Design and Synthesis of 6‐O‐Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid β Aggregation

Abstract Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid β (Aβ) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non‐protein components of Aβ deposits that not only modulates Aβ aggregation, but also acts as a receptor for Aβ fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aβ could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6‐O‐phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP‐mediated Aβ fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.


General Procedures
All reactions were performed under an atmosphere of nitrogen/argon in flame-dried glassware. Solvents were distilled in the standard way, and commercial reagents were used without any purification unless otherwise stated. Anhydrous solvents like CH2Cl2, Et2O, DMF, and Et3N were dried in a standard way. TLC was performed on glass plates pre-coated with Silica Gel 60 F254 (0.25 mm, E. Merck). TLC plates were visualized by exposure to ultraviolet light (UV-254 nm) and/or detection was executed by spraying with a solution of Ce(NH4)2(NO3)6, (NH4)6Mo7O24, and H2SO4 in water and subsequent heating on a hot plate. Specific rotations were taken at ambient conditions and reported in 10 -1. deg . cm 2. g -1 ; the sample concentrations are in g . dL -1 . Flash column chromatography was carried out on Silica Gel 60 (230-400 mesh, E. Merck) or MP. 1 H NMR spectra were recorded on 600 MHz instrument at ambient temperature. Data were recorded as follows: chemical shift in ppm from the solvent resonance employed as the internal standard (CDCl3 at 7.26 ppm, and CD3OD at 3.31 ppm and 4.78 ppm), multiplicity (s = singlet, d = doublet; t = triplet; q = quartet; st = septet; m = multiplet; br = broad), coupling constant (Hz), integration. 13 C NMR spectra were measured on 150 MHz spectrometer. Chemical shifts were recorded in ppm S6 (150 MHz,CDCl3)

Compound 22.
To a solution of compound 21 (4.4 g, 3.0 mmol) in a 1/1 ratio of methanol/dichloromethane (20 mL) was added sodium methoxide (24 mg, 0.45 mmol) at 0 °C under nitrogen atmosphere. The ice bath was removed, and the resulting solution was kept stirring at room temperature for 18 h.

Compound 24.
To a solution of compound 23 (10.5 g, 7.82 mmol) in N,N-dimethylformamide (100 mL) was added sulfur trioxide/triethylamine (8.5 g, 46.9 mmol) at room temperature. The reaction flask was heated to 60 °C, and the mixture was kept stirring at the same temperature for 18 h. The reaction flask was cooled down to room temperature, water (100 mL) and sodium bicarbonate (20.0 g) were consecutively added to the solution, and the mixture was continuously stirred for 3 h. The resulting mixture was concentrated in vacuo, and the crude mass was extracted by dichloromethane (2 x 150 mL). The combined organic layer was washed with sat. NaHCO3(aq), dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (MeOH/CHCl3 = 1/20  1/15  1/10  1/5) on silica gel to yield the 2-sulfate 24 (8.3 g, 75%). 1

Compound 25.
A 7/3 ratio of hydrogen fluoride/pyridine (2 mL) was slowly added to a solution of compound 24 (2.0 g, 1.4 mmol) in tetrahydrofuran (8 ml) and pyridine (8 ml) at 0 °C under nitrogen atmosphere. The reaction solution was stirred for 3 d, silica gel (10 g) was added to quench the reaction, and the mixture was kept stirring for 1 h. The whole mixture was filtered, and the solid was washed with a 1/1 ratio of chloroform/methanol (20 mL). The filtrate was concentrated in vacuo, and the crude residue was purified by flash column chromatography (MeOH/CHCl3 = 1/20  1/15  1/10  1/4) on silica gel to furnish the 6'-alcohol 25 (1.0 g, 60%). 1

