Synthesis of bivalent lactosides based on terephthalamide, N,N′-diglucosylterephthalamide, and glycophane scaffolds and assessment of their inhibitory capacity on medically relevant lectins

pubs.acs.org/joc

Synthesis of Bivalent Lactosides Based on Terephthalamide,

N,N⁰-Diglucosylterephthalamide, and Glycophane Scaffolds and

Assessment of Their Inhibitory Capacity on Medically Relevant Lectins

†

Rosaria Leyden, Trinidad Velasco-Torrijos, Sabine Andre, Sebastien Gouin,

†

ꢀ ^‡

Hans-Joachim Gabius,^‡and Paul V. Murphy*^,†,§

^†School of Chemistry and Chemical Biology, Centre for Synthesis and Chemical Biology, University College

Dublin, Belfield, Dublin 4, Ireland, ^‡Institute of Physiological Chemistry, Faculty of Veterinary Medicine,

€

Ludwig-Maximilians-University Munich, Veterinarstrasse 13, 80539 Munich, Germany, and

^§School of Chemistry, National University of Ireland, Galway

paul.v.murphy@nuigalway.ie

Received August 5, 2009

Glycan recognition by lectins initiates clinically relevant processes such as toxin binding or tumor

spread. Thus, the development of potent inhibitors has a medical perspective. Toward this goal, we

report thesynthesisofbothrigid and flexible bivalent lactosideson scaffoldsthatinclude secondaryand

tertiary terephthalamides and N,N⁰-diglucosylterephthalamides. Construction of these compounds

involved Schmidt-Michel glycosidation, and amide coupling or Ugi reactions of relevant glycosyl

amines in key steps. A glycocluster based on a rigid glycophane was also prepared from coupling of a

glucuronic acid derivative and p-xylylenediamine with subsequent ring-closing metathesis. Finally, a

more flexible bivalent lactoside was produced from lactosyl azide with use of the copper-catalyzed

azide-alkyne cycloaddition. Distances between lactose residues were analyzed computationally as

were their orientations for the relatively rigid subset of compounds. Distinct spacing properties were

revealed by varying the structure of the scaffold or by varying the location of the lactose residue on the

scaffold. To relate these features to bioactivity a plant toxin and human adhesion/growth-regulatory

galectins were used as sensors in three types of assay, i.e. measuring agglutination of erythrocytes,

bindingtoglycans of a surface-immobilizedglycoprotein, orbinding to humancells. Methodologically,

the common hemeagglutination assay was found to be considerably less sensitive than both solid-phase

and cell assays. The bivalent compounds were less effective at interfering with glycoprotein binding to

the plant toxin than to human lectins. Significantly, a constrained compound was identified that

displayed selectivity in its inhibitory potency between galectin-3 and its proteolytically processed form.

Conversely, compounds with a high degree of flexibility showed notable ability to protect human cells

from plant toxin binding. The applied conjugation chemistry thus is compatible with the long-term aim

to produce potent and selective lectin inhibitors.

* To whom correspondence should be addressed. Fax: þ353-91-525700

9010 J. Org. Chem. 2009, 74, 9010–9026

Published on Web 11/09/2009

DOI: 10.1021/jo901667r

r

2009 American Chemical Society

Leyden et al.

JOCArticle

Introduction

efficiently interfere with binding of certain effectors to cell

surface glycans.

Carbohydrates have ideal properties to generate an un-

rivalled number of oligomers. They are capable of engaging

in molecular recognition events in biological systems, under-

lying the concept of the sugar code.¹Indeed, glycan chains

presented on cell surface glycoconjugates are versatile bio-

chemical signals by serving as direct contact sites for recep-

tors (lectins) that bind to carbohydrates (trans-interactions)

and as potent molecular switches regulating aspects of

protein accessibility and functionality (cis-interactions).^1,2

Besides the structure of the headgroup of a glycan chain its

presentation and local density govern the affinity in inter-

molecular interactions, as recently revealed in the case of

physiological core substitutions in N-glycans which trigger

significant alterations in glycan conformations and, impor-

tantly, in lectin affinity.³Distinct changes in glycan display

including microdomain formation in natural systems can

thus lead to induction of cellular responses such as anoikis/

apoptosis or autoimmune suppression via lectin binding.⁴To

underscore the emerging clinical relevance a tumor suppres-

sor and responses in inflammation can apparently work

on this level by remodelling cell surface glycan profiles and

even lectin expression to result in host defense/antima-

lignancy effects.^4c,dClinically disadvantageous, lectin bind-

ing to glycans is also involved in disease progression.

Herewith, it gives an incentive for drug design. Considering

the multiple, medically relevant activities of cell surface

glycans in concert with lectins in regulating, for example,

adhesion, migration, proliferation, or tissue invasion or

serving as docking sites for plant/bacterial toxins,¹it clearly

is an attractive goal to develop new molecules which can

Of current interest to us toward this goal is a series of

bivalent glycoclusters of varying degree of scaffold flexibil-

ity, altering ligand spacing with a view to profiling the

structure-bioactivity relationships. As a first step, we re-

cently described the synthesis and biological evaluation of a

series of bivalent mannosides based on terephthalamide and

glycocyclophane scaffolds, the latter being derived from

phenylene-1,4-diamines.⁵Initial testing for bioactivity with

leguminous lectins in cell-based assays showed that presen-

tation of the mannose residues on a rigid macrocyclic scaf-

fold can lead to enhanced inhibitory capacity.⁵Evidently, the

mannose moieties maintained ligand-binding capacity for

the tested plant lectins. Thus, this proof-of-principle result

encourages further efforts with a headgroup different from

that of mannose and medically relevant lectins.

Due to its positioning at branch ends of glycan chains and

frequent involvement in lectin binding β-galactose satisfies the

requirement for biological relevance.^1,2Testing of bivalent

scaffolds presenting β-galactosides (e.g., lactosides) in assays

with a biohazardous toxin and with human lectins was there-

fore envisaged to provide a medical perspective. Consequently,

we were interested to establish synthetic routes that would

allow bivalent lactosides based on macrocyclic and terephtha-

lamide scaffolds to be generated. As follows, the syntheses of

ligands (Chart 1) with β-lactose conjugated to terephthala-

mide scaffolds⁶(1-2), N⁰,N⁰-diglucosylterephthalamides

(3-5), and a rigid macrocyclic glycocyclophane 8 are

presented. Preparations of 6 and 7 with increased flexibility

compared to the conformationally arrested compound 8 are

included as was the generation of the triazole derivative 9.

Geometrical aspects of presentation of the β-lactose units in

the more rigid compounds were then comparatively inferred

by computational methods.

Bioactivity of the sugar headgroup and the impact of struc-

tural properties were next examined in the commonly used

hemeagglutination test and also in a solid-phase binding assay.

The latter assay was based on evaluating the ability of synthetic

compounds to inhibit binding of lectins to a natural glycopro-

tein (asialofetuin (ASF) with three N-glycosylation sites prefer-

entially occupied by complex-type triantennary N-glycans).

Since any clinical application would be based on impairing

lectin binding to cells, we also determined the extent of lectin

binding to native human cells as a function of the presence of the

new compounds. Lectin selection for the assays was based on

the aim to protect cells from toxicity, in addition to evaluating

the potential of the test panel to interfere with progression of

disease in malignancy due to activity of distinct adhesion/

growth-regulatory galectins.⁷In detail, the binding of Viscum

album agglutinin (VAA) and human galectins-3 and -4 to the

surface-presented glycoprotein was monitored in the absence/

presence of a test compound, using free lactose as an internal

standard of inhibitory potency. Galectin-3 has two special

features in this respect: it can pentamerize in the presence of a

(1) Gabius, H.-J., Ed. The Sugar Code. Fundamentals of glycosciences;

Wiley-VCH: Weinheim, Germany, 2009.

(2) (a) Sharon, N.; Lis, H. In Glycosciences: Status and Perspectives; Gabius,

H.-J., Gabius, S., Eds.; Chapman & Hall: London, UK, 1997; pp 133-162. (b)

ꢀ

von der Lieth, C.-W.; Siebert, H.-C.; Koꢁzar, T.; Burchert, M.; Frank, M.; Gilleron,

M.; Kaltner, H.; Kayser, G.; Tajkhorshid, E.; Bovin, N. V.; Vliegenthart, J. F. G.;

Gabius, H.-J. Acta Anat. (Basel) 1998, 161, 91–109. (c) Gabius, H.-J.; Siebert,

ꢀ

H.-C.; Andre, S.; Jimenez-Barbero, J.; R€udiger, H. ChemBioChem 2004, 5, 740–

764. (d) Gabius, H.-J. Crit. Rev. Immunol. 2006, 26, 43–79. (e) Gabius, H.-J.

ꢀ

Biochem. Soc. Trans. 2008, 36, 1491–1496. (f) Andre, S.; Koꢁzar, T.; Kojima, S.;

Unverzagt, C.; Gabius, H.-J. Biol. Chem. 2009, 390, 557–566. (g) Honke K.;

Taniguchi, N. In The Sugar Code. Fundamentals of glycosciences; Gabius, H.-J.,

Ed.; Wiley-VCH: Weinheim, Germany, 2009; pp 385-401. (h) Ledeen, R. W.;

Wu, G. In The Sugar Code. Fundamentals of glycosciences; Gabius, H.-J., Ed.;

Wiley-VCH: Weinheim, Germany, 2009; pp 495-516. (i) Zuber, C.; Roth, J. In

The Sugar Code. Fundamentals of glycosciences; Gabius, H.-J., Ed.; Wiley-

VCH: Weinheim, Germany, 2009; pp 87-110.

ꢀ

(3) (a) Andre, S.; Unverzagt, C.; Kojima, S.; Dong, X.; Fink, C.; Kayser,

K.; Gabius, H.-J. Bioconjugate Chem. 1997, 8, 845–855. (b) Unverzagt, C.;

ꢀ

Andre, S.; Seifert, J.; Kojima, S.; Fink, C.; Srikrishna, G.; Freeze, H. H.;

Kayser, K.; Gabius, H.-J. J. Med. Chem. 2002, 45, 478–491. (c) Andre, S.;

ꢀ

Unverzagt, C.; Kojima, S.; Frank, M.; Seifert, J.; Fink, C.; Kayser, K.; von

der Lieth, C.-W.; Gabius, H.-J. Eur. J. Biochem. 2004, 217, 118–134. (d)

ꢀ

Andre, S.; Kozar, T.; Schuberth, R.; Unverzagt, C.; Kojima, S.; Gabius, H.-

J. Biochemistry 2007, 46, 6984–6995.

ꢁꢀ

ꢀ

(4) (a) Kopitz, J.; von Reitzenstein, C.; Andre, S.; Kaltner, H.; Uhl, J.;

Ehemann, V.; Cantz, M.; Gabius, H.-J. J. Biol. Chem. 2001, 276, 35917–

35923. (b) Rappl, G.; Abken, H.; Muche, J. M.; Sterry, W.; Tilgen, W.;

ꢀ

Andre, S.; Kaltner, H.; Ugurel, S.; Gabius, H.-J.; Reinhold, U. Leukemia

2002, 16, 840–845. (c) Andre, S.; Sanchez-Ruderisch, H.; Nakagawa, H.;

ꢀ

€

Buchholz, M.; Kopitz, J.; Forberich, P.; Kemmner, W.; Bock, C.; Deguchi,

K.; Detjen, K. M.; Wiedenmann, B.; von Knebel Doeberitz, M.; Gress, T.

M.; Nishimura, S.-I.; Rosewicz, S.; Gabius, H.-J. FEBS J. 2007, 274, 3233–

3256. (d) Marathe, D. D.; Chandrasekaran, E. V.; Lau, J. T. Y.; Matta, K. L.;

ꢀ

(5) Andre, S.; Velasco-Torrijos, T.; Leyden, R.; Gouin, S.; Tosin, M.;

Murphy, P. V.; Gabius, H.-J. Org. Biomol. Chem. 2009, 7, 4715–4725.

ꢀ

Neelamegham, S. FASEB J. 2008, 22, 4154–4167. (e) Patsos, G.; Andre, S.;

(6) (a) Avalos, M.; Babiano, R.; Carretero, M. J.; Cintas, P.; Higes, F. J.;

ꢀ

V.; Bradley, H.; Tosin, M.; Pitt, N.; Fitzpatrick, G. M.; Glass, W. K. J. Org.

Chem. 2003, 68, 5692–5704. (c) Tosin, M.; Gouin, S. G.; Murphy, P. V. Org.

Lett. 2005, 7, 211–214. (d) Bradley, H.; Fitzpatrick, G.; Glass, W. K.; Kunz,

H.; Murphy, P. V. Org. Lett. 2001, 3, 2629.

Roeckel, N.; Gromes, R.; Gebert, J.; Kopitz, J.; Gabius, H.-J. Glycobiology

2009, 19, 726–734. (f) Schwartz-Albiez, R. In The Sugar Code. Fundamentals of

glycosciences; Gabius, H.-J., Ed.; Wiley-VCH: Weinheim, Germany, 2009; pp

447-467. (g) Wang, J.; Lu, Z.-H.; Gabius, H.-J.; Rohowsky-Kochan, C.; Ledeen,

R. W.; Wu, G. J. Immunol. 2009, 182, 4036–4045.

Jimenez, J. L.; Palacios, J. C. Tetrahedron 1998, 54, 615–628. (b) Murphy, P.

J. Org. Chem. Vol. 74, No. 23, 2009 9011

Leyden et al.

JOCArticle

CHART 1. β-Lactose-Based Glycoclusters

multivalent ligand and it is physiologically truncated by matrix

metalloproteinases-2/-9 to yield a trimmed (truncated) version

with diminished propensity for oligomer formation.⁸Galectin-3

and its truncated derivative were thus tested to reveal any

difference in sensitivity to the glycocompounds and a selective

inhibitor was identified. The obtained results demonstrate the

bioactivity of this panel of glycoclusters as well as reveal a

notable influence of assay system, compound, lectin, and matrix

properties on inhibitory potency.

