Modular synthesis of heparan sulfate oligosaccharides for structure-activity relationship studies

Abstract

Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides. To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease.

Figure 1

Orthogonal protecting groups for disaccharide building blocks

Figure 2

Modular mono- and disaccharide building blocks

Figure 3

Putative synthetic HS ligands for BACE-1

Figure 4

Dose response inhibition curves for selected compounds compared to porcine mucosal heparin in the FRET peptide cleavage assay (solid squares, heparin; triangles, compound 83; circles, compound 77 and open squares, compound 81).

Scheme 1

Synthesis of 2-azido-2-deoxy-α-D-glucopyranoside acceptors (a) TDSCl, imidazole, DCM (90%); (b) CCl₃CN, DBU, DCM (90%); (c) HO(CH₂)₅N(Bn)Cbz, DCM:Et₂O, TMSOTf, molecular sieves, −20°C (α-anomer, 62%) (d) i) NaOMe, MeOH; ii) PhCH(OMe)₂, CSA, DMF (29: 92% and 30: 76%, 2 steps); (e) i) BnBr, Ag₂O, molecular sieves, DCM; ii) DCM:TFA:H₂O (31: 95%, 2 steps); (f) NaH, BnBr, DMF; ii) DCM:TFA:H₂O (32: 93%, 2 steps); (g) AcOH, 2-chloro-1-methyl-1-pyridinium iodide, DABCO, DCM (5: 65%, 7: 68%), (h) LevOH, 2-chloro-1-methyl-1-pyridinium iodide, DABCO, DCM (6: 86%, 8: 82%)

Scheme 2

Synthesis of thioethyl gluco- and idosyl donor. (a) i) AcOH, Ac₂O, TFA, r.t; ii) EtSH, BF₃.Et₂O, DCM, 0°C to room temperature; (b) i) NaOMe, MeOH; ii) PhCH(OMe)₂, CSA, DMF, 70%; (c) Ac₂O, Py, r.t, 85%; (d) LevOH, DCC, DMAP, DCM, 72%; (e) (i) NaOMe, MeOH; ii) PhCH(OMe)₂, p-TsOH, DMF, 61% (2 steps); iii) (Bu₂Sn)O, MeOH, 75–80°C, BnBr, CsF, DMF, 16 hr, r.t, 61%; (c) Ac₂O, Py, 60%: 1: 93%, 3: 85%; (d) LevOH, DCC, DMAP, DCM, r.t, 2: 89% 4: 70%

Scheme 3

Synthesis of glucuronyl and idouronyl disaccharides. (a) NIS, TMSOTf, 0°C, DCM (37: 81%, 38: 75%, 39: 92%, 40: 75%. 49: 90% 50: 66%, 51: 95%, 52: 80%); (b) i) EtSH, p-TsOH, DCM or DCM:TFA:H₂O; ii) TEMPO, BAIB, DCM, H₂O, 1 h; iii) CH₂N₂, THF (3 steps 65–85%); (e) i) FmocCl, Py,DMAP, 0°C to room temperature; (ii)! HF.Py,18 h; iii) K₂CO₃, CCl₃CN, DCM (70–90%)

Scheme 4

(a) Synthesis of hexasaccharide 69: (a) TMSOTf, −20°C to +5°C, 4°A sieves, 61: 64%; 63: 65%; (b) Et₃N, DCM, 82%; (c) NH₂NH₂.HOAc, toluene/EtOH, 90%; (d) Py.SO₃, DMF, 80%; (e) i) Et₃N, DMF; ii) LiOH, H₂O₂, THF; ii) 4M NaOH, MeOH (58%, 3 steps), 80%; (f) PMe₃, THF, NaOH, 65%; (g) N-sulfation: Py.SO₃, MeOH, Et₃N, 0.1 M NaOH, 50%; (i) i) Pd/C, H₂, MeOH:H₂O; ii) Pd(OH)₂/C, H₂, H₂O, 67%

Scheme 5

(a) Synthesis of library of tetrasaccharides. (a) TMSOTf, DCM, −20°C to 5°C, molecular sieves (70: 61%, 71: 62%, 72: 64%, 73: 62%, 74: 51%, 75: 59%)