Compound 27.
To a solution of compound 26 (293 mg, 0.2 mmol) in tetrahydrofuran (2.5 mL) were consecutively added a 1.0 N solution of lithium hydroxide in water (5.0 mL) and a 37% solution of hydrogen peroxide in water (1 mL) at room temperature. The mixture was stirred at 37 o C for 18 h, then the reaction flask was cooled down to room temperature. The reaction was quenched and neutralized S12 with acetic acid till pH 7, and the resulting mixture was concentrated in vacuo. The crude compound was dissolved in tetrahydrofuran (5 mL) at room temperature, a 0.1 N sodium hydroxide aqueous solution (0.2 mL) and a 1 M solution of trimethylphosphine in tetrahydrofuran (0.2 mL, 0.2 mmol) were sequentially added into the reaction flask, and the mixture was continuously stirred for 14 h. The reaction was quenched by 0.1 N HCl(aq) (0.2 mL), the mixture was concentrated in vacuo, and the residue was purified by flash column chromatography (MeOH/CHCl3 = 1/9  1/4) on silica gel followed by Na + ion exchange column (AG 50W-X8 cation exchange resin) using methanol as eluent to obtain the crude 2'-amino product. This amine compound was dissolved in methanol (3 mL

Compound 28.
A mixture of compound 27 (10.3 mg, 6.7 mol) and Degussa type 20% Pd(OH)2/C (78 mg) in methanol (2 mL) and pH 7 phosphate buffer (0.15 mL) was purged with nitrogen for 10 min at room temperature. The reaction flask was then equipped with a hydrogen balloon. After stirring for 3 d, the whole mixture was filtered through a pad of Celite®, washed with methanol, and concentrated in vacuo. The residue was purified by Sephadex G-25 column followed by AG 50W-X8 cation exchange column (Na + form) using water as eluent to furnish the disaccharide 6'phpsphate 28 (4.1 mg, 78%). 1

Compound 29.
Compound 25 (107 mg, 90.4 mol) was dissolved in pyridine (2 mL) at room temperature under nitrogen atmosphere. Diethyl phosphoryl chloride (76 L, 525 mol) was added to the solution, and the reaction mixture was kept stirring at the same temperature for 2 h. The resulting solution S14 was diluted with dichloromethane (10 mL), and the mixture was quenched with sat. sodium bicarbonate. The organic layer was washed with water, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography MeOH/CHCl3 = 1/20  1/15  1/10  1/7  1/5) on silica gel to afford compound 29 (106 mg, 89%). 1

Compound 30.
Compound 29 (98 mg, 74 μmol) was dissolved in tetrahydrofuran (1.0 mL) at room temperature. A 1.0 N solution lithium hydroxide in water (1.9 mL) and a 37% solution of hydrogen peroxide in S15 water (0.37 mL) were sequentially added to the reaction solution. The mixture was warmed up and stirred at 37 o C for 18 h. After cooling to room temperature, the reaction was neutralized by acetic acid till pH 7, and the resulting mixture was concentrated in vacuo. The crude compound was dissolved in tetrahydrofuran (1 mL) at room temperature, a 0.1 N sodium hydroxide aqueous solution (74 μL) and a 1 M solution of trimethylphosphine in tetrahydrofuran (74 μL, 74 μmol) were sequentially added into the reaction flask, and the mixture was continuously stirred for 14 h.
The reaction was quenched by 0.1 N HCl(aq) (74 μL), the mixture was concentrated in vacuo, and the residue was purified by flash column chromatography (MeOH/CHCl3 = 1/9  1/4) on silica gel followed by Na + ion exchange column (AG 50W-X8 cation exchange resin) using methanol as eluent to obtain the crude 2'-amino product. This amine compound was dissolved in methanol (1   mL