(7) (a) Gabius, H.-J. Biochimie 2001, 83, 659–666. (b) Gabius, H.-J.;

ꢀ

Darro, F.; Remmelink, M.; Andre, S.; Kopitz, J.; Danguy, A.; Gabius, S.;

Salmon, I.; Kiss, R. Cancer Invest. 2001, 19, 114–126. (c) Nagy, N.; Legendre,

Results and Discussion

ꢀ

H.; Engels, O.; Andre, S.; Kaltner, H.; Wasano, K.; Zick, Y.; Pector, J.-C.;

Decaestecker, C.; Gabius, H.-J.; Salmon, I.; Kiss, R. Cancer 2003, 97, 1849–

Synthesis of Glycoclusters. Synthetic routes were first es-

tablished to produce 1-9. It was envisaged that 1 and 2 could

be prepared from lactosyl amine 10. Fittingly, coupling of 10

with terephthaloyl chloride in the presence of triethyla-

ꢀ

1858. (d) Lahm, H.; Andre, S.; Hoeflich, A.; Kaltner, H.; Siebert, H.-C.;

Sordat, B.; von der Lieth, C.-W.; Wolf, E.; Gabius, H.-J. Glycoconj. J. 2004,

20, 227–238. (e) Delacour, D.; Gouyer, V.; Zanetta, J.-P.; Drobecq, H.;

Leteurtre, E.; Grard, G.; Moreau-Hannedouche, O.; Maess, E.; Pons, A.;

ꢀ

Andre, S.; Le Bivic, A.; Gabius, H.-J.; Manninen, A.; Simons, K.; Huet, G. J.

Cell Biol. 2005, 169, 491–501. (f) Jimenez, M.; Saiz, J. L.; Andre, S.; Gabius,

ꢀ

mine and subsequent Zemplen deacetylation gave 1. Since

ꢀ

the glycosyl amine 10 also contained a small amount of the

R-anomer, a mixture of diamides was obtained from the

coupling reaction. The bivalent compounds 11 and a lesser

amount of 12 containing R-glycosyl amides were thus also

obtained after HPLC-based separation of the deacetylated

diamide mixture (Scheme 1). Next, the Ugi reaction of 10 in

the presence of formaldehyde, methyl isocyanoacetate, and

ꢀ

H.-J.; Solı

´

Siebert, H.-C.; Gabius, H.-J.; Solı

s, D. Glycobiology 2005, 15, 1386–1395. (g) Jimenez, M.; Andre, S.;

s, D. Glycobiology 2006, 16, 926–937. (h)

´

ꢀ

Villalobo, A.; Nogales-Gonzales, A.; Gabius, H.-J. Trends Glysci. Glyco-

technol. 2006, 18, 1–37. (i) Langbein, S.; Brade, J.; Badawi, J. K.; Hatzinger,

ꢀ

M.; Kaltner, H.; Lensch, M.; Specht, K.; Andre, S.; Brinck, U.; Alken, P.;

Gabius, H.-J. Histopathology 2007, 51, 681–690. (j) Moisa, A.; Fritz, P.; Eck,

€

A.; Wehner, H.-D.; Murdter, T.; Simon, W.; Gabius, H.-J. Anticancer Res.

2007, 27, 2131–2140. (k) Andre, S.; Sansone, F.; Kaltner, H.; Casnati, A.;

Kopitz, J.; Gabius, H.-J.; Ungaro, R. ChemBioChem 2008, 9, 1649–1661. (l)

Saussez, S.; Decaestecker, C.; Mahillon, V.; Cludts, S.; Cappouillez, A.;

ꢀ

Chevalier, D.; Kaltner, H.; Andre, S.; Toubeau, G.; Leroy, X.; Gabius, H.-J.

Laryngoscope 2008, 118, 1583–1590. (m) Cada, Z.; Smetana, K., Jr.; Lacina,

ꢀ

L.; Plzakova, Z.; Stork, J.; Kaltner, H.; Russwurm, R.; Lensch, M.; Andre,

S.; Gabius, H.-J. Folia Biol (Praha) 2009, 55, 145–52. (n) Stechly, L.; Morelle,

W.; Dessein, A.-F.; Andre, S.; Grard, G.; Trinel, D.; Dejonghe, H.-J.;

Leteurtre, E.; Drobecq, H.; Trugnan, G.; Gabius, H.-J.; Huet, G. Traffic

2009, 10, 438–450.

(8) (a) Herrmann, J.; Turck, C. W.; Atchison, R. E.; Huflejt, M. E.;

Poulter, L.; Gitt, M. A.; Burlingame, A. L.; Barondes, S. H.; Leffler, H. J.

ꢀ

Biol. Chem. 1993, 268, 26704–26711. (b) Ahmad, N.; Gabius, H.-J.; Andre,

S.; Kaltner, H.; Sabesan, S.; Roy, R.; Liu, B.; Macaluso, F.; Brewer, C. F. J.

ꢀ

Biol. Chem. 2004, 279, 10841–10847. (c) Dam, T. K.; Gabius, H.-J.; Andre,

S.; Kaltner, H.; Lensch, M.; Brewer, C. F. Biochemistry 2005, 44, 12564–

12571. (d) Kubler, D.; Hung, D.-W.; Dam, T. K.; Kopitz, J.; Andre, S.;

Kaltner, H.; Lohr, M.; Manning, J. C.; He, L.; Wang, H.; Middelberg, A.;

Brewer, C. F.; Reed, J.; Lehmann, W.-D.; Gabius, H.-J. Biochim. Biophys.

Acta 2008, 1780, 716–722.

ꢀ

terephthalic acid and subsequent Zemplen deacetylation re-

ꢁ

sulted in the bivalent compound 2. It differs from 1 in that

tertiary amides are present as opposed to secondary amides.

In the next step, approaches to bivalent lactosides based

on N,N⁰-diglucopyranosylterephthalamide scaffolds were

studied. The Schmidt-Michel glycoside coupling reaction

of trichloroacetimidate 14 with 13 with TMSOTf as catalyst

and subsequent reaction of the trisaccharide product with

SnCl₄and TMSN₃generated azide 15. Catalytic hydrogena-

tion led to the glycosyl amine and subsequent coupling with

terephthaloyl chloride followed by deacetylation as before

gave 3 where the lactosyl units are attached to both 6-O

atoms of the scaffold (Scheme 2).

ꢁ

ꢀ

€

ꢀ

9012 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

SCHEME 1. Synthesis of 1, 2, and 11

SCHEME 2. Synthesis of 3

The synthesis of 4 was achieved via the glucopyrano-

side 16. Glycosidation of the acetylated lactosyl trichlo-

roacetimidate donor 14 opened the way to the desired

trisaccharide 17. Acetolysis gave the glycosyl acetate deriva-

tive and its subsequent reaction with azidotrimethylsilane

in the presence of SnCl₄the desired β-azide 18. Reaction

of 18 as described for 15 produced bivalent compound 4

(Scheme 3). The lactose units are covalently linked to the

two 4-O atoms of the N,N⁰-diglucosylterephthalamide

scaffold.

To prepare other analogues which would be available by

using the 3-O atoms of the scaffold as an attachment site, a

respective strategy was developed. Toward this aim, glyco-

sidation reactions of the glycosyl acceptor 20 with donor 14

were not successful, which contrasted with what we observed

for the other acceptors (Schemes 2 and 3). An orthoester

byproduct, often detected in glycosidation reactions, was

obtained instead of the desired glycosidic product. To ad-

dress this problem the benzoylated lactosyl trichloroaceti-

midate 19 was prepared and its reaction with 20 investigated,

leading to the successful formation of the desired β-glycoside

21 in good yield. Hydrolysis of the acetonide groups, sub-

sequent acetylation followed by reaction of the product with

SnCl₄and TMSN₃, and then catalytic hydrogenation gave

the amine 22. Treatment of amine 22 with terephthaloyl

chloride as before and ensuing removal of the acyl protecting

groups resulted in 5 (Scheme 4).

With this series of bivalent lactosides in hand we next

added compounds to the panel based on scaffolds that were

amides of glucuronic acid. Consequently, the acceptor 23,

which we had used previously to prepare mannosides, on

reaction with acetylated lactose donor 14 gave the undesired

orthoester 24. Application of the benzoylated donor 19 in the

glycosidation yielded the suitable trisaccharide intermediate,

which was then converted to 25 after selective removal of the

allyl ester with Pd(0) catalysis (Scheme 5).⁹In our previous

work we had synthesized the bivalent mannosides via cou-

pling of a glucuronic acid derivative with phenylene-1,4-

diamine. The products of coupling of phenylene-1,4-diamine

to the acid 25 were analyzed indicating that only one

(9) Kunz, H.; Waldmann, H. Angew. Chem., Int. Ed. 1984, 23, 71–72.

J. Org. Chem. Vol. 74, No. 23, 2009 9013

Leyden et al.

JOCArticle

SCHEME 3. Synthesis of 4

SCHEME 4. Synthesis of 5

trisaccharide residue had been added to one amine group

(Scheme 5). It is presumed that formation of the first amide

group leads to a reduction in nucleophilicity of the second

amine group of the phenylene-1,4-diamine residue preclud-

ing formation of a second amide. We thus investigated the

preparation of diamides from 1,4-di(aminomethyl)benzene

(p-xylylenediamine), where electron-withdrawing effects

were expected to have been lessened. Conversion of 25

to its acid chloride with a view to using this for the

amide synthesis led to the formation of the glycosyl chlo-

ride 26, tentatively explained by the mechanistic proposal

outlined in Scheme 6. The activation of O-glycosidic linkages

by manipulation of remote carboxylic acid groups has

already been delineated and favorably utilized in glyco-

sidation reactions.¹⁰Herein, a glycosyl carbenium ion is

proposed to be generated via the acid chloride, the for-

mer which is subsequently trapped by chloride ion

(Scheme 6). Eventually, the desired coupling reaction en

route to the diamide was achieved by activation of the acid

25 with HOBt, DIPEA, and HATU in the presence of the

p-xylylenediamine. Removal of the acetate and benzoate

protecting groups gave the flexible divalent compound 6

(Scheme 5).

Having established conditions for the formation of 6, we

next worked toward the synthesis of bivalent lactosides

based on a glycocyclophane scaffold derived from the p-

xylylenediamine. The acceptor 28 was therefore prepared

from 27 as described previously⁵and its reaction with 19 and

subsequent chemoselective deallylation with Pd(0) gave 29.

Reaction of 29 with p-xylylenediamine in the presence of

HOBt, HATU, and DIPEA generated the protected divalent

compound 30 (Scheme 7), a suitable precursor for ring

closure metathesis (RCM).

(10) Kim, K. S.; Kim, J. H.; Yong, Lee, J.; Lee, Y. J.; Park, J. J. Am.

Chem. Soc. 2001, 123, 8477–8481.

9014 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

SCHEME 5. Synthesis of 6

SCHEME 6. Proposed Route of Formation of 26

SCHEME 7. Synthesis of 30

The subsequent RCM of 30 with the Grubbs-I catalyst

gave a mixture of macrocyclic isomers containing E- and

Z-alkene groups, and unreacted 30 was also recovered.

Hydrogenation of the mixture produced 31 and 32. These

compounds were readily separated by chromatography. The

removal of the protecting groups from 31 and 32 was the last

step of the route providing the acyclic derivative 7 and the

rigid macrocyclic derivative 8 (Scheme 8).

Finally, the bivalent lactoside 9 was prepared from 34 and

the lactosyl azide 33 with the copper-catalyzed azide-alkyne

cycloaddition reaction.¹¹To facilitate purification of 9,

the product from the click reaction was acetylated, puri-

fied by chromatography, and subsequently deacetylated

(Scheme 9). Having herewith completed the synthetic part,

we next proceeded to calculate geometrical aspects of ligand

positioning in the bivalent systems.

Analysis of Ligand Spacing. The use of rigid scaffolds

presenting ligands can avoid entropic penalties that occur

upon binding to a receptor, particularly if they are preorga-

nized into a constellation favorable for binding. If the two

sugar headgroups react with different lectin molecules,

then orientation and spacing may also have a bearing on

bioactivity. The aim to relate structure to function ex-

plains our interest to calculate interligand spatial relation-

ships for the bivalent lactosides. At the outset, the panel

of compounds was separated into two categories based on

their relative flexibility. Two parameters were chosen to define

the interligand spacing: the distances between the two Glc C-1

atoms of the lactose residues was defined as the interlactose

distance whereas the torsion Gal C4-Glc C1-Glc C1-Gal

C4 corresponding to the lactose residues was defined as the

lactose orientation (see Figure 2D). Since the β-lactosides 1-5

and 8 were judged to have fewer degrees of freedom when

compared to 6, 7, and 9, conformational analysis was focused

on the former structures. When calculating the spatial rela-

tionships between the two lactose residues in compounds

1-5 and 8, we used Macromodel for model building and

all subsequent computations.¹²Experimentally observed

(11) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.

2001, 40, 2004–2021. (b) Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108,

2952–3015. (c) Leyden, R.; Murphy, P. V. Synlett 2009, 1949–1950.

(12) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;

Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput.

Chem. 1990, 11, 440–467.

J. Org. Chem. Vol. 74, No. 23, 2009 9015

Leyden et al.

JOCArticle

SCHEME 8. Synthesis of 7 and 8

SCHEME 9. Synthesis of 9

glycosidic torsion angles (φ = O5-C1-O1-Cx; ψ =

C1-O1-Cx-C(xþ1); ω = O5-C5-C6-O1) were used to

guide the building¹³of model structures establishing the

structure from which conformational searching commenced.

In detail, the glycosidic torsions from a published crystal

structure of a β-lactoside¹⁴(φ=-88; ψ=-161) defined the

corresponding torsions in the lactose residues of the model

structures. To complete the models of 3-5 additional torsions

for the glycosidic linkage between the glucose residue of the

scaffold and the glucose residue of the lactose needed to be

defined; the φ, ψ, and ω values were again obtained from X-

ray crystal structural data. For the β-^D-Glc(1-6)-β-D-Glc

linkage in 3 the φ, ψ, and ω were -59°, -155°, and -61°,

respectively;¹⁵for the β-D-Glc(1-4)-β-D-Glc linkage in 4, the

φ and ψ used were -76° and 132°;¹⁶for the β-D-Glc(1-3)-β-D-

Glc linkage in 4, the φ and ψ used were -94° and 78°.¹⁷

(14) Qingfeng, P.; Noll, B. C.; Serianni, A. S. Acta Cryst. C 2005, 61, 674–

677.

(15) Arene, F.; Newman, A.; Longchambon, F. C. R. Acad. Sci. 1979,

288, 331–334.

(16) Chu, S. S.; Jeffrey, G. A. Acta Cryst. B 1968, 24, 830–8.

(17) Noguchi, K.; Okyama, K.; Kitamura, S.; Takeo, K. Carbohydr. Res.

1992, 237, 33–42.

Equally relevant studies on the structural orientations of

N-glycosyl benzamides had been carried out previously,

providing a basis to define the preferred torsional parameters

9016 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

FIGURE 1. The ten lowest energy structures for 1, 3, and 8 generated by conformational search with Macromodel are superimposed. For 1 and

3 structures were obtained where the terephthalamide torsion was cis or trans and these are shown separately.

FIGURE 2. Structural profiles of low-energy conformational isomers of 1-5 and 8 (A-C) and definition of parameters (D). Data for 2 were

obtained from stochastic molecular dynamics simulations for the isomer where the two carbonyl groups of the terephthalamide residue were trans.

