 3.2 Hz, 1H,Gal-H4), 4.01-4.13 (m, 4H, Gal′-H3, Sia-H5, Sia-H6, OCH2-Hb),4.32 (d, J ) 7.5 Hz, 1H, Gal-H1), 4.39, 4.43 (A, B of AB, J )11.5 Hz, 2H, C6H4CH2), 4.50 (dd, J ) 7.6, 11.6 Hz, 1H, Gal′-H6a), 4.60 (d, J ) 7.8 Hz, 1H, Gal′-H1), 4.61 (m, 1H, Gal′-H6b),4.75, 4.78 (A, B of AB, 2J ) 10.7 Hz, 2H, C6H4CH2), 4.94 (ddd,J ) 4.6, 10.0, 12.0 Hz, 1H, Sia-H4), 5.24 (d, J ) 9.5 Hz, 1H,NH), 5.28-5.34 (m, 2H, Sia-H7, Sia-H8), 6.82-6.86, 7.21-7.23,7.34-7.36, 7.39-7.42, 7.53-7.56, 8.01-8.03 (m, 13H, C6H5, 2C6H4). 13C NMR (125 MHz, CDCl3): δ -1.5 (SiMe3), 18.4 (SiCH2),20.7, 20.8, 21.1, 22.1 (4 COCH3), 37.4 (Sia-C3), 49.4 (Sia-C5),51.0 (Sia-C9), 53.2, 55.2, 55.2 (3 OCH3), 63.2 (Gal′-C6), 67.1(OCH2), 67.6, 67.6 (Gal′-C4, Sia-C7), 68.5 (Sia-C4), 68.6 (Gal-C4), 68.9 (Sia-C8), 69.3 (Gal′-C2), 69.5 (Gal-C6), 72.1 (Gal′-C5),72.8 (Sia-C6), 73.1 (C6H4CH2), 73.2 (Gal-C5), 74.5 (C6H4CH2),76.1 (Gal′-C3), 77.5 (Gal-C2), 84.0 (Gal-C3), 97.5 (Sia-C2), 102.8(Gal-C1), 103.8 (Gal′-C1), 113.6, 113.7, 128.4, 129.2, 129.6, 129.6,129.8, 130.3, 133.3, 159.1 (18C, C6H5, 2 C6H4), 166.2, 168.4, 170.0,170.1, 170.3, 170.9 (6 CO). Anal. Calcd for C58H78N4O24Si: C,56.03; H, 6.32; N, 4.51. Found: C, 55.81; H, 6.32; N, 4.35.

2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyra-nosynate)-(2f3)-(2,4-di-O-acetyl-6-O-benzoyl-�-D-galactopyr-anosyl)-(1f3)-4-O-acetyl-2,6-di-O-(p-methoxybenzyl)-�-D-galactopyranoside (14). To a stirred solution of 13 (87.0 mg, 70.0µmol) in pyridine (0.4 mL) at 0 °C was added Ac2O (0.2 mL). Themixture was stirred at rt for 16 h under argon and then quenchedby addition of MeOH (1 mL). The solution was concentrated andcoevaporated with toluene (3 × 5 mL). The residue was purifiedby silica gel chromatography (10% gradient EtOAc in toluene) toafford 14 (83.0 mg, 87%) as a colorless foam.

[R]D -8.6, (c 0.66, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.00(s, 9H, SiMe3), 0.99-1.04 (m, 2H, SiCH2), 1.67 (t, J ) 12.5 Hz,1H, Sia-H3a), 1.84, 1.92, 2.00, 2.02, 2.09, 2.11, 2.15 (7s, 21H, 7COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz, 1H, Sia-H3b), 3.09 (dd, J )5.8, 13.6 Hz, 1H, Sia-H9a), 3.44-3.65 (m, 7H, Gal-H2, Gal-H5,Gal-H6, Sia-H6, Sia-H9b, OCH2-Ha), 3.73, 3.78, 3.79 (3s, 9H, 3OCH3), 3.86 (m, 1H, Gal-H3), 3.98-4.05 (m, 3H, Gal′-H5, Sia-H5, OCH2-Hb), 4.16 (dd, J ) 7.9, 11.0 Hz, 1H, Gal′-H6a), 4.36(d, J ) 7.8 Hz, 1H, Gal-H1), 4.38-4.44 (m, 3H, Gal′-H6b,C6H4CH2), 4.54 (dd, J ) 3.3, 10.2 Hz, 1H, Gal′-H3), 4.65, 4.76(A, B of AB, J ) 10.6 Hz, 2H, C6H4CH2), 4.83 (d, J ) 8.0 Hz,1H, Gal′-H1), 4.85 (m, 1H, Sia-H4), 5.05 (d, J ) 3.3 Hz, 1H, Gal′-H4), 5.07 (d, J ) 10.4 Hz, 1H, NH), 5.11 (dd, J ) 8.0, 10.2 Hz,1H, Gal′-H2), 5.33 (dd, J ) 2.6, 8.7 Hz, 1H, Sia-H7), 5.40-5.43(m, 2H, Gal-H4, Sia-H8), 6.84-6.87, 7.23-7.25, 7.32-7.33,7.40-7.43, 7.53-7.56, 8.02-8.04 (m, 13H, C6H5, 2 C6H4). 13CNMR (125 MHz, CDCl3): δ -1.6 (SiMe3), 18.4 (SiCH2), 20.7, 20.7,

20.8, 20.9, 21.2, 23.08 (7C, 7 COCH3), 36.9 (Sia-C3), 49.0 (Sia-C5), 51.2 (Sia-C9), 53.0, 55.2 (3C, 3 OCH3), 61.2 (Gal′-C6), 67.2(Gal′-C4), 67.7 (2C, Sia-C7, OCH2), 68.6, 69.1 (Gal-C4, Sia-C8),69.2 (Gal-C6), 69.5 (Sia-C4), 70.2 (Gal′-C2), 70.4 (Gal′-C5), 71.5(Gal′-C3), 72.2 (Sia-C6), 72.9 (Gal-C5), 73.2, 74.7 (2 C6H4CH2),78.3 (Gal-C2), 80.2 (Gal-C3), 96.7 (Sia-C2), 101.0 (Gal′-C1), 103.0(Gal-C1), 113.5, 113.7, 128.2, 129.2, 129.3, 129.6, 129.7, 133.0,130.1, 131.1, 159.0, 159.2 (18C, C6H5, 2 C6H4), 165.6, 167.9, 169.7,170.2, 170.2, 170.4 (9C, 9 CO).

2-(Trimethylsilyl)ethyl (Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-benzamido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonu-lopyranosynate)-(2f3)-(2,4-di-O-acetyl-6-O-benzoyl-�-D-galac-topyranosyl)-(1f3)-4-O-acetyl-2,6-di-O-(4-methoxybenzyl)-�-D-galactopyranoside (15). Compound 14 (79.3 mg, 57.9 µmol) andbenzoylchloride (27.0 µL, 0.232 mmol) were dissolved in DCE (5mL) and stirred at rt under argon. Ph3P (33.0 mg, 0.127 mmol) inDCE (1 mL) was added after 5 min. The reaction was stirred at rtfor 16 h and then diluted with DCM (10 mL) and washed withsaturated aq NaHCO3 (2 × 10 mL) and H2O (10 mL). The organiclayer was dried (Na2SO4), filtered, and concentrated in vacuo. Theresidue was purified by silica gel chromatography (0.25% gradientMeOH in DCM) to afford 15 (47.5 mg, 57%) as a colorless foam.

[R]D +2.3, (c 0.90, CHCl3). 1H NMR (500 MHz, CDCl3): δ 0.00(s, 9H, SiMe3), 0.98-1.07 (m, 2H, SiCH2), 1.70 (t, J ) 12.4 Hz,1H, Sia-H3a), 1.84, 1.95, 2.00, 2.08, 2.09, 2.10 (7s, 21H, 7 COCH3),2.58 (dd, J ) 4.6, 12.7 Hz, 1H, Sia-H3b), 2.82 (dt, J ) 3.9, 15.0Hz, 1H, Sia-H9a), 3.42-3.49 (m, 2H, Gal-H6), 3.53 (dd, J 2.6,10.8 Hz, 1H, Sia-H6), 3.59-3.63 (m, 3H, Gal-H2, Gal-H5, OCH2-Ha), 3.71, 3.77, 3.78 (3s, 9H, 3 OCH3), 4.00-4.05 (m, 2H, Gal′-H5, OCH2-Hb), 4.11 (q, J ) 10.4 Hz, 1H, Sia-H5), 4.18 (dd, J )7.6, 11.0 Hz, 1H, Gal′-H6a), 4.31-4.38 (m, 2H, Gal′-H6b, Sia-H9b), 4.36 (d, J ) 7.8 Hz, 1H, Gal-H1), 4.39, 4.42 (A, B of AB,J ) 11.2 Hz, 2H, C6H4CH2), 4.66, 4.76 (A, B of AB, J ) 10.3 Hz,2H, C6H4CH2), 4.67 (m, 1H, Gal′-H3), 4.85 (ddd, J ) 4.6, 10.5,12.2 Hz, 1H, Sia-H4), 4.91 (d, J ) 7.9 Hz, 1H, Gal′-H1), 5.07 (d,J ) 2.7 Hz, 1H, Gal′-H4), 5.10 (dd, J ) 7.9, 10.1 Hz, 1H, Gal′-H2), 5.13 (d, J ) 10.4 Hz, 1H, NH), 5.20 (dd, J ) 2.6, 9.2 Hz,1H, Sia-H7), 5.34 (dt, J ) 3.2, 9.2 Hz, 1H, Sia-H8), 5.39 (d, J )3.6 Hz, 1H, Gal-H4), 6.82-6.88, 7.23-7.26, 7.31-7.34, 7.39-7.55,7.77-7.79, 8.01-8.03 (m, 18H, 2 C6H5, 2 C6H4). 13C NMR (125MHz, CDCl3): δ -1.5 (SiMe3), 18.5 (SiCH2), 20.7, 20.9, 20.9, 21.3,23.1 (7 COCH3), 37.5 (Sia-C3), 38.3 (Sia-C9), 48.2 (Sia-C5), 53.0,55.2, 55.2 (3 OCH3), 61.3 (Gal′-C6), 67.3 (Gal′-C4), 67.7 (OCH2),67.9 (Sia-C7), 68.5 (Sia-C8), 69.3 (Gal-C6), 69.3 (Sia-C4), 69.8(Gal-C4), 70.3, 70.4 (Gal′-C2, Gal′-C5), 71.7 (Gal′-C3), 72.1 (Sia-C6), 73.0 (Gal-C5), 73.2, 74.7 (2 C6H4CH2), 78.3 (Gal-C2), 80.1(Gal-C3), 96.7 (Sia-C2), 101.0 (Gal′-C1), 103.0 (Gal-C1), 113.6,113.7, 113.8, 126.9, 128.2, 128.4, 128.5, 128.6, 129.3, 129.4, 129.5,129.7, 129.8, 130.1, 130.1, 131.5, 131.9, 133.0, 134.2, 159.1, 159.2(24C, 2 C6H5, 2 C6H4), 165.6, 165.7, 167.1, 167.7, 169.6, 170.1,170.3, 170.7, 170.9, 171.7 (10 CO). Anal. Calcd for C71H90N2O28Si:C, 58.91; H, 6.27; N, 1.94. Found: C, 58.31; H, 6.24; N, 1.67.

