Clustering around the octasilsesquioxane scaffold via photoinduced free-radical thiol-ene and thiol-yne

coupling. Observation of a striking glycoside cluster effect

Samuele Staderini1, Alberto Marra1, Nathalie Berthet2, Pascal Dumy2, Olivier Renaudet2 and Alessandro Dondoni1

1Dipartimento di Chimica, Laboratorio di Chimica Organica, Università di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy,2Département de Chimie Moléculaire, UMR CNRS 5250, Université Joseph Fourier, 570 Rue de la Chimie, 38041, Grenoble, France

A new polyhedral oligomeric silsesquioxane (POSS) derivatives possessing a periphery of eight PEGylated chains functionalized with terminal allyl or propargyl groups was synthesized starting from the commercially available octavinyl POSS. The photoinduced free-radical coupling of these octaallyl and octapropargyl POSS derivatives with various sugar thiols enabled the preparation of globular octavalent and hexadecavalent glycoclusters. The affinities of some selected densely glycosylated clusters

toward specific lectins were measured by the Enzyme-Linked Lectin Assay (ELLA).

ProductNumber of sugar units (n)

IC50 (µM)

Relative Potency (rp; IC50 mono / IC50 cluster)

rp / n

1 1422±129 1 1

8 40,4±0,7 35,2 4,4

1 328±27 1 1

8 6,8±0,9 48,2 6

1 28000±2500 1 1

8 0,003±0,0006 9,3 x 106 106

R1S

R2 R1S

R2

SR1

R1S

R1SH

R1S

R2

R1SH

R1S

R2

SR1

R2

R2S

R1R2S

R1

R2SH

R2S

R1

OMe

O

OH

HOHO

OH

O

OH

HOHO

HOS

OMe

O

OH

HOHO

OH

O

OH

HOHO

OH

S

ELLA results for octavalent clusters Concanavallin A

As indicated in table 1 both glycoclusters (gluco- and manno-) showed modest inhibitory properties with IC50 values of 40 and 7 µM respectively, which correspond to a relative potency (rp) of 35 and 48 in reference to the corresponding monosaccharide. When reported to the number of sugar units (rp/n) the inhibition enhancement was 4.4 (gluco) and 6-fold (manno) higher, indicating a weak glycoside cluster effect.

Wheat Germ AgglutininA similar assay was performed with WGA and PEGylated POSS-based GlcNAc octavalent cluster while GlcNAc was used as monovalent reference..GlcNAc cluster showed a strong inhibition effect (IC50 = 3 nM) compared to the monosaccharidic GlcNAc (rp = 9,3 x 106 ; rp/n = 106).These unprecedented values for the inhibition of WGA by a synthetic glycocluster clearly indicated a strong multivalent effect, very likely due to a chelate binding model.Indeed WGA is a dimeric lectin containing a total of eight binding sites separated by approximately 14 Å. These structural features appear fully compatible with the tridimensional orientation and the length of the spacers linking the GlcNAc moieties to the silsesquioxane platform. Therefore, the multiple and simultaneous interactions of the sugar ligands with the WGA binding sites take place efficiently.

OHO

OH

HOHO

AcHN

SO

OH

HOHO

AcHNPEG-POSS Product

Number of sugar units (n)

IC50 (µM)

Relative Potency (rp; IC50 mono / IC50 cluster)

rp / n

16 4,4±0,6 479 30

16 0,179±0,011 2564 160

16 0,002±5-5 14,4 x 106 9 x 105

O

OH

HOHO

HOS

S

PEG-POSS

O

OH

HOHO

HO

O

OH

HOHO

HO

S

S

PEG-POSS

O

OH

HOHO

HO

OOH

HOHO

AcHNS

S

PEG-POSS

OOH

HOHO

AcHN

ELLA results for octavalent clustersMannosylated and glucosylated clusters were submitted to the ELLA experiments using Concanavalin A as above. Mannosylated cluster showed good inhibitory properties with an IC50 of 0.179 µM, which corresponds to a relative potency (rp) of 2564 respect to the monovalent reference. This rp value was higher (53-fold) than that found for the corresponding octavalent glycocluster (rp = 48.2). When reported to the number of sugar unit (rp/n = 160), the inhibition enhancement was 27-fold higher, indicating a moderate glycoside cluster effect. Similarly, the hexadecavalent glucosylated cluster showed stronger inhibition properties (IC50 = 4.4 µM; rp = 479) than the octavalent counterpart (IC50 = 40.4 µM; rp = 35.2).Also the hexadecavalent N-acetyl-D-glucosamine cluster showed remarkably strong inhibition properties toward WGA when submitted to similar ELLA experiments (IC50 = 2 nM), although the rp (14.4 × 106) and rp/n (9.0 × 105) values were only slightly higher or even lower, respectively, than those found for the corresponding octavalent cluster.

Table 1

Table 2

We have succeeded in preparing densely glycosylated silsesquioxanes via TEC and TYC, they featuring eight or sixteen carbohydrate fragments appended to the POSS scaffold through thioether linkages. Pure compounds were isolated in very good yields although some material was lost upon chromatography over Sephadex LH-20. Notably, the compounds are S- and C-glycosides, and therefore are expected to be endowed with enhanced chemical stability and enzymatic resistance. Finally, it has to be also pointed out that all isolated products were mixtures of diastereoisomers very likely in 1:1 ratio due to the lack of stereoselectivity of the thiyl radical addition to the vinyl thioether intermediate. No attempts were made to separate individual stereoisomers as this matter was beyond the scope of the present work. Therefore, it cannot ruled out that separation or stereoselective synthesis of pure stereoisomers could allow the identification of even stronger and more selective lectin ligands.Moreover the obtained products showed a inhibitory glycoside cluster effect on two appropriate lectins. This effect is weak or moderate for gluco and manno derivatives on ConA for both ctavalent and hexadecavalent clusters and became extremely high for GlcNAc on WGA. This suggest that cluster geometry and protein structure play a crucial role in the multivalent effect interaction.

References:1. Lo Conte M., Staderini S., Chambery A., Berthet N., Dumy P., Renaudet O., Marra A., Dondoni A. Org. Biomol. Chem., 2012, 10, 3269-32772. Dondoni A., Massi A., Nanni P., Roda A. Chem. Eur. J., 2009, 15, 11444-114493. Bossu I., Berthet N., Dumy P., Renaudet O., Org. Biomol. Chem., 2011, 9, 1948-19594. Minozzi M., Monesi A., Nanni D., Spagnolo P., Marchetti N., Massi A. J. Org. Chem.,2011, 76, 450-459