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

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.

Scheme 1. Synthesis of Glycosyl Acceptor 7

Reagents and conditions: (i) hydrazine acetate, DMF; (ii) TBDMSCl, imidazole, DMF, room temperature; (iii) 0.1 mol L^–1 NaOMe in MeOH, room temperature; (iv) PhCH(OMe)₂, p-TSA, acetonitrile, room temperature; (v) levulinic acid, DCC, DMAP, CH₂Cl₂, room temperature; (vi) Et₃N·3HF, THF, room temperature; (vii) CCl₃CN, DBU, CH₂Cl₂, 0°C; (viii) 3-azidopropan-1-ol, TMSOTf, CH₂Cl₂, −30°C; (ix) hydrazine acetate, pyridine, room temperature.

Scheme 2. Synthesis of Glycosyl Acceptor 10 and Glycosyl Donor 12

Reagents and conditions: (i) TMSOTf, CH₂Cl₂, 0°C; (ii) hydrazine acetate, pyridine, room temperature; (iii) Et₃N·3HF, THF, room temperature; (iv) CCl₃CN, DBU, CH₂Cl₂, 0°C.

Scheme 3. Synthesis of Chondroitin Sulfate Precursors 13 and 15

Reagents and conditions: (i) TMSOTf, CH₂Cl₂, −30°C.

Scheme 4. Synthesis of Chondroitin Sulfate Precursors (18–23) and RP HPLC Monitoring of the Reactions

Reagents and conditions: (i) thiourea, pyridine–EtOH (1:1, v/v), 2 h at 80°C; (ii) SO₃·TMA, DMF, overnight at 50°C; (iii) 0.1% aq TFA, 5 h at r.t.; (iv) Ac₂O, pyridine, overnight at r.t.; (v) 80% aq AcOH, 1 h at 100°C. RP HPLC conditions: an analytical RP HPLC column (250 × 4.6 mm, 5 μm), flow rate: 1.0 mL min^–1, detection at 220 nm, profiles a, d, and g: a gradient elution from 20 to 100% MeCN in 0.1 mol L^–1 aqueous triethylammonium acetate (0–25 min), profiles b, c, e, f, and h: a gradient elution from 0 to 50% MeCN in 0.1 mol L^–1 aqueous triethylammonium acetate (0–25 min), then from 50% to 100% MeCN in 0.1 mol L^–1 aqueous triethylammonium acetate (25–30 min), profiles of crude product (16–23) mixtures described.

Scheme 5. Synthesis of Carboxyrhodamine-Labeled CSs (24–26, 29, 30)

Reagents and conditions: (i) 0.1 mol L^–1 aq NaOH, 3 h at 55°C; (ii) conc. aq NH₃, 4 days at 55°C; (iii) Ac₂O, Et₃N, aq MeCN; (iv) conc. aq NH₃, 5 h at 55°C; (v) dibenzylcyclooctyne-PEG4-5/6-carboxyrhodamine (2 equiv), DMF, overnight at room temperature; (vi) 80% aq AcOH, 1 h at 100°C; (vii) SO₃·TMA, DMF, overnight at 50°C. RP HPLC conditions: an analytical RP HPLC column (250 × 4.6 mm, 5 μm), flow rate: 1.0 mL min^–1, detection at 501 nm, a gradient elution from 0 to 100% MeCN in 0.1 mol L^–1 aqueous triethyammonium acetate over 30 min. Notes: (a) An RP HPLC profile of the crude product (24) mixture; (b–f) RP HPLC profiles of the purified products (24–26, 29, 30). Retention times (t_r) of the product peaks: 24: 18.8 min, 25: 19.3 min, 26: 19.4 min, 29: 22.0 min, 30: 21.8 min.

Scheme 6. Synthesis of CS–Oligonucleotide Conjugates (32–34)

Reagents and conditions: (i) 0.1 mol L^–1 aq NaOH, 3 h at 55°C; (ii) conc. aq NH₃, 4 days at 55°C; (iii) Ac₂O, Et₃N, aq MeCN; (iv) conc. aq NH₃, 5 h at 55°C; (v) 5′-cyclooctyne-modified 2′-deoxy oligoribonucleotide 31 (1.5 equiv), H₂O, overnight at 55°C.; RP HPLC conditions: an analytical RP HPLC column (250 × 4.6 mm, 5 μm), flow rate: 1.0 mL min^–1, detection at 260 nm, a gradient elution from 0 to 70% MeCN in 0.1 mol L^–1 aqueous triethylammonium acetate over 30 min. An RP HPLC profile of the crude (a–c) and homogenized (d–f) products (32: t_r = 13.4 min, 33: t_r = 13.0 min, 34: t_r = 13.2 min).

Figure 1

Labeled CSs (29 and 30) images. Cultured hippocampal neurons from rat brain at 7 days in vitro were treated with CS conjugates (29 or 30) at 50 or 10 nM unconjugated dye (DBCO-PEG4–5/6-Carboxyrhodamine 110) or with DMSO for 10 days as indicated. Cells were fixed with 4% cold paraformaldehyde in phosphate buffered saline and stained with Hoechst-33342. Confocal images of Z-sections through nuclei are shown. Signal for the conjugates or the dye is shown in green while nuclei are highlighted in blue. Scale = 10 μm.