2-(Trimethylsilyl)ethyl (Sodium 5-Acetamido-9-benzamido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-�-D-galactopyranosyl-(1f3)-�-D-galactopyranoside (16).To a stirred solution of 15 (23.0 mg, 15.9 µmol) in DCM (1.8 mL)were added DDQ (10.8 mg, 47.7 µmol) and H2O (0.1 mL). Themixture was stirred at rt for 3 h, and then the precipitate was filteredoff and washed with DCM (2 × 5 mL). The combined filtrateswere washed with H2O (2 × 5 mL). The organic layer was dried(Na2SO4), filtered, and concentrated under reduced pressure. Theresidue was purified by silica gel chromatography (0.5% gradientMeOH in DCM) to afford a white foam (16.2 mg), which wasdissolved in MeOH (1.8 mL) and treated with freshly prepared 1M NaOMe/MeOH (0.2 mL). The mixture was stirred at rt for 5 hunder argon, and then H2O (0.5 mL) was added and stirringcontinued for 3 h. The solution was neutralized with 0.5% HCl,concentrated, and the residue purified by reversed-phase chroma-tography (RP-18, 5% gradient MeOH in H2O), Dowex ion-exchangechromatography (Na+ type), and P2 size exclusion chromatography


to afford 16 as a white solid (10.1 mg, 74%) after a finallyophilization from water.

[R]D +2.2, (c 0.67, H2O). 1H NMR (500 MHz, D2O): δ 0.02 (s,9H, SiMe3), 0.96 (td, J ) 5.2, 12.9 Hz, SiCH2-Ha), 1.05 (td, J )5.5, 12.9 Hz, SiCH2-Hb), 1.80 (t, J ) 12.1 Hz, Sia-H3a), 2.00 (s,3H, COCH3), 2.76 (dd, J ) 4.6, 12.2 Hz, 1H, Sia-H3b), 3.55-3.66(m, 6H, Gal-H2, Gal-H5, Gal′-H2, Gal′-H5, Sia-H7, Sia-H9a),3.67-3.78 (m, 9H, Gal-H3, Gal-H6, Gal′-H6, Sia-H4, Sia-H6, Sia-H9b, OCH2-Ha), 3.87 (t, J ) 10.2 Hz, 1H, Sia-H5), 3.94 (d, J )3.1 Hz, 1H, Gal′-H4), 3.99-4.07 (m, 2H, Sia-H8, OCH2-Hb), 4.11(dd, J ) 3.1, 9.8 Hz, 1H, Gal′-H3), 4.14 (d, J ) 3.3 Hz, 1H, Gal-H4), 4.37 (d, J ) 8.0 Hz, 1H, Gal-H1), 4.64 (d, J ) 7.8 Hz, 1H,Gal′-H1), 7.53-7.56, 7.61-7.64, 7.78-7.80 (m, 5H, C6H5). 13CNMR (125 MHz, D2O): δ -2.2 (SiMe3), 17.9 (SiCH2), 22.4(COCH3), 40.1 (Sia-C3), 43.1 (Sia-C9), 52.0 (Sia-C5), 61.2, 61.3(Gal-C6, Gal′-C6), 67.8 (Gal′-C4), 68.6 (OCH2), 68.8 (Gal-C4),69.8 (Sia-C4), 70.1, 70.1 (3C, Gal-C2, Gal′-C2, Sia-C7), 70.7 (Sia-C8), 75.0 (2C, Gal-C5, Gal′-C5), 75.3 (Sia-C6), 76.1 (Gal′-C3),83.1 (Gal-C3), 100.2 (Sia-C2), 102.1 (Gal-C1), 104.5 (Gal′-C1),127.5, 129.2, 132.5, 134.1 (6C, C6H5), 171.6, 174.1 (2 CO). HRMSCalcd for C35H55N2O19Si [M - Na]-: 835.3168; Found: 835.3164.

(2,3,4,6-Tetra-O-benzoyl-�-D-galactopyranosyl)-(1f3)-1,5-an-hydro-4,6-O-benzylidene-2-deoxy-D-lyxo-hexitol (48). A stirredsuspension of 33 (500 mg, 2.11 mmol), donor 826 (3.20 g, 2.54mmol), and activated powdered molecular sieves 4 Å (1.00 g) inDCM (20 mL) was cooled to -20 °C under argon. Then NIS (950mg, 4.22 mmol) was added and stirring continued for 2 h at -20°C. After the addition of TfOH (24.2 µL, 0.275 mmol), stirringwas continued for 1 h at -20 °C. The reaction was quenched withNEt3 (2.5 mL) and filtered through a pad of celite. The celite waswashed with DCM (3 × 10 mL), and the combined filtrates weresuccessively washed with 20% aq Na2S2O3 (2 × 15 mL) and H2O(10 mL). The organic layer was dried (Na2SO4), filtered, andconcentrated under reduced pressure. The residue was purified bysilica gel chromatography (petrol ether/EtOAc 2:1 to 1:1) to afford48 (1.50 g, 87%) as a colorless foam.

[R]D +92.9 (c 1.05, CH2Cl2). 1H NMR (500 MHz, CDCl3): δ1.65 (m, 1H, Hex-H2a), 2.08 (m, 1H, Hex-H2b), 3.14 (m, 1H, Hex-H5), 3.33 (ddd, J ) 1.7, 12.2, 13.8 Hz, 1H, Hex-H1a), 3.80 (m,2H, Hex-H6), 3.83 (m, 1H, Hex-H3), 3.93 (m, 1H, Hex-H1b), 4.14(dd, J ) 1.4, 1.8 Hz, 1H, Hex-H4), 4.28 (m, 1H, Gal-H5), 4.34(m, 1H, Gal-H6a), 4.64 (dd, J ) 1.4, 11.3 Hz, 1H, Gal-H6b), 5.07(d, J ) 7.9 Hz, 1H, Gal-H1), 5.43 (s, 1H, PhCH), 5.55 (dd, J )3.5, 9.5 Hz, 1H, Gal-H3), 5.79 (dd, J ) 7.9, 9.7 Hz, 1H, Gal-H2),5.92 (m, 1H, Gal-H4), 7.18-7.20, 7.25-7.38, 7.41-7.42, 7.72-7.73,7.82-7.84, 7.95-7.97, 8.01-8.03 (m, 25H, 5 C6H5). 13C NMR(125 MHz, CDCl3): δ 25.0 (Hex-C2), 61.3 (Gal-C6), 64.7 (Hex-C1), 67.2 (Gal-C4), 68.7 (2C, Hex-C5, Gal-C2), 69.3 (Hex-C6),70.5 (Gal-C5), 70.7 (Hex-C4), 74.1 (Gal-C3), 75.0 (Hex-C3), 98.4(Gal-C1), 99.3 (PhCH), 125.0, 127.0, 127.3, 127.4, 127.5, 127.6,127.7, 128.0, 128.4, 128.6, 128.8, 128.8, 129.1, 132.2, 132.3, 132.5,132.7 (30C, 5 C6H5), 164.7, 165.0 (4C, 4 CO). ESI-MS calcd forC47H42NaO13 [M + Na]+, 837.25; found, 837.33.

(2,3,4,6-Tetra-O-benzoyl-�-D-galactopyranosyl)-(1f3)-1,5-an-hydro-2-deoxy-D-lyxo-hexitol (49). Compound 48 (948 mg, 1.16mmol) was dissolved in 80% aq acetic acid (20 mL) and stirredfor 3 h at 60 °C. The solvent was removed in vacuo and the residuecoevaporated with toluene (2 × 10 mL). The remaining solid waspurified by chromatography on silica gel (DCM/MeOH, 20:1 to10:1) to give 49 (705 mg, 83%) as a colorless foam.

[R]D +109 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ 1.49(m, 1H, Hex-H2a), 1.93 (m, 1H, Hex-H2b), 2.10, 2.58 (2s, 2H, 2OH), 3.22 (m, 1H, Hex-H5), 3.28 (m, 1H, Hex-H1a), 3.45 (d, J )11.4 Hz, 1H, Hex-H6a), 3.70-3.74 (m, 2H, Hex-H3, Hex-H6b),3.91-3.94 (m, 2H, Hex-H1b, Hex-H4), 4.30 (m, 1H, Gal-H5), 4.43(dd, J ) 5.3, 11.6 Hz, 1H, Gal-H6a), 4.57 (dd, J ) 7.5, 11.6 Hz,1H, Gal-H6b), 4.89 (d, J ) 7.9 Hz, 1H, Gal-H1), 5.55 (dd, J )3.4, 10.4 Hz, 1H, Gal-H3), 5.72 (dd, J ) 8.1, 10.2 Hz, 1H, Gal-H2), 5.92 (m, 1H, Gal-H4), 7.17-7.20, 7.30-7.59, 7.70-7.72,7.86-7.88, 7.95-7.97, 8.02-8.05 (m, 20H, 4 C6H5). 13C NMR(125 MHz, CDCl3): δ 26.1 (Hex-C2), 62.2 (Gal-C6), 63.4 (Hex-

C6), 65.6 (Hex-C1), 68.0 (2C, Hex-C4, Gal-C4), 69.7 (Gal-C2),71.3 (Gal-C3), 71.8 (Gal-C5), 77.9 (Hex-C5), 78.7 (Hex-C3), 100.3(Gal-C1), 128.3, 128.5, 128.6, 128.7, 128.8, 129.1, 129.2, 129.6,129.7, 129.7, 130.0, 133.4, 133.4, 133.5, 133.7 (24C, 4 C6H5), 165.3,165.5, 165.6, 166.0 (4 CO). ESI-MS calcd for C40H38NaO13 [M +Na]+, 749.22; found, 749.28.