Compound 32.
A mixture of the donor 20 (6.8 g, 5.4 mmol), the acceptor 31 (6.8 g, 5.1 mmol), and freshly activated 3Å MS (14.0 g) in dichloromethane (140 mL) was stirred at room temperature for 30 min under nitrogen atmosphere. The reaction flask was cooled down to -78 °C, and Niodosuccinimide (1.4 g, 6.2 mmol) was added to the solution. After stirring for 30 min, trifluoromethanesulfonic acid (0.18 mL, 2.0 mmol) was added to the reaction mixture, and the resulting solution was gradually warmed up to -40 °C for 3 h. The reaction was quenched by 10% Na2S2O3(aq) and sat. NaHCO3(aq). The crude reaction mixture was filtered through a pad of Celite® followed by washed with dichloromethane. After separation, the organic layer was washed with water, dried over anhydrous MgSO4, filtered, concentrated in vacuo. The crude product was S19 To a solution of compound 32 (4.4 g, 3.0 mmol) in a 1/1 ratio of methanol/dichloromethane (20 mL) was added sodium methoxide (1.9 g, 36 mmol) at 0 °C under nitrogen atmosphere. The mixture was gradually warmed up to room temperature, and the reaction was kept stirring for 18 h. The reaction mixture was neutralized with DOWEX 50 (H + exchange resin), filtered, and the solvent was removed under reduced pressure. The crude compound was dissolved in ethyl acetate (100 mL) followed by washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was dissolved in a 2/1 ratio of dichloromethane/water (35 mL) at room temperature. (2,2,6,oxyl (TEMPO, 86 mg, 0.55 mmol) and bis(acetoxy)iodobenzene (BAIB, 2.2 g, 6.8 mmol) were consecutively added to the solution, and the reaction mixture was kept stirring for 16 h. The reaction was quenched with 10 % Na2S2O3(aq), and the organic layer was washed with water, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/Hex = 1/20  1/5) on silica gel to provide the lactone 33 (2.7 g, 70% in two steps). 1

Compound 35.
Compound 34 (200 mg, 91.6 mol) was dissolved in N,N-dimethylformamide (2.0 mL) at room temperature, sulfurtrioxide•triethylamine (199 mg, 1.09 mmol) was added to the solution, and the reaction mixture was warmed up and kept stirring at 60 °C for 18 h. The reaction flask was cooled down to room temperature, water (2 mL) and sodium bicarbonate solid (400 mg) were added to the solution, and the resulting mixture was continuously stirred for 3 h. The solvent was removed at the reduced pressure, and the crude residue was dissolved in dichloromethane (20 mL) followed by washed with sat. NaHCO3(aq) and water. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (methanol/chloroform = 1/20  1/15  1/10  1/5) on silica gel to furnish the O-sulfonated product (128 mg, 75%). This sulfate compound (0.10 g, 42 μmol) was dissolved in tetrahydrofuran (0.8 mL) and pyridine (0.8 mL) at room temperature. The reaction flask was cooled down to 0 °C, a ~70%/~30% ratio of hydrogen fluoride/pyridine (0.6 mL) was slowly added to the solution, and the mixture was kept stirring for 3 d. The reaction was quenched by silica gel (1 g), and the mixture was continuously stirred for 1 h. The whole mixture was filtered, and the solid part was washed with 1/1 ratio of methanol/chloroform (5 mL). The filtrate was concentrated in vacuo, and the crude residue was purified by flash column chromatography (methanol/chloroform = 1/20  1/15  1/10  1/4) on silica gel to give the diol 35 (60 mg, 76%).

Measuring Aβ Fibril Formation
Fibril formation of Aβ was measured by means of a ThT assay. Aβ (10 µM) in phosphate-buffered saline (PBS, pH 7.4) was incubated in the presence of 10 µM ThT and heparin (40 µg/mL) or HS/heparin derivatives (100 µM) at 37 °C with agitation on an orbital rotator at 10 rpm.
Fluorescence of ThT was recorded from 460 to 600 nm with an excitation wavelength of 445 nm.

AFM Analysis
Aβ assemblies for morphologic analysis by using an atomic force microscope (AFM) were prepared by incubating Aβ (10 µM) in PBS in the presence of heparin (40 µg/mL) without or with a HS derivative (100 µM) at 37 °C for 24 h. Ten µL of each fibril solution in PBS was diluted with 45 µL of distilled water and spotted on a freshly cleaved mica (Nilaco Corp., Tokyo, Japan), after which the mica was washed with 20 µL of distilled water. Sample images were obtained under ambient conditions at room temperature by using NanoScope IIIa Tapping Mode AFM (Veeco Instrument, Plainview, NY) and a single-crystal microcantilever OMCLAC160TS-R3 (Olympus, Tokyo, Japan) at a scan rate of 0.5 Hz in tapping mode.

Statistical analysis
The data were analyzed using an ordinary two-way analysis of variance (ANOVA) with Dunnett's test or Tukey's range test using Prism software (GraphPad Software, La Jolla, CA, USA).
Differences were regarded as significant for P < 0.05.