Thiol‐ene and thiol‐yne‐based synthesis of glycodendrimers as nanomolar inhibitors of wheat germ agglutinin

Thiol-Ene and Thiol-Yne-Based Synthesis of Glycodendrimers as

Nanomolar Inhibitors of Wheat Germ Agglutinin

Mattia Ghirardello,1 Kim €Oberg,2 Samuele Staderini,1 Olivier Renaudet,3 Nathalie Berthet,3

Pascal Dumy,4 Yvonne Hed,2 Alberto Marra,4 Michael Malkoch,2 Alessandro Dondoni5

1Dipartimento di Scienze Chimiche e Farmaceutiche, Universit�a di Ferrara, Via Fossato di Mortara 17, 44121 Ferrara, Italy2Division of Coating Technology, KTH The Royal Institute of Technology, School of Chemical Science and Engineering,

Teknikringen 56-58, SE-10044 Stockholm, Sweden3D�epartement de Chimie Mol�eculaire, UMR CNRS 5250, Universit�e Joseph Fourier, 570 Rue de la chimie, BP 53, 38041 Grenoble

cedex 9, France4Institut des Biomol�ecules Max Mousseron (IBMM), UMR 5247, Universit�e Montpellier 2, Ecole Nationale Sup�erieure de Chimie

de Montpellier, 8 Rue de l’Ecole Normale, 34296 Montpellier cedex 5, France5Interdisciplinary Center for the Study of Inflammation, Universit�a di Ferrara, Via Borsari 46, 44100 Ferrara, Italy

Correspondence to: O. Renaudet (E-mail: [email protected]) or A. Marra (E-mail: [email protected]) or

M. Malkoch (E-mail: [email protected]) or A. Dondoni (E-mail: [email protected])

Received 31 March 2014; accepted 24 May 2014; published online 00 Month 2014

DOI: 10.1002/pola.27262

ABSTRACT: Alkene and alkyne functional polyester-based den-

drimers of generation 1 to 4 have been prepared and reacted

under free-radical conditions with 2-acetamido-2-deoxy-1-thio-b-D-

glucose (GlcNAc-SH). As the alkene-dendrimers underwent the

addition of one thiyl radical per ene group whereas each yne

group of alkyne-dendrimers was saturated by two thiyl radicals, a

collection of glycodendrimers with glycan density ranging from

six to ninety-six GlcNAc per dendrimer was obtained. The recogni-

tion properties of the prepared glycodendrimers toward the wheat

germ agglutinin (WGA) were evaluated by enzyme-linked lectin

assay (ELLA). The eight glycodendrimers were excellent ligands

showing IC50 values in the nanomolar range and relative potencies

per sugar unit up to 2.27 e6 when compared to monosaccharidic

GlcNAc used as monovalent reference. VC 2014 Wiley Periodicals,

Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 00, 000–000

KEYWORDS: carbohydrates; chiral; dendrimers; photochemistry;

radical reactions

INTRODUCTION Specific, reversible interactions between car-bohydrates at the cell surface (glycolipids and glycoproteins)and their protein receptors (lectins) play a crucial role in amultitude of biological and pathological processes includingfertilization, intercellular communication, viral or bacterialinfection, inflammation, tumor cell metastasis, and immuneresponse.1 Thus, analysis of carbohydrate-protein interac-tions and development of inhibitors/probes of these interac-tions are at the forefront of modern glycobiology. In thiscontext, it is important to take into account that lectins typi-cally bind monovalent carbohydrate ligands with low affinityand poor selectivity.2 In nature, this low intrinsic affinity iscompensated by the architecture of the lectin itself, bearingclustered binding sites, and by the presence of multiple cop-ies of lectin on the same surface (e.g., the viral envelope).3

Moreover, also the carbohydrate ligands are present as clus-ters on the bacterial or cell membrane. The resulting multi-valent complexes have more affinity, called avidity, and

selectivity than the combination of individual binding events,that is the whole interaction is greater than the sum of itsparts. This effect is known as the glycoside cluster effect.4

Therefore, multivalency has been a leading feature fordesigning inhibitors or probes of lectins. Consequently,researchers have been elaborating on synthetic oligosaccha-rides and their conjugation to a variety of multivalent scaf-folds including cyclopeptides,5 glycoproteins,6 dendrimersand dendrons,7 polymers,8 polymeric and gold nanopar-ticles,9 fullerenes,10 calixarenes,11 cyclodextrins,12 DNA,13

and silsesquioxanes.14 Each multivalent platforms displaysglycans differently with varying spacing, orientation, density,flexibility, and overall architecture. Along with the broadwindow of possible structural representations come the chal-lenges of identifying the scaffold that can provide optimalarrangement for a particular glycan-lectin interaction, partic-ularly when the spacing and geometries of the lectin bindingsites are not known. Therefore, the construction of

Additional Supporting Information may be found in the online version of this article.

VC 2014 Wiley Periodicals, Inc.

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JOURNAL OFPOLYMER SCIENCE WWW.POLYMERCHEMISTRY.ORG ARTICLE

glycoclusters with a known number of glycans that are con-fined to the surface of new scaffolds and evaluation of theiraffinity for specific lectins appears to be an empirical yet via-ble approach toward the discovery of new inhibitors. Basedon these premises, we herein report the exploitation of twodifferent photoinduced hydrothiolation click reactions: thefree-radical thiol-ene coupling (TEC)15 and thiol-yne coupling(TYC)16 for the introduction of an exact number of N-acetyl-glucosamine (GlcNAc) residues at the periphery of 2,2-bis(methylol)propanoic acid (bis-MPA)17 based polyester den-drimers, ranging from generation 1 to 4. The resulting librarycomprises glycodendrimers with variable glycan density of6–96 groups and their binding properties were evaluatedtoward the plant lectin wheat germ agglutinin (WGA) byEnzyme-Linked Lectin Assay (ELLA) experiments.