(2,3,4,6-Tetra-O-benzoyl-�-D-galactopyranosyl)-(1f3)-1,5-an-hydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (50).To a solution of 49 (700 mg, 0.963 mmol) and biphenyl-4-carbaldehyde (351 mg, 1.93 mmol) in MeCN (25 mL) was addedp-toluene sulfonic acid monohydrate (18.0 mg, 0.096 mmol) at rt.The mixture was stirred for 23 h at 70 °C before NEt3 (1 mL) wasadded. The solvents were removed and the residue was purified bysilica gel chromatography (petrol ether/EtOAc/NEt3, 85:15:1 to 25:75:1) to give the acetal intermediate (563 mg). The acetal wasdissolved in THF (15 mL) and subsequently treated with Me3N ·BH3

(185 mg, 2.53 mmol) and anhyd AlCl3 (505 mg, 3.79 mmol) withstirring under argon. After 24 h, the reaction was quenched withH2O (5 mL) followed by 0.1 M aq HCl (5 mL) and the solutionextracted with DCM (3 × 50 mL). The combined organic phaseswere washed with saturated aq NaHCO3 (30 mL), dried (Na2SO4),filtered, and concentrated under reduced pressure. The residue waspurified by silica gel chromatography (petrol ether/EtOAc/NEt3,66:33:1 to 25:75:1) to afford 50 (475 mg, 55%) as a colorless foam.

[R]D +69.5 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.52 (m, 1H, Hex-H2a), 2.00 (m, 1H, Hex-H2b), 2.46 (d, J ) 1.2Hz, 1H, OH), 3.30 (m, 1H, Hex-H1a), 3.42 (m, 1H, Hex-H5), 3.47(dd, J ) 4.1, 9.9 Hz, 1H, Hex-H6a), 3.63 (dd, J ) 7.4, 9.8 Hz, 1H,Hex-H6b), 3.76 (m, 1H, Hex-H3), 3.91-3.94 (m, 2H, Hex-H1b,Hex-H4), 4.28 (m, 1H, Gal-H5), 4.39 (dd, J ) 6.0, 11.4 Hz, 1H,Gal-H6a), 4.44, 4.51 (A, B of AB, J ) 12.1 Hz, 2H, BiphCH2),4.57 (dd, J ) 7.1, 11.4 Hz, 1H, Gal-H6b), 4.90 (d, J ) 7.9 Hz,1H, Gal-H1), 5.55 (dd, J ) 3.5, 10.4 Hz, 1H, Gal-H3), 5.72 (dd, J) 8.0, 10.3 Hz, 1H, Gal-H2), 5.92 (d, J ) 3.4 Hz, 1H, Gal-H4),7.16-7.19, 7.25-7.38, 7.42-7.51, 7.55-7.58, 7.70-7.72, 7.85-7.87,7.94-7.95, 8.02-8.04 (m, 29H, C6H4, 5 C6H5). 13C NMR (125MHz, CDCl3): δ 26.0 (Hex-C2), 62.0 (Gal-C6), 65.6 (Hex-C1),67.6, 67.9 (Hex-C4, Gal-C4), 69.7 (Gal-C2), 70.6 (Hex-C6), 71.4(Gal-C3), 71.6 (Gal-C5), 73.3 (BiphCH2), 77.5 (Hex-C5), 78.4(Hex-C3), 100.0 (Gal-C1), 127.1, 127.2, 127.2, 128.2, 128.3, 128.3,128.5, 128.5, 128.6, 128.7, 128.7, 128.9, 129.1, 129.6, 129.8, 130.0,133.4, 133.4, 133.7, 137.1 (36C, C6H4, 5 C6H5), 165.3, 165.5, 166.0(4C, 4CO). ESI-MS calcd for C53H48NaO13 [M + Na]+, 915.30;found, 915.34.

�-D-Galactopyranosyl-(1f3)-1,5-anhydro-2-deoxy-D-lyxo-hex-itol (51). A solution of 50 (470 mg, 0.526 mmol) in MeOH (15mL) was treated with freshly prepared 1 M NaOMe/MeOH (1.5mL) and stirred at rt for 19 h under argon. Then the mixture wasneutralized with Dowex 50 × 8 ion-exchange resin (H+ type),filtrated, and concentrated. The residue was purified by reversed-phase chromatography (RP-18 column, 5% gradient MeOH inwater) to afford 51 (205 mg, 82%) as a colorless foam.

[R]D +2.25 (c 0.80, MeOH). 1H NMR (500 MHz, CD3OD): δ1.79 (dd, J ) 3.8, 12.3 Hz, 1H, Hex-H2a), 1.97 (m, 1H, Hex-H2b),3.47 (m, 1H, Hex-H1a), 3.48 (dd, J ) 3.3, 9.7 Hz, 1H, Gal-H3),3.52 (m, 1H, Gal-H5), 3.54 (dd, J ) 7.5, 9.7 Hz, 1H, Gal-H2),3.57 (m, 1H, Hex-H5), 3.66 (dd, J ) 4.7, 10.2 Hz, 1H, Hex-H6a),3.70 (dd, J ) 7.2, 10.1 Hz, 1H, Hex-H6b), 3.71 (dd, J ) 5.1, 11.4Hz, 1H, Gal-H6a), 3.75 (dd, J ) 7.0, 11.4 Hz, 1H, Gal-H6b), 3.82(d, J ) 3.0 Hz, 1H, Gal-H4), 3.92 (ddd, J ) 3.0, 4.7, 11.7 Hz, 1H,Hex-H3), 3.98 (d, J ) 2.6 Hz, 1H, Hex-H4), 4.00 (m, 1H, Hex-H1b), 4.40 (d, J ) 7.5 Hz, 1H, Gal-H1), 4.59, 4.62 (A, B of AB,J ) 12.0 Hz, 2H, BiphCH2), 7.31-7.34, 7.41-7.44, 7.60-7.62(m, 9H, C6H4, C6H5). 13C NMR (125 MHz, CD3OD): δ 27.0 (Hex-C2), 62.6 (Gal-C6), 66.9 (Hex-C1), 69.4 (Hex-C4), 70.3 (Gal-C4),71.6 (Hex-C6), 72.5 (Gal-C2), 74.1 (BiphCH2), 74.7 (Gal-C3), 76.7(Gal-C5), 77.8 (Hex-C3), 79.0 (Hex-C5), 102.9 (Gal-C1), 127.9,128.0, 128.3, 129.4, 129.9, 138.7 (12C, C6H4, C6H5). ESI-MS calcdfor C25H32NaO9 [M + Na]+, 499.19; found, 499.20.


(3,4-O-Isopropylidene-�-D-galactopyranosyl)-(1f3)-1,5-anhy-dro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (52). Toa solution of 51 (195 mg, 0.409 mmol) and 2,2-dimethoxypropane(75.2 µL, 0.614 mmol) in acetone (15 mL) was added p-toluenesulfonic acid monohydrate (7.8 mg, 0.041 mmol). The mixture wasstirred for 3.5 h at 70 °C before NEt3 (0.5 mL) was added. Thesolvents were removed in vacuo and the residue was purified bysilica gel chromatography (DCM/MeOH, 50:1 to 10:3) to give 52(166 mg, 78%) and the 4,6-O-isopropylidene (33.3 mg, 16%) ascolorless foams.

[R]D +20.8 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.34, 1.52 (2s, 6H, C(CH3)2), 1.66 (s, 1H, OH), 1.72 (m, 1H, Hex-H2a), 2.08 (m, 1H, Hex-H2b), 2.94 (s, 1H, OH), 3.46 (m, 1H, Hex-H1a), 3.52 (m, 1H, Hex-H5), 3.60 (t, J ) 7.7 Hz, 1H, Gal-H2),3.70 (dd, J ) 5.0, 10.0 Hz, 1H, Hex-H6a), 3.74 (dd, J ) 6.6,10.0 Hz, 1H, Hex-H6b), 3.80 (m, 1H, Hex-H3), 3.82 (dd, J ) 3.8,11.5 Hz, 1H, Gal-H6a), 3.87 (m, 1H, Gal-H5), 3.95 (dd, J ) 7.0,11.4 Hz, 1H, Gal-H6b), 4.03 (s, 1H, Hex-H4), 4.07-4.11 (m, 2H,Hex-H1b, Gal-H3), 4.16 (d, J ) 4.7 Hz, 1H, Gal-H4), 4.34 (d, J )8.1 Hz, 1H, Gal-H1), 4.59, 4.63 (A, B of AB, J ) 12.1 Hz, 2H,BiphCH2), 7.32-7.36, 7.40-7.45, 7.56-7.59 (m, 9H, C6H5, C6H4).13C NMR (125 MHz, CDCl3): δ 26.2 (Hex-C2), 26.6, 28.3(C(CH3)2), 62.5 (Gal-C6), 65.6 (Hex-C1), 68.1 (Hex-C4), 70.2(Hex-C6), 73.0 (Gal-C2), 73.6 (BiphCH2), 73.7 (Gal-C4), 73.8 (Gal-C5), 77.0 (Hex-C5), 78.1 (Hex-C3), 78.9 (Gal-C3), 100.8 (Gal-C1), 105.9 (C(CH3)2), 127.2, 127.3, 127.5, 128.3, 128.7, 136.8,140.5, 140.8 (12C, C6H4, C6H5). ESI-MS calcd for C28H36NaO9

[M + Na]+, 539.23; found, 539.25.(2,6-Di-O-acetyl-3,4-O-isopropylidene-�-D-galactopyranosyl)-

(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (53). To a solution of 52 (161 mg, 0.312mmol) in pyridine (5 mL) were subsequently added acetic anhydride(442 µL, 4.68 mmol) and DMAP (10 mg) at 0 °C under argon.The mixture was stirred for 21 h at rt and then concentrated invacuo. The residue was coevaporated with toluene (3 × 5 mL),dissolved in DCM (20 mL), and washed with 0.1 N HCl (10 mL)and saturated NaHCO3 (10 mL). The organic phase was dried(Na2SO4), filtered, and concentrated in vacuo to give 53 (188 mg,94%) as a colorless foam.

