Chapter 2: Methods for Short- and Long-Term Glycan Engineering
2.5 Materials and Methods for HaloTag Experiments
2.5.1 Synthetic Methods
N-Boc-2-(2-hydroxyethoxy)ethylamine (2-10). Compounds 2-10 and 2-11 were synthesized as previously described.31 A solution of di-tert-butyl dicarbonate (Boc2O) (2.18 g, 10.0 mmol, 1 eq) in anhydrous methanol (2 mL) was added dropwise to a stirring solution of 2-(2-aminoethoxy)ethanol (1.05 g, 9.99 mmol) in anhydrous methanol (20 mL) at 0 °C. The mixture was stirred for 30 min at 0
°C and then warmed to RT. After 2 h, the solution was diluted with DCM (30 mL), washed with ddH2O (3 x 25 mL) and brine (1 x 25 mL), dried over MgSO4, filtered, and concentrated to afford a colorless oil 2-10 (1.85 g, 91%). 1H NMR (500 MHz, CDCl3): δ 1.42 (s, 9H, CH3), 3.30 (t, J = 5.1 Hz, 2H, CH2N), 3.49-3.58 (m, 4H, CH2OCH2), and 3.68-3.75 (m, 2H, CH2OH). MS (ESI) calcd. for C4H11NO2+ [M + H+] 206.14, found 206.10.
N-Boc-1-(2-(2-aminoethoxy)ethoxy)-6-chlorohexane (2-11). A solution of 2-10 (0.34 g, 1.7 mmol) in DMF (2 mL) was added dropwise to a stirring solution of 60% NaH (0.095 g, 2.4 mmol, 1.4 eq) in DMF (30 mL) at 0 °C. The mixture was stirred for 1 h at 0 °C, after which 1-chloro-6-iodohexane (0.30 mL, 2.0 mmol, 1.2 eq) was added. Stirring was continued for 24 h at RT, and then the reaction was
58 quenched with 1 M HCl and extracted with DCM, ddH2O (6 x 25 mL) and brine (1 x 25 mL), dried over MgSO4, filtered, and concentrated to afford a yellow oil.
Purification by silica gel flash chromatography using 3:1 Hex/EtOAc gave 2-11 as a colorless oil (0.20 g, 37%). 1H NMR (500 MHz, CDCl3): δ 1.33-1.40 (m, 2H, CH2), 1.43 (s, 9H, CH3), 1.44-1.48 (m, 2H, CH2), 1.55-1.64 (m, 2H, CH2), 1.73- 1.80 (m, 2H, CH2), 3.30 (t, J = 5.2 Hz, 2H, CH2N), 3.45 (t, J = 6.7 Hz, 2H, CH2Cl), 3.49-3.57 (m, 6H, CH2O), and 3.57-3.61 (m, 2H, CH2O). MS (ESI) calcd. for C14H29ClNO5+ [M + H+] 324.20, found 324.21.
2,5-Dioxopyrrolidin-1-yl 4-oxopentanoate (2-12). N-Hydroxysuccinimide (NHS;
0.55 g, 4.8 mmol, 1.1 eq) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride (0.91 g, 4.8 mmol, 1.1 eq) were added to a stirring solution of levulinic acid (0.50 g, 4.3 mmol) in anhydrous DCM at 0 °C. The mixture was warmed to RT and stirred for 2 h, at which time the reaction was diluted with DCM, extracted with ddH2O (3 x 25 mL) and brine (1 x mL), dried over MgSO4, filtered, and concentrated to afford a white fluffy solid 2-12 (0.69 g, 75%). 1H NMR (500 MHz, CDCl3): δ 2.21 (s, 3H, CH3), 2.83 (s, 4H, CH2CH2-succinimide), and 2.86-2.92 (m, 4H, CH2CH2-levulinate). MS (ESI) calcd. for C9H11NO5+ [M + H+] 214.08, found 214.10.
N-(2-(2-(6-Chlorohexyloxy)ethoxy)ethyl)-4-oxopentanamide (2-13). Trifluoroacetic acid (TFA) (0.14 mL, 1.8 mmol, 6 eq) was added dropwise to a stirring solution of 2-11 (0.10 g, 0.31 mmol) in anhydrous DCM (5 mL). The mixture was stirred at
RT for 4 h, then azeotroped with toluene, and concentrated to afford the free amine as a yellow oil, which was used without further purification. This intermediate (0.025 g, 0.074 mmol) was stirred with N,N-diisopropylethylamine (DIPEA; 0.076 mL, 0.436 mmol, 6 eq) in anhydrous DCM (3 mL) for 20 min at RT, to which 2-12 (0.016 g, 0.075 mmol, 1 eq) was added and stirred overnight.