EXPERIMENTAL

Flash column chromatography was performed on silica gel60 (40–63 mm). Optical rotations were measured at 206 2�C in the stated solvent; [a]D values are given in deg mL g21

dm21. 1H NMR (400 MHz) and 13C NMR spectra (100 MHz)were recorded in the stated solvent at room temperatureunless otherwise specified. In the 1H NMR spectra reportedbelow, the n and m values quoted in geminal or vicinalproton-proton coupling constants Jn,m refer to the number ofthe corresponding sugar protons. MALDI-TOF mass spectros-copy was conducted on a Bruker UltraFlex with a SCOUT-MTP Ion Source (Bruker Daltonics) equipped with a N2-laser(337 nm), a gridless ion source and a reflector. The laserintensity was set to the lowest value possible to acquirehigh-resolution spectra. The spectra were acquired usingreflector mode with an acceleration of 25 kV to the extentpossible; however for compounds over 20 kDa a linear modewas required. The instrument was calibrated using Spheri-CalTM calibrants purchased from Polymer Factory SwedenAB. A THF solution of either 9-nitroanthracene or 2,5-dihy-droxybenzoic acid (DHB) (10 mg mL21) doped with sodiumtrifluoroacetate was used as the matrix. The obtained spectrawere analyzed with FlexAnalysis Bruker Daltonics version2.2. Size exclusion chromatography measurements were per-formed on a TOSOH EcoSEC HLC-8320GPC system equippedwith an EcoSEC RI detector and three columns (PSS PFG 5mm; Microguard, 100 Å, and 300 Å) (Mw resolving range:300–100,000 Da) from PSS GmbH, using DMF (0.2 mLmin21) with 0.01 M LiBr as the mobile phase at 50 �C. Aconventional calibration method was created using narrowlinear poly(methyl methacrylate) standards. Corrections forflow rate fluctuations were made using toluene as an inter-nal standard. PSS WinGPC Unity software version 7.2 wasused to process data. The photoinduced thiol-ene and thiol-yne reactions were carried out in a glass vial located 2.5 cmaway from the household UVA lamp apparatus equippedwith four 15 W tubes (1.5 3 27 cm each). The commerciallyavailable photoinitiator 2,2-dimethoxy-2-phenylacetophenone(DMPA) was used without further purification. The den-drimers 1–4 were purchased from Polymer Factory SwedenAB, Stockholm, Sweden. Horseradish peroxidase-labelled

Triticum vulgaris lectin WGA-HRP, bovine serum albumin(BSA), and SIGMAFAST O-phenylenediamine dihydrochloride(OPD) were purchased from Sigma-Aldrich. The 2-acetamido-2-deoxy-D-glucose-polyacrylamide (D-GlcNAc-PAA) was obtainedfrom Lectinity Holding, Moscow.

TMP-G1-ene6 (5)To a solution of TMP-G1-OH6 1 (1.23 g, 2.55 mmol) in pyri-dine (3.7 mL) and anhydrous CH2Cl2 (10 mL) were added4-dimethylaminopyridine (DMAP) (375 mg, 3.07 mmol) and3-[2-(allyloxy)ethoxycarbonyl]propanoic anhydride (7.71 g,19.95 mmol). The mixture was stirred at room temperaturefor 16 h, then diluted with H2O (4 mL) and stirred for 16 hto destroy the excess of anhydride. The mixture wasextracted with CH2Cl2 (100 mL) and the organic phase waswashed with 10% aqueous NaHSO4 (5 3 20 mL) and 10%aqueous Na2CO3 (2 3 20 mL), then dried (MgSO4) and con-centrated. The residue was eluted from a column of silica gelcolumn with n-heptane-AcOEt (from 4:1 to 1:4) to give 5(1.66 g, 41%) as a syrup.

1H NMR (CDCl3): d 5.85 (ddt, 6H, J5 5.8, 10.5, 17.6 Hz,6 CH@CH2), 5.27–5.21 and 5.17–5.13 (2 m, 12H, 6 CH@CH2),4.22–4.16 (m, 24H, 3 CH3C(CH2O)2, 6 CO2CH2CH2O), 4.02 (s,6H, CH3CH2C(CH2O)3), 3.98–3.96 (m, 12H, 6 CH2CH@CH2),3.61–3.58 (m, 12H, 6 AllOCH2), 2.63–2.56 (m, 24H,6 C(O)CH2CH2CO2), 1.44 (q, 2H, J5 7.7 Hz, CH2CH3), 1.20 (s,9H, 3 CH3C), 0.87 (t, 3H, CH2CH3).

13C NMR (CDCl3): d 172.1,171.7, 134.4, 117.4, 72.1, 72.0, 67.8, 65.3, 63.9, 63.7, 46.5,41.5, 28.8, 22.8, 17.8, 7.3. MALDI-TOF MS m/z Calcd. forC75H110NaO36 (M1Na)1 1609.667, found 1609.978.

TMP-G2-ene12 (6)The TMP-G2-OH12 2 (1.00 g, 0.85 mmol) was functionalizedas described for the preparation of 5 to give 6 (1.49 g, 52%)as a syrup.

1H NMR (CDCl3): d 5.86 (ddt, 12H, J5 5.8, 10.5, 17.6 Hz,12 CH@CH2), 5.28–5.22 and 5.18–5.14 (2 m, 24H, 12CH@CH2), 4.28–4.13 (m, 60H, 9 CH3C(CH2O)2, 12 CO2CH2-

CH2O), 4.06 (s, 6H, CH3CH2C(CH2O)3), 4.00–3.97 (m, 24H,12 CH2CH@CH2), 3.61–3.59 (m, 24H, 12 AllOCH2), 2.64–2.57(m, 48H, 12 C(O)CH2CH2CO2), 1.50 (q, 2H, J5 7.7 Hz,CH2CH3), 1.24 (s, 9H, 3 CH3C), 1.19 (s, 18H, 6 CH3C), 0.90 (t,3H, CH2CH3).

13C NMR (CDCl3): d 172.1, 171.9, 171.7, 134.5,117.4, 72.1, 67.8, 65.2, 64.1, 63.9, 46.8, 46.4, 41.5, 28.9, 17.8,17.6, and 7.5. MALDI-TOF MS m/z Calcd. for C159H230NaO78

(M1Na)1 3412.50, found 3411.79.