[R]D +27.7 (c 1.01, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.32, 1.55 (2s, 6H, C(CH3)2), 1.76 (dd, J ) 3.6, 12.6 Hz, 1H, Hex-H2a), 1.96 (m, 1H, Hex-H2b), 2.07 (s, 9H, 3 COCH3), 3.49-3.54(m, 3H, Hex-H1a, Hex-H6), 3.65 (m, 1H, Hex-H5), 3.88 (m, 1H,Hex-H3), 3.95 (dt, J ) 1.5, 6.1 Hz, 1H, Gal-H5), 4.10-4.16 (m,3H, Hex-H1b, Gal-H3, Gal-H4), 4.32 (d, J ) 6.1 Hz, 2H, Gal-H6), 4.43 (d, J ) 8.1 Hz, 1H, Gal-H1), 4.50, 4.60 (A, B of AB, J) 11.9 Hz, 2H, BiphCH2), 4.93 (m, 1H, Gal-H2), 5.32 (d, J ) 3.0Hz, 1H, Hex-H4), 7.32-7.36, 7.39-7.45, 7.55-7.59 (m, 9H, C6H5,C6H4). 13C NMR (125 MHz, CDCl3): δ 20.8, 20.9, 20.9 (3 COCH3),26.3 (C(CH3)2), 27.0 (Hex-C2), 27.6 (C(CH3)2), 63.2 (Gal-C6), 66.0(Hex-C1), 68.7 (Hex-C4), 69.9 (Hex-C6), 70.9 (Gal-C5), 72.5 (Gal-C2), 73.4 (Gal-C3), 73.4 (BiphCH2), 73.8 (Hex-C3), 76.7 (Gal-C4), 77.3 (Hex-C5), 97.7 (Gal-C1), 110.8 (C(CH3)2), 127.1, 127.2,127.2, 128.4, 128.7, 136.8, 140.7, 140.9 (12C, C6H4, C6H5), 169.3,170.2, 170.8 (3 CO).ESI-MS calcd for C34H42NaO12 [M + Na]+,665.26; found, 665.35.

(2,6-Di-O-acetyl-�-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (54).Compound 53 (180 mg, 0.280 mmol) was suspended in 80% aqacetic acid (20 mL) and stirred for 2.5 h at 80 °C. The solvent wasremoved in vacuo and the residue coevaporated with toluene (3 ×5 mL). The remaining solid was purified by silica gel chromatog-raphy (DCM/MeOH, 50:1 to 20:1) to give 54 (136 mg, 81%) as acolorless foam.

[R]D +17.8 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.76 (dd, J ) 3.8, 12.7 Hz, 1H, Hex-H2a), 1.95 (m, 1H, Hex-H2b),2.07, 2.10 (2s, 9H, 3 COCH3), 3.03, 3.13 (2s, 2H, 2 OH), 3.47-3.53(m, 3H, Hex-H1a, Hex-H6), 3.60 (dd, J ) 2.8, 9.8 Hz, 1H, Gal-H3), 3.63-3.67 (m, 2H, Hex-H5, Gal-H5), 3.86 (d, J ) 3.1 Hz,1H, Gal-H4), 3.91 (m, 1H, Hex-H3), 4.11 (m, 1H, Hex-H1b), 4.26(dd, J ) 6.3, 11.5 Hz, 1H, Gal-H6a), 4.35 (dd, J ) 6.7, 11.5 Hz,

1H, Gal-H6b), 4.48 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.49, 4.60 (A, Bof AB, J ) 12.0 Hz, 2H, BiphCH2), 4.93 (dd, J ) 7.9, 9.8 Hz, 1H,Gal-H2), 5.32 (d, J ) 2.9 Hz, 1H, Hex-H4), 7.33-7.36, 7.39-7.45,7.55-7.59 (m, 9H, C6H5, C6H4). 13C NMR (125 MHz, CDCl3): δ20.8, 20.9, 20.9 (3 COCH3), 27.1 (Hex-C2), 62.3 (Gal-C6), 65.8(Hex-C1), 68.6 (Gal-C4), 69.0 (Hex-C4), 69.8 (Hex-C6), 72.1, 72.2(Gal-C3, Gal-C5), 72.7 (Gal-C2), 73.3 (BiphCH2), 73.9 (Hex-C3),77.0 (Hex-C5), 98.5 (Gal-C1), 127.1, 127.2, 127.3, 128.4, 128.7,136.7, 140.7, 140.9 (12C, C6H4, C6H5), 170.6, 170.8, 171.1 (3 CO).ESI-MS calcd for C31H38NaO12 [M + Na]+, 625.23; found, 625.22.

(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,6-di-O-acetyl-�-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (55). A stirredsuspension of 54 (70.0 mg, 0.116 mmol), donor 1227 (146 mg, 0.290mmol), and activated powdered molecular sieves 3 Å (1.50 g) inMeCN (7 mL) was cooled to -40 °C under argon. Then NIS (131mg, 0.580 mmol) was added and stirring continued for 1.5 h at-40 °C. After the addition of TfOH (6.1 µL, 0.073 mmol), stirringwas continued for 19 h at -40 °C. The reaction was diluted withDCM (15 mL) and filtered through a pad of celite. The celite waswashed with DCM (3 × 5 mL), and the combined filtrates weresuccessively washed with 20% aq Na2S2O3 (2 × 5 mL) and H2O(10 mL). The organic layer was dried (Na2SO4), filtered, andconcentrated under reduced pressure. The residue was purified bysilica gel chromatography (0.33% gradient MeOH in DCM) toafford the �-sialoside 55 (20.8 mg) as an inseparable mixture withglycal and the pure R-sialoside 55 (75.4 mg, 61%) as a colorlessfoam.

[R]D +1.63 (c 0.72, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.81 (m, 1H, Hex-H2a), 1.86-1.90 (m, 4H, Sia-H3a, COCH3), 1.95(m, 1H, Hex-H2b), 2.03, 2.04, 2.06, 2.11, 2.15, 2.18 (6s, 18H, 6COCH3), 2.46 (s, 1H, OH), 2.65 (dd, J ) 4.4, 12.7 Hz, 1H, Sia-H3b), 3.22 (dd, J ) 6.7, 13.5 Hz, 1H, Sia-H9a), 3.42 (m, 1H, Gal-H4), 3.48-3.52 (m, 4H, Hex-H1a, Hex-H6, Sia-H9b), 3.64-3.65(m, 2H, Hex-H5, Gal-H5), 3.80 (s, 3H, OCH3), 3.90 (dd, J ) 3.6,7.3 Hz, 1H, Hex-H3), 3.94 (dd, J ) 2.4, 10.8 Hz, 1H, Sia-H6),4.06-4.12 (m, 2H, Hex-H1b, Sia-H5), 4.25-4.27 (m, 3H, Gal-H3, Gal-H6), 4.50, 4.60 (A, B of AB, J ) 12.0 Hz, 2H, BiphCH2),4.63 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.79 (m, 1H, Sia-H4), 5.03 (dd,J ) 8.1, 9.8 Hz, 1H, Gal-H2), 5.15 (d, J ) 10.2 Hz, 1H, NH), 5.31(m, 1H, Hex-H4), 5.32 (dd, J ) 2.4, 8.8 Hz, 1H, Sia-H7), 5.48 (m,1H, Sia-H8), 7.32-7.35, 7.39-7.44, 7.55-7.58 (m, 9H, C6H4,C6H5). 13C NMR (125 MHz, CDCl3): δ 20.8, 21.2, 23.1 (7C, 7COCH3), 27.1 (Hex-C2), 37.7 (Sia-C3), 49.0 (Sia-C5), 51.6 (Sia-C9), 53.1 (OCH3), 62.9 (Gal-C6), 65.8 (Hex-C1), 67.3 (Gal-C4),68.1 (Sia-C7), 68.7 (2C, Sia-C4, Sia-C8), 69.0 (Hex-C4), 69.1 (Gal-C2), 70.0 (Hex-C6), 71.8 (Gal-C5), 72.6 (Sia-C6), 73.3 (Gal-C3),73.5 (BiphCH2), 74.0 (Hex-C3), 77.1 (Hex-C5), 96.8 (Sia-C2), 98.5(Gal-C1), 127.0, 127.1, 127.2, 128.3, 128.7, 136.8, 140.6, 140.9(12C, C6H4, C6H5), 168.5, 169.3, 169.9, 170.3, 170.4, 170.6, 170.9(8C, 8 CO). ESI-MS calcd for C49H62N4NaO22 [M + Na]+, 1081.38;found, 1081.54.

(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-azido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,4,6-tri-O-acetyl-�-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (56). To asolution of 55 (108 mg, 0.102 mmol) in pyridine (3 mL) weresubsequently added acetic anhydride (96.0 µL, 1.02 mmol) andDMAP (3 mg) at 0 °C under argon. The mixture was stirred for17 h at rt, quenched by addition of MeOH (1 mL), and concentratedin vacuo. The residue was coevaporated with toluene (3 × 5 mL)and then purified by silica gel chromatography (0.25% gradientMeOH in DCM) to afford 56 (89.5 mg, 80%) as a colorless foam.

[R]D +3.34 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.70 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.84 (m, 1H, Hex-H2a), 1.85(s, 3H, COCH3), 1.97 (m, 1H, Hex-H2b), 2.00, 2.01, 2.06, 2.09,2.10, 2.17, 2.21 (7s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz,1H, Sia-H3b), 3.19 (dd, J ) 6.6, 13.5 Hz, 1H, Sia-H9a), 3.47-3.53(m, 4H, Hex-H1a, Hex-H6, Sia-H9b), 3.63 (m, 1H, Hex-H5), 3.64(dd, J ) 2.8, 10.7 Hz, 1H, Sia-H6), 3.82 (m, 1H, Gal-H5), 3.85 (s,


3H, OCH3), 3.89 (m, 1H, Hex-H3), 4.01 (m, 2H, Gal-H6), 4.08 (q,J ) 10.5 Hz, 1H, Sia-H5), 4.09 (m, 1H, Hex-H1b), 4.48 (dd, J )3.4, 10.2 Hz, 1H, Gal-H3), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz,2H, BiphCH2), 4.69 (d, J ) 7.9 Hz, 1H, Gal-H1), 4.85 (ddd, J )4.6, 10.5, 12.1 Hz, 1H, Sia-H4), 4.89 (d, J ) 3.1 Hz, 1H, Gal-H4),4.98 (dd, J ) 7.9, 10.2 Hz, 1H, Gal-H2), 5.18 (d, J ) 10.3 Hz,1H, NH), 5.31 (d, J ) 3.1 Hz, 1H, Hex-H4), 5.33 (dd, J ) 2.7, 8.8Hz, 1H, Sia-H7), 5.50 (ddd, J ) 3.3, 6.6, 8.7 Hz, 1H, Sia-H8),7.32-7.35, 7.38-7.44, 7.54-7.58 (m, 9H, C6H4, C6H5). 13C NMR(125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8, 20.8, 21.4, 23.1 (8C,8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3), 48.9 (Sia-C5), 51.6 (Sia-C9), 53.2 (OCH3), 61.8 (Gal-C6), 65.8 (Hex-C1), 67.4 (Gal-C4),68.0 (Sia-C7), 68.6 (Sia-C8), 68.8 (Hex-C4), 69.1 (Sia-C4), 69.5(Gal-C2), 70.1 (Hex-C6), 70.7 (Gal-C5), 71.2 (Gal-C3), 72.2 (Sia-C6), 73.3 (BiphCH2), 74.5 (Hex-C3), 77.2 (Hex-C5), 96.7 (Sia-C2), 98.6 (Gal-C1), 127.1, 127.1, 127.2, 128.3, 128.7, 136.8, 140.6,140.9 (12C, C6H4, C6H5), 167.9, 169.1, 169.8, 170.3, 170.4, 170.4,170.7, 170.9 (9C, 9 CO). ESI-MS calcd for C51H64N4NaO23 [M +Na]+, 1123.39; found, 1123.48.

