Peptidomimetic Star Polymers for Targeting Biological Ion Channels

Four end-functionalized star polymers that could attenuate the flow of ionic currents across biological ion channels were first de novo designed computationally, then synthesized and tested experimentally on mammalian K+ channels. The 4-arm ethylene glycol conjugate star polymers with lysine or a tripeptide attached to the end of each arm were specifically designed to mimic the action of scorpion toxins on K+ channels. Molecular dynamics simulations showed that the lysine side chain of the polymers physically occludes the pore of Kv1.3, a target for immuno-suppression therapy. Two of the compounds tested were potent inhibitors of Kv1.3. The dissociation constants of these two compounds were computed to be 0.1 μM and 0.7 μM, respectively, within 3-fold to the values derived from subsequent experiments. These results demonstrate the power of computational methods in molecular design and the potential of star polymers as a new infinitely modifiable platform for ion channel drug discovery.

pIRES2-EGFP (Clontech, USA) for coexpression with GFP. Plasmids containing each of the six channels were subsequently transfected into HEK293 cells using the SofastTM Transfection Reagent (Sunma). HEK293 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen), and incubated at 37 °C with 10% fetal bovine serum, 1% penicillin/streptomycin. Cells were transfected using FuGENE transfection reagent (Roche Diagnostics) and used for

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The structure of 11 was further confirmed by the 1 H NMR in Fig K in S1 File. The Boc-protecting groups on 11 were then removed using TFA for 6 h at room temperature to give 12.
16 was designed with a longer spacer (i.e. two EG units per arm) within the second generational layer (Scheme C in S1 File). Compound 13 was synthesized through the CuAAC coupling of 6 and 2 to produce 13, and then conversion of the HO-group on 14 to an alkyne (see Schemes C in S1 File). The CuAAC reaction between 14 and 10 afforded 15 (crude) in quite a low purity of 35.6 % as determined by the LND fit (Fig N in S1 File). The majority of the impurity was due to 14 (45%), which was used in excess to the azide core 10 (6.3% remaining). There was only a small amount of 1, 2 and 3-arm products, suggesting the excess of 14 drove the reaction to the 4-arm product 15.
After preparative SEC, the purity of 15 increased to 97% with only 3% of the 3-arm product in S1 File). The impurities consisted of the starting compounds and formation of 1-arm to 3-arm star products. After preparative SEC, the purity increased to 97% with 3% of 3-arm star remaining.
The MALDI-ToF further confirmed the high purity with only one major peak after preparative SEC shows that a number of species were formed after ionization through the MALDI.
These species were consistent with the various oxidized species from the sulfur on the methionine group ( Fig V in S1 File). Deprotection of Boc-groups on 30 using TFA gave 31 with near quantitative conversion.

Size Exclusion Chromatography (RI-SEC)
All polymer samples were dried prior to analysis in a vacuum oven for 2 days at 25 o C. The dried polymer was dissolved in tetrahydrofuran (THF) to a concentration of 1 mg mL -1 and then filtered Calibration was performed using narrow molecular weight PSTY standards (PDIRI≤1.1) ranging from 500 to 2 million g mol -1 . Data acquisition was performed using Empower software, and molecular weights were calculated relative to polystyrene standards.

Nuclear Magnetic Resonance (NMR)
All NMR spectra were recorded on either a Bruker DRX 400 or 500 MHz spectrometer using an external lock (CDCl3), and all spectra were referenced to the residual nondeuterated solvent (CHCl3). The frequencies were 400 or 500 MHz for 1 H NMR spectra, and 100 or 125 MHz for 13 C NMR spectra.

Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-ToF) Mass Spectrometry
MALDI-ToF MS spectra were obtained using a Bruker MALDI-ToF autoflex III smart beam

Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethanol (2)
9 A mixture of 2-(2-(2-chloroethoxy)ethoxy)ethanol (1, 5 g, 2.976 x 10 -2 mol), NaN3 (19.3 g, 2.976 x 10 -1 mol), NaI (0.9 g, 6.0 x 10 -3 mol) and H2O (30 mL) was added into a 100 mL flask and was then placed into a 60 o C oil bath. The reaction was allowed to react for 16 h with stirring. After removal of the solvent under reduced pressure, the product was diluted in 50 mL DCM, washed with saturated brine (2 x 20 mL) and the organic phase was dried over anhydrous MgSO4. The solvent was concentrated under reduce pressure. 4.47 g viscous oil 2 was obtained with the yield of 86% and used for next step directly without further purification.       Then, the solution of 14 was added via syringe pump, at a flow rate of 0.003 mL min -1 , and after feeding the reaction was allowed to react for 2 h. The copper salts were removed by passage through activated basic alumina. The solvent was removed under reduced pressure. The crude product was purified using a Varian Pro-Star preparative SEC system equipped with a manual injector, differential refractive index detector, and single wave-length ultra-violet visible detector.

Synthesis of OH-EG-
Flow rate was maintained 10 mL min -1 and HPLC grade THF was used as the eluent. Separations were achieved using a PL gel 10 mm 1x103 Å, 300 mm x 25 mm preparative SEC column held at

Synthesis of 4-arm star-like EG-(Lysine-Boc)2 conjugate (24)
A solution of 23 (13.6 mg, 9. and after feeding the reaction was allowed to react for 2 h. The copper salts were removed by passage through activated basic alumina. The solvent was removed under reduced pressure. The crude product was purified using a Varian Pro-Star preparative SEC system equipped with a manual injector, differential refractive index detector, and single wave-length ultra-violet visible detector.
Flow rate was maintained 10 mL min -1 and HPLC grade THF was used as the eluent. Separations were achieved using a PL gel 10 mm 1x103 Å, 300 mm x 25 mm preparative SEC column held at

Synthesis of 4-arm star-like EG-MKF-Boc conjugate (30)
A solution of 29 (0.184 g, 2.327 x 10 -4 mol) in toluene (20 mL) was purged with argon for 60 min to remove oxygen. Meanwhile, a mixture of 10 (0.027 g, 4.655 x 10 -5 mol), PMDETA (5.3 mg, 3.007 x 10 -5 mol), toluene (20 mL) was degassed by argon for 60 min, to the deoxygenated mixture above, CuBr (4.3 mg, 3.007 x 10 -5 mol) was added under positive argon flow. Then, the solution of 30 was added via syringe pump, at a flow rate of 0.06 mL min -1 , and after feeding the reaction was allowed to react for 2 h. The copper salts were removed by passage through activated basic alumina.
The solvent was removed under reduced pressure. The crude product was purified using a Varian Pro-Star preparative SEC system equipped with a manual injector, differential refractive index detector, and single wave-length ultra-violet visible detector. Flow rate was maintained 10 mL min -1 and HPLC grade THF was used as the eluent. Separations were achieved using a PL gel 10 mm