1H NMR (CDCl3): d 5.88 (ddt, 24H, J5 5.8, 10.5, 17.6 Hz,24 CH@CH2), 5.29–5.23 and 5.19–5.15 (2 m, 48H, 24CH@CH2), 4.32–4.14 (m, 132H, 21 CH3C(CH2O)2, 24 CO2CH2-

CH2O), 4.10 (s, 6H, CH3CH2C(CH2O)3), 4.02–3.99 (m, 48H,24 CH2CH@CH2), 3.63–3.60 (m, 48H, 24 AllOCH2), 2.66–2.58

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(m, 96H, 24 C(O)CH2CH2CO2), 1.53 (q, 2H, J5 7.7 Hz,CH2CH3), 1.29 (s, 9H, 3 CH3C), 1.24 (s, 18H, 6 CH3C), 1.21 (s,36H, 12 CH3C), 0.93 (t, 3H, CH2CH3).

13C NMR (CDCl3)selected data: d 172.2, 172.0, 171.8, 171.7, 134.6, 117.5,72.2, 67.9, 65.2, 64.0, 46.8, 46.7, 46.4, 28.9, 17.9, and 17.7.MALDI-TOF MS m/z Calcd. for C327H470NaO162 (M1Na)1

7016.18, found 7013.98.


1H NMR (CDCl3) selected data: d 5.86 (ddt, 48H, J5 5.8,10.5, 17.6 Hz, 48 CH@CH2), 5.27–5.21 and 5.17–5.13 (2 m,96H, 48 CH@CH2), 4.32–4.13 (m, 276H, 45 CH3C(CH2O)2, 48CO2CH2CH2O), 3.99–3.96 (m, 96H, 48 CH2CH@CH2), 3.61–3.58 (m, 96H, 48 AllOCH2), 2.63–2.55 (m, 192H,48 C(O)CH2CH2CO2), 1.29 (s, 9H, 3 CH3C), 1.25 (s, 18H, 6CH3C), 1.23 (s, 36H, 12 CH3C), 1.19 (s, 72H, 24 CH3C).

13CNMR (CDCl3) selected data: d 172.2, 172.0, 171.7, 171.6,134.6, 117.3, 72.1, 67.9, 65.0, 63.9, 46.6, 46.3, 28.9, 17.8, and17.6. MALDI-TOF MS m/z Calcd. for C663H950NaO330

(M1Na)1 14,223.55, found 14,233.78.

TMP-G1-yne6 (9)To a solution of TMP-G1-OH6 1 (1.31 g, 2.71 mmol) in pyri-dine (4 mL) and anhydrous CH2Cl2 (12 mL) were addedDMAP (400 mg, 3.27 mmol) and 3-(propargyloxycarbonyl)-propanoic anhydride (6.25 g, 21.24 mmol). The mixture wasstirred at room temperature for 16 h, then diluted with H2O(4 mL) and stirred for 16 h to destroy the excess of anhy-dride. The mixture was extracted with CH2Cl2 (100 mL) andthe organic phase was washed with 10% aqueous NaHSO4

(5 3 20 mL) and 10% aqueous Na2CO3 (2 3 20 mL), thendried (MgSO4) and concentrated. The residue was elutedfrom a column of silica gel column with n-heptane-AcOEt(from 4:1 to 1:4) to give 9 (2.35 g, 66%) as a syrup.

1H NMR (CDCl3): d 4.65 (d, 12H, J5 2.4 Hz, 6 CH2CB CH), 4.21and 4.18 (2 d, 12H, J5 12.0 Hz, 3 CH3C(CH2O)2), 4.02 (s, 6H,CH3CH2C(CH2O)3), 2.65–2.57 (m, 24H, 6 C(O)CH2CH2CO2), 2.48(t, 6H, 6 CBCH), 1.45 (q, 2H, J5 7.7 Hz, CH2CH3), 1.21 (s, 9H, 3CH3C), 0.88 (t, 3H, CH2CH3).

13C NMR (CDCl3): d 172.1, 171.5,171.4, 77.6, 75.2, 65.4, 63.7, 60.4, 52.3, 46.5, 41.5, 28.7, 22.9,21.1, 17.8, 14.2, and 7.4. MALDI-TOF MS (matrix: DHB) m/zCalcd. for C63H74NaO30 (M1Na)1 1333.416, found 1333.394.

TMP-G2-yne12 (10)The TMP-G2-OH12 2 (1.00 g, 0.85 mmol) was functionalizedas described for the preparation of 9 to give 10 (1.71 g,71%) as a syrup.

1H NMR (CDCl3): d 4.61 (d, 24H, J5 2.4 Hz, 12 CH2CBCH),4.23 and 4.18 (2 d, 12H, J5 11.5 Hz, 3 CH3C(CH2O)2), 4.16and 4.12 (2 d, 24H, J5 11.3 Hz, 6 CH3C(CH2O)2), 4.03 (s, 6H,CH3CH2C(CH2O)3), 2.63–2.55 (m, 48H, 12 C(O)CH2CH2CO2),2.47 (t, 12H, 12 CBCH), 1.47 (q, 2H, J5 7.7 Hz, CH2CH3),1.21 (s, 9H, 3 CH3C), 1.16 (s, 18H, 6 CH3C), 0.88 (t, 3H,

CH2CH3).13C NMR (CDCl3): d 171.8, 171.6, 171.4, 171.3,

77.5, 77.4, 75.2, 75.1, 65.2, 64.0, 60.3, 52.1, 46.7, 46.3, 41.3,31.8, 28.9, 28.6, 23.0, 22.6, 21.0, 17.7, 17.5, 14.1, and 7.4.MALDI-TOF MS (matrix: DHB) m/z Calcd. for C135H158NaO66

(M1Na)1 2857.890, found 2857.906.


