Chemical synthesis and functionalization of clickable glycosylphosphatidylinositol anchors

Abstract

Glycosylphosphatidylinositol (GPI) anchorage is a common posttranslational modification of eukaryotic proteins. Chemical synthesis of structurally defined GPIs and GPI derivatives is a necessary step toward understanding the properties and functions of these molecules in biological systems. In this work, the synthesis of several functionalized GPI anchors was accomplished using the para-methoxybenzyl (PMB) group for permanent hydroxyl protection, which allowed the incorporation of functionalities that are incompatible with permanent protecting groups traditionally used in carbohydrate synthesis. A flexible convergent-divergent assembly strategy enabled efficient access to a diverse set of target structures, including "clickable" Alkynyl-GPIs 1 and 2 and Azido-GPI 3. For global deprotection, a one-pot reaction was employed to afford the target GPIs in excellent yields (85-97%). Fully deprotected clickable GPIs 2 and 3 were readily conjugated to imaging and affinity probes via Cu(I)-catalyzed and Cu-free strain-promoted [3+2] cycloaddition, respectively, resulting in GPI-Fluor 4 and GPI-Biotin 5.

Fig. 1

GPI structure and anchoring function.

Fig. 2

Synthetic targets.

Fig. 3

Possible mechanism of 1,6-anhydro sugar formation.

Scheme 1

Convergent-divergent synthetic strategy.

Scheme 2

(A) Failed preparation of 8 using initial 1,6-O-differentiation. (B) Synthesis of 8 via delayed 1,6-O-differentiation. (a) NaH, AllBr, DMF, 81%. (b) AcCl, CH₂Cl₂, MeOH, 98%. (c) NaH, PMBCl, DMF, 73%. (d) Ti(Oi-Pr)₄, cyclohexylmagnesium chloride, THF, Et₂O, 85%. (e) Bu₂SnO, toluene, reflux; AllBr, CsF, DMF, 72%. (f) (1S)-(−)-camphanic chloride, DMAP, Et₃N, CH₂Cl₂, 46%. (g) 1 M NaOH, MeOH, THF, 95%.

Scheme 3

Synthesis of donors 9 and 30. (a) AllOH, SnCl₄, MS 4 Å, CH₂Cl₂, 81%. (b) NaOMe, MeOH. (c) anisaldehyde dimethyl acetal, CSA, DMF, 77% for 22, 78% for 28 (two steps). (d) NaH, PMBCl, DMF, 97% for 23, 95% for 29. (e) NaBH₃CN, dry HCl in Et₂O, MS 4 Å, THF. (f) TBSOTf, 2,6-lutidine, CH₂Cl₂, 60% for 24, 72% for 30 (two steps). (g) [Ir(COD)(PMePh₂)₂]PF₆, H₂, THF; then HgCl₂, HgO, acetone, H₂O, 85%. (h) Cl₃CCN, DBU, CH₂Cl₂, 83%. (i) BnNH₂, THF, 87%. (j) DAST, CH₂Cl₂, 98%.

Scheme 4

Synthesis of pseudodisaccharide 35. (a) 1H-tetrazole, CH₃CN, CH₂Cl₂; then t-BuOOH, 0 °C. (b) Et₃N·3HF, THF, CH₃CN, 54% (two steps).

Scheme 5

Synthesis of mannose building blocks 10–12. (a) Bu₂SnO, toluene, reflux; PMBCl, CsF, DMF. (b) NaH, AllBr, DMF, 68% (two steps). (c) DIBAL-H, CH₂Cl₂, 82%. (d) NaOMe, MeOH. (e) NaH, PMBCl, DMF, 76% (two steps). (f) AcOH-H₂O (1:1), 70%. (g) Cl₃CCN, DBU, CH₂Cl₂, 77%. (h) AllOH, BF₃·OEt₂, MS 4 Å, CH₂Cl₂, 66% (two steps from d-mannose). (i) NaOMe, MeOH. (j) PMTrtCl, pyridine, 66% (two steps). (k) NaH, PMBCl, DMF, 73%. (l) AcOH, H₂O, CH₂Cl₂, 94%. (m) TBSCl, pyridine, 88%. (n) PdCl₂, AcOH, NaOAc, CH₂Cl₂, H₂O, 82%. (o) Cl₃CCN, DBU, CH₂Cl₂, 73%.