Data for other compounds was obtained from low-energy structures within 12 kJ/mol of minimum obtained by systematic conformational searches.

for glycosyl amide residues of 1-5. Thus N-glycosyl benza-

mides that contain secondary amides adopt the Z-configura-

tion (or trans amide), whereas those that are tertiary amides

prefer the E-configuration (cis amide). The glycosyl amide

torsion defined by H1-C1-N-H was constrained at a value

of 171° (anti-conformation); this is close to the average of

J. Org. Chem. Vol. 74, No. 23, 2009 9017

Leyden et al.

JOCArticle

the angles observed (168.2° to 175.0°) in a number of X-ray

crystal structures of related secondary glycosyl amides and

supported by ¹H NMR (J_H1-NH≈ 9.0 Hz) in solution.¹⁸The

two carbonyl groups of the terephthalamide residues were

constructed with a trans coplanar arrangement (dihedral

defined by OdC;CdO 180 ( 60°) for 1 and 3-5. During

modeling of 2, the aromatic groups were not coplanar with the

carbonyl groups for reasons discussed previously.⁶All these

parameters combined facilitated the establishment of the

initial models of 1-5. A starting structure of the glycocyclo-

phane derivative 8 was also established and together with the

models of 1, 2, and 3-5 was used to start systematic con-

formational searches; for 1 and 2 stochastic molecular dy-

namics was also explored. The AMBER (assisted model

building with energy refinement) force field and the GB/SA

continuum for water in Macromodel were generally applied.

For the conformational searches all isomers within 12 kJ/mol

of the global minimum were retained and used in the subse-

quent analysis. The terephthalamides containing secondary

amides (1, 3-5) consistently gave low-energy structures where

the carbonyl groups of the terephthalamides were either cis or

trans (shown for low-energy structures of 1 and 3 in Figure 1).

This torsion, defined by OdC;CdO atoms of the terephtha-

lamide residue, is referred to as the terephthalamide torsion

subsequently. Our analysis (Figure 2A,C) for all the low-

energy structures generated showed how the terephtalamide

torsion affected interlactose distance and lactose orientation.

For 1 the interlactose distance was constrained between 10.1

and 10.4 A for all low-energy structures. Increased flexibility

was observed for 3 where interlactose distance ranged from

13.0 to 18.8 A, whereas intermediate flexibility was observed

for 4 (19.1-20.7 A) and 5 (16.3-17.2 and 18.0-18.4 A). In all

low-energy conformers of 8 (Figure 1), which was the most

rigid structure, the glucuronic acid residues inherent in the

macrocycle adopted a cis relationship (or a U-shape) with

respect to the benzene residue, as was also found in related

macrocycles derived from phenylene-1,4-diamine. The inter-

lactose distance was firmly constrained between 15.4 and

15.5 A in 8. Conformational searches on 2 led to highly folded

low-energy structures which were deemed unlikely to be

accessed by lectins and which possibly were artifacts. Thus,

a stochastic molecular dynamics simulation instead explored

the conformational space for 2 and commenced from a

structure where the carbonyl groups of the terephthalamide

residue were trans (OdC;CdO torsion ∼90°). In the molec-

ular dynamics runs a temperature of 300 K, time step of 1.5 fs,

equilibration time of 1.0 ps, and simulation time of 2 ns were

set and 500 structures were sampled over the duration of the

simulation. The result derived from the sampled structures is

also included in Figure 2. The interlactose distance was found

to be constrained between 7.2 and 8.1 A for 2. Similar

stochastic molecular dynamics simulations were also per-

formed for 1 (not shown), coming up with reasonable correla-

tions between the interlactose distances and lactose

orientations of structures generated in the dynamics simula-

tions and in conformational searching.

and lactose orientation (Figure 2B), which further demon-

strated the unique spatial profile for each molecule. This

analysis was also useful to depict the constraints imposed on

each of the molecules. For example, for 5 there are two low-

energy clusters: the first corresponds to a set of conformers

with a lactose orientation that varies from -50° to -22° and

a corresponding interlactose distance of 16.3-17.2 A; the

second cluster is a set of conformers where the lactose

orientation varies between 84° and 139° and a corresponding

interlactose distance of 18.0-18.4 A. In general, the analysis

shows that, while the subset of glycoclusters can adopt a

range of interlactose distances (∼7-21 A) as well as a very

large range of interlactose orientations from -180° to þ180°,

not all regions of space definable by both these parameters

are occupied. Running a similar conformational search for

the triazole derivative 9 indicated that it should be more

flexible than the selected rigid subset. For 9 the interlactose

distance was shown to vary from 9 to 17 A and the lactose

orientation varied between -165° and þ 165°, suggesting it

can have spacing arrangements not accessible to 1-5 and 8.

This would be the case also for 6 and 7. If bioactive, it will

thus be a key question whether flexible compounds such as 6

and 9 or less flexible substances will exhibit relatively higher

potency for interfering with lectin activity. Having prepared

the panel of glycoclusters and examined their conforma-

tional properties, the next step was therefore to ascertain the

implied bioactivity of the carbohydrate headgroups and, if

possible, to establish structure-activity profiles for inhibi-

tory lectin binding.

Inhibition Studies. At the outset, we performed hemeag-

glutination tests classical to lectin research.¹Using trypsin-

treated, glutaraldehyde-fixed rabbit erythrocytes, the activ-

ity of lactose or the glycoclusters was assessed for their ability

to inhibit lectin-mediated hemeagglutination in a total vo-

lume of 50 μL. Lactose (but not controls such as mannose)

was effective at blocking lectin activity at concentrations of 2

and 0.8 mM, respectively, when using the plant toxin (25 ng/

50 μL) and galectin-3 (2 μg/50 μL). The sugar units in

compound 9 matched the activity of free lactose but no

inhibitory activity was seen for any other test substance up

to a concentration of 25 mM (given as sugar concentration).

To infer whether the setting of this assay, where cell agglu-

tination was the measured parameter, may have a bearing on

sensitivity to pick up compound activity, we next employed

an assay that measured the ability of a glycocluster to inhibit

binding, in this case to a surface-immobilized glycoprotein.

In the solid-phase setting, the extent of the lectin-glycopro-

tein interaction and inhibition thereof are determined spec-

trophotometrically.

To set up optimal conditions for this assay the coating

density and lectin concentration were systematically tested

until the dependence of signal intensity on lectin concentra-

tion was in the linear range. The signal was shown to be

consistently dependent on the protein-carbohydrate inter-

action, as revealed by haptenic inhibition with lactose.

Under these subsaturating conditions, the sensitivity to

determine inhibitory potency of the synthetic compounds

was optimal. As seen in Figure 3, glycoclusters from the

synthetic panel blocked lectin binding to the glycans of the

glycoprotein with higher efficiency than free lactose. Ob-

viously, the conjugation chemistry did not impair the bio-

activity, and attachment to a scaffold could enhance the

In addition, plots were generated for each set of confor-

mers showing the relationship between interlactose distance

(18) Rawe, S. L.; Doyle, D.; Zaric, V.; Rozas, I.; McMahon, K.; Tosin,

M.; Mueller Bunz, H.; Murphy, E. P.; O’ Boyle, K. M.; Murphy, P. V.

Carbohydr. Res. 2006, 341, 1370–1390.

9018 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

FIGURE 3. Inhibition of binding of biotinylated human galectin-3 (A) and human galectin-4 (B) to the glycans of surface-immobilized

asialofetuin by lactose (b) and the lactose-presenting compounds 2 (O), 6 (Δ), and 8 (0) (see Table 1 for IC₅₀values). The inhibitor

concentration given is normalized to lactose for direct comparison. Assays were routinely done in triplicates for up to five independent series

with standard deviations not exceeding 17%.

TABLE 1. IC₅₀Values (mM per lactose residue) for Blocking Binding

of Lectins to Surface-Immobilized ASF

inhibitory potency of lactose. Titrations were used to deter-

mine the concentration required to reach 50% inhibition

(IC₅₀; Table 1). The inhibitory effects were most pronounced

for the proteolytically truncated form of human galectin-3

and for galectin-4, when using ASF as matrix. The minimal

interlactose distance observed for compound 2 translated

into potent inhibition in these two cases. In comparison, the

plant toxin (VAA), where the two readily accessible lectin

sites of the 2γ subdomains are separated by 87 A,^7gshowed

no notable response to cluster formation or scaffold rigidi-

fication. Even more important, the unprocessed form of

galectin-3 was much less sensitive to the presence of 2. Thus,

inhibitory capacity depended on the type of lectin and scaffold

used to present the lactose as well as lectin-site presentation (e.

g., galectin-3 vs. truncated galectin-3). It might also depend on

matrix properties. To address this issue the solid-phase assay

was carried out with a glycoprotein with only one N-glycosy-

lation site, this strictly bearing bi- instead of triantennary N-

glycans, i.e. serum amyloid P component (SAP). The effect of

using SAP as the matrix was thus to reduce the glycan

microdensity. In this case, enhancements of inhibitory activity

were measured for galectin-3, with all compounds being at

least 5-fold more active than free lactose and substances 4-9

being the most potent reaching up to a 50-fold activity

increase. In contrast, toxin binding remained less susceptible.

Considering this aspect and attaining increased physiological

relevance, we next probed binding of the plant toxin and

galectin-3 to the surface of native cells, in this case human B-

lymphoblastoid Croco II cells. Interference by the glycoclus-

ters was quantified.

All measurements were routinely performed with aliquots

of the same cell batch and passage, prolonged culture periods

with inevitable impact on cell features were hereby avoided.

The extent of carbohydrate-dependent cell binding was

experimentally determined by fluorescent staining in FACS-

can analysis, obtaining the percentage of positive cells and

signal intensity on a logarithmic scale as read-out, as pre-

sented in Figure 4. The binding of the labeled lectin to the cell

surface shifted the curve representing the staining profile of

each lectin-exposed cell population from the position of the

control (given as the shaded area in each figure), and the

presence of an effective inhibitor will move the curve back into

the direction of the control. Optimal conditions with a sub-

saturating lectin concentration and a lactose concentration in

lactose

units per

compd molecule

VAA^agalectin-3 galectin-3

galectin-4

[5 μg

[0.5 μg

[5 μg

(truncated)

[30 μg mL^-1

mL^-1

]

mL^-1

]

mL^-1

]

1

2

3

4

5

6

7

8

2

1

0.7

1.4

1.3

0.9

0.8

0.3

0.9

1.2

0.4

1.0

0.7

1.85

1.6

2.1

0.7

1.8

0.6

1.4

1.6

1.2

1.8

2.7

0.15

0.06

0.15

0.6

0.15

5

0.3

0.08

1.1

0.12

0.3

0.12

0.3

1.1

9

11

lactose

0.15

0.08

0.6

0.15

2.5

^aViscum album agglutinin/toxin.

the linear range of signal intensity were identified first by

systematic testing (Figure 4A,B). Routinely, measurements

were done in triplicates with up to three independent series. As

exemplarily illustrated in Figure 4C-F, compounds 6 and 9

were considerably more active than free lactose to block plant

toxin binding to the cells, which correlated reasonably well

with the binding data obtained from the solid-phase assay. In

the case of galectin-3, the measurements (scans not shown)

came up with a broadly similar trend as observed in the solid-

phase assay, and 8 was less potent than 6 and 9 in this setting.

In detail, five compounds superseded the activity of free

lactose. With standard deviations of less than 11%, the

difference between the flexible and rigid compounds 6 and 8

seen in solid-phase assays remained with respective values of

34.8vs. 39.8relativeto49.9forlactosein mean intensity(at0.5

mM sugar concentration), whereas compounds 2 and 3 were

less potent than lactose with intensity values of 51.8 and 61.6.

Compound 9 also resulted in marked inhibition to 35.1 in

intensity. Thus, the substances 6 and 9 are particularly

effective to reduce lectin association to this type of native

human cells. Flexibility of bivalent compounds thus appears

to be a favorable factor when aiming to inhibit the toxin and

galectin-3.

Summary and Conclusions

We have reported the synthesis of different types of

bivalent lactosides based on terephthalamides and glyco-

cyclophanes as well as other analogues. Rather rigid

J. Org. Chem. Vol. 74, No. 23, 2009 9019

Leyden et al.

JOCArticle

end point disclosed remarkable bioactivity. These experi-

ments added substantial support to the concept that both

terephthalamide and glycocyclophanes¹⁹can be considered

as suitable scaffolds for presentation of carbohydrate li-

gands.^20,21Either introducing structural changes to a scaf-

fold (e.g., compare 1, 2, and 8) or strategically grafting a

lactose residue to different locations of a common scaffold

(compare 3-5) led to diverse interlactose spacing. These

changes in geometry can apparently factor into the observed

alterations for inhibitory capacity, especially for the trun-

cated form of galectin-3 and the tandem-repeat-type galectin

(galectin-4). In addition, increasing rigidity of a scaffold by

macrocyclisation (6 and 7 < 8) also affects lectin reactivity to

bivalent scaffolds, with a trend toward activity loss in assays

with the plant toxin as compounds become more rigid.

Protection of cells from association with the plant toxin

was best achieved with the flexible compound 6, in accord

with the data from solid-phase assays. The relatively in-

creased activity for the flexible derivatives could be due to

their ability to access favorable ligand spacings not possible

for the more rigid compounds. In addition, the chemical

characteristics of the spacer and its environment may also

play a role in enhancing binding properties, as does the

choice of glycoprotein used as matrix and the cell surface,

rendering it difficult to draw unambiguous a priori con-

clusions.

The structural analysis indicates that it is unlikely that

compounds exert their inhibition by binding to multiple

carbohydrate recognition sites on one lectin as would be

the case, for example, for the glycoside cluster effect of

triantennary N-glycans homing in on the hepatic asialogly-

coprotein receptor; it seems more likely that the effects would

be due to bivalent compounds binding to more than one

lectin molecule, possibly promoting lectin clustering. At any

rate, the sugar headgroups maintained bioactivity after

conjugation and attained differential reactivity to the tested

human lectins, as seen especially for compound 2 and the

truncated form of galectin-3 and galectin-4 with a more than

10-fold and even higher increase of sugar activity relative to

free lactose. This gain in activity seen in solid-phase assays

could be due to constraint of the lactose residues into a

favorable presentation at minimal interlactose distance, a

benefit of using a scaffold based on the tertiary terephthala-

mide. The differential activity of 2 between galectin-3 and its

naturally derived truncated form illustrates the potential to

exploit physiological changes in binding-site presentation of

a lectin for selective inhibition. Synthesis of a bivalent

structure 11 incorporating an R-lactosyl amide linkage led

to notable activity against the two galectins noted above but

FIGURE 4. Illustration of binding of labeled plant toxin to cells of

the human B-lymphoblastoid line Croco II as a function of lectin

concentration (A), of the presence of lactose as haptenic inhibitor

(B), and of the compounds at a fixed concentration of 2 mM lactose

per assay (C-F). Controls in the absence of lectin and in the absence

of inhibitor are given by the shaded area and the solid black line.