1H NMR (CDCl3): d 4.66 (d, 48H, J5 2.4 Hz, 24 CH2CBCH),4.30–4.18 (m, 36H, 9 CH3C(CH2O)2), 4.20 and 4.16 (2 d,48H, J5 11.3 Hz, 12 CH3C(CH2O)2), 4.09 (s, 6H,CH3CH2C(CH2O)3), 2.66–2.60 (m, 96H, 24 C(O)CH2CH2CO2),2.51 (t, 24H, 24 CBCH), 1.54 (q, 2H, J5 7.7 Hz, CH2CH3),1.29 (s, 9H, 3 CH3C), 1.23 (s, 18H, 6 CH3C), 1.20 (s, 36H, 12CH3C), 0.92 (t, 3H, CH2CH3).

13C NMR (CDCl3): d 172.0,171.63, 171.58, 171.4, 77.7, 77.4, 75.3, 65.3, 65.1, 53.6, 52.3,46.8, 46.7, 46.4, 28.8, 18.6, 17.8, 17.6, and 7.6. MALDI-TOFMS (matrix: DHB) m/z Calcd. for C279H326NaO138 (M1Na)1

5910.53, found 5911.00.


1H NMR (CDCl3): d 4.68 (d, 96H, J5 2.4 Hz, 48 CH2CBCH),4.36–4.12 (m, 186H, 45 CH3C(CH2O)2, 1 CH3CH2C(CH2O)3),2.68–2.62 (m, 192H, 48 C(O)CH2CH2CO2), 2.54 (t, 48H, 48CBCH), 1.50 (q, 2H, J5 7.7 Hz, CH2CH3), 1.33 (s, 9H, 3CH3C), 1.28 (s, 18H, 6 CH3C), 1.26 (s, 36H, 12 CH3C), 1.22 (s,72H, 24 CH3C), 0.90 (t, 3H, CH2CH3).

13C NMR (CDCl3)selected data: d 172.1, 171.8, 171.7, 171.6, 77.9, 77.4, 75.4,65.4, 52.4, 46.8, 46.5, 28.9, 17.9, and 17.7. MALDI-TOF MS(matrix: DHB) m/z Calcd. for C567H662NaO282 (M1Na)1

12,012.24, found 12,017.85.

TMP-G1-GlcNAc6 (14)To a solution of TMP-G1-ene6 5 (50 mg, 31.5 mmol), thiol 13(90 mg, 0.38 mmol), and DMPA (2.9 mg, 11.3 mmol) in DMF(300 lL), partially concentrated under vacuum (ca., 0.1mbar) to remove the traces of Me2NH, was slowly addedH2O (200 lL). The mixture was irradiated (kmax 365 nm)under vigorous stirring at room temperature for 1 h andthen concentrated. The residue was eluted from a column ofSephadex LH-20 (2 3 50 cm, d 3 h) with 1:1 MeOH-H2O togive 14 (56 mg, 59%) as a white powder; [a]D 5 28.6 (c 0.4,H2O).

1H NMR (D2O) selected data: d 4.58 (d, 6H, J1,2 5 10.5 Hz, 6H-1), 4.31–4.17 (m, 24H, 3 CH3C(CH2O)2, 6 CO2CH2CH2O),4.11 (s, 6H, CH3CH2C(CH2O)3), 2.83–2.76 and 2.73–2.66(2 m, 12H, 6 CH2S), 2.72–2.63 (m, 24H, 6 C(O)CH2CH2CO2),2.01 (s, 18H, 6 Ac), 1.94–1.80 (m, 12H, 6 CH2CH2CH2), 1.25(s, 9H, 3 CH3C), 0.92 (t, 3H, J5 7.7 Hz, CH2CH3).

13C NMR(D2O): d 173.96, 173.92, 173.5, 84.2, 79.9, 75.1, 69.7, 69.2,68.0, 65.9, 64.0, 60.9, 54.7, 46.5, 29.0, 28.79, 28.72, 26.8,



22.2, 17.1, and 6.8. MALDI-TOF MS m/z Calcd. forC123H200N6NaO66S6 (M1Na)1 3032.079, found 3032.226.

TMP-G2-GlcNAc12 (15)The dendrimer TMP-G2-ene12 6 (51 mg, 15.0 mmol) wasallowed to react with the thiol 13 as described for the prep-aration of 14 to give, after column chromatography onSephadex LH-20 (1:1 MeOH-H2O), 15 (46 mg, 49%) as awhite powder; [a]D 5 210.2 (c 0.5, H2O).

1H NMR (D2O) selected data: d 4.58 (d, 12H, J1,2 5 10.5 Hz,12 H-1), 2.82–2.75 and 2.72–2.65 (2 m, 24H, 12 CH2S),2.71–2.62 (m, 48H, 12 C(O)CH2CH2CO2), 2.00 (s, 36H, 12Ac), 1.91–1.80 (m, 24H, 12 CH2CH2CH2), 1.30 (s, 9H, 3CH3C), 1.23 (s, 18H, 6 CH3C).

13C NMR (D2O) selected data:d 173.9, 173.8, 173.3, 84.2, 79.9, 75.2, 69.7, 69.2, 68.0, 64.0,60.9, 54.7, 48.8, 46.4, 29.0, 28.7, 26.7, 22.2, and 17.1.MALDI-TOF MS m/z Calcd. for C255H410N12NaO138S12(M1Na)1 6255.198, found 6255.467.


1H NMR (D2O) selected data: d 4.58 (d, 24H, J1,2 5 10.5 Hz,24 H-1), 4.33–4.10 (m, 138H, 21 CH3C(CH2O)2, 24 CO2CH2-

CH2O, 1 CH3CH2C(CH2O)3), 2.83–2.75 and 2.72–2.64 (2 m,48H, 24 CH2S), 2.71–2.60 (m, 96H, 24 C(O)CH2CH2CO2), 2.01(s, 72H, 24 Ac), 1.92–1.80 (m, 48H, 24 CH2CH2CH2), 1.31 (s,9H, 3 CH3C), 1.26 (s, 18H, 6 CH3C), 1.22 (s, 36H, 12 CH3C).13C NMR (D2O) selected data: d 174.3, 88.7, 80.1, 74.7, 69.3,60.5, 54.0, and 22.1. MALDI-TOF MS m/z Calcd. forC519H830N24NaO282S24 (M1Na)1 12,710.75, found 12,712.49.