(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-benzamido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,4,6-tri-O-acetyl-�-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (57).Compound 56 (40.0 mg, 36.3 µmol) and benzoyl chloride (16.9µL, 0.145 mmol) were dissolved in DCE (3 mL) and stirred at rtunder argon. Ph3P (20.8 mg, 79.9 µmol) in DCE (1 mL) was addedafter 5 min. The reaction was stirred at rt for 18 h and then dilutedwith DCM (10 mL) and washed with saturated aq NaHCO3 (5 mL)and H2O (5 mL). The organic layer was dried (Na2SO4), filtered,and concentrated in vacuo. The residue was purified by silica gelchromatography (0.5% gradient MeOH in DCM) to afford 57 (27.9mg, 65%) as a colorless foam.

[R]D +13.3 (c 0.73, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.72 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.83 (m, 1H, Hex-H2a), 1.86(s, 3H, COCH3), 1.96 (m, 1H, Hex-H2b), 2.01, 2.06, 2.09, 2.15,2.18, 2.19 (6s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.7 Hz, 1H,Sia-H3b), 2.90 (dt, J ) 3.8, 15.1 Hz, 1H, Sia-H9a), 3.44-3.51 (m,3H, Hex-H1a, Hex-H6), 3.58 (dd, J ) 2.6, 10.8 Hz, 1H, Sia-H6),3.61 (m, 1H, Hex-H5), 3.83 (s, 3H, OCH3), 3.84 (m, 1H, Gal-H5),3.88 (m, 1H, Hex-H3), 4.01 (m, 2H, Gal-H6), 4.06 (m, 1H, Hex-H1b), 4.15 (q, J ) 10.4 Hz, 1H, Sia-H5), 4.39 (ddd, J ) 2.8, 8.7,15.1 Hz, 1H, Sia-H9b), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz, 2H,BiphCH2), 4.55 (dd, J ) 3.4, 10.2 Hz, 1H, Gal-H3), 4.72 (d, J )7.9 Hz, 1H, Gal-H1), 4.86 (ddd, J ) 4.6, 10.5, 12.0 Hz, 1H, Sia-H4), 4.90 (d, J ) 3.0 Hz, 1H, Gal-H4), 4.98 (dd, J ) 8.0, 10.1 Hz,1H, Gal-H2), 5.20 (d, J ) 10.3 Hz, 1H, NH), 5.24 (dd, J ) 2.6,9.4 Hz, 1H, Sia-H7), 5.30 (d, J ) 2.5 Hz, 1H, Hex-H4), 5.43 (dt,J ) 3.2, 9.4 Hz, 1H, Sia-H8), 6.90 (dd, J ) 4.0, 8.5 Hz, 1H, NH),7.32-7.34, 7.39-7.46, 7.50-7.58, 7.79-7.80 (m, 14H, C6H4, 2C6H5). 13C NMR (125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8, 20.8,21.1, 21.4, 23.1 (8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3), 38.5(Sia-C9), 49.1 (Sia-C5), 53.1 (OCH3), 61.8 (Gal-C6), 65.8 (Hex-C1), 67.5 (Gal-C4), 67.9 (Sia-C7), 68.5 (Sia-C8), 68.9 (Hex-C4),69.3 (Sia-C4), 69.6 (Gal-C2), 70.1 (Hex-C6), 70.6 (Gal-C5), 71.4(Gal-C3), 72.0 (Sia-C6), 73.3 (BiphCH2), 74.3 (Hex-C3), 77.2 (Hex-C5), 96.7 (Sia-C2), 98.4 (Gal-C1), 126.9, 127.1, 127.1, 127.2, 128.3,128.6, 128.7, 134.2, 136.8, 140.6, 140.9 (18C, C6H4, 2 C6H5), 167.3,167.8, 169.3, 170.3, 170.4, 170.4, 171.0, 171.7 (10C, 10 CO). ESI-MS calcd for C58H70N2NaO24 [M + Na]+, 1201.42; found, 1201.76.

(Methyl 5-Acetamido-4,7,8-tri-O-acetyl-9-(4-chlorobenzamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-(2,4,6-tri-O-acetyl-�-D-galactopyranosyl)-(1f3)-4-O-acetyl-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (58). According to the procedure described for the synthesisof 57, a solution of 56 (40.0 mg, 36.3 µmol) and p-chlorobenzoylchloride (18.6 µL, 0.145 mmol) in DCE (3 mL) was treated withPh3P (20.8 mg, 79.9 µmol) in DCE (1 mL) for 17 h. After workup,the crude product was purified by silica gel chromatography (0.25%gradient MeOH in DCM) to afford 58 (33.0 mg, 75%) as a colorlessfoam.

[R]D +12.6 (c 1.00, CHCl3). 1H NMR (500 MHz, CDCl3): δ1.73 (t, J ) 12.4 Hz, 1H, Sia-H3a), 1.80 (m, 1H, Hex-H2a), 1.86(s, 3H, COCH3), 1.95 (m, 1H, Hex-H2b), 2.01, 2.03, 2.07, 2.09,2.14, 2.18, 2.19 (7s, 21H, 7 COCH3), 2.58 (dd, J ) 4.6, 12.6 Hz,1H, Sia-H3b), 2.87 (dt, J ) 3.6, 15.0 Hz, 1H, Sia-H9a), 3.47-3.52(m, 3H, Hex-H1a, Hex-H6), 3.57 (dd, J ) 2.6, 10.8 Hz, 1H, Sia-H6), 3.64 (m, 1H, Hex-H5), 3.82 (s, 3H, OCH3), 3.83 (m, 1H, Gal-H5), 3.90 (m, 1H, Hex-H3), 4.02 (m, 2H, Gal-H6), 4.08 (m, 1H,Hex-H1b), 4.16 (q, J ) 10.5 Hz, 1H, Sia-H5), 4.38 (ddd, J ) 2.9,8.8, 15.1 Hz, 1H, Sia-H9b), 4.50, 4.59 (A, B of AB, J ) 12.0 Hz,2H, BiphCH2), 4.52 (dd, J ) 3.5, 10.1 Hz, 1H, Gal-H3), 4.72 (d,J ) 8.0 Hz, 1H, Gal-H1), 4.86 (ddd, J ) 4.6, 10.7, 12.0 Hz, 1H,Sia-H4), 4.90 (d, J ) 3.0 Hz, 1H, Gal-H4), 4.98 (dd, J ) 8.0, 10.1Hz, 1H, Gal-H2), 5.19 (d, J ) 10.5 Hz, 1H, NH), 5.21 (dd, J )2.6, 9.7 Hz, 1H, Sia-H7), 5.31 (d, J ) 2.7 Hz, 1H, Hex-H4), 5.42(dt, J ) 3.0, 9.7 Hz, 1H, Sia-H8), 6.91 (dd, J ) 4.0, 8.6 Hz, 1H,NH), 7.32-7.35, 7.39-7.44, 7.55-7.58, 7.73-7.75 (m, 13H, 2C6H4, C6H5). 13C NMR (125 MHz, CDCl3): δ 20.6, 20.7, 20.7, 20.8,20.8, 21.1, 21.4, 23.1 (8 COCH3), 26.9 (Hex-C2), 37.4 (Sia-C3),38.4 (Sia-C9), 49.2 (Sia-C5), 53.1 (OCH3), 61.8 (Gal-C6), 65.8(Hex-C1), 67.5 (Gal-C4), 67.8 (Sia-C7), 68.3 (Sia-C8), 68.9 (Hex-C4), 69.3 (Sia-C4), 69.6 (Gal-C2), 70.1 (Hex-C6), 70.6 (Gal-C5),71.4 (Gal-C3), 72.0 (Sia-C6), 73.3 (BiphCH2), 74.3 (Hex-C3), 77.2(Hex-C5), 96.7 (Sia-C2), 98.4 (Gal-C1), 127.1, 127.2, 127.2, 128.3,128.7, 128.9, 132.5, 136.9, 140.6, 140.9 (18C, 2 C6H4, C6H5), 166.3,169.2, 170.3, 170.4, 170.4, 170.9, 171.0 (10C, 10 CO). ESI-MScalcd for C58H69ClN2NaO24 [M + Na]+, 1235.38; found, 1235.42.

(Sodium 5-Acetamido-9-benzamido-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-�-D-galactopyrano-syl-(1f3)-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (59). To a solution of 57 (24.0 mg, 20.4 µmol) inMeOH (3 mL) was added freshly prepared 1 M NaOMe/MeOH(0.3 mL). The mixture was stirred at rt under argon for 6 h, andthen water (0.5 mL) was added and the mixture stirred for another15 h at rt. The solution was neutralized with Dowex 50×8 ion-exchange resin (H+ type), filtrated, and concentrated. The residuewas purified by reversed-phase chromatography (RP-18 column,5% gradient MeOH in water), Dowex 50×8 ion-exchange chro-matography (Na+ type), and P2 size exclusion chromatography toafford 59 (14.3 mg, 79%) as a colorless solid after a finallyophilization from water.