Quantitative data on the percentage of positive cells and mean

fluorescence intensity are listed in each panel. Lectin concentrations

at 0.05, 0.1, 0.2, 0.5, and 1 μg/mL resulting in signals of increasing

level (A); assays at 0.4 μg/mL lectin with increasing concentrations

of lactose at 1, 2, 4, 20, and 40 mM document corresponding

decreases of level of signal (B). Using a constant concentration of

2 mM normalized to the sugar content the panel of test substances

was assayed: compounds 2 (thin gray line), 1 (dashed black line),

and 4 (black line) in panel C where data for positive and negative

controls are listed as bold numbers, compounds 3 (dashed black

line), 5 (thin gray line), and 6 (black line) in panel D, compounds 8

(dashed black line) and 9 (thin gray line) in panel E, and compounds

7 (dashed black line) and 11 (thin gray line) in panel F.

compounds were characterized computationally with respect

to geometry of presentation of the sugar headgroups. Bioac-

tivity of the glycocompounds was systematically tested in

hemeagglutination as well as solid-phase and cell assays.

Haemagglutination as a test system proved rather insensi-

tive, where only one compound was found to be active as

inhibitor of lectin-dependent erythrocyte agglutination. In

contrast, measuring the extent of lectin binding to glycopro-

teins on a solid phase or on cell surfaces as the experimental

(20) Hirschmann, R. Angew. Chem., Int. Ed. 1991, 30, 1278.

(21) The application of carbohydrates and carbohydrate-derived struc-

tures as scaffolds for presentation of recognition groups in chemical biology

and medicinal chemistry has recently been reviewed. (a) Yamazaki, N.;

ꢀ

Kojima, S.; Bovin, N. V.; Andre, S.; Gabius, S.; Gabius, H.-J. Adv. Drug

Delivery Rev. 2000, 43, 225–244. (b) Murphy, P. V.; O’ Brien, J. L.; Gorey-

Feret, L. J.; Smith, A. B., III Bioorg. Med. Chem. Lett. 2002, 12, 1763–66. (c)

Meutermans, W.; Le, G. T.; Becker, B. ChemMedChem 2006, 1, 1164–1194.

(d) Murphy, P. V.; Dunne, J. L. Curr. Org. Synth. 2006, 3, 403–437. (e) Velter,

I.; La Ferla, B.; Nicotra, F. J. Carbohydr. Chem. 2006, 25, 97–138. (f)

Murphy, P. V. Eur. J. Org. Chem. 2007, 4177–4187. (g) Murphy P. V.;

Velasco-Torrijos, T. In Glycoscience-Chemistry and Chemical Biology, 2nd

ed.;. Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer-Verlag: Berlin-

Heidelberg, Germany, 2008; p 997. (h) Chabre, Y. M.; Roy, R. In The Sugar

Code. Fundamentals of glycosciences; Gabius, H.-J.,Ed.; Wiley-VCH: Wein-

heim, Germany, 2009; pp 53-70.

(19) For applications of both cyclic peptides and cyclophanes as scaffolds

for multivalent ligand display see: (a) Hayashida, O.; Hamachi, I. J. Org.

Chem. 2004, 69, 3509–3516. (b) Wittmann, V.; Seeberger, S. Angew. Chem.,

Int. Ed. 2000, 39, 4348–4352. (c) Ohta, T.; Miura, N.; Funitani, N.;

Nakajima, F.; Niikura, K.; Sadamoto, R.; Guo, C.-T.; Suzuki, T.; Suzuki,

Y.; Yasuo, M.; Monde, K.; Nishimura, S.-I. Angew. Chem., Int. Ed. 2003, 42,

5186–5189.

9020 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

not the plant toxin. In sum, the conjugation chemistry is

compatible with the long-term aim to develop inhibitors with

selectivity to certain members among a family of medically

relevant human lectins. Inhibitory efficiency was obtained

with bivalent compounds, offering the perspective to in-

crease ligand density. Also of note for further work, fine-

specificity differences between homologous lectins could be

exploited by respective tailoring of the structure of the

headgroup, hereby reducing cross-reactivity.

(0.222 g, 98%) that was purified further by semipreparative

HPLC to give 2 as an interconverting mixture (83:38) of EE and

EZ isomers;⁶[R]_Dþ18 (c 1.22, H₂O); ¹H NMR (600 MHz, D₂O,

30 °C) data for EE isomer: δ 7.70 (s, 4H), 4.85 (d, J = 8.9 Hz,

2H), 4.42 (d, J = 7.7 Hz, 2H), 4.37 (d, J = 14.5 Hz, 4H), 4.12 (d,

4H), 3.97 (dd, J = 12.5 Hz, J = 1.5 Hz, 2H), 3.90 (d, J = 3.1 Hz,

2H), 3.83-3.72 (m, 10H), 3.73-3.62 (m, 10H), 3.57-3.47 (m,

6H); selected ¹H NMR data for the EZ isomer: δ 7.67 (d, J = 6.0

Hz, 2H), 7.61 (d, J = 5.9 Hz, 2H), 5.77 (d, J = 9.2 Hz, 2H, H-1),

4.48 (d, J = 7.7 Hz, 2H, H-1⁰); ¹³C NMR (125 MHz, D₂O) data

for EE isomer: δ 174.4, 172.2 (CO₂Me, CONH), 136.1 (C),

127.9, 103.1, 87.5, 77.8, 73.0, 75.6, 74.3, 72.7, 71.1, 70.1, 68.8

(each CH), 61.3, 60.3 (each CH₂), 53.1 (CH₃), 45.0, 41.6 (each

CH₂); IR (KBr) 3456-3305 (br s), 1743, 1655, 1444, 1383, 1225,

1078 cm^-1; LRMS (ESI) 1069.5 [M - H]^-, 1093.1 [M þ Na]^þ;

HRMS (ESI) found 1069.3503 [M - H]^-, C₄₂H₆₁N₄O₂₈requires

1069.3472.

2,3,4,6-Tetra-O-acetyl-β-^D-galactopyranosyl-(1f4)-2,3,6-tri-

O-acetyl-β-D-glucopyranosyl-(1f6)- 2,3,4-tri-O-acetyl-1-azido-

1-deoxy-β-^D-glucopyranose 15. Trichloroacetamide 14²³(65 mg,

84 μmol), acceptor 13 (29.2 mg, 84 μmol),²⁴and molecular sieves

4 A (166 mg) were dried at high vacuum for 2 h. Dry dichlor-

omethane was then added (2 mL) and the solution was stirred for

2 h at rt. TMSOTf (1.54 μL, 8.4 μmol) was added and the mixture

was stirred for 30 min at rt. Satd NaHCO₃was then added and the

stirring continued for a further 30 min. The mixture was then

filtered through Celite, washing with dichloromethane (5 mL).

The solvent was removed and chromatography of the residue

(EtOAc-cyclohexane, 3:2) gave a protected trisaccharide inter-

mediate (42 mg, 52%) as a white powder. This protected trisac-

charide (0.966 g, 1 mmol) was dissolved in dry CH₂Cl₂(20 mL)

under N₂. Azidotrimethylsilane (340 μL, 2.5 mmol,) and tin(IV)

chloride (60 μL, 0.5 mmol) were added and the mixture was stirred

for 15 min. Solid NaHCO₃(0.5 g) was then added and stirring

continued for an additional 30 min. The reaction mixture was then

diluted with dichloromethane (20 mL), washed with satd NaH-

CO₃(2 mL) and water (30 mL), dried (MgSO₄), and filtered, then

the solvent was removed under vacuum. Chromatography of the

residue (cyclohexane-EtOAc, 4:6) afforded 15 as a white solid

(0.78 g, 82%); mp 104 °C; [R]²⁰_D-12 (c 0.5, CHCl₃); ¹H NMR

(300 MHz, CDCl₃) δ (ppm) 5.35 (d, J = 3.9 Hz, 1H), 5.19 (t, J =

9.3 Hz, 1H), 5.19 (t, J = 9.0 Hz, 1H), 5.11 (dd, J = 7.8 Hz, J =

10.2 Hz, 1H), 4.96 (dd, J = 10.2 Hz, J = 3.3 Hz, 1H), 4.94-4.86

(m, 4H), 4.63 (d, J = 9.0 Hz, 1H), 4.55-4.48 (m, 3H), 4.15-4.05

(m, 3H), 3.90-3.73 (m, 4H), 3.80 (t, J = 9.3 Hz, 1H), 3.63-3.56

(m, 2H), 3.52 (dd, J = 10.8 Hz, J = 6.0 Hz, 1H), 2.15, 2.13, 2.10,

2.07, 2.06, 2.05 (2s), 2.03, 2.00, 1.97 (each s); ¹³C NMR (75 MHz,

CDCl₃) δ (ppm) 170.4, 170.3, 170.2, 170.0, 169.5, 169.3, 169.2

(each CO), 101.2, 100.6 (each CH), 87.8 (CH), 76.2, 75.4,

72.9, 72.7, 71.5, 71.1, 71.0, 69.2, 68.6 (each CH), 68.1 (CH₂),

66.7 (CH), 61.9, 60.9 (each CH₂), 20.9, 20.7, 20.6 (each CH₃);

HRMS (ESI) found 972.2709 [M þ Na^þ], C₃₈H₅₁N₃NaO₂₅

requires 972.2709.

Experimental Section

N,N⁰-Di(β-D-galactopyranosyl-(1f4)-β-D-glucopyranosyl)tere-

phthalamide 1 and N-(β-^D-Galactopyranosyl-(1f4)-β-D-glucopy-

ranosyl)-N⁰-(β-D-galactopyranosyl-(1f4)-R-D-glucopyranosyl)te-

rephthalamide 11. The lactosyl amine 10²²(0.473 g, 0.744 mmol)

and DIPEA (0.16 mL, 0.967 mmol) in dry THF (10 mL) were

added dropwise at rt into freshly recrystallized terephthaloyl

chloride (68 mg, 0.335 mmol) in dry THF. After 2 h the

solvent was removed and chromatography of the residue

(EtOAc-cyclohexane, gradient elution, 1:1 to 3:2 to 2:1) gave

a mixture of diamides as a white amorphous solid (0.432 g,

92%). Deacetylation of the diamide mixture (0.187 g, 0.133

mmol) gave a 79:20:1 mixture of 1, 11, and 12 (0.108 g,

quantitative yield). Semipreparative HPLC (isocratic elution

with water-CH₃CN, 99:1, flow rate 10 mL/min) was used to

separate 1, 11, and 12. Analytical data for 1: [R]_Dþ58.7 (c 0.7,

H₂O); ¹H NMR (500 MHz, D₂O, 30 °C) δ 7.94 (s, 4H), 5.24 (d,

J = 9.2 Hz, 2H), 4.48 (d, J = 7.8 Hz, 2H), 3.96 (d, J = 12.0 Hz,

2H), 3.93 (d, J = 3.3 Hz, 2H), 3.85 (dd, J = 12.3 Hz, J = 2.3 Hz,

2H), 3.83-3.71 (m, 12H), 3.67 (dd, J = 10.0 Hz, J = 3.4 Hz,

2H), 3.62 (t, J = 9.0 Hz, 2H), 3.57 (dd, J = 9.9 Hz, J = 7.9 Hz,

2H); ¹³C NMR (125 MHz, D₂O) δ 171.2 (CONH), 136.8 (C),

128.2 (CH), 103.1, 80.0, 78.0, 76.8, 75.6, 75.4, 72.8, 71.7, 71.2,

68.8 (each CH), 61.3, 60.1 (each CH₂); IR (KBr) 3481-3259 (br

signal), 1657, 1545, 1383, 1298, 1082, 1041 cm^-1; HRMS (ESI)

found 811.2609 [M - H]^-, C₃₂H₄₇N₂O₂₂requires 811.2620, and

found 835.2626 [M þ Na]^þ, C₃₂H₄₈N₂NaO₂₂requires 835.2596.

Analytical data for 11: [R]_Dþ42.6 (c 0.96, H₂O); ¹H NMR (500

MHz, D₂O, 25 °C) δ 7.93 (2 d, J = 8.4 Hz, 2H), 5.83 (d, J = 4.6

Hz, 1H), 5.27 (d, J = 9.2 Hz, 1H), 4.51 (dd, J = 7.8 Hz, J = 4.1

Hz, 1H), 4.00 (br s, 1H), 3.98 (br s, 1H), 3.96 (s, 1H), 3.89 (s, 2H),

3.86-3.73 (m, 6H), 3.72-3.68 (m, 1H), 3.66 (t, J = 9.0 Hz, 1H),

3.59 (dt, J = 10.4 Hz, J = 7.9 Hz, 1H); ¹³C NMR (125 MHz,

D₂O) δ 172.0, 170.9 (each CO), 136.9, 136.2 (each C), 128.0,

127.8, 102.8, 79.7, 77.1, 78.0, 77.7, 76.5, 75.3, 75.1, 72.5, 71.7,

71.6, 71.4, 70.9, 69.2, 68.5 (each CH), 61.0, 59.8 (each CH₂);

HRMS (ESI) found 835.2635 [M þ Na]^þ, C₃₂H₄₈N₂NaO₂₂

requires 835.2596.

N,N⁰-Di(β-D-galactopyranosyl-β-(1f4)-β-D-glucopyranose)-

N,N⁰-di[(1-methoxycarbonyl)methylamino-2-oxoethyl]terephthala-

mide 2. Terephthalic acid (73 mg, 0.44 mmol), amine 10 (0.558 g,

0.878 mmol), and formaldehyde (80 μL of a 37% soln,

1.023 mmol) were suspended in MeOH (13 mL) and the mixture

was stirred at rt for 1 h. Methyl isocyanoacetate (80 μL, 0.878

mmol) was then added and the mixture was stirred at rt for 18 h.