Scheme 6

Synthesis of trimannoside 7. (a) TMSOTf (cat.), MS 4 Å, CH₂Cl₂, 0 °C. (b) K₂CO₃, MeOH, 66% (two steps). (c) 12, TMSOTf (cat.), MS 4 Å, Et₂O, 0 °C, 76%.

Scheme 7

Key glycosylation and phosphorylation reactions. (a) p-TolSCl, AgOTf, TTBP, wet CH₂Cl₂, 50% (68% BRSM). (b) Cl₃CCN, DBU, CH₂Cl₂, 92%. (c) 35, TMSOTf (cat.), MS 4 Å, Et₂O. (d) Et₃N·3HF, THF, CH₃CN, 83% (two steps). (e) 47, 1H-tetrazole, CH₂Cl₂, CH₃CN; then t-BuOOH, 0 °C, 81% (two steps). (f) [Ir(COD)(PMePh₂)₂]PF₆, H₂, THF; then HgCl₂, HgO, acetone, H₂O, 90%.

Scheme 8

Synthesis of first generation Alkynyl-GPI 1. (a) excess succinic anhydride, DMAP, CH₂Cl₂, MS 4 Å, 67%. (b) propargyl amine, EDCI, HOBt, CH₂Cl₂, DMF, 75%. (c) Zn, AcOH, CH₂Cl₂, 1 h; DBU, CH₂Cl₂ 1 h; CH₂Cl₂-TFA (9:1), 30 min, 85% (0.40 mg, three steps).

Scheme 9

Synthesis of second generation Alkynyl-GPI 2. (a) Ti(Oi-Pr)₄, cyclopentylmagnesium chloride, THF, Et₂O, 82%. (b) NaH, propargyl bromide, DMF, 78%. (c) para-nitrobenzenesulfenyl chloride, AgOTf, TTBP, wet CH₂Cl₂, 77%. (d) Cl₃CCN, DBU, CH₂Cl₂, 90%. (e) 35, TMSOTf (cat.), MS 4 Å, Et₂O. (f) Et₃N·3HF, THF, CH₃CN, 82% (two steps). (g) 47, 1H-tetrazole, CH₂Cl₂, CH₃CN; then t-BuOOH, −40 °C, 72% (two steps). (h) Zn, AcOH, CH₂Cl₂, 1 h; DBU, CH₂Cl₂ 1 h; CH₂Cl₂-TFA (9:1), 30 min, 93% (1.03 mg, three steps).

Scheme 10

Synthesis of modified pseudodisaccharide 60. (a) Zn, AcOH, CH₂Cl₂. (b) FmocCl, NaHCO₃, 1,4-dioxane-H₂O (10:1), 78% (two steps). (c) 34, 1H-tetrazole, CH₂Cl₂, CH₃CN; then t-BuOOH, 0 °C. (d) Et₃N·3HF, THF, CH₃CN, 67% (two steps).

Scheme 11

Synthesis of second generation Azido-GPI 3. (a) NaH, tert-butyl bromoacetate, DMF, 77%. (b) LiAlH₄, THF, 77%. (c) MsCl, DIPEA, CH₂Cl₂. (d) NaN₃, DMF, 90 °C, 82% (two steps). (e) NIS, AgOTf, TTBP, wet CH₂Cl₂, 71%. (f) Cl₃CCN, DBU, CH₂Cl₂, 96%. (g) 60, TMSOTf (cat.), MS 4 Å, CH₂Cl₂. (h) Et₃N·3HF, THF, CH₃CN, 30% (two steps, 88% BRSM). (i) 47, 1H-tetrazole, CH₂Cl₂, CH₃CN; then t-BuOOH, 0 °C, 61% (two steps). (j) DBU, CH₂Cl₂ 1 h; CH₂Cl₂-TFA (9:1), 30 min, 97% (0.76 mg, two steps).

Scheme 12

Click reactions of GPIs 2 and 3 to form GPI-conjugates 4 and 5. (a) 5 equivalents Azide-Fluor 488 (65), CuSO₄, sodium ascorbate, 37 °C, THF-MeOH-H₂O (1:1:1). (b) BARAC-biotin (66), CHCl₃-MeOH-H₂O (3:3:1).