Synthesis of Azide-Modified Chondroitin Sulfate Precursors: Substrates for "Click"- Conjugation with Fluorescent Labels and Oligonucleotides.

Satish Jadhav,Vijay Gulumkar,Prasannakumar Deshpande,Eleanor T. Coffey,Pasi Virta,Harri Lönnberg

doi:10.1021/acs.bioconjchem.8b00317

Abstract

Azidopropyl-modified precursors of chondroitin sulfate (CS) tetrasaccharides have been synthesized, which, after facile conversion to final CS structures, may be conjugated with alkyne-modified target compounds by a one-pot “click”-ligation. RP HPLC was used for the monitoring of the key reaction steps (protecting group manipulation and sulfation) and purification of the CS precursors (as partially protected form, bearing the O-Lev, O-benzoyl, and N-trichloroacetyl groups and methyl esters). Subsequent treatments with aqueous NaOH, concentrated ammonia, and acetic anhydride (i.e., global deprotection and acetylation of the galactosamine units) converted the precursors to final CS structures. The azidopropyl group was exposed to a strain-promoted azide–alkyne cycloaddition (SPAAC) with a dibenzylcyclooctyne-modified carboxyrhodamine dye to give labeled CSs. Conjugation with a 5′-cyclooctyne-modified oligonucleotide was additionally carried out to show the applicability of the precursors for the synthesis of biomolecular hybrids.

Highlights

Chondroitin sulfate (CS) is a linear sulfated polysaccaharide that plays pivotal roles in many biological processes such as cell division, neuronal development, viral invasion, cancer metastasis, and spinal cord injury.1−5 CS is composed of repeating βD-glucuronic acid (GlcA) and N-acetyl-β-D-galactosamine (GalNAc) units arranged in the sequence by GlcA-β(1 → 3)GalNAc-β(1 → 4) glycosidic bonds with variable high and low sulfation patterns
Glycosylation of simple alcohols with monomeric N-acyl galactosamines may give the expected βanomer as a major product, but the extent of the α-anomer may become significant in glycosylation of D-glucuronic acid-derived acceptors and with disaccharidic N-acyl galactosamine donors
RP HPLC was used for the monitoring of the key reaction steps and purification of the CS precursors

Summary

■ INTRODUCTION

Chondroitin sulfate (CS) is a linear sulfated polysaccaharide that plays pivotal roles in many biological processes such as cell division, neuronal development, viral invasion, cancer metastasis, and spinal cord injury.− CS is composed of repeating βD-glucuronic acid (GlcA) and N-acetyl-β-D-galactosamine (GalNAc) units arranged in the sequence by GlcA-β(1 → 3)GalNAc-β(1 → 4) glycosidic bonds with variable high and low sulfation patterns. The required protecting group manipulation and sulfation were carried out on a 6 μmol scale and the key reaction steps were monitored by RP HPLC The labeled CS tetrasaccharide and CS disaccharide were synthesized from 9 and 13 using exactly the same procedures: The benzylidene protections were removed and the exposed hydroxyl groups were sulfated as described to 23 from 16 above. As the global deprotection/N-acylation step above proved virtually quantitative (cf RP HPLC analysis of crude product mixture of 24, a/Scheme 5), the applicability of the azidopropyl modified CS precursors (18, 21, and 23, Scheme 4) was evaluated to gain 5′-CS-oligonucleotide conjugates (32−34, Scheme 6). These results suggest that may be taken up via receptor-mediated endocytosis in neurons, whereas is not

■ CONCLUSIONS

■ EXPERIMENTAL PROCEDURES

■ REFERENCES