The reaction was then heated to 45 °C for 6 h and then cooled to

rt and left to stir for a further 18 h and then solvent was removed

under diminished pressure. Chromatography of the residue

(gradient elution, EtOAc to EtOAc-MeOH, 98:2), obtained

after removal of solvent, gave the protected intermediate as a

N,N⁰-Di(β-D-galactopyranosyl-(1f4)-O-β-D-glucopyrano-

syl-(1f6)-β-^D-glucopyranosyl)terephtalamide 3. Catalytic hy-

drogenation of the azide 15 (0.102 g, 0.105 mmol) gave the

desired glycosyl amine intermediate as a white solid (96 mg,

99%). Reaction of this amine (0.132 g, 0.143 mmol) with

terephthaloyl chloride (11 mg, 0.0715 mmol) as described for

10 gave, after chromatography (cyclohexane-EtOAc, 1:4 to

1:3), the protected diamide (0.114 g, 40%). Deacetylation (0.1 g,

0.57 mmol) gave 3 as a white powder (quantitative yield); [R]²⁰

D

ꢀ

white amorphous solid (0.352 g, 48%). Zemplen deacetylation

of this intermediate (0.352 g, 0.212 mmol) gave a yellow powder

1

þ2 (c 0.2, H₂O); H NMR (300 MHz, CDCl₃) δ (ppm) 7.96

(s, 4H), 5.25 (d, J = 8.4 Hz, 2H), 4.55 (d, J = 8.1 Hz, 2H), 4.45

€

ꢀ

(22) (a) Peto, C.; Batta, G.; Gyorgydeak, Z.; Sztaricskai, F., Liebigs Ann.

Chem. 1991, 5, 505-507. (b) Aravind, S.; Park, W. K. C.; Brochu, S.; Roy, R.

Tetrahedron Lett. 1994, 35, 7739–7742.

(23) Li, H.; Cai, M.-S.; Li, Z.-J. Carbohydr. Res. 2000, 328, 611–615.

(24) Lee, G. S.; Lee, Y.-J.; Choi, S. Y.; Park, Y. S.; Yoon, K. B. J. Am.

Chem. Soc. 2000, 122, 12151–12157.

J. Org. Chem. Vol. 74, No. 23, 2009 9021

Leyden et al.

JOCArticle

(d, J = 7.8 Hz, 2H), 4.25 (d, J = 10.5 Hz, 2H), 3.37-4.00 (m,

32H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 173.7 (CO), 139.3

(C), 130.8, 105.8, 105.2, 82.8, 81.3, 79.5, 79.3, 78.2, 77.6, 77.1,

75.6, 75.4, 74.5, 73.8 (each CH), 72.1 (CH₂), 71.4 (CH), 63.8,

62.9 (CH₂); HRMS (ESI) found 1159.3596 [M þ H^þ], C₄₄H₆₈-

N₂NaO₃₂requires 1159.3653.

This intermediate (23.5 mg, 10 μmol) was dissolved in dry

MeOH (1 mL). Potassium carbonate was then added (15 mg),

and the resulting solution was stirred for 20 h at rt. The solvent

was removed under reduced pressure and water was added (2 mL).

Amberlite IR-120(plus) was added, until the pH reached 6.

Water was then removed by lyophilization to obtain 4 (white

2,3,4,6-Tetra-O-acetyl-β-^D-galactopyranosyl-(1f4)-2,3,6-tri-

O-acetyl-β-D-glucopyranosyl-(1f4)-2,3,6-tri-O-benzoyl-1-O-

methyl-r-^D-glucopyranose 17. Glycosidation coupling of 16

with 14 as described for 13 gave, after chromatography

(EtOAc-cyclohexane, 35:75 to 40:60), 17 (48%); mp 121

°C; [R]²⁰_Dþ51 (c 0.5, CH₂Cl₂); ¹H NMR (300 MHz, CDCl₃)

δ (ppm) 8.08 (d, J = 7.2 Hz, 2H), 8.00 (d, J = 7.5 Hz, 2H),

7.93 (d, J = 7.2 Hz, 2H), 7.62 (m, 1H), 7.50 (t, J = 7.2 Hz,

4H), 7.35 (m, 4H), 6.03 (t, J = 9.6 Hz, 1H), 5.29 (d, J = 3.3

Hz, 1H), 5.09 (m, 2H), 5.06 (t, 1H, J = 9.3 Hz, 1H), 4.99 (dd,

1H, J = 7.8 Hz, J = 10.5 Hz, 1H), 4.89-4.77 (m, 2H), 4.75 (d,

1H, J = 12.0 Hz), 4.68 (d, 1H, J = 7.8 Hz), 4.44 (dd, 1H, J =

12.0 Hz, J = 5.1 Hz, 1H), 4.27 (d, 1H, J = 7.8 Hz, 1H),

4.23-4.19 (m, 1H), 4.84 (dd, J = 10.2 Hz, J = 3.6 Hz, 1H),

4.50-4.46 (m, 2H), 4.11-3.97 (m, 4H), 3.79 (t, J = 6.9 Hz,

1H), 3.67-3.59 (m, 2H), 3.28 (m, 1H), 3.42 (s, 3H), 2.11, 2.04,

2.02, 1.92 (each s, each CH₃); ¹³C NMR (75 MHz, CDCl₃) δ

(ppm): 170.4, 170.2, 170.1 (2s), 169.8, 169.7, 169.0, 166.1,

166.0, 165.0, 133.4 (each C), 130-128.4 (15 ꢀ CH), 101.1,

100.1, 96.9, 75.6, 73.0, 72.7, 72.4, 72.2, 71.1, 70.7, 70.4, 69.0

(each CH), 62.8, 61.8, 60.9 (each CH₂), 55.6 (CH), 20.9, 20.8,

20.7, 20.6 (each CH₃); HRMS (ESI) found 1147.3267 [M þ

Na^þ], C₅₄H₆₀NaO₂₆requires 1147.3271.

2,3,4,6-Tetra-O-acetyl-β-^D-galactopyranosyl-(1f4)-2,3,6-tri-

O-acetyl-β-^D-glucopyranosyl-(1f4)-2,3,6-tri-O-benzoyl-β-D-glu-

copyranosyl Azide 18. Trisaccharide 17 (1.17 g, 1.00 mmol) was

dissolved in acetic anhydride (10 mL) and an acetic anhydri-

de-sulfuric acid mixture (1:50, 20 mL) was added and the

mixture was stirred for 2 h at rt. Dichloromethane was then

added (100 mL) and the organic layer washed with water (100

mL), satd NaHCO₃(100 mL), and water (100 mL), dried

(MgSO₄), and filtered. The solvent was removed under dimin-

ished pressure. Chromatography of the residue (cyclo-

hexane-EtOAc, 7:3) gave the glycosyl acetate intermediate as

a white solid (0.81 g, 70%). Reaction of this glycosyl acetate (0.8

g, 0.69 mmol) with azidotrimethylsilane (0.4 g, 3.45 mmol) and

SnCl₄as described for the preparation of 15 gave the title

compound (0.6 g, 77%); mp 102 °C; [R]²⁰_Dþ7 (c 0.3, CH₂Cl₂);

¹H NMR (300 MHz, CDCl₃) δ (ppm) 8.09 (d, J = 7.2 Hz, 2H),

8.00 (d, d, J = 7.2 Hz, 2H), 7.91 (d, J = 7.2 Hz, 2H), 7.63 (m,

1H), 7.53-7.48 (m, 4H), 7.37-7.27 (m, 4H), 5.75 (t, J = 9.3 Hz,

1H), 5.33 (t, J = 9.0 Hz, 1H), 5.29 (s, 1H), 5.05 (t, 1H, J = 9.0

Hz, 1H), 4.98 (dd, J = 9.0 Hz, J = 7.5 Hz, 1H), 4.90-4.80 (m,

4H), 4.65 (d, J = 7.8 Hz, 1H), 4.44 (dd, J = 12.0 Hz, J = 4.8 Hz,

1H), 4.26 (d, J = 7.8 Hz, 1H), 4.15 (dd, J = 9.0 Hz, 1H), 4.04 (m,

4H), 3.95 (d, J = 10.5 Hz, 1H), 3.77 (t, J = 6.9 Hz, 1H), 3.59 (t,

J = 6.9 Hz, 1H), 3.50 (dd, 1H, J = 10.5 Hz, J = 5.1 Hz), 3.28 (m,

1H), 2.11, 2.00, 1.97, 1.93 (each s, 7 ꢀ CH₃); ¹³C NMR (75 MHz,

CDCl₃) δ (ppm) 170.4, 170.2, 170.1, 170.0, 169.8, 169.6, 169.0,

166.0, 165.2, 165.0, 133.6 (each C), 130-128.5 (15 ꢀ CH), 101.0,

100.3, 88.0, 76.1, 75.6, 75.3, 72.9 (2s), 72.8, 72.0, 71.5, 71.0, 70.7,

69.0, 66.6 (each CH), 62.4, 61.8, 60.8 (each CH₂), 20.9, 20.8,

20.7, 20.6 (each CH₃); HRMS (ESI) found 1158.3213 [M þ

Na^þ], C₅₃H₅₇N₃NaO₂₅requires 1158.3179.

powder) in quantitative yield; mp 187 °C; [R]²⁰-4 (c 0.1,

D

CH₂Cl₂); ¹H NMR (300 MHz, D₂O) δ (ppm) 7.94 (s, 4H), 5.27

(d, J = 9.0 Hz, 2H), 4.54 (d, J = 8.1 Hz, 2H), 4.48 (d, J = 7.8 Hz,

2H), 4.00-3.54 (m, 36H), 3.41 (t, J = 8.7 Hz, 2H); ¹³C NMR (75

MHz, CDCl₃) δ (ppm) 173.8, 139.4 (each C), 130.8, 105.8, 105.2,

82.0, 81.0, 80.8, 79.4, 78.2, 77.9, 77.7, 77.0, 75.7, 75.4, 74.4, 73.8,

71.4 (each CH), 63.9, 62.8, 62.7 (each CH₂); HRMS (ESI) found

1159.3639 [M þ Na^þ], C₄₄H₆₈N₂NaO₃₂requires 1159.3653.

2,3,4,6-Tetra-O-benzoyl-β-D-galactopyranosyl-(1f4)-2,3,6-

tri-O-benzoyl-β-^D-glucopyranosyl-(1f3)-1,2:5,6-di-O-isopropy-

lidene-β-^D-glucofuranoside 21. Glycosidation coupling of tri-

chloroacetimidate 19 (1.08 g, 0.89 mmol) with 1,2:5,6-di-O-

isopropylidene-R-^D-glucofuranose 20 (0.193 g, 0.742 mmol)

as described for 13 gave, after chromatography (cyclo-

hexane-EtOAc, gradient elution, 4:1 to 3:1), 21 as a white foam

(0.69 g, 71%); R_f0.36 (cyclohexane-EtOAc, 2:1); [R]²⁰_Dþ33.2

(c 2.5, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.02-7.94 (m, 10

H), 7.90 (d, J = 7.3 Hz, 2H), 7.72 (dd, J = 8.4, J = 1.1 Hz, 2H),

7.65-7.45 (m, 8H), 7.44-7.28 (m, 9H), 7.23-7.14 (m, 4H), 5.79

(t, J = 9.4 Hz, 1H), 5.75-5.70 (m, 2H), 5.46 (d, J = 3.7 Hz, 1H),

5.41 (dd, J = 6.7, 3.0 Hz, 1H), 5.40 (dd, J = 6.4, 2.9 Hz, 1H),

4.89 (d, J = 7.9 Hz, 1H), 4.82 (d, J = 7.9 Hz, 1H), 4.59 (dd, J =

12.1 Hz, J = 1.5 Hz, 1H), 4.49 (dd, J = -12.2 Hz, J = 4.2 Hz,

1H), 4.32-4.17 (m, 5H), 3.97 (dd, J = 8.6 Hz, J = 6.5 Hz, 1H),

3.92 (dd, J = 8.5 Hz, J = 6.0 Hz, 1H), 3.91 (t, J = 6.4, 1H), 3.82

(ddd, J = 9.9 Hz, J = 3.9 Hz, J = 1.8 Hz, 1H), 3.75 (d, J = 6.7

Hz, 2H), 1.39, 1.32, 1.13, 1.09 (each s, each 3H); ¹³C NMR (125

MHz, CDCl₃) δ 165.8, 165.5, 165.4, 165.3, 165.2, 164.8 (2s)

(each C), 133.5, 133.4 (2s), 133.3, 133.2, 129.7 (4s), 129.6 (2s),

129.5, 129.4 (2s), 129.1, 128.9, 128.7, 128.6 (3s), 128.5, 128.3,

128.2 (each CH), 111.9, 108.5 (each C), 104.9, 101.1, 99.8, 82.7,

81.4, 80.4, 75.9, 73.4, 73.0, 72.7, 71.7 (2s), 71.4, 69.9, 67.5 (each

CH), 67.0, 66.1, 62.0 (each CH₂), 26.6, 26.5, 25.9, 25.0 (each

CH₃); IR (KBr) 3064, 2985, 1730, 1603, 1453, 1269, 1093, 1070

cm^-1; HRMS (ESI) found 1335.3987 [M þ Na]^þ, C₇₃H₆₈NaO₂₃

requires 1335.4049.

2,3,4,6-Tetra-O-benzoyl-β-^D-galactopyranosyl-(1f4)-2,3,6-

tri-O-benzoyl-β-^D-glucopyranosyl-(1f3)-2,4,6-tri-O-acetyl-β-D-

glucopyranosyl Amine 22. A solution of 21 (0.59 g, 0.45 mmol) in

80% acetic acid (100 mL) was heated at 100 °C for 16 h. The

solvents were then removed under diminished pressure. Metha-

nol (2 ꢀ 10 mL) and CH₂Cl₂(2 ꢀ 10 mL) were evaporated from

the residue to give the desired trisaccharide intermediate as a

white foam (quantitative yield, HRMS (ESI): 1255.3361 [M þ

Na]^þ). This intermediate was dissolved in pyridine (5 mL) and

acetic anhydride (5 mL) and the mixture was stirred for 15 h. The

volatile reagents were removed and toluene (2 ꢀ 10 mL) and

CH₂Cl₂(2 ꢀ 10 mL) were evaporated from the residue. Chro-

matography (cyclohexane-EtOAc, gradient elution, 3.5:1 to

1:1) gave the protected glycosyl acetate intermediate as a white

amorphous solid (0.51 g, 81% over two steps, mixture of

anomers, R:β= 3:2). Reaction of this glycosyl acetate (0.492 g,

0.351 mmol) with azidotrimethylsilane (0.155 mL, 0.878 mmol)

as described for preparation of 15 gave, after chromatography

(cycohexane-EtOAc, gradient elution, 3:1 to 1:1), the glycosyl

azide precursor to 22 as a white foam (0.294 g, 61%). Catalytic

hydrogenation of this glycosyl azide (0.252 g, 0.182 mmol) as

described for 15 gave, after chromatography (cyclohexane-

EtOAc, 1:1 to EtOAc-CH₂Cl₂, 9:1), 22 as a white foam

(0.179 g, 72%); R_f0.44 (EtOAc-CH₂Cl₂, 9:1); [R]²⁰_Dþ12.6 (c

N,N⁰-Di(β-D-galactopyranosyl-(1f4)-β-D-glucopyranosyl-

(1f4)-β-^D-glucopyranosyl)terephtalamide 4. Catalytic hydroge-

nation of azide 18 (0.21 g, 0.176 mmol) as described for 15

gave an amine intermediate. Reaction of the amine (0.25 g,

0.22 mmol) with terephthaloyl chloride (44 mg, 0.22 mmol) as

described for the preparation of 15 and following chromatog-

raphy (EtOAc-cyclohexane, 60:40 to 55:45) yielded the pro-

tected divalent compound as a white powder (63 mg, 25%).