1H NMR (D2O) selected data: d 4.59 (d, 48H, J1,2 5 10.5 Hz,48 H-1), 4.26–4.12 (m, 282H, 45 CH3C(CH2O)2, 48 CO2CH2-

CH2O, 1 CH3CH2C(CH2O)3), 2.83–2.75 and 2.72–2.64 (2 m,96H, 48 CH2S), 2.72–2.60 (m, 192H, 48 C(O)CH2CH2CO2),2.01 (s, 144H, 48 Ac), 1.92–1.80 (m, 96H, 48 CH2CH2CH2).13C NMR (D2O) selected data: d 84.2, 79.9, 75.2, 69.7, 69.2,68.1, 63.9, 60.9, 54.7, 46.3, 29.1, 26.7, 22.3, and 17.2.MALDI-TOF MS m/z Calcd. for C1047H1670N48NaO570S48(M1Na)1 25,612.67, found 25,776.81.

TMP-G1-GlcNAc12 (18)A solution of the dendrimer TMP-G1-yne6 9 (16 mg, 12.2mmol), thiol 13 (69 mg, 0.29 mmol), and DMPA (2.2 mg, 8.8mmol) in AcOEt (50 mL), DMF (300 lL), partially concen-trated under vacuum (ca., 0.1 mbar) to remove the traces of

Me2NH, and H2O (50 mL) was irradiated (kmax 365 nm)under vigorous stirring at room temperature for 1 h andthen concentrated. The residue was eluted from a column ofSephadex LH-20 (2 3 50 cm, d 3 h) with 1:1 MeOH-H2O togive 18 (35.5 mg, 70%) as a syrup.

1H NMR (D2O) selected data: d 4.59, 4.56, 4.53, and 4.47 (4 d,12H, J1,25 10.5 Hz, 12 H-1), 4.37–4.25 (m, 6H, 3 OCH2CH2S),4.19–4.07 (m, 18H, 3 CH3C(CH2O)2, 3 OCH2CH2S), 4.01–3.95(bs, 6H, CH3CH2C(CH2O)3), 3.03–2.85 (m, 12H, 6 CH2S), 2.61–2.49 (m, 24H, 6 C(O)CH2CH2CO2), 1.87 (bs, 36H, 12 Ac), 1.13(s, 9H, 3 CH3C), 0.80 (t, 3H, J5 7.5 Hz, CH2CH3). MALDI-TOFMS (matrix: THAB) m/z Calcd. for C159H254N12NaO90S12(M1Na)1 4181.53, found 4182.30.

TMP-G2-GlcNAc24 (19)The dendrimer TMP-G2-yne12 10 (17 mg, 6.0 lmol) wasallowed to react with the thiol 13 (68 mg, 0.29 mmol) in 4:1DMF-H2O (500 mL) as described for the preparation of 18 togive, after column chromatography on Sephadex LH-20 (1:1MeOH-H2O), 19 (40 mg, 78%) as a syrup.

1H NMR (D2O) selected data: d 4.70, 4.67, 4.60, and 4.53 (4d, 24H, J1,2 5 10.5 Hz, 24 H-1), 3.12–2.98, 2.96–2.88, and2.85–2.77 (3 m, 24H, 12 CH2S), 2.68–2.54 (m, 48H,12 C(O)CH2CH2CO2), 1.94 (bs, 72H, 24 Ac), 1.24 (s, 9H, 3CH3C), 1.16 (s, 18H, 6 CH3C). MALDI-TOF MS (matrix: THAB)m/z Calcd. for C327H518N24NaO186S24 (M1Na)1 8554.23,found 8555.10.

TMP-G3-GlcNAc48 (20)The dendrimer TMP-G3-yne24 11 (17 mg, 2.9 lmol) wasallowed to react with the thiol 13 (66 mg, 0.28 mmol) in 4:1DMF-H2O (500 mL) as described for the preparation of 18 togive, after column chromatography on Sephadex LH-20 (1:1MeOH-H2O), 20 (43 mg, 86%) as a syrup.

1H NMR (D2O) selected data: d 4.76, 4.73, 4.68, and 4.61 (4bd, 48H, J1,2 5 10.5 Hz, 48 H-1), 3.22–3.07, 3.05–2.96, and2.92–2.83 (3 m, 48H, 24 CH2S), 2.03 (bs, 144H, 48 Ac),1.34–1.20 (3 bs, 63H, 21 CH3C). Anal. Calcd. forC663H1046N48O378S48.100H2O: C, 41.74; H, 6.58; N, 3.52; S,8.07. Found: C, 41.48; H, 6.46; N, 3.22; S, 7.27.

TMP-G4-GlcNAc96 (21)The dendrimer TMP-G4-yne48 12 (10 mg, 0.83 lmol) wasallowed to react with the thiol 13 (38 mg, 0.16 mmol) in2:1:1 DMF-H2O-MeOH (400 mL) as described for the prepara-tion of 18 to give, after column chromatography on Sepha-dex LH-20 (1:1 MeOH-H2O), 21 (19 mg, 65%) as a syrup.

1H NMR (D2O) selected data: d 2.76–2.57 (m, 192H,48 C(O)CH2CH2CO2), 2.05 (bs, 288H, 96 Ac), 1.37–1.20 (m,135H, 45 CH3C) Anal. Calcd. for C1335H2102N96O762S96.80H2O:C, 44.28; H, 6.30; N, 3.71; S, 8.50. Found: C, 44.38; H, 6.38;N, 3.26; S, 9.00.