After 24 h, the reaction was diluted with DCM (20 mL) and extracted with ddH2O (3 x 25 mL) and brine (1 x 25 mL), dried over MgSO4, filtered, and concentrated to afford a yellow oil. Purification by silica gel flash chromatography using 97:3 DCM/MeOH gave 2-13 as a colorless oil (0.016 g, 67%). 1H NMR (500 MHz, CDCl3): δ 1.33-1.41 (m, 2H, CH2), 1.42-1.49 (m, 2H, CH2), 1.56-1.65 (m, 2H, CH2), 1.73-1.81 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.43 (t, J = 6.6 Hz, 2H, CH2C(O)), 2.79 (t, J = 6.6 Hz, 2H, CH2C(O)), 3.41-3.49 (m, 4H, CH2Cl and CH2N), 3.51-3.56 (m, 4H, CH2O), 3.56-3.63 (m, 4H, CH2O), 6.15 (bs, 1H, NH).
MS (ESI) calcd. for C15H28ClNO4+ [M + H+] 322.18, found 322.20.
Fluorescein-chloroalkane linker conjugate (F-CL). To a solution of 2-13 (2.6 mg, 8.1 µmol) in 0.5 mL anhydrous MeOH was added 5-(((2- (carbohydrazino)methyl)thio)acetyl)aminofluorescein (F) in ddH2O (4.0 mg, 8.1 µmol, 1 eq; C-356, Life Technologies). The mixture was stirred for 12 h at RT in the dark, concentrated, and purified by silica gel flash chromatography in the dark using a 97:3 DCM/MeOH mixture to afford the desired compound as an orange solid (4.8 mg, 75%). 1H NMR (500 MHz, D2O): δ 1.26-1.33 (m, 2H, CH2), 1.33-
60 1.39 (m, 2H, CH2), 1.40 (s, 3H, CH3), 1.44-1.52 (m, 2H, CH2), 1.63-1.70 (m, 2H, CH2), 3.32-3.55 (m, 20H, CH2), 6.42-6.48 (m, 2H, Ar-H) 6.49-6.60 (m, 4H, Ar-H), 7.07 (dd, J = 8.3, 2.9 Hz, 1H, Ar-H), 7.73-7.81 (m, 1H, Ar-H), and 8.21-8.30 (m, 1H, Ar-H). MS (ESI) calcd. for C39H43ClN4O10S-2Na+ [M-2 + Na+] 817.23, found 817.25.
Biotinylated heparan sulfate (B-HS). To a stirring solution of EZ-Link NHS-PEG4- biotin (0.10 g, 0.17 mmol; 21363, Thermo Scientific) in dry DCM (5 mL) was added a solution of ethylenediamine (0.022 mL, 0.33 mmol, 2 eq) and triethylamine (TEA; 0.15 mL, 1.1 mmol, 6 eq) in dry DCM (1 mL). The mixture immediately turned cloudy upon addition and was then stirred for 1.5 h at RT.
The precipitate was filtered, and the reaction was concentrated to afford the conjugated amine as a white solid (0.068 g, 76%), which was used without further purification. De-6-O-sulfated HS (7.0 mg, 0.58 µmol) was dissolved in ddH2O (1 mL), followed by the addition of cyanogen bromide in ddH2O (5.0 mg, 47 µmol, excess). The pH was adjusted to 11.0 using 0.2 M NaOH and stirred for 10 min.
The mixture was then desalted on a PD-10 Sephadex column using 0.2 M sodium borate (pH 8.0). The HS fractions were pooled (3 mL) and immediately stirred with the biotin-conjugated amine (5.0 mg, 9.4 µmol, excess) overnight (12 h). The mixture was then flash frozen, lyophilized, redissolved in ddH2O (0.5 mL), and purified using a G-25 Sephadex column. The desired fractions were pooled, flash frozen, and lyophilized to afford B-HS as a white solid (99% recovery). 1H NMR
(600 MHz, D2O): δ 3.29 (bs, 1H), 3.72-3.91 (m, 5H), 4.04-4.13 (m, 1H), 4.18-4.32 (m, 1H), 4.33-4.44 (m, 1H), 4.84 (bs, 1H), 5.26 (bs, 1H, IdoA C-1), 5.42 (bs, 1H, GlcN C-1); substoichiometric: 1.30 (bs, 2H, CH2-biotinPEG4), 1.43 (bs, 2H, CH2- biotinPEG4), 1.50-1.52 (m, 2H, CH2-biotinPEG4), 2.05 (bs, 3H, NHAc), 2.29 (t, 2H, CH2-biotinPEG4), 2.52-2.60 (m, 2H, CH2-biotinPEG4), 2.78-2.82 (m, 2H, C(O)CH2-biotinPEG4) 2.99-3.03 (m, 1H, CH-biotinPEG4), 3.32-3.54 (m, 22H, CH2O- and CH2N-biotinPEG4). Biotin(PEG)4 incorporation was estimated to be 0.9 molecules per polysaccharide.