1

1.04, CHCl₃); H NMR (500 MHz, CDCl₃) δ 8.04-7.95 (m,

8H), 7.90-7.82 (m, 4H), 7.71 (dd, J = 8.2 Hz, J = 1.0 Hz, 2H),

9022 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

7.64-7.56 (m, 3H), 7.55-7.28 (m, 14H), 7.20 (t, J = 7.9 Hz,

2H), 7.13 (t, J = 7.8 Hz, 2H), 5.79 (t, J = 9.5 Hz, 1H), 5.71-5.67

(m, 2H), 5.37 (dd, J = 9.8 Hz, J = 8.0 Hz, 1H), 5.32 (dd, J =

10.3 Hz, J = 3.4 Hz, 1H), 4.93 (t, J = 9.6 Hz, 1H), 4.85 (d, J =

7.9 Hz, 1H), 4.80 (d, J = 7.9 Hz, 1H), 4.68 (t, J = 9.4 Hz, 1H),

4.58 (dd, J = 12.2 Hz, J = 1.4 Hz, 1H), 4.47 (dd, J = 12.3 Hz,

J = 4.1 Hz, 1H), 4.29 (t, J = 9.5 Hz, 1H), 4.07-4.04 (m, 2H),

3.95 (d, J = 9.0 Hz, 1H), 3.87-3.76 (m, 4H), 3.66 (tt, J = 10.1

Hz, J = 5.2 Hz, 1H), 3.47 (td, J = 10.1 Hz, J = 3.6 Hz, 1H),

2.05, 1.92, 1.72 (each s, each CH₃); ¹³C NMR (125 MHz, CDCl₃)

δ 171.0, 169.7, 168.4, 165.9, 165.8, 165.7, 165.6, 165.5, 165.4,

165.0 (each C), 133.9, 133.8 (2s), 133.7, 133.5 (2s), 13.4, 130.2,

130.1, 130.0, 129.9 (3s) (each CH), 129.7 (CH), 129.7 (2s), 129.5,

129.1 (each C), 128.9 (each CH), 128.9 (C), 128.8 (CH), 128.8

(C), 128.5 (2s), 128.4 (each CH), 101.4, 101.2, 85.4, 79.5, 75.9,

73.9, 73.3, 73.1, 73.0, 72.2, 72.0, 71.7, 70.2, 68.7, 67.8 (each CH),

62.8, 61.3 (each CH₂), 21.2, 21.0, 20.6 (each CH₃); IR (film on

NaCl) 2958, 1730, 1375, 1267, 1092, 1068, 1028 cm^-1; HRMS

(ESI) found 1358.4141 [M þ H]^þ, C₇₃H₆₈NO₂₅requires

1358.4080.

(C), 132.1 (2s), 131.9 (2s), 129.9, 129.8, 129.7 (2s), 129.6 (2s),

129.5 (each CH), 129.5, 129.4, 129.3, 128.9 (each C), 128.7 (CH),

128.7 (C), 128.6, 128.5 (2s), 128.4, 128.2, 79.0, 76.0, 75.5, 73.0,

72.8, 72.0, 71.9, 71.7, 71.4, 69.9, 69.5, 67.5 (each CH), 62.5, 61.0

(CH₂), 20.6, 20.5 (each CH₃); IR (film on NaCl) 3483-3305

(br s), 2121, 1746, 1619, 1429, 1376, 1232, 1046, 901 cm^-1

;

LRMS (ES) 1355.2 [M - H]^-; HRMS (ESI) found 1378.3523

[M þ Na]^þ, C₇₁H₆₁NaN₃O₂₅requires 1378.3492.

1,4-Di[(β-^D-galactopyranosyl-(1f4)-β-D-glucopyranosyl)-(1f3)-

1-azido-1-deoxy-β-^D-glucopyranuronamidomethyl]benzene 6.

Acid 25 (117 mg, 0.88 mmol), HOBt (27 mg, 0.201 mmol),

p-xylylenediamine (5 mg, 0.04 mmol), and DIPEA (0.018 mL,

0.1 mmol) were dissolved in dry DMF (0.8 mL) at 0 °C under N₂.

HATU (43 mg, 0.11 mmol) was added and the reaction was

stirred for 1 h at 0 °C and then overnight at rt. Ethyl acetate was

added and the mixture was washed with H₂O (2 ꢀ 10 mL), the

organic layer was separated, dried (MgSO₄), and filtered, and

the solvent was removed. Chromatography of the residue

(toluene-EtOAc, 2:1) gave the intermediate protected diamide

as a pale yellow solid (72 mg, 65%). To this protected diamide

(33 mg, 0.012 mmol) in MeOH-CH₂Cl₂(2.5 mL, 4:1) was

slowly added a solution of NaOMe (0.1 mL, 25 M in MeOH)

and the resulting mixture was stirred for 3 h at 0 °C. Solid CO₂

was added, the mixture was stirred for 20 min, and the solvent

was then removed under diminished pressure. The residue was

dissolved in water and then lyophilized to give 6 as a white

powder. ¹H NMR (600 MHz, D₂O) δ 7.32 (s, 4H), 4.85 (d,

J = 8.9 Hz, 2H), 4.80 (d, J = 8.0 Hz, 2H), 4.46 (s, 4H), 4.44 (d,

J = 7.9 Hz, 2H), 4.05 (d, J = 9.9 Hz, 2H), 3.99 (dd, J = 12.2 Hz,

J = 1.7 Hz, 2H), 3.93 (d, J = 3.2 Hz, 2H), 3.85 (t, J = 9.1 Hz,

2H), 3.83-3.70 (m, 10H), 3.69-3.64 (m, 6H), 3.63-3.59 (m,

2H), 3.55 (dd, J = 9.0 Hz, J = 8.9 Hz, 4H), 3.40 (dd, J = 8.3 Hz,

J = 8.8 Hz, 2H); ¹³C NMR (150 MHz, D₂O) δ 169.9, 136.9 (each

C), 127.7, 103.2, 102.7, 90.2, 83.4, 78.7, 76.9, 75.6, 75.1, 74.4,

73.4, 72.8, 72.5, 71.2, 69.8, 68.8 (each CH), 61.3, 60.4, 42.8 (each

CH₂); IR (KBr) 3483-3305 (br s), 2121, 1660, 1385, 1277, 1070

cm^-1; LRMS (ES) 1185.4 [M - H]^-; HRMS (ESI) found

1209.3837 [M þ Na]^þ, C₄₄H₆₆N₈NaO₃₀requires 1209.3783,

and found 1185.3833 [M - H]^-, C₄₄H₆₅N₈O₃₀requires

1185.3807.

N,N⁰-Di(β-D-galactopyranosyl-(1f4)-β-D-glucopyranosyl-

(1f3)-β-^D-glucopyranosyl)terephthalamide 5. Reaction of the

amine 22 (48.5 mg, 0.036 mmol) with terephthaloyl chloride as

described for 10 gave, after chromatography (EtOAc-cyclo-

hexane, gradient elution, 1:1 to 3:2), the protected diamide as a

white amorphous solid (28.6 mg, 56%). Deprotection of the

ꢀ

diamide (27 mg, 0.01 mmol) with the Zemplen procedure gave 5

(5.6 mg, 52%) as a white solid after semipreparative HPLC;

[R]²⁰þ512 (c 0.16); ¹H NMR (500 MHz, D₂O, 30 °C) δ 7.93

D

(s, 4H), 5.24 (d, J = 9.3 Hz, 2H), 4.83 (d, J = 8.0 Hz, 2H), 4.45

(d, J = 7.8 Hz, 2H), 3.99 (dd, J = 12.2 Hz, J = 1.8 Hz, 2H), 3.92

(d, J = 2.9 Hz, 2H), 3.91-3.86 (m, 2H), 3.84-3.74 (m, 10H),

3.74-3.61 (m, 14H), 3.61-3.57 (m, 2H), 3.54 (dd, J = 9.9 Hz,

J = 7.8 Hz, 2H), 3.42 (dd, J = 9.3 Hz, J = 8.0 Hz, 2H); ¹³C

NMR (125 MHz, D₂O) δ 171.2, 136.7 (each C), 128.1, 103.2,

102.8, 80.0, 85.2, 78.6, 77.7, 75.6, 75.1, 74.5, 73.4, 72.8, 71.7,

71.2, 68.8, 68.0 (each CH), 61.3, 60.8, 60.3 (each CH₂); IR (KBr)

3473-3313 (br s), 1657, 1552, 1385, 1071, 1043 cm^-1; HRMS

(ESI) found 1135.3638 [M - H]^-, C₄₄H₆₇N₂O₃₂requires

1135.367.

2,3,4,6-Tetra-O-benzoyl-β-^D-galactopyranosyl-(1f4)-2,3,6-

tri-O-benzoyl-β-^D-glucopyranosyl)-(1f3)-2,4-di-O-acetyl-1-azi-

do-1-deoxy-β-^D-glucopyranuronic Acid 25. Reaction of the tri-

chloroacetimidate 19 (1.64 g, 1.35 mmol) with acceptor 23^5,19c

(0.745 g, 2.18 mmol) as described for 13 gave, after chromatog-

raphy (cyclohexane-toluene-EtOAc, 3:2:1), the glycosidic

product as a white foam (0.62 g, 33%). To this intermediate

(0.264 g, 0.189 mmol) in dry CH₃CN (2 mL) at 0 °C were added

Pd(Ph₃)₄(0.022 g, 0.019 mmol) and pyrrolidine (0.016 mL, 0.189

mmol), respectively, with stirring for 1 h. The mixture was then

filtered through Celite and the solvent was removed. The residue

was dissolved in EtOAc and the organic layer was washed with

H₂O (2 ꢀ 15 mL). The water layer was then washed with EtOAc

(3 ꢀ 15 mL) and the organic layers combined, dried (MgSO₄),

and filtered, then solvent was removed under diminished pres-

sure to give 25 (0.238 g, 92%); [R]²⁰_Dþ6.6 (c 2.55, CHCl₃); ¹H

NMR (600 MHz, CDCl₃) δ 8.02-7.92 (m, 7H), 7.86 (dd, J =

13.4 Hz, J = 7.6 Hz, 4H), 7.71 (d, J = 7.4 Hz, 2H), 7.67 (dd, J =

12.0 Hz, J = 7.2 Hz, 2H), 7.64-7.28 (m, 16H), 7.20 (t, J = 7.8

Hz, 2H), 7.12 (t, J = 7.8 Hz, 2H), 5.76 (t, J = 9.4 Hz, 1H),

5.70-5.66 (m, 2H), 5.37 (d, J = 8.1 Hz, 1H), 5.31 (dd, J = 10.3

Hz, J = 3.4 Hz, 1H), 5.14-5.02 (br s, 1H), 4.84-4.81 (m, 1H),

4.79 (overlapping d, J = 7.9 Hz, 2H), 4.54 (d, J = 11.0 Hz, 1H),

4.46 (dd, J = 12.2 Hz, J = 3.9 Hz, 1H), 4.31 (broad d, J = 8.1

Hz, 1H), 4.27 (t, J = 9.4 Hz, 1H), 3.94-3.71 (m, 5H), 3.66 (dd,

Allyl 2,3,4,6-Tetra-O-acetyl-r-^D-mannopyranosyl-(1f3)-2,4-

di-O-acetyl-1-O-allyl-r-^D-glucopyranuronate 28. Deacetylation

of 27^19b(940 mg, 2.61 mmol) gave allyl R-D-glucopyranosiduro-

nic acid as a yellow foam (589 mg, 96%). This intermediate (1.56

g, 6.30 mmol) was suspended in acetic anhydride (23 mL) in the

presence of molecular sieves and stirred in a preheated bath at 85

°C for 2 h. The solvent was coevaporated with toluene to give a

brown oil that was dried under high vacuum. The oil was

dissolved in cyclohexane-EtOAc (1:1) then filtered, and the

filtrate was passed through a short column of silica gel eluting

with cyclohexane-EtOAc (1:1). This gave a 6,3-lactone inter-

mediate as a yellow oil (1.63 g, 86%), which was used in the next

reaction without further purification. Allyl alcohol (2.5 mL,

36.7 mmol) was added to a solution of the 6,3 lactone inter-

mediate (1.63 g, 5.33 mmol) in dry THF (8 mL) in the presence of

NaOAc (217 mg, 2.65 mmol) and molecular sieves at rt and

under N₂. The mixture was stirred at rt for 24 h then filtered, and

the filtrate was evaporated under reduced pressure to give a

brown oil. This oil was dissolved in dichloromethane (30 mL)

and washed with brine. The aq phase was further extracted with

dichloromethane (2 ꢀ 15 mL) and the combined organic

extracts were dried (MgSO₄) and filtered, then the solvent was

removed under reduced pressure. Chromatography of the re-

sidue (cyclohexane-EtOAc, 2:1) gave 28 as a white solid (1.03 g,

75%); R_f₁0.45 (cyclohexane-EtOAc, 1:1); [R]_Dþ20.8 (c 0.18,

CHCl₃); H NMR (CDCl₃, 300 MHz) δ 5.98-5.78 (m, 2H),

5.39-5.20 (m, 4H), 5.17-5.15 (d, J = 3.6 Hz, 1H), 5.11-5.04 (t,

J = 9.6 Hz, 1H), 4.83-4.79 (dd, J = 3.9 Hz, J = 10.2 Hz, 1H),

J = 11.1 Hz, J = 6.5 Hz, 1H), 1.85, 1.70 (each s, each CH₃); ¹³

C

NMR (150 MHz, CDCl₃) δ 170.4, 169.2, 168.4, 165.7, 165.5,

165.4 (2s), 165.2 (2s), 164.8, 133.6 (each C), 133.5 (CH), 133.2

J. Org. Chem. Vol. 74, No. 23, 2009 9023

Leyden et al.

JOCArticle

4.69-4.55 (m, 2H), 4.31-4.28 (d, J = 10.2, 1H), 4.25-4.09 (m

and overlapping t, J = 9.6 Hz, 2H), 4.06-3.99 (m, 1H,), 2.45 (d,

J = 5.4 Hz, 1H), 2.13, 2.08 (each s, 3H, each CH₃); ¹³C NMR

(CDCl₃, 75 MHz) δ 170.7, 170.4, 167.8 (each C), 133.0, 131.2

(each CH), 118.5, 118.2 (each CH₂), 95.4, 72.9, 72.0, 69.4 (each

CH), 69.1 (CH₂), 68.7 (CH), 66.6 (CH₂), 20.9, 20.8 (each CH₃);

IR (film from dichloromethane) 3480, 2996, 1748, 1374, 1230,

1050 cm^-1; HRMS (ESI) found 359.1349 [M þ H]^þ, C₁₆H₂₃O₉

requires 359.1342.