[R]D -4.56 (c 0.41, MeOH). 1H NMR (500 MHz, CD3OD): δ1.73 (m, 1H, Hex-H2a), 1.75 (t, J ) 11.9 Hz, 1H, Sia-H3a), 1.92(m, 1H, Hex-H2b), 1.98 (s, 3H, COCH3), 2.86 (dd, J ) 3.9, 12.1Hz, 1H, Sia-H3b), 3.39-3.43 (m, 2H, Hex-H1a, Sia-H7), 3.49-3.53(m, 3H, Hex-H5, Gal-H5, Sia-H9a), 3.58 (dd, J ) 7.8, 9.5 Hz, 1H,Gal-H2), 3.62-3.76 (m, 7H, Hex-H6, Gal-H6, Sia-H4, Sia-H5, Sia-H6), 3.78 (dd, J ) 2.9, 13.4 Hz, 1H, Sia-H9b), 3.88-3.96 (m, 4H,Hex-H1b, Hex-H3, Hex-H4, Gal-H4), 4.00-4.05 (m, 2H, Gal-H3,Sia-H8), 4.45 (d, J ) 7.7 Hz, 1H, Gal-H1), 4.57, 4.61 (A, B ofAB, J ) 12.1 Hz, 2H, BiphCH2), 7.31-7.34, 7.41-7.47, 7.51-7.54,7.59-7.61, 7.83-7.84 (m, 14H, C6H4, 2 C6H5). 13C NMR (125MHz, CD3OD): δ 22.6 (COCH3), 26.9 (Hex-C2), 42.0 (Sia-C3),44.4 (Sia-C9), 53.8 (Sia-C5), 62.8 (Gal-C6), 66.9 (Hex-C1), 69.0(Gal-C4), 69.3 (Hex-C4), 69.4 (Sia-C4), 70.8 (Gal-C2), 71.3 (Sia-C8), 71.8 (Hex-C6), 72.1 (Sia-C7), 74.0 (BiphCH2), 74.7 (Sia-C6),76.6 (Gal-C5), 77.6 (Gal-C3), 77.9 (Hex-C3), 79.0 (Hex-C5), 101.2(Sia-C2), 102.7 (Gal-C1), 127.9, 128.0, 128.3, 128.3, 129.5, 129.6,129.9, 132.6, 135.8, 138.6, 141.9, 142.1 (18C, C6H4, 2 C6H5), 170.3,175.4 (3C, 3 CO). HRMS calcd for C43H54N2NaO17 [M + H]+,893.3320; found, 893.3323.

(Sodium 5-Acetamido-9-(4-chlorobenzamido)-3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyranosynate)-(2f3)-�-D-galac-topyranosyl-(1f3)-1,5-anhydro-6-O-(biphenyl-4-yl-methyl)-2-deoxy-D-lyxo-hexitol (60). According to the procedure describedfor the synthesis of 59, a solution of 58 (19.0 mg, 15.7 µmol) inMeOH (3 mL) was treated with 1 M NaOMe/MeOH (0.3 mL).After 6 h, water (0.5 mL) was added and the mixture stirred foranother 16 h. After workup, the crude product was purified byreversed-phase chromatography (RP-18 column, 5% gradient MeOHin water), Dowex 50×8 ion-exchange chromatography (Na+ type),


and P2 size exclusion chromatography to afford 60 (10.9 mg, 75%)as a colorless solid after a final lyophilization from water.

[R]D -4.94 (c 0.55, MeOH). 1H NMR (500 MHz, CD3OD): δ1.72 (m, 1H, Hex-H2a), 1.88 (t, J ) 12.0 Hz, 1H, Sia-H3a), 1.94(m, 1H, Hex-H2b), 1.97 (s, 3H, COCH3), 2.78 (dd, J ) 4.0, 12.5Hz, 1H, Sia-H3b), 3.41-3.45 (m, 2H, Hex-H1a, Sia-H7), 3.51-3.56(m, 3H, Hex-H5, Gal-H5, Sia-H9a), 3.60 (dd, J ) 7.9, 9.5 Hz, 1H,Gal-H2), 3.64 (dd, J ) 4.7, 10.3 Hz, 1H, Gal-H6a), 3.66-3.74(m, 4H, Hex-H6, Gal-H6b, Sia-H6), 3.76-3.84 (m, 3H, Sia-H4,Sia-H5, Sia-H9b), 3.87 (ddd, J ) 2.9, 4.4, 11.6 Hz, 1H, Hex-H3),3.93 (m, 1H, Gal-H4), 3.96-4.00 (m, 3H, Hex-H1b, Hex-H4, Sia-H8), 4.07 (dd, J ) 2.6, 9.6 Hz, 1H, Gal-H3), 4.46 (d, J ) 7.7 Hz,1H, Gal-H1), 4.57, 4.61 (A, B of AB, J ) 12.0 Hz, 2H, BiphCH2),7.31-7.33, 7.41-7.44, 7.46-7.48, 7.59-7.61, 7.82-7.84 (m, 13H,2 C6H4, C6H5). 13C NMR (125 MHz, CD3OD): δ 22.7 (COCH3),27.0 (Hex-C2), 41.2 (Sia-C3), 44.8 (Sia-C9), 53.7 (Sia-C5), 62.5(Gal-C6), 66.9 (Hex-C1), 68.9 (Sia-C4), 69.3 (Hex-C4), 69.4 (Gal-C4), 70.7 (Gal-C2), 71.3 (Sia-C8), 71.7 (Hex-C6), 72.0 (Sia-C7),74.0 (BiphCH2), 75.1 (Sia-C6), 76.4 (Gal-C5), 77.7 (Gal-C3), 78.1(Hex-C3), 79.0 (Hex-C5), 100.8 (Sia-C2), 102.8 (Gal-C1), 127.9,128.0, 128.3, 129.4, 129.7, 129.9, 130.1, 134.4, 138.7, 141.9, 142.2(18C, 2 C6H4, C6H5), 169.4, 175.1 (3C, 3 CO). HRMS calcd forC43H53ClN2NaO17 [M + H]+, 927.2930; found, 927.2926.

In Vitro Binding Assay. A recombinant protein consisting ofthe N-terminal three domains of MAG and the Fc part of humanIgG (Fc-MAGd1-3) was produced by expression in CHO cells andaffinity purification on protein A-agarose as described.36 For thehapten inhibition assay a modification of the previously describedassay17a was used. Instead of human erythrocytes as a target forsialic acid-dependent binding of MAG, commercially availablemicrotiter plates with immobilized Neu5Ac (Lundonia, Lund,Sweden) were used. In brief, to each well 10 µL of an oligosac-charide solution was added followed by 20 µL of Fc-MAGd1-3,which had been precomplexed with an anti-Fc antibody labeledwith alkaline phosphatase. After an overnight incubation at 4 °C,unbound Fc-MAG complexes were removed by washing and theamount of bound Fc-MAG was determined via the alkalinephosphatase with fluorescein diphosphate as substrate. For eacholigosaccharide, at least eight concentrations were used to determinethe concentration required for 50% inhibition (IC50). To comparethe results from different assays, compound 4 was included in eachtest and used as a reference to calculate the relative inhibitorypotencies (rIP). At least three independent titrations were performedfor each compound.

Surface Plasmon Resonance Experiments (Biacore). Surfaceplasmon resonance experiments were performed on a Biacore 3000machine using CM5 chips. Goat antihuman IgG antibody (Fcspecific, Sigma) was immobilized following the standard BiacoreEDC/NHS immobilization procedure. For this, 2 µL of a Fc-antibody solution of 2.1 mg/mL (in 50 mM phosphate buffer, pH7.0) was added to 98 µL of 10 mM acetate buffer (pH 5.5). Theimmobilization yielded between 4000 and 5000 resonance units.A sample and a reference surface were prepared sequentially. Forcapturing Fc-MAGd1-3 onto the sample surface, Fc-MAGd1-3 inHBS-EP buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.0005% surfactant P20; Biacore) was injected. Ligandswere dissolved in HBS-EP buffer. Eight ligand concentrations of a2-fold dilution series starting from 50 µM were prepared. Thesamples were injected in a randomized order. Five blank bufferinjections were performed before triplicate measurements and onebetween each single experiment. The flow rate was 10 µL/min forthe immobilization, 1 µL/min for the capturing of MAG, and 20µL/min for the injection of ligands. For the determination of KD

values and for the kinetics analysis, standard Biacore software,version 3.2, was used.

Acknowledgment. We gratefully acknowledge the financialsupport by the Swiss National Science Foundation (200020-120628) and the Volkswagen Foundation (center project grant“conformational control of biomolecular functions”).

Supporting Information Available: Synthesis of compounds6-11 and 21-47; HRMS data, HPLC traces, and 1H NMR spectrafor the target compounds 16, 27-29, 37, 41-43, 45, 47, 59, and60. This material is available free of charge via the Internet at http://pubs.acs.org.

References(1) Ramon y Cajal, S. Degeneration and Regeneration of the NerVous

System; Oxford University Press: London, 1928.(2) (a) Tello, F. La influencia del neurotropismo en la regeneration de

los centros nerviosos. Trab. Lab. InVest. Biol. 1911, 9, 123–129. (b)Davies, S. J.; Goucher, D. R.; Doller, C.; Silver, J. Robust Regenerationof Adult Sensory Axons in Degenerating White Matter of the AdultRat Spinal Cord. J. Neurosci. 1999, 19, 5810–5822.

(3) (a) Caroni, P.; Savio, T.; Schwab, M. E. Central Nervous SystemRegeneration: Oligodendrocytes and Myelin as Nonpermissive Sub-strates for Neurite Growth. Prog. Brain Res. 1988, 78, 363–370. (b)Filbin, M. T. Myelin-associated Inhibitors of Axonal Regeneration inthe Adult Mammalian CNS. Nat. ReV. Neurosci. 2003, 4, 703–713.(c) Sandvig, A.; Berry, M.; Barrett, L. B.; Butt, A.; Logan, A. Myelin-,Reactive Glia-, and Scar-Derived CNS Axon Growth Inhibitors:Expression, Receptor Signaling, and Correlation with Axon Regenera-tion. Glia 2004, 46, 225–251. (d) He, Z.; Koprivica, V. The NogoSignaling Pathway for Regeneration Block. Annu. ReV. Neurosci. 2004,27, 341–368.

(4) (a) Caroni, P.; Schwab, M. E. Antibody Against Myelin-associatedInhibitor of Neurite Growth Neutralizes Nonpermissive SubstrateProperties of CNS White Matter. Neuron 1988, 1, 85–96. (b) Caroni,P.; Schwab, M. E. Two membrane protein fractions from rat centralmyelin with inhibitory properties for neurite growth and fibroblastspreading. J. Cell Biol. 1988, 106, 1281–1288. (c) Chen, M. S.; Huber,A. B.; van der Haar, M. E.; Frank, M.; Schnell, L.; Spillmann, A. A.;Christ, F.; Schwab, M. E. Nogo-A Is a Myelin-Associated NeuriteOutgrowth Inhibitor and an Antigen for Monoclonal Antibody IN-1.Nature 2000, 403, 434–439. (d) GrandPre, T.; Nakamura, F.; Varta-nian, T.; Strittmatter, S. M. Identification of the Nogo Inhibitor ofAxon Regeneration as a Reticulon Protein. Nature 2000, 403, 439–444.