ELLA96-well microtiter Nunc-Immuno plates (Maxi-Sorp) werecoated with PAA-GlcNAc (100 mL per well, diluted from a


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stock solution of 5 mg mL21 in 50 mM carbonate buffer pH9.6) for 1 h at 37 �C. The wells were then washed withT-PBS [3 3 100 mL well21, PBS pH 7.4 containing 0.05%(v/v) Tween 20]. This washing procedure was repeated aftereach incubation step. The coated microtiter plates were thenblocked with BSA in PBS (3%, w/v, 1 h at 37 �C, 100 mL perwell). Serial two-fold dilutions of each inhibitor was pre-incubated 1 h at 37 �C in PBS (60 mL per well) in the pres-ence of WGA-HRP (60 mL) at the desired concentration. Theabove solutions (100 mL) were then transferred to theblocked microtiter plates which were incubated for 1 h at 37�C. After incubation, the plates were washed with T-PBS (33 100 mL per well) then the color was developed using OPD(100 mL per well, 0.4 mg mL21 in 0.05 M phosphate-citratebuffer) and urea hydrogen peroxide (0.4 mg mL21). Thereaction was stopped after 10 min by adding H2SO4 (30%,v/v, 50 mL per well) and the absorbance was measured at490 nm. The percentage of inhibition was plotted against thelogarithm of the concentration of the sugar derivatives. Thesigmoidal curves were fitted and the concentrations at 50%inhibition of binding of WGA-HRP to PAA-GlcNAc coatedplates were determined (IC50). The percentages of inhibitionwere calculated as given in the equation below, whereA5 absorbance. The IC50 values were systematically per-formed in triplicate.

% Inhibition5 [(A(no inhibitor) 2A(with inhibitor))/A(no inhibitor)]3 100

RESULTS AND DISCUSSION

A wide variety of glycodendrimers with different architectureand generation have been constructed by covalent attach-ments of glycan arrays on the periphery of dendritic scaf-folds.7(d) From the large number of different yet efficientligation tools that have been employed, the copper-catalyzedversion of the Huisgen azide-alkyne cycloaddition (CuAAC)has occupied an important role. As an alternative strategy,we recently reported on the synthesis of glycodendrimers byphotochemically induced addition of sugar thiols to thealkene groups displayed at the periphery of a fourth genera-tion dendrimer scaffold.18 Our choice to use the TEC clickreaction19 for grafting of the carbohydrate residues was dic-tated by two main reasons, namely the need to overcomethe serious limitation of CuAAC arising from the contamina-tion of the product by significant amounts of copper-basedcatalyst20 and the intent to replace the rigid and potentiallyimmunogenic21 triazole linker with a flexible, sterically non-demanding and non-immunogenic21(b) thioether linkage. It isnow well established that free-radical TEC has several desir-able attributes (high chemoselectivity and regioselectivity aswell as complete atom economy and unique tolerance to oxy-gen and moisture) and it is considered a valuable metal-freeligation tool for conjugation of sensitive molecules such ascarbohydrates, peptides, and proteins.22 Notably, TEC occursat room temperature in aqueous solvents under neutral con-ditions and is promoted by irradiation at wavelength closeto visible light. Thus, based on these premises we selected

TEC as a tool for the installation of GlcNAc residues at thesurface of aliphatic bis-MPA dendrimers of generation 1–4.The choice of these dendrimers as scaffolds was based ontheir biocompatibility23 and the facile approach to intro-duce allyl groups necessary for the UV initiated TEC withthe thiol functional sugars. Additionally, the free-radicalTYC was sought out as a complementary sister reaction tothe TEC. This reaction is emerging as an efficient chemose-lective metal-free ligation tool for glycoconjugate synthe-sis.24 TYC leads to the formation of bis-addition products,that is, bis-thioethers, via exclusive 1,2-addition mode oftwo thiyl radicals across the alkyne triple bond. Therefore,by using TYC the constructed glycodendrimers will displaya carbohydrate density twice of that resulting from the den-dritic scaffolds that undergo TEC. Finally, it has to be notedthat both TEC and TYC allow the attachment of the sugarresidues to the dendritic scaffold by thioglycosidic linkages.The latter, featuring CAS bond lengths and CASAC bondangles similar to those of O-glycosides,25 are stable towardchemical and enzymatic hydrolysis.26 The beneficial effectof sulfur chemistry in glycodendrimers has been recentlyhighlighted.27

In this context, a library of glycodendrimers was affordedthrough a facile two-step approach using a set of commer-cially available hydroxyl functional polyester-based bis-MPAdendrimers of four different generations, which originatefrom a tris(hydroxymethyl)propane (TMP) core,23 that is,TMP-G1-OH6 1, TMP-G2-OH12 2, TMP-G3-OH24 3, and TMP-G4-OH48 4 (Fig. 1). All dendrimers were separately treatedwith excess (1.3 equiv./OH group) of 3-[2-(allyloxy)ethoxy-carbonyl]propanoic anhydride in the presence of pyridineand DMAP to give the corresponding allylated dendrimersTMP-G1-ene6 5, TMP-G2-ene12 6, TMP-G3-ene24 7, TMP-G4-ene48 8 in 41–68% isolated yields (Fig. 1). Following asimilar strategy, treatment of the hydroxylated dendrimers1–4 with 3-(propargyloxycarbonyl)propanoic anhydride(1.3 equiv./OH group) afforded the propargylated den-drimers TMP-G1-yne6 9, TMP-G2-yne12 10, TMP-G3-yne2411, TMP-G4-yne48 12 in 41–71% yields (Fig. 1). The TMP-GX-enen 5–8 and TMP-GX-ynen 9–12 dendrimers werecharacterized by 1H and 13C NMR spectroscopy (Figs. 2and 3) and MALDI-TOF mass spectrometry (see“Experimental” section). The latter analysis clearly demon-strated that for each dendrimer all the hydroxyl functionswere converted into the corresponding allyl or propargylreactive groups.

With the two sets of alkene and alkyne functional den-drimers 5–8 and 9–12 in hand, synthetic efforts weredirected towards the introduction of sugar units via TEC andTYC. To this end, the known28 2-acetamido-2-deoxy-1-thio-b-D-glucose 13 (D-GlcNAc-SH) was selected for construction ofglycodendrimers and the evaluation of their binding efficacytoward the WGA, a lectin from Triticum vulgaris that is spe-cific for D-GlcNAc. Both photoinduced TEC and TYC couplingswere efficiently carried out under benign conditions



employed for glycodendrimer synthesis.18 Typically, DMF-H2O solutions of individual allylated or propargylated den-drimers TMP-GX-enen 5–8 and TMP-GX-ynen 9–12, respec-tively, and excess of thiol 13 (2 equiv. of 13/ene group and4 equiv. of 13/yne group) in the presence of DMPA as initia-tor were irradiated (kmax 5 365 nm) for 1 h to give the gly-codendrimers 14–17 and 18–21 in 49–63% and 65–86%isolated yield, respectively (Fig. 4).