2H), 5.34-5.32 (dd, J = 10.2 Hz, J = 3.0 Hz, 2H), 5.21-5.14

(m, 4H), 5.03-5.00 (t, J = 9.6, 2H), 5.00 (d, J = 3.6 Hz, 2H),

4.87 (d, J = 7.8 Hz, 2H), 4.81 (d, J = 8.4 Hz, 2H), 4.58-4.54 (m,

4H), 4.48-4.46 (dd, J = 4.5 Hz, J = 13.2 Hz, 2H), 4.38-4.30

(m, 4H), 4.30-4.27 (t, J = 9.6 Hz, 2H), 4.18-4.15 (t, J = 9.6 Hz,

2H), 4.13 (d, J = 10 Hz, 2H), 4.09-4.05 (m, 2H), 3.89-386 (m,

2H), 3.82-3.77 (m, 6H), 3.68-3.66 (m, 2H), 1.82, 1.78 (each s,

3H); ¹³C NMR (CDCl₃, 150 MHz) δ (ppm) 169.5, 169.4, 167.3,

165.7, 165.6, 165.5, 165.4, 165.2, 165.1, 164.8, 137.0 (each C),

133.6, 133.5 (2s), 133.4, 133.2 (each CH), 133.2 (C), 133.1, 132.7,

129.9, 129.8, 129.7 (2s) (each CH), 129.6 (2s) (C), 129.6, 129.5

(2s) (each CH), 129.4, 129.3, 128.9 (each C), 128.7 (2s) (each

CH), 128.6 (C), 128.5, 128.3 (2s), 128.2 (each CH), 118.5 (CH₂),

101.2, 100.9, 94.5, 75.7, 75.6, 73.0, 72.9, 72.8, 72.1, 71.7, 71.5,

69.9, 69.5 (each CH), 69.2 (CH₂), 68.7, 67.6 (each CH),

62.4, 61.1, 43.7 (each CH₂), 20.5, 20.4 (each CH₃); IR (film

from dichloromethane) 3432, 3343, 3064, 2944, 1729, 1687,

1585, 1527, 1452, 1371, 1263, 1099, 1051 cm^-1; HRMS (ESI)

found 1421.4327 [M þ 2H]^2þ, C₁₅₆H₁₄₀N₂O₅₀requires m/2z

1421.4315.

2,3,4,6-Tetra-O-benzoyl-β-^D-galactopyranosyl-(1f4)-2,3,6-

tri-O-benzoyl-β-D-glucopyranosyl-(1f3)-2,4-di-O-acetyl-1-O-

allyl-r-^D-glucopyranuronic Acid 29. Glycoside coupling reac-

tion of acceptor 28 (1.31 g, 3.65 mmol) and trichloroacetimi-

date 19 (3.22 g, 2.65 mmol) as described for 13 gave, after

chromatography (eluant cyclohexane-toluene-EtOAc 3:2:1

to 5:4:2), the protected trisaccharide intermediate as a white

solid (3.11 g, 83%). To this protected trisaccharide (1.45 g,

1.027 mmol) in dry acetonitrile (8 mL), cooled on an ice bath

and under N₂, tetrakis(triphenylphosphine)palladium (117

mg, 0.10 mmol) was added followed by pyrrolidine (85 μL,

1.032 mmol). The reaction mixture was stirred on an ice bath

for 1 h and then the solvent was removed under diminished

pressure. The residue was dissolved in dichloromethane and

washed with 0.1 M HCl and brine and then dried (MgSO₄) and

filtered, then the solvent was removed under diminished

pressure. The residue was filtered through a short column of

silica gel (eluant: dichloromethane then MeOH) to give 29 as a

pale yellow solid (980 mg, 70%); R_f0.15 (dichloromethane-

MeOH, 95:5); [R]²⁰_Dþ4.26 (c 0.26, CHCl₃); ¹H NMR (CDCl₃,

600 MHz) δ (ppm) 8.00-7.96 (m, 8H), 7.88 (d, J = 7.2 Hz,

2H), 7.79 (d, J = 6 Hz, 2H), 7.72 (d, J = 7.2 Hz, 2H),

7.62-7.56 (m, 2H), 7.50-7.31 (m, 15H), 7.21-7.19 (t, J =

7.8 Hz, 2H), 7.14-7.12 (m, 2H), 5.77-5.67 (m, 4H), 5.36-5.32

(m, 2H), 5.18-5.15 (d, J = 17 Hz, 1H), 5.11-4.94 (m, 3H),

4.86 (d, J = 6.6 Hz, 1H), 4.81 (d, J = 7.8 Hz, 1H), 4.60-4.53

(m, 2H), 4.47-4.45 (m, 1H), 4.27-4.24 (t, J = 8.4 Hz, 1H),

4.11-4.01 (m, 3H), 3.90-3.69 (m, 5H), 1.76, 1.67 (each s, 3H);

¹³C NMR (CDCl₃, 100 MHz) δ 169.4, 165.7, 165.6, 165.5,

165.4, 165.2, 164.8 (each C), 133.5, 133.2, 129.9, 129.8, 129.7,

129.6 (2s) (each CH), 129.6 (C), 129.5, 129.4, 129.2, 128.9,

128.7, 128.6, 128.5, 128.2 (each CH), 118.0 (CH₂), 101.1,

100.9, 95.0, 75.6, 73.1, 72.7, 72.1, 71.7, 71.4, 69.8 (each CH),

68.9 (CH₂), 67.5 (CH), 62.4, 61.0 (each CH₂), 20.5, 20.4 (each

CH₃); IR (film from dichloromethane) 3598, 3374, 1729, 1602,

1423, 1371, 1267, 1103, 1050 cm^-1; HRMS (ESI) found

1393.3729 [M þ Na]^þ, C₇₄H₆₆O₂₆requires 1393.3740.

Macrocyclic Compound 31 and Acyclic Compound 32. A

degassed solution of the diamide 30 (629 mg, 0.22 mmol) in

dry dichloromethane (190 mL, 1.15 mM) and under N₂was

treated with Grubbs-I catalyst (53 mg, ∼9%) for 60 h. The

solvent was evaporated, then the resulting black solid was

dissolved and subjected to fractionation through a short column

of silica gel (toluene-EtOAc, 2:1) yielding a mixture of products

that was used in the next reaction without further purification.

Three cycles of catalytic hydrogenation of the mixture (530 mg)

using the H-Cube (70 °C) hydrogenation apparatus with Pd as

catalyst and EtOAc as solvent and subsequent chromatography

(toluene-cyclohexane-EtOAc, 3:1:1 to 1:1:1) gave both 31 (265

mg, 43%) and 32 (233 mg, 44%). Analytical data for 32: R_f0.75

1

(toluene-EtOAc 1:1); [R]_Dþ2.86 (c 0.24, CHCl₃); H NMR

(CDCl₃, 600 MHz) δ 8.02-7.96 (m, 14H), 7.86 (d, J = 7.2 Hz,

4H), 7.82 (d, J = 7.2 Hz, 4H), 7.71 (d, J = 7.2 Hz, 4H),

7.63-7.60 (t, J = 7.2 Hz, 4H), 7.57-7.55 (t, J = 7.8 Hz, 2H),

7.52-7.30 (m, 28H), 7.21-7.19 (t, J = 7.8 Hz, 4H) overlapping

with 7.20 (s, 4H), 7.16-7.12 (m, 6H), 6.52-6.49 (t, J = 5.4 Hz,

2H, NH), 5.80-5.77 (t, J = 9.6 Hz, 2H), 5.72-5.68 (m, 4H),

5.37-5.34 (dd, J = 8.4 Hz, J = 10.2 Hz, 2H), 5.34-5.31 (dd,

J = 5.4 Hz, J = 10.2 Hz, 2H), 5.01-4.98 (t, J = 9 Hz, 2H), 4.96

(d, J = 3.0 Hz, 2H), 4.88 (d, J = 7.8 Hz, 2H), 4.81 (d, J = 7.8 Hz,

2H), 4.59-4.57 (m, 2H), 4.52-4.50 (dd, J = 3.6 Hz, J = 9.8 Hz),

4.49-4.46 (dd, J = 3.6 Hz, J = 12 Hz, 2H), 4.37-4.27 (m, 6H),

4.14-4.09 (m, 4H), 3.82-376 (m, 6H), 3.68-3.65 (m, 2H),

3.54-3.50 (m, 2H), 3.26-3.22 (m, 2H), 1.82, 1.78 (each s, 3H),

1.53-1.47 (m, 4H), 0.85-0.83 (t, J = 6.0 Hz, 6H); ¹³C NMR

(CDCl₃, 150 MHz) δ 169.5 (2 s), 167.4, 165.7, 165.6, 165.5,

165.4, 165.2, 165.1, 164.8, 137.0, 133.6 (each C), 133.5 (2s), 133.2

(each CH), 133.2, 133.1 (each C), 129.9, 129.8, 129.7 (3s), 129.5

(CH), 129.4, 129.3, 128.9 (C), 128.7(2s), 128.6, 128.3 (3s) (each

CH), 101.2, 100.9, 95.2, 75.8, 75.6, 73.2, 73.0, 72.8, 72.1, 71.8,

71.5 (each CH), 70.5 (CH₂), 69.9, 69.6, 68.5, 67.6 (each CH),

62.5, 61.1, 42.7, 22.4 (each CH₂), 20.5, 20.4, 10.4 (each CH₃); IR

(film from dichloromethane) 3394, 3264, 2967, 2856, 1731, 1681,

1,4-Di[2,3,4,6-tetra-O-benzoyl-β-^D-galactopyranosyl-(1f4)-

2,3,6-tri-O-benzoyl-β-D-glucopyranosyl-(1f3)-2,4-di-O-acetyl-

1-O-allyl-r-^D-glucopyranuronamidomethyl]benzene 30. To acid

29 (980 mg, 0.71 mmol) in dry DMF (7.5 mL) and under N₂were

added HOBt monohydrate (134 mg, 0.98 mmol) followed by p-

xylylenediamine (41 mg, 0.3 mmol). Diisopropylethylamine

(180 μL, 1.07 mmol) was added and the mixture became cloudy,

then it was cooled on an ice bath and HATU (350 mg, 0.92

mmol) was added. The reaction mixture was allowed to attain rt

and was then stirred for 15 h. Dichloromethane was added and

the mixture was washed with water. The aq phase was extracted

with dichloromethane and the combined organic extracts were

dried (MgSO₄) and filtered and the solvent was removed under

diminished pressure. Chromatography (toluene-EtOAc, 2.5:1

and EtOAc) gave 30 as a white solid (629 mg, 74%); R_f0.55

(toluene-EtOAc, 1:1); [R]²⁰_Dþ2.41 (c 0.25, CHCl₃); ¹H NMR

(CDCl₃, 600 MHz) δ (ppm) 8.02-7.97 (m, 14H), 7.87 (d, J = 7.2

Hz, 4H), 7.81 (d, J = 7.2 Hz, 4H), 7.71 (d, J = 7.2 Hz, 4H),

7.62-7.59 (m, 4H), 7.57-7.55 (m, 2H), 7.51-7.30 (m, 24H),

7.26-7.23 (t, J = 6 Hz, 4H), 7.20 (s, 4H) overlapping 7.20-7.11

(m, 10H), 6.52-6.50 (t, J = 6 Hz, 2H), 5.80-5.77 (t, J = 9.6 Hz,

2H), 5.77-5.68 (m, 6H), 5.36-5.34 (dd, J = 8.4 Hz, J = 9.3 Hz,

1585, 1521, 1452, 1369, 1265, 1176, 1085, 1070, 1025 cm^-1

;

HRMS (ESI) found 1423.4458 [M þ 2H]^2þ, C₁₅₆H₁₄₄N₂O₅₀

requires 1423.4472. Anal. Calcd for C₁₅₆H₁₄₄N₂O₅₀: C 65.82, H

5.10, N 0.98. Found: C 65.51, H 5.26, N 0.83. Analytical data for

31: R_f= 0.7 (toluene-EtOAc 1:1); [R]_Dþ0.61 (c 0.18, CHCl₃);

¹H NMR (CDCl₃, 600 MHz) δ 8.02-7.96 (m, 14H), 7.87 (d, J =

7.2 Hz, 4H), 7.83 (d, J = 7.2 Hz, 4H), 7.71 (d, J = 7.2 Hz, 4H),

7.62-7.60 (t, J = 6.6 Hz, 4H), 7.58-7.54 (m, 2H), 7.52-7.31

(m, 28H), 7.27-7.24 (t, J = 7.8 Hz, 4H), 7.26 (s, 4H), 7.21-7.11

(m, 6H), 6.74-6.72 (dd, J = 3.6 Hz, J = 9.6 Hz, 2H), 5.79-5.76

(t, J = 9.0 Hz, 2H), 5.69-5.65 (m, 4H), 5.38-5.36 (dd, J = 7.9

Hz, J = 10.0 Hz, 2H), 5.35-5.32 (dd, J = 3.3 Hz, J = 8.6 Hz,

9024 J. Org. Chem. Vol. 74, No. 23, 2009

Leyden et al.