Biotinylated heparan sulfate with chloroalkane linker (B-HS-CL). A solution of 2-13 (4.0 mg, 12 µmol, excess) and NaBH3CN (2.0 mg, 32 µmol, excess) in 1:1 MeOH:ddH2O (400 µL) was added to B-HS (2.0 mg, 0.17 µmol) in ddH2O (500 µL). MeOH (approximately 300 µL) was added until the reaction turned from cloudy to colorless, and the mixture was stirred for 12 h at RT, concentrated, and purified by gel filtration chromatography using G-25 Sephadex resin. The pooled fractions containing the polysaccharide were lyophilized to yield a white powder (99% recovery). 1H NMR (600 MHz, D2O): δ 3.24 (bs, 1H), 3.62-3.89 (m, 5H), 4.04-4.14 (m, 1H), 4.17-4.32 (m, 1H), 4.33-4.44 (m, 1H), 5.24-5.45 (m, 2H, IdoA C-1, GlcN C-1); substoichiometric: 1.07 (d, J = 6.3 Hz, 3H, CH3-CL), 1.50-1.54 (m, 2H), 2.01 (s, 3H, NHAc), 2.18-2.23 (m, 6H), 2.34-2.45 (m, 2H), 2.75 (s, 2H), 2.99- 3.10 (m, 2H). CL incorporation was estimated to be 1.8 CL molecules per polysaccharide.
62 General procedure for chloroalkane heparin/heparan sulfate derivatives. In a typical procedure, 2-13 (4.0 mg, 12 µmol, excess) was dissolved in MeOH and ddH2O (1:1, 400 µL) with NaBH3CN (2.0 mg, 32 µmol, excess) and added to HS, de-HS, deO-HS, 6-deO-HS, or 2-deO-HS (2.0 mg, 0.17 µmol) in ddH2O (500 µL). MeOH (approximately 300 µL) was added until the reaction turned from cloudy to colorless, and the mixture was stirred for 12 h at RT, concentrated, and purified by gel filtration chromatography using G-25 Sephadex resin. The pooled fractions containing the polysaccharides were lyophilized to yield white powders (99%
recovery of polysaccharide in all cases). CL incorporation was estimated to be between 0.4 to 4 molecules per polysaccharide. HS-CL: 1H NMR (600 MHz, D2O):
δ 3.29 (bs, 1H), 3.78-3.91 (m, 2H), 4.06 (bs, 1H), 4.12 (bs, 1H), 4.20-4.31 (m, 2H), 4.33-4.44 (m, 2H), 4.91 (bs, 1H), 5.27-5.46 (m, 2H, IdoA C-1, GlcN C-1);
substoichiometric: 1.19 (dd, J = 6.3, 0.9 Hz, 3H, CH3-CL), 1.67-1.77 (m, 2H), 2.06 (bs, NHAc), 2.20-2.31 (m, 6H), 2.38-2.45 (m, 2H), 2.73 (s, 2H), 2.77-2.86 (m, 2H). de-HS-CL: 1H NMR (600 MHz, D2O): δ 3.77-3.94 (m, 4H), 3.96-4.12 (m, 2H), 4.19-4.38 (m, 2H), 4.92 (bs, 1H), 5.08-5.23 (m, 2H, IdoA C-1), 5.40 (bs, 1H, GlcN C-1); substoichiometric: 1.19 (d, J = 6.2 Hz, 3H, CH3-CL), 1.68-1.77 (m, 2H), 2.02 (bs, 3H, NHAc), 2.41 (t, J = 6.8 Hz, 2H), 2.66 (s, 2H), 2.79 (t, J = 6.8 Hz, 2H). deO-HS-CL: 1H NMR (600 MHz, D2O): δ 3.26 (bs, 1H), 3.62-3.77 (m, 4H), 3.78-3.93 (m, 3H), 4.06 (bs, 1H), 4.12 (bs, 1H), 4.95 (bs, 1H, IdoA C-1), 5.38 (bs, 1H, GlcN C-1); substoichiometric: 1.19 (d, J = 6.3 Hz, 3H, CH3-CL), 1.70-1.75 (m, 2H), 2.04 (bs, 3H, NHAc), 2.21-2.26 (m, 6H), 2.40-2.43 (m, 2H), 2.66 (s, 2H),
2.78 (t, J = 6.9 Hz, 2H). 6-deO-HS-CL: 1H NMR (600 MHz, D2O): δ 3.26 (bs, 1H), 3.64-3.95 (m, 5H), 4.05 (bs, 1H), 4.26 (bs, 1H), 4.36 (bs, 1H), 4.93 (bs, 1H), 5.28- 5.41 (m, 2H, IdoA C-1, GlcN C-1); substoichiometric: 1.19 (dd, J = 6.3, 1.7 Hz, 3H, CH3-CL), 1.69-1.76 (m, 2H), 2.06 (bs, 3H, NHAc), 2.18-2.30 (m, 6H), 2.41 (td, J = 6.9, 1.7 Hz, 3H), 2.66 (d, J = 1.7 Hz, 2H), 2.79 (td, J = 6.9, 1.5 Hz, 2H). 2-deO-HS- CL: 1H NMR (600 MHz, D2O): δ 3.26 (bs, 1H), 3.60-3.93 (m, 5H), 3.94-4.29 (m, 3H), 4.35 (bs, 1H), 5.30-5.45 (m, 2H, GlcN C-1, IdoA C-1); substoichiometric:
1.19 (d, J = 6.3 Hz, 3H, CH3-CL), 1.69-1.77 (m, 2H), 2.05 (bs, 3H, NHAc), 2.21- 2.34 (m, 6H), 2.44 (t, J = 6.8 Hz, 2H), 2.72-2.83 (m, 4H).