The 1H NMR analysis of the crude mixtures revealed thetotal disappearance of ene (5–6 ppm) and yne (ca., 2.5 ppm)functional groups after the photoinduced TEC or TYC reac-tions (Fig. 5). The glycodendrimers were separated from

the unreacted thiol 13 and the corresponding disulfidebyproduct by column chromatography on Sephadex LH-20using a 1:1 MeOH-H2O solution as the eluent. The structureof the TEC adducts 14–17, containing from 6 to 48 GlcNACunits, was confirmed by MALDI-TOF MS analysis (see“Experimental” section). Interestingly, gel permeation chro-matography (GPC) analysis revealed molecular weights anddispersities significantly above the expected values. Thesehigher values are hypothesized to arise from a clusteringeffect of the glycodendrimers through extensive hydrogenbonding. In fact, assessing solutions of TMP-G2-GlcNAc12 15in the LiBr-doped DMF eluent after 1 day and 1 month indi-cated further, yet not complete, dissolution and a molecular

FIGURE 1 Alkene (5–8) and alkyne (9–12) functional dendrimers prepared from TMP-GX-OHn 1–4.


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weight approaching the theoretical MW of 6232 g mol21

(Fig. 6). In the case of the TYC adducts 18–21, the MALDI-TOF mass spectra (matrix: THAB) showed molecular weightsin good agreement with the calculated ones only for theTMP-G1-GlcNAc12 18 and TMP-G2-GlcNAc24 19 adducts (see“Experimental” section). As the MALDI spectra for the G3and G4 glycodendrimers 20 (Mw 5 17,277) and 21(Mw 5 34,768), respectively, could not be obtained, thesecompounds were characterized by consistent elemental anal-ysis of their hydrated forms (found values for carbon andhydrogen within 0.3% of the calculated values).

The library of synthesized glycodendrimers featured sugardensities ranging from six units per molecule for the TMP-G1-GlcNAc6 14 up to an impressive 96 units per moleculefor the TMP-G4-GlcNAc96 21 obtained by TYC. Because ofthe high density of peripheral carbohydrate residues and theglobular structure, the latter glycodendrimer can be com-pared to an “artificial glycoprotein.”29 Having in hand sub-

stantial amounts of the eight glycodendrimers 14–21 whoseelements of diversity were dendritic generation and densityof the attached sugar residues, we set out to study theirbinding properties toward the WGA, a 36-kDa homodimericlectin from Triticum vulgaris, which is known to be specificfor N-acetylneuraminic acid (Neu5Ac) and 2-acetamido-2-deoxy-D-glucose (D-GlcNAc).30 Each monomer of WGA isorganized into four domains (A–D) containing adjacent“primary” (B and C domains) and “secondary” (A and Ddomains) binding sites, separated by approximately 14 Å,with different affinities for GlcNAc. Whereas binding proper-ties of various glycodendrimers toward specific lectins havebeen reported,7(d),31 studies regarding the use of WGA wereonly limited to a few small size glycoclusters.32 Thus, as inour recent studies of silsesquioxane14(c,e) and cyclodecapep-tide-based33 glycocluster-WGA interaction, the binding affin-ities of glycoconjugates 14–21 toward WGA weresystematically evaluated by competitive ELLA. These assaysprovide a fast method to evaluate lectin binding properties

FIGURE 2 1H NMR spectra (400 MHz, CDCl3) of allylated dendrimers 5 (a), 6 (b), 7 (c), and 8 (d).

FIGURE 3 1H NMR spectra (400 MHz, CDCl3) of propargylated dendrimers 9 (a), 10 (b), 11 (c), and 12 (d).



of either natural or unnatural ligands in solution. Microtiterplates are coated with a reference ligand (a glycoprotein, apolysaccharide or a glycopolymer) and the binding inhibitionof an enzyme-labelled lectin to the immobilized reference

ligand by the soluble ligands to be tested is evaluated. TheIC50 values that are determined correspond to the concentra-tion of glycoclusters necessary to prevent 50% of thebinding.

FIGURE 4 Glycodendrimers 14–17 and 18–21 prepared via thiol-ene and TYCs, respectively.


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All compounds showed excellent inhibitory properties of thebinding of horseradish peroxidase-labelled WGA (WGA-HRP)to a D-GlcNAc-PAA conjugate with IC50 values in the 0.26–27 nM range (Table 1). In the thiol-ene series, the ligandTMP-G1-GlcNAc6 14, featuring the lowest valency, showed a106-fold binding improvement (relative potency, rp) withrespect to monosaccharidic D-GlcNAc used as reference,which corresponds to a relative potency reported to thenumber of sugars (rp/n) of 1.7 3 105. Interestingly, extend-ing the carbohydrate clustering to 12 (15) and 24 sugarunits (16) led to the expected decrease of the IC50 values(from 27 to 2.7 and 4.5 nM, respectively) which, however,was accompanied by only a moderate enhancement of theinhibition (5- and 1.5-fold) in a per sugar basis compared tothe hexavalent ligand 14 (e.g., 16: rp/n5 260,000, 14: rp/n5 170,000). A strong effect was achieved with TMP-G4-GlcNAc48 17, which showed an IC50 value of 0.26 nM, muchlower than those recently reported by us for other multiva-lent glycoconjugates synthesized by TEC (Fig. 7), that is, thetetravalent cyclodecapeptide-based cluster33 22 (1.5 nM),the octavalent14(c) 23 (3 nM) and hexadecavalent14(e) 24(2 nM) silsesquioxane GlcNAc-clusters (Table 1). Therefore,

by comparing the IC50 values, it appears that the very largeligand 17, bearing 48 GlcNAc units (Mw 5 25,589), was thesole glycodendrimer of the thiol-ene series able to bind tothe WGA more efficiently than the cyclopeptide (22) as wellas the silsesquioxane (23, 24) GlcNAc-clusters featuringmuch lower valency (4–16) and molecular weight (2247–6060 Da). However, the relative potency found for 17 (rp/n5 2.27 3 106) was only 13-fold higher than that observedfor the hexavalent TMP-G1-GlcNAc6 14 (1.7 3 105) andaround two-fold higher than that found for the octavalent(23) and hexadecavalent (24) silsesquioxane-based glycoclus-ters. Conversely, the glycodendrimer 17 was endowed withless than half of the relative potency (rp/n) showed by thecyclopeptide-based cluster 22 (2.27 3 106 vs. 4.87 3 106).