JOCArticle

2H), 4.99-4.96 (m, 4H), 4.86 (d, J = 7.7 Hz, 2H), 4.85-4.77 (m,

4H), 4.55-4.51 (m, 4H), 4.49-4.45 (m, 2H), 4.30-4.27 (t, J =

9.6 Hz, 2H), 4.09-4.06 (t, J = 9.0 Hz, 2H), 3.95 (d, J = 10.8 Hz,

2H), 3.82-374 (m, 8H), 3.69-3.65 (m, 2H), 3.27-3.23 (m, 2H),

3.19-3.14 (m, 2H), 1.85, 1.76 (each s, each 3H), 1.48-1.42 (m,

2H), 1.31-1.22 (m, 2H); ¹³C NMR (CDCl₃, 150 MHz) δ 169.5

(2s, 167.3, 166.0, 165.7, 165.6, 165.3, 165.2, 165.0, 164.8 (each

CO), 138.2, 133.6 (C), 133.5 (2s, CH), 133.4 (C), 133.3 (CH),

133.2, (C), 129.9, 129.8, 129.7 (2s), 129.6 (2s), 129.5 (2s), 129.4

(each CH), 129.2 (C), 128.9, 128.7, 128.6 (3s), 128.3 (2s), 128.2

(each CH), 127.9 (C), 127.8 (CH), 127.8, 127.7 (each C), 101.0,

100.9, 96.1, 75.8, 75.5, 73.0, 72.9, 72.8, 72.1, 71.7, 71.5, 69.9

(each CH), 69.4 (CH₂), 69.3, 67.9, 67.6 (each CH), 62.4, 61.1,

42.2, 25.56 (each CH₂), 20.4, 20.3 (each CH₃); IR (film from

dichloromethane) 3598, 3374, 1729, 1602, 1423, 1371, 1267,

1103, 1050, 3405, 3315, 3062, 2980, 2921, 2856, 1731, 1683,

1600, 1523, 1452, 1399, 1315, 1263, 1067, 1027 cm^-1; HRMS

(ESI) found m/2z 1408.4218 [M þ 2H]^2þ, C₁₅₄H₁₃₈N₂O₅₀

requires 1408.4237.

off-white foam (0.517 g, 76% over two steps). Deacetylation of

this peracetate (0.314 g, 0.208 mmol) in MeOH-CH₂Cl₂(10

mL, 4:1) as described for 6 gave, after lyophilization, 9 as a

brown solid (0.191 g, quantitative); [R]_Dþ38.5 (c 1.0, H₂O); ¹H

NMR (500 MHz, D₂O) δ 8.24 (s, 2H), 6.97 (s, 4H), 5.75 (d, J =

9.2 Hz, 2H, H-1), 5.16 (s, 4H), 4.49 (d, J = 7.8 Hz, 2H), 4.03 (t,

J = 9.1 Hz, 2H), 3.95-3.91 (m, 4H), 3.89-3.81 (m, 8H),

3.81-3.70 (m, 6H), 3.67 (dd, J =10.0 Hz, J = 3.4 Hz, 2H),

3.57 (dd, J = 9.9 Hz, J = 7.8 Hz, 2H); ¹³C NMR (125 MHz,

D₂O) δ 152.5, 144.0 (each CH), 124.7 (C), 117.1, 103.2, 87.5,

78.0, 77.7, 75.6, 74.8, 72.8, 72.3, 71.2, 68.8 (each CH), 62.2, 61.3,

60.0 (each CH₂); IR (KBr) 3502-3197 (br s), 1643, 1508, 1460,

1385, 1225, 1126, 1070 cm^-1; LRMS (ESI) 943.1 [M þ Na]^þ;

HRMS (ESI) found 921.3257 [M þ H]^þ, C₃₆H₅₃N₆O₂₂requires

921.3213, and found 919.3021 [M - H]^-, C₃₆H₅₃N₆O₂₂requires

919.3056.

Molecular Modeling. The AMBER force field in Macromodel

and the GB/SA continuum²⁵for water were generally applied in

all calculations. Systematic conformational searches with the

SUMM method in Macromodel were carried out with 10 000

structures being generated starting from the initial model struc-

tures. Both glycosyl amide torsions (H₁-C₁-N-H) in the

divalent molecules 1, 3-5 were constrained to 171° with a force

constant of 2000 applied throughout the calculations. Each

conformer generated was minimized to convergence and all

structures within 12 kJ/mol of the global minimum for each

compound were retained and included in the analysis shown in

Figure 2. Conformational searches with 2 led to highly folded

low-energy structures which seemed unlikely to bind to lectins.

Thus stochastic molecular dynamics were also carried out for 2

beginning from an initial structure where the carbonyl group of

the terephthalamide residue was trans (OdC;CdO torsion

∼90°). During the dynamics simulations a temperature of

300 K, time step of 1.5 fs, equilibration time of 1.0 ps, and

simulation time 2 ns were applied and 500 structures were selected

randomly over the duration of the simulation. The structures

obtained from the conformational searches and dynamics simu-

lations were analyzed in the same manner. In general the lactose

glycosidic torsions were consistent in all structures generated. For

example, the average observed values for 1 from the conforma-

tional search were -72.0° ( 5.3° for φ and 132.1° ( 1.6° for ψ.

Lectins and Activity Assays. Extracts of dried mistletoe leaves

and of pellets of bacteria after recombinant production were the

sources for lectin purification by affinity chromatography on

lactosylated Sepharose 4B as crucial step.^7g,k,26Proteolytic

truncation by collagenase-dependent cleavage at the Tyr106/

Gly107 and Glu229/Ile230 peptide bonds in human galectin-3

was performed by a standard protocol.^4a,8dPurity control by

one- and two-dimensional gel electrophoresis preceded biotiny-

lation under activity-preserving conditions and the following

activity controls by hemeagglutination with trypsin-treated and

glutaraldehyde-fixed rabbit erythrocytes as well as binding to

glycoproteins and human tumor cells.^26a,27Haemagglutination

1-4-Di[β-^D-galactopyranosyl-(1f4)-β-D-glucopyranosyl-(1f3)-

1-O-n-propyl-r-^D-glucopyranuronamido]benzene 7. Removal of

the protecting groups from 32 (48 mg, 0.017 mmol) as described

for 6 gave, after semipreparative HPLC (gradient elution:

H₂O-CH₃CN, 99:1 to 96:4) and lyophilization, 7 (10 mg,

1

49%); [R]_Dþ2.19 (c 0.10, H₂O); H NMR (D₂O, 35 °C, 600

MHz) δ 7.30 (s, 4H), 4.98 (d, J = 3.6 Hz, 2H), 4.74-4.73 (d, J =

7.8 Hz, 2H), 4.47-4.40 (m, 6H), 4.15-4.13 (d, J = 10.2 Hz, 2H),

3.99-3.97 (dd, J = 2.4 Hz, J = 12.6 Hz, 2H), 3.95-3.93

(m, 4H), 3.82-3.75 (m, 8H), 3.74-3.71 (m, 2H), 3.69-3.63

(m, 10H), 3.62-3.61 (m, 2H), 3.55-3.52 (m and t overlapping,

J = 9.6 Hz, 4H), 3.41-3.38 (m, 2H), 1.66-1.60 (m, 4H),

0.92-0.90 (t, J = 7.8 Hz, 6H); ¹³C NMR (D₂O, 150 MHz) δ

171.1, 137.0 (each C), 127.7, 103.2, 102.9, 98.7, 81.9, 78.7, 75.6,

75.1, 74.5, 73.5, 72.8, 71.8, 71.2, 70.9 (each CH), 70.9 (CH₂), 70.3

(CH₂), 68.9 (CH), 61.3, 60.4, 42.8, 22.4 (each CH₂), 10.2 (CH₃);

HRMS (ESI) found 1220.4763 [M þ H]^þ, C₅₀H₈₀N₂O₃₂requires

1220.4694.

Glycophane 8. Removal of the protecing groups from 31 (62

mg, 0.021 mmol) as described for formation of 6 gave 8, after

semipreparative HPLC (gradient elution: H₂O-CH₃CN, 99:1

to 96:4) and lyophilization, as a white solid (17 mg, 66%); [R]_D

þ2.0 (c 0.17, H₂O); ¹H NMR (D₂O, 35 °C, 500 MHz) δ 7.30 (s,

4H), 4.93 (d, J = 2.0 Hz, 2H), 4.72-4.70 (d, J = 7 Hz, 2H),

4.51-4.48 (d, J = -14.0 Hz, 2H), 4.43-4.42 (d, J = 8.0 Hz,

2H), 4.28-4.25 (d, J = -14.0 Hz, 2H), 3.98-3.95 (m, 4H), 3.90

(d, J = 2.0 Hz, 2H), 3.85-3.83 (d, J = 9.0 Hz, 2H), 3.80-3.74

(m, 6H), 3.72-3.64 (m, 12H), 3.59-3.56 (m, 2H), 3.54-3.51 (m,

4H), 3.47-3.42 (m, 2H), 3.40-3.35 (m, 2H), 1.60-1.53 (m, 2H),

1.48-1.39 (m, 2H); ¹³C NMR (D₂O, 125 MHz) δ 170.5, 138.2

(each C), 128.4, 103.2, 102.8, 99.2, 82.0, 78.6, 75.6, 75.1, 74.5,

73.4, 72.8, 71.7, 71.2, 70.9, 69.9 (each CH), 69.7 (CH₂), 68.9

(CH), 61.3, 60.4, 42.7, 25.5 (each CH₂); LRMS (ESI) 1189.2

[M - H]^-, 593.6 [M - 2H]^2-; HRMS (ESI) found 1190.4271

[M þ H]^þ, C₄₈H₇₄N₂O₃₂requires 1190.4225;

1,4-Di[1-(β-^D-galactopyranosyl-(1f4)-β-D-glucopyranosyl)-

1,2,3-triazol-4-ylmethyloxy]benzene 9. Lactosyl azide 3323a (331

mg, 0.901 mmol) was dissolved in CH₃CN-H₂O (1:1, 4 mL),

then p-bispropargyloxybenzene^11c34 (84 mg, 0.451 mmol) and

(25) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am.

Chem. Soc. 1990, 112, 6127–6129.

(26) (a) Gabius, H.-J.; Engelhardt, R.; Rehm, S.; Cramer, F. J. Natl.

Cancer Inst. 1984, 73, 1349–1357. (b) Gabius, H.-J.; Engelhardt, R.; Cramer,

€

F.; Batge, R.; Nagel, G. A. Cancer Res. 1985, 45, 253–257. (c) Gabius, H.-J.

sodium ascorbate (0.22 mL of 0.4 M) followed by CuSO₄5H₂O

3

Anal. Biochem. 1990, 189, 91–94. (d) Gabius, H.-J.; Walzel, H.; Joshi, S. S.;

Kruip, J.; Kojima, S.; Gerke, V.; Kratzin, H.; Gabius, S. Anticancer Res.

ꢀ

1992, 12, 669–676. (e) Andre, S.; Kaltner, H.; Furuike, T.; Nishimura, S.-I.;

Gabius, H.-J. Bioconjugate Chem. 2004, 15, 87–98.

(0.22 mL of 0.2 M) were subsequently added and the mixture

was stirred for 12 h, after which it was diluted with H₂O (15 mL)

and extractd with EtOAc (2 ꢀ 5 mL). After lyophilization of the

aq solution the pale green powder (0.397 g) that was obtained

was added to pyridine-Ac₂O (1:1, 10 mL) and the mixture was

stirred overnight. The volatile reagents were then removed

under diminished pressure and toluene (3 ꢀ 10 mL) was eva-

porated from the residue. Chromatography (EtOAc-cyclo-

hexane, gradient elution 1:1 to 2:1) gave the peracetate as an

(27) (a) Gabius, H.-J.; Wosgien, B.; Hendrys, M.; Bardosi, A. Histochem-

ꢀ

ꢁꢀ

ꢀ

istry 1991, 95, 269–277. (b) Purkrabkova, T.; Smetana, K., Jr.; Dvorankova,

ꢀ

€

ꢀ

B.; Holı

Klıma, J.; Smetana, K.; Motlı

(c) Andre, S.; Pei, Z.; Siebert, H.-C.; Ramstrom, O.; Gabius, H.-J. Bioorg.

´

kova, Z.; Bock, C.; Lensch, M.; Andre, S.; Pytlik, R.; Liu, F.-T.;

´

k, J.; Gabius, H.-J. Biol. Cell 2003, 95, 535–545.

ꢀ

€

ꢀ

Med. Chem. 2006, 14, 6314–6326. (d) Andre, S.; Maljaars, C. E. P.; Halkes,

K. M.; Gabius, H.-J.; Kamerling, J. P. Bioorg. Med. Chem. Lett. 2007, 17,

793–798.

J. Org. Chem. Vol. 74, No. 23, 2009 9025

Leyden et al.

JOCArticle

assays were performed with 2-fold serial dilutions of sugar in

triplicates with at least five independent test series. For studies in

vitro, cells of the human B lymphoblastoid line Croco II were

processed in aliquots of the same batch in triplicates and at least

three independent series with incubation of lectin-containing

solution at 4 °C for 30 min as labeling step and with streptavidin-

R-phycoerythrin as reporter conjugate.^27c,dControls included

omission of the labeling step to determine the level of lectin-

independent background staining and application of noncog-

nate sugar to infer osmolarity effects. The solid-phase assay with

spectrophotometric signal detection at 490 nm was performed

under optimized conditions as described previously.²⁸In detail,

the plastic surface of the microtiter plate wells was coated with

ligand by incubation of 50 μL of phosphate-buffered saline

(pH 7.2) containing the lectin-reactive glycoprotein (serum

amyloid P component: 0.5 μg; asialofetuin; 0.5 μg) at 4 °C

overnight. Following blocking residual sites for protein adsorp-

tion by an incubation with 100 μL of buffer containing 1% (w/v)

carbohydrate-free bovine serum albumin for 1 h at 37 °C and

washing, 50 μL of solution with biotinylated lectin was applied

at a concentration resulting in signal intensity within the linear

range (please see top part of Table 1 for details), and bound

lectin was detected by its biotin label using streptavidin-perox-

idase conjugate (0.5 μg/mL for 1 h at 37 °C; Sigma, Munich,

Germany) and the chromogenic substrates o-phenylenediamine

(1 mg/mL) and H₂O₂(1 μL/mL). Up to five series of indepen-

dent experiments were run in triplicates with standard devia-

tions not exceeding 17%.

Acknowledgment. Generous funding was provided by

Science Foundation Ireland, the European Commission

through Marie Curie IntraEuropean Fellowships ((MEIF-

CT-2003-500748, 514958, to S.G.G. and T.V.T.), a Marie

Curie Research Training Network grant (contract no. CT-

2005-19561), the Programme for Research in Third-Level

Institutions (PRTLI), administered by the HEA (of Ireland),

the research initiative LMUexcellent, and the Verein zur

€

Forderung des biologisch-technologischen Fortschritts in

der Medizin e. V. (Heidelberg, Germany). Inspiring discus-

sions with Drs. B. Friday and S. Namirha are gratefully

acknowledged.

Supporting Information Available: Experimental details,

analytical data for intermediates, and ¹H and ¹³C NMR spectra.

This material is available free of charge via the Internet at http://

pubs.acs.org.

ꢀ

(28) (a) Andre, S.; Ortega, P. C.; Perez, M. A.; Roy, R.; Gabius, H.-J.

Glycobiology 1999, 9, 1253–1261. (b) Andre, S.; Kojima, S.; Prahl, I.; Lensch,

ꢀ

M.; Unverzagt, C.; Gabius, H.-J. FEBS J. 2005, 272, 1986–1998.

9026 J. Org. Chem. Vol. 74, No. 23, 2009

Article Doi

Synthesis of bivalent lactosides based on terephthalamide, N,N′-diglucosylterephthalamide, and glycophane scaffolds and assessment of their inhibitory capacity on medically relevant lectins

DOI: 10.1021/jo901667r

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jo901667r

Full text of DOI:10.1021/jo901667r

Products guided by the article

R&D Labs maybe for 1198402-15-1

Relevant to this article

Hot Product