(5) (a) Kottis, V.; Thibault, P.; Mikol, D.; Xiao, Z. C.; Zhang, R.;Dergham, P.; Braun, P. E. Oligodendrocyte-Myelin Glycoprotein(OMgp) is an Inhibitor of Neurite Outgrowth. J. Neurochem. 2002,82, 1566–1569. (b) Wang, K. C.; Koprivica, V.; Kim, J. A.;Sivasankaran, R.; Guo, Y.; Neve, R. L.; He, Z. Oligodendrocyte-Myelin Glycoprotein is a Nogo Receptor Ligand that Inhibits NeuriteOutgrowth. Nature 2002, 417, 941–944.

(6) (a) McKerracher, L.; David, S.; Jackson, D. L.; Kottis, V.; Dunn, R. J.;Braun, P. E. Identification of Myelin-Associated Glycoprotein as aMajor Myelin-Derived Inhibitor of Neurite Growth. Neuron 1994, 13,805–811. (b) Mukhopadhyay, G.; Doherty, P.; Walsh, F. S.; Crocker,P. R.; Filbin, M. T. A novel role for myelin-associated glycoproteinas an inhibitor of axonal regeneration. Neuron 1994, 13, 757–767. (c)Vinson, M.; Strijbos, P. J. L. M.; Rowles, A.; Facci, L.; Moore, S. E.;Simmons, D. L.; Walsh, F. S. Myelin-Associated GlycoproteinInteracts with Ganglioside GT1b: A Mechanism for Neurite OutgrowthInhibition. J. Biol. Chem. 2001, 276, 20280–20285.

(7) Fournier, A. E.; GrandPre, T.; Strittmatter, S. M. Identification of aReceptor Mediating Nogo-66 Inhibition of Axonal Regeneration.Nature 2001, 409, 341–346.

(8) (a) Wang, K. C.; Kim, J. A.; Sivasankaran, R.; Segal, R.; He, Z. P75Interacts with the Nogo Receptor as a Co-receptor for Nogo, MAG,and OMgp. Nature 2002, 420, 74–78. (b) Wong, S. T.; Henley, J. R.;Kanning, K. C.; Huang, K. H.; Bothwell, M.; Poo, M. M. A p75NTRand Nogo Receptor Complex Mediates Repulsive Signaling by Myelin-Associated Glycoprotein. Nat. Neurosci. 2002, 5, 1302–1308.

(9) Mi, S.; Lee, X.; Shao, Z.; Thill, G.; Ji, B.; Relton, J.; Levesque, M.;Allaire, N.; Perrin, S.; Sands, B.; Crowell, T.; Cate, R. L.; McCoy,J. M.; Pepinsky, R. B. LINGO-1 Is a Component of the Nogo-66Receptor/p75 Signaling Complex. Nat. Neurosci. 2004, 7, 221–228.

(10) (a) Yang, L. J.-S.; Zeller, C. B.; Shaper, N. L.; Kiso, M.; Hasegawa,A.; Shapiro, R. E.; Schnaar, R. L. Gangliosides Are Neuronal Ligandsfor Myelin-Associated Glycoprotein. Proc. Natl. Acad. Sci. U.S.A.1996, 93, 814–818. (b) Vyas, A. A.; Patel, H. V.; Fromholt, S. E.;Heffer-Lauc, M.; Vyas, K. A.; Dang, J.; Schachner, M.; Schnaar, R. L.Gangliosides Are Functional Nerve Cell Ligands for Myelin-Associ-ated Glycoprotein (MAG), an Inhibitor of Nerve Regeneration. Proc.Natl. Acad. Sci. U.S.A. 2002, 99, 8412–8417.

(11) Yamashita, T.; Higuchi, H.; Tohyama, M. The p75 Receptor Trans-duces the Signal from Myelin-Associated Glycoprotein to Rho. J. Cell.Biol. 2002, 157, 565–570.

(12) (a) Kelm, S.; Pelz, A.; Schauer, R.; Filbin, M. T.; Tang, S.; de Bellard,M. E.; Schnaar, R. L.; Mahoney, J. A.; Hartnell, A.; Bradfield, P.;


Crocker, P. R. Sialoadhesin, Myelin-Associated Glycoprotein andCD22 Define a New Family of Sialic Acid-Dependent AdhesionMolecules of the Immunoglobulin Superfamily. Curr. Biol. 1994, 4,965–972. (b) Crocker, P. R.; Clark, E. A.; Filbin, M.; Gordon, S.;Jones, Y.; Kehrl, J. H.; Kelm, S.; Le Douarin, N.; Powell, L.; Roder,J.; Schnaar, R. L.; Sgroi, D. C.; Stamenkovic, K.; Schauer, R.;Schachner, M.; van den Berg, T. K.; van der Merwe, P. A.; Watt,S. M.; Varki, A. Siglecs: A Family of Sialic Acid Binding Lectins.Glycobiology 1998, 8, Glycoforum 2 v-vi. .

(13) McKerracher, L. Ganglioside Rafts as MAG Receptors that MediateBlockade of Axon Growth. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,7811–7813.

(14) (a) Hotta, K.; Ishida, H.; Hasegawa, A. Synthetic Studies on Sialogly-coconjugates 66: First Total Synthesis of a Cholinergic Neuron-SpecificGanglioside GQ1bR. J. Carbohydr. Chem. 1995, 14, 491–506. (b) Ito,H.; Ishida, H.; Ando, S.; Kiso, M. Total Synthesis of a CholinergicNeuron-Specific Ganglioside GT1aR: A High Affinity Ligand forMyelin-Associated Glycoprotein (MAG). Glycoconjugate J. 1999, 16,585–598. (c) Ito, H.; Ishida, H.; Kiso, M. A Highly Efficient TotalSynthetic Route to R-Series Gangliosides: GM1R, GD1R, and GT1R.J. Carbohydr. Chem. 2001, 20, 207–228.

(15) (a) Collins, B. E.; Yang, L. J.-S.; Mukhopadhyay, G.; Filbin, M. T.;Kiso, M.; Hasegawa, A.; Schnaar, R. L. Sialic acid specificity ofmyelin-associated glycoprotein binding. J. Biol. Chem. 1997, 272,1248–1255. (b) Collins, B. E.; Kiso, M.; Hasegawa, A.; Tropak, M. B.;Roder, J. C.; Crocker, P. R.; Schnaar, R. L. Binding Specificities ofthe Sialoadhesin Family of I-Type Lectins. J. Biol. Chem. 1997, 272,16889–16895. (c) Kelm, S. Ligands for Siglecs. In MammalianCarbohydrate Recognition Systems; Crocker, P. R., Ed.; Springer:Berlin, 2001; pp 153-176.

(16) Crocker, P. R.; Kelm, S.; Hartnell, A.; Freeman, S.; Nath, D.; Vinson,M.; Mucklow, S. Sialoadhesin and Related Cellular RecognitionMolecules of the Immunoglobulin Superfamily. Biochem. Soc. Trans.1996, 24, 150–156.

(17) (a) Strenge, K.; Schauer, R.; Bovin, N.; Hasegawa, A.; Ishida, H.;Kiso, M.; Kelm, S. Glycan Specificity of Myelin-Associated Glyco-protein and Sialoadhesin Deduced from Interactions with SyntheticOligosaccharides. Eur. J. Biochem. 1998, 258, 677–685. (b) Collins,B. E.; Ito, H.; Sawada, N.; Ishida, H.; Kiso, M.; Schnaar, R. L.Enhanced Binding of the Neural Siglecs, Myelin-Associated Glyco-protein, and Schwann Cell Myelin Protein to Chol-1 (R-Series)Gangliosides and Novel Sulfated Chol-1 Analogs. J. Biol. Chem. 1999,274, 37637–37643. (c) Sawada, N.; Ishida, H.; Collins, B. E.; Schnaar,R. L.; Kiso, M. Ganglioside GD1R Analogues as High-AffinityLigands for Myelin-Associated Glycoprotein (MAG). Carbohydr. Res.1999, 316, 1–5. (d) Ito, H.; Ishida, H.; Collins, B. E.; Fromholt, S. E.;Schnaar, R. L.; Kiso, M. Systematic synthesis and MAG-bindingactivity of novel sulfated GM1b analogues as mimics of Chol-1 (R-series) gangliosides: highly active ligands for neural siglecs. Carbo-hydr. Res. 2003, 338, 1621–1639.

(18) (a) Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H.-J.; Strenge, K.;Schauer, R. Functional Groups of Sialic Acids Involved in Bindingto Siglecs (Sialoadhesins) Deduced from Interactions with SyntheticAnalogues. Eur. J. Biochem. 1998, 255, 663–672. (b) Kelm, S.;Brossmer, R. Siglec Inhibitors. PCT Patent WO 03/000709A2, 2003;(c) Schwizer, D.; Gathje, H.; Kelm, S.; Porro, M.; Schwardt, O.; Ernst,B. Antagonists of the Myelin-Associated Glycoprotein: A New Classof Tetrasaccharide Mimics. Bioorg. Med. Chem. 2006, 14, 4944–4957.(d) Shelke, S. V.; Gao, G.-P.; Mesch, S.; Gathje, H.; Kelm, S.;Schwardt, O.; Ernst, B. Synthesis of Sialic Acid Derivatives as Ligandsfor the Myelin-Associated Glycoprotein (MAG). Bioorg. Med. Chem.2007, 15, 4951–4965. (e) Gao, G.-P.; Smiesko, M.; Schwardt, O.;Gathje, H.; Kelm, S.; Vedani, A.; Ernst, B. Mimetics of the Tri- andTetrasaccharide Epitope of GQ1bR as Myelin-Associated Glycoprotein(MAG) Ligands. Bioorg. Med. Chem. 2007, 15, 7459–7469.

(19) Yang, L. J. S.; Lorenzini, I.; Vajn, K.; Mountney, A.; Schramm, L. P.;Schnaar, R. L. Sialidase Enhances Spinal Axon Outgrowth in Vivo.Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11057–11062.

(20) Vyas, A. A.; Blixt, O.; Paulson, J. C.; Schnaar, R. L. Potent GlycanInhibitors of Myelin-Associated Glycoprotein Enhance Axon Out-growth in Vitro. J. Biol. Chem. 2005, 280, 16305–16310.

(21) Vyas, A. A.; Schnaar, R. L. Brain Gangliosides: Functional Ligandsfor Myelin Stability and the Control of Nerve Regeneration. Biochimie2001, 83, 677–682.

