Example 12
Synthesis of Polyethylene Glycol Dendrimer (26K)
The syntheses of PEG dendrimer was done in two steps. First the building of the PEG dendron blocks was completed and second the blocks were joined to create the dendrimer structure.
i. Preparation of Dendron Building Block:
Et-G1-NHBoc. L-lysine ethyl ester dihydrochloride (0.253 g, 1.025 mmol) and SCM-PEG-NHBoc 2K (4.71 g, 2.36 mmol) were dissolved in dichloromethane (170 ml). After addition of TEA (0.714 ml, 5.12 mmol), the mixture was stirred overnight at room temperature. The reaction mixture was quenched with 51 mL of 0.1N HCl solution and stirred with of NaCl (5.1 g). Two layers were separated and the aqueous phase was extracted with dichloromethane (50 mL). The combined organic phases were dried over Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to give crude product as a waxy solid. The crude material was dissolved in water and passed through an Amberlite column and then an ion-exchange column using both DEAE Sepharose FF and SP Sepharose FF. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide Et-G1-NHBoc (3.4 g, 84% yield). 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (t, 3H, —OCH2CH3), 1.44 ppm (s, 18H, —NHBoc), 4.01 ppm (m, 4H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.32 ppm (q, 2H, —OCH2CH3), 4.59 ppm (q, 1H, —CH(CO2Et)NH—).
CO2H-G1-NHBoc Et-G1-NHBoc (0.975 g, 0.247 mmol) was dissolved in water (6.2 ml) and stirred overnight with 0.1 N NaOH (5 ml, 0.5 mmol). The mixture was acidified by adding 0.5 mL of 1N HCl, charged with 1.8 g of NaCl (15% w/v), and then stirred with 10 mL of DCM. The two layers were separated and the aqueous phase was extracted with 8 mL of DCM. The combined organic phases were dried over Na2SO4, filtered, concentrated, and dried in vacuo to give CO2H-G1-NHBoc (0.928 g, 96% yield) as a pale yellow waxy powder. The completion of the hydrolysis was confirmed by 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) revealed the disappearance of ester proton peaks, shown at 1.28 and 4.32 ppm (—OCH2CH3)
Et-G1-NH2.2TFA Et-G1-NHBoc (2.42 g, 0.613 mmol) was dissolved in dichloromethane (15.33 ml) and stirred with TFA (2.36 ml, 30.7 mmol) for 1 hour at room temperature. Most of the volatiles were removed using a rotary evaporator to give ˜4.5 g of thick red extract. The crude product was stirred with 30 mL of diethyl ether to give a sticky powder and a slightly cloudy suspension. After decanting the liquid, the residue was stirred with 30 mL of diethyl ether. After decanting the solution, the pale white powder (waxy) was dried overnight in vacuo. The crude product was dissolved in 25 mL of dichloromethane and then washed with brine (20 mL), dried over Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to give Et-G1-NH2.2TFA (2.10 g, 86% yield). The completion of the deprotection was confirmed by the disappearance of -Boc group proton peak, shown at 1.44 ppm (s, 18H, —NHBoc).
CO2H-G1-Ethynyl HOBT (0.209 g, 1.362 mmol) was dried by azeotropic distillation using acetonitrile. To the residue was added a solution of 4-pentynoic acid (0.125 g, 1.277 mmol) in dichloromethane (20 ml). DCC (0.264 g, 1.277 mmol) was added and the mixture was stirred for 10 minutes to give a cloudy solution. A solution of Et-G1-NH2.2TFA (1.69 g, 0.426 mmol) with TEA (0.356 ml, 2.55 mmol) in dichloromethane (20 ml) was added. After stirring for 18 hours, the reaction mixture was filtered using a syringe filter and quenched with 0.1N HCl. All the organic volatiles were removed using a rotary evaporator and passed through an Amberlite column and then an ion-exchange column using DEAE Sepharose FF. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide Et-G1-Ethynyl.
Hydrolysis of Et-G1-Ethynyl The ethyl ester product was dissolved in water and the pH of the solution was adjusted to 13 using 0.5 N NaOH. After stirring overnight, the mixture was acidified to pH 3 and purified on an Amberlite column and an ion-exchange column using DEAE Sepharose FF to give 1.14 g (69% yield) of CO2H-G1-Ethynyl as the desired product. 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 2.03 (m, 2H, —CH2CH2CCH), 2.42 (t, 4H, —CH2CH2CCH), 2.53 (t, 4H, —CH2CH2CCH), 3.98-4.16 ppm (m, 4H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.62 ppm (q, 1H, —CH(CO2Et)NH—).
ii. Construction of Dendrimer via a Convergent Pathway
Et-G2-NHBoc
HOBT (0.035 g, 0.227 mmol) was dried by azeotropic
distillation using acetonitrile (20 mL). To the residue was added a
solution of CO2H-G1-NHBoc (0.890 g, 0.227 mmol) in
dichloromethane (15 ml). DCC (0.047 g, 0.227 mmol) was added and the
mixture was stirred for 3 hours. After addition of Et-G1-NH2.2TFA
(0.410 g, 0.103 mmol) and TEA (0.086 ml, 0.620 mmol), the reaction
mixture was stirred overnight at room temperature. The mixture was
filtered using a syringe filter and quenched with 0.1N HCl. All the
organic volatiles were removed using a rotary evaporator. The resulting
aqueous solution was passed through an Amberlite column and then an
ion-exchange column using both DEAE Sepharose FF and SP Sepharose FF.