FIGURE 5 1H NMR spectra (400 MHz, D2O) of GlcNAc thiol 13 (a), propargylated dendrimer 9 (b) (in CDCl3), crude glycodendrimer

18 (c), and glycodendrimer 18 after purification on Sephadex LH-20 column (d).

FIGURE 6 GPC-traces of TMP-G2-GlcNAc12 15 after 1 day

(gray) and 1 month (black) in DMF containing LiBr (0.01 M).

TABLE 1 ELLA data for the inhibition of the binding of WGA-

HRP to PAA-GlcNAc by the glycodendrimers 14-–21 and the

previously reported glycoclusters 22-–24 (Figure 7)a

Compound nb IC50 (nM) rpc rp/nd

D-GlcNAc 1 2.8 e7 6 2 e6 1 1

TMP-G1-GlcNAc6 14 6 27 6 10 1.04 e6 170,000

TMP-G2-GlcNAc12 15 12 2.7 6 0.4 10.3 e6 860,000

TMP-G3-GlcNAc24 16 24 4.5 6 1.5 6.4 e6 260,000

TMP-G4-GlcNAc48 17 48 0.26 6 0.2 109 e6 2,270,000

TMP-G1-GlcNAc12 18 12 8.30 6 0.4 3.37 e6 280,000

TMP-G2-GlcNAc24 19 24 1.39 6 0.2 2.01 e6 840,000

TMP-G3-GlcNAc48 20 48 1.46 6 0.3 19.2 e6 400,000

TMP-G4-GlcNAc96 21 96 1.06 6 0.2 26.4 e6 270,000

Cyclopeptide 22 4 1.5 6 0.3 19.5 e6 4,875,000

Silsesquioxane 23 8 3.0 6 0.6 9.3 e6 1,162,000

Silsesquioxane 24 16 2.0 6 0.5 14.4 e6 900,000

a Each experiment was realized in triplicate.b Number of sugar unit in the glycoconjugate.c Relative potency 5 IC50(monosaccharide)/IC50(glycodendrimer).d Relative potency/number of sugar units.



The multivalent ligands of the thiol-yne series, that is, 18–21, displaying twice the amount of sugar units in respect tothe same generation glycodendrimers obtained by TEC, alsorevealed IC50 values in the nanomolar range (1.06–8.30 nM)with rp/n values ranging from 2.7 3 105 to 8.4 3 105 (Table1). However, the latter values did not improve when thevalency increased, but, on the contrary, decreased signifi-cantly in the case of the highest valency glycodendrimers 20and 21. Moreover, upon comparison of the rp/n values foundfor the couples of thiol-ene and thiol-yne based glycoden-drimers displaying the same number of GlcNAc moieties (i.e.,15 and 18, 16 and 19, 17 and 20), it appears that thehigher ligand density (number of sugars per volume or MWunit of dendrimeric scaffold) did not enhance the inhibitionpotency toward the WGA, the only exception being the cou-ple 16 and 19 (260,000 vs. 840,000). Finally, the ELLAexperiments indicated that none of the four glycodendrimers

18–21 displayed a relative potency per sugar unit (rp/n)higher than that observed for the hexadecavalentsilsesquioxane-based glycocluster 24 prepared by TYC (Table1). It is worth noting that compounds 18–21 were actuallycomplex mixtures of diastereomers due to the lack of stereo-selectivity of the TYC.24(f) In fact, a new stereocenter isformed after the addition of the second sugar unit to eachintermediate vinylthioether arm of the dendrimer. Therefore,the possibility of different binding toward WGA displayed bythe various stereoisomers of the same glycodendrimer can-not be ruled out. Anyway, all these data suggest that thepolyester-based dendrimer display sugar units in a favour-able orientation to ensure extremely strong multivalentinteractions with the lectin binding sites. Nevertheless,increasing ligand density and, to a lesser extent, ligandvalency in this dendritic framework led to moderateimprovement or even reduced lectin interaction, as alreadyobserved in previous assays carried out with nanomolar lec-tin ligands.7(d),31 It should be noted that ELLA experimentsmeasure the ability of a ligand to inhibit the binding of a lec-tin to an immobilized glycopolymer. Therefore, the IC50 valueis only indicative of the binding potency of the ligand to thelectin in reference to the immobilized compound. In order tofully assess the binding properties of the glycodendrimers14–21 toward the WGA, assays such as Isothermal TitrationCalorimetry or Surface Plasmon Resonance should beperformed.

CONCLUSIONS

Photoinduced TEC and TYC demonstrated their power andfidelity for the introduction of GlcNAc residues at the periph-ery of four generation dendrimers to give globular glycoden-drimers holding the sugar fragments by flexible and notencumbering thioglycosidic linkages. The ELLA-based bioas-says demonstrated excellent binding properties of all thesynthesized glycodendrimers toward the WGA as proved bytheir IC50 values in the nanomolar or, in one case, sub-nanomolar range.

ACKNOWLEDGMENTS

The authors thank the Universit�a di Ferrara and the EcoleNationale Sup�erieure de Chimie de Montpellier for financialsupport, and the Laboratoire de mesures physiques (Universit�eMontpellier 2) for MS and elemental analyses.

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