(22) Bhunia, A.; Schwardt, O.; Gathje, H.; Gao, G.-P.; Kelm, S.; Benie,A. J.; Hricovini, M.; Peters, T.; Ernst, B. Consistent BioactiveConformation of the Neu5AcR(2f3)Gal Epitope Upon Lectin Binding.ChemBioChem 2008, 9, 2941-2945.

(23) Schwardt, O.; Gao, G.-P.; Visekruna, T.; Rabbani, S.; Gassmann, E.;Ernst, B. Substrate Specificity and Preparative Use of RecombinantRat ST3Gal III. J. Carbohydr. Chem. 2004, 23, 1–26.

(24) Bock, N.; Kelm, S. Binding and Inhibition Assay for Siglecs. InMethods in Molecular Biology, Vol. 347; Brockhausen, I., Ed.;Humana Press Inc: Totowa NJ, 2006, pp 359-376.

(25) Murase, T.; Kameyama, A.; Kartha, K. P. R.; Ishida, H.; Kiso, M.;Hasegawa, A. Synthetic Studies on Sialoglycoconjugates 5. Facile,Regio- and Stereoselective Synthesis of Ganglioside GM4 and ItsPosition Isomer. J. Carbohydr. Chem. 1989, 8, 265–283.

(26) Jansson, K.; Frejd, T.; Kihlberg, J.; Magnusson, G. Boron TrifluorideEtherate as an Effective Reagent for the Stereoselective One-PotConversion of Acetylated 2-(Trimethylsilyl)ethyl Glycosides into Sugar1,2-trans-Acetates. Tetrahedron Lett. 1986, 27, 753–756.

(27) (a) Fugedi, P.; Garegg, P. J. A Novel Promoter for the EfficientConstruction of 1,2-trans Linkages in Glycoside Synthesis, UsingThioglycosides as Glycosyl Donors. Carbohydr. Res. 1986, 149, c9-c12. (b) Kanie, O.; Kiso, M.; Hasegawa, A. Studies on the Thiogly-cosides of N-Acetylneuraminic Acid. Part 5. Glycosylation UsingMethyl Thioglycosides of N-Acetylneuraminic Acid and Dimethyl-(methylthio)sulfonium Triflate. J. Carbohydr. Chem. 1988, 7, 501–506.

(28) Mesch, M.; Shelke, S.; Spreafico, M.; Strasser, D.; Cutting, B.;Schwardt, O.;Gathje, H.; Kelm, S.; Ernst, B., unpublished results.

(29) Boullanger, P.; Maunier, V.; Lafont, D. Syntheses of AmphiphilicGlycosylamides from Glycosyl Azides without Transient Reductionto Glycosylamines. Carbohydr. Res. 2000, 324, 97–106.

(30) Shin, S.-Y.; Gathje, H.; Schwardt, O.; Gao, G.-P.; Ernst, B.; Kelm,S.; Meyer, B. Binding Epitopes of Gangliosides to their NeuronalReceptor, Myelin-Associated Glycoprotein, from Saturation TransferDifference NMR. ChemBioChem 2008, 9, 2946–2949.

(31) David, S.; Hanessian, S. Regioselective Manipulation of HydroxylGroups via Organotin Derivatives. Tetrahedron 1985, 41, 643–663.

(32) Paulsen, H.; Himpkamp, P.; Peters, T. Building Units of Oligosac-charides LXIX. Synthesis of 1,6-Anhydromuramyl Peptides. LiebigsAnn. Chem. 1986, 664–674.

(33) (a) Blatter, G.; Beau, J.-M.; Jacquinet, J.-C. The Use of 2-Deoxy-2-trichloroacetamido-D-glucopyranose Derivatives in Syntheses of Oli-gosaccharides. Carbohydr. Res. 1994, 260, 189–202. (b) Belot, F.;Jacquinet, J.-C. Intermolecular Aglycon Transfer of a Phenyl 1-Thioga-lactosaminide Derivative Under Trichloroacetimidate GlycosylationConditions. Carbohydr. Res. 1996, 290, 79–86.

(34) Compound 34 was prepared in analogy to Kameyama, A.; Ishida, H.;Kiso, M.; Hasegawa, A. Synthetic Studies on Sialoglycoconjugates12. Total Synthesis of Sialyl Neolactotetraosyl Ceramide. J. Carbo-hydr. Chem. 1989, 8, 799–804.

(35) Sherman, A. A.; Mironov, Y. V.; Yudina, O. N.; Nifantiev, N. E. ThePresence of Water Improves Reductive Openings of BenzylideneAcetals with Trimethylaminoborane and Aluminium Chloride. Car-bohydr. Res. 2003, 338, 697–703.

(36) Crocker, P. R.; Kelm, S. Methods for Studying the Cellular BindingProperties of Lectin-like Receptors. In Weir’s Handbook of Experi-mental Immunology, 5th ed.; Herzenberg, L. A., Weir, D. M., Eds.;Blackwell: Cambridge, MA, 1996; Vol. 4, pp 166.1-166.11.

(37) (a) Rich, R. L.; Myszka, D. G. Advances in Surface PlasmonResonance Biosensor Analysis. Curr. Opin. Biotechnol. 2000, 11, 54–61. (b) Morton, T. A.; Myszka, D. G. Kinetic Analysis of Macromo-lecular Interactions Using Surface Plasmon Resonance Biosensors.Methods Enzymol. 1998, 295, 268–294. (c) Nagata, K.; Handa, H.Real-Time Analysis of Biomolecular Interactions: Applications ofBiacore; Springer-Verlag: Berlin, 2000.

(38) Shi, J.; Blundell, T. L.; Mizuguchi, K. FUGUE: Sequence-structurehomology recognition using environment-specific substitution tablesand structure-dependent gap penalties. J. Mol. Biol. 2001, 310, 243–257.

(39) Deane, C. M.; Kaas, Q.; Blundell, T. L. SCORE: predicting the coreof protein models. Bioinformatics 2001, 17, 541–550.

(40) Deane, C. M.; Kaas, Q.; Blundell, T. L. CODA: a combined algorithmfor predicting the structurally variable region of protein models. ProteinSci. 2001, 10, 599–612.

(41) Tripos Inc., St. Louis, MO, 2007.(42) Case, D. A.; Pearlman, D. A.; Caldwell, J. W. et al. AMBER 7;

University of California: San Francisco, 2002.(43) Mohamadi, F.; Richards, N. G.; Guida, W.; Liskamp, R.; Lipton, M.;

Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. MacroModelsanintegrated software system for modeling organic and bioorganicmolecules using molecular mechanics. J. Comput. Chem. 1990, 11,440–467.

(44) Weiner, S. J.; Kollmann, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.;Alagona, G., Jr.; Weiner, P. A new force field for molecular-mechanical simulation of nucleic acids and proteins. J. Am. Chem.Soc. 1984, 106, 765–784.

(45) Stewart, J. J. P. MOPACsA semi-empirical molecular orbital program.J. Comput.-Aided Mol. Des. 1990, 4, 1–105.

(46) May, A. P.; Robinson, R. C.; Vinson, M.; Crocker, P. R.; Jones, E. Y.Crystal Structure of the N-terminal Domain of Sialoadhesin in


Complex with 3′-Sialyllactose at 1.85 Å Resolution. Mol. Cell 1998,1, 719–728.

(47) Humphrey, W.; Dalke, A.; Schulten, K. VMDsVisual MolecularDynamics. J. Mol. Graphics 1996, 14, 33–38.

(48) (a) Kolb, H. C.; Ernst, B. Development of tools for the design ofselectin antagonists. Chem.sEur. J. 1997, 3, 1571–1578. (b) Jahnke,W.; Kolb, H. C.; Blommers, M. J. J.; Magnani, J. L.; Ernst, B.Comparison of the Bioactive Conformations of Sialyl LewisX and aPotent Sialyl LewisX Mimic. Angew. Chem., Int. Ed. Engl. 1997, 36,2603–2607. (c) Bamford, M. J.; Bird, M.; Gore, P. M.; Holmes, D. S.;Priest, R.; Prodger, J. C.; Saez, V. Synthesis and Biological Activityof Conformationally Constrained Sialyl Lewis X Analogs withReduced Carbohydrate Character. Bioorg. Med. Chem. Lett. 1996, 6,239–244. (d) Topfer, A.; Kretzschmar, G.; Schuth, S.; Sonnentag, M.Malonic Acid Derivatives as Sialyl Lewis X Mimetics. Bioorg. Med.Chem. Lett. 1997, 7, 1317–1322.

(49) (a) Bernardi, A.; Potenza, D.; Capelli, A. M.; Garcıa-Herrero, A.;Canada, F. J.; Jimenez-Barbero, J. Second-Generation Mimics ofGanglioside GM1 Oligosaccharide: A Three-Dimensional View ofTheir Interactions with Bacterial Enterotoxins by NMR and Compu-tational Methods. Chem.sEur. J. 2002, 8, 4597–4612. (b) Bernardi,

A.; Arosio, D.; Potenza, D.; Sanchez-Medina, I.; Mari, S.; Canada,F. J.; Jimenez-Barbero, J. Intramolecular Carbohydrate-AromaticInteractions and Intermolecular van der Waals Interactions Enhancethe Molecular Recognition Ability of GM1 Glycomimetics for CholeraToxin. Chem.sEur. J. 2004, 10, 4395–4406.

(50) (a) Markgren, P.-O.; Schaal, W.; Hamalainen, M.; Karlen, A.; Hallberg,A.; Samuelsson, B.; Danielson, U. H. Relationships between Structureand Interaction Kinetics for HIV-1 Protease Inhibitors. J. Med. Chem.2002, 45, 5430–5439. (b) Shuman, C. F.; Hamalainen, M. D.;Danielson, U. H. Kinetic and Thermodynamic Characterization ofHIV-1 Protease Inhibitors. J. Mol. Recognit. 2004, 17, 106–119.

(51) (a) Bieberich, E.; Liour, S. S.; Yu, R. K. Mammalian gangliosidesialidases: preparation and activity assays. Methods Enzymol. 2000,312B, 339–358. (b) Kopitz, J.; Sinz, K.; Brossmer, R.; Cantz, M. PartialCharacterization and Enrichment of a Membrane-Bound SialidaseSpecific for Gangliosides from Human Brain Tissue. Eur. J. Biochem.1997, 248, 527–534.

JM801058N


Examination of the Biological Role of the α(2→6)-Linked Sialic Acid in Gangliosides Binding to...

Documents

Transcript of Examination of the Biological Role of the α(2→6)-Linked Sialic Acid in Gangliosides Binding to...