The resulting aqueous solution was charged with NaCl (15% w/v) and
extracted with dichloromethane. The combined organic phases were dried
over anhydrous Na2SO4, filtered, concentrated
using a rotary evaporator, and dried in vacuo to provide Et-G2-NHBoc
(0.879 g, 74% yield). Ion-exchange analysis on both DEAE and SP column
revealed all neutral species. 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (m, 3H, —OCH2CH3), 1.44 ppm (s, 36H, —NHBoc), 3.98-4.04 ppm (m, 12H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.19 ppm (m, 2H, —OCH2CH3), 4.59 ppm (q, 1H, —CH(CO2Et)NH—).
Et-G2-NH2.4HCl Et-G2-NHBoc (0.877 g, 0.076 mmol) was stirred with 20 mL of methanolic HCl (5 ml, 15.20 mmol) for 1 hour at room temperature. All the volatiles were removed under vacuum. The residue was dissolved in 30 mL of dichloromethane and washed with 25 mL of brine solution. The organic solution was dried over Na2SO4, filtered, concentrated, and dried in vacuo to give Et-G2-NH2.HCl (0.883 g, quantitative yield). 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (m, 3H, —OCH2CH3), 3.94-4.04 ppm (m, 12H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.17 ppm (m, 2H, —OCH2CH3). The completion of deprotection was confirmed by disappearance of t-Boc proton peak at 1.44 ppm (s, 36H, —NHBoc).
Et-G3-Ethynyl HOBT (0.051 g, 0.332 mmol) was dried by azeotropic distillation using 30 mL of acetonitrile. To the residue was added a solution of CO2H-G1-Ethynyl (1.133 g, 0.292 mmol) in dichloromethane (33 ml). DCC (0.060 g, 0.292 mmol) was added and the mixture was stirred for 2 hours at room temperature to give a cloudy solution. After addition of Et-G2-NH2HCl (0.75 g, 0.066 mmol) and TEA (0.074 ml, 0.532 mmol), the mixture was stirred for 16 hours at room temperature. The mixture was quenched with 6 mL of 0.1 N HCl. All the organic volatiles were removed using a rotary evaporator and the remaining aqueous solution was diluted with 15 mL of water. The resulting aqueous solution was passed through an Amberlite column and then an ion-exchange column using both DEAE Sepharose FF and SP Sepharose FF to remove excess acid dendron species and amino species resulting from incomplete reaction. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide pale yellow solids. Further purification was performed by stirring with 30 mL of diethyl ether for 30 minutes, filtering on a glass frit, and drying to give Et-G3-Ethynyl (1.221 g, 69% yield) as pale yellow crystalline material. Ion-exchange analysis on both DEAE and SP column revealed all neutral species. 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (m, 3H, —OCH2CH3), 2.03 (m, 2H, —CH2CH2CCH), 2.43 (t, 16H, —CH2CH2CCH), 2.53 (t, 16H, —CH2CH2CCH), 3.98-4.03 ppm (m, 28H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.17 ppm (m, 2H, —OCH2CH3), 4.40 ppm (q, 6H, —CH(CO—)NH—). 4.62 ppm (q, 1H, —CH(CO2Et)-NH—).
https://www.google.com/patents/US8383093
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The syntheses of PEG dendrimer was done in two steps. First the building of the PEG dendron blocks was completed and second the blocks were joined to create the dendrimer structure.
i. Preparation of Dendron Building Block:
Et-G1-NHBoc. L-lysine ethyl ester dihydrochloride (0.253 g, 1.025 mmol) and SCM-PEG-NHBoc 2K (4.71 g, 2.36 mmol) were dissolved in dichloromethane (170 ml). After addition of TEA (0.714 ml, 5.12 mmol), the mixture was stirred overnight at room temperature. The reaction mixture was quenched with 51 mL of 0.1N HCl solution and stirred with of NaCl (5.1 g). Two layers were separated and the aqueous phase was extracted with dichloromethane (50 mL). The combined organic phases were dried over Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to give crude product as a waxy solid. The crude material was dissolved in water and passed through an Amberlite column and then an ion-exchange column using both DEAE Sepharose FF and SP Sepharose FF. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide Et-G1-NHBoc (3.4 g, 84% yield). 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (t, 3H, —OCH2CH3), 1.44 ppm (s, 18H, —NHBoc), 4.01 ppm (m, 4H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.32 ppm (q, 2H, —OCH2CH3), 4.59 ppm (q, 1H, —CH(CO2Et)NH—).
CO2H-G1-NHBoc Et-G1-NHBoc (0.975 g, 0.247 mmol) was dissolved in water (6.2 ml) and stirred overnight with 0.1 N NaOH (5 ml, 0.5 mmol). The mixture was acidified by adding 0.5 mL of 1N HCl, charged with 1.8 g of NaCl (15% w/v), and then stirred with 10 mL of DCM. The two layers were separated and the aqueous phase was extracted with 8 mL of DCM. The combined organic phases were dried over Na2SO4, filtered, concentrated, and dried in vacuo to give CO2H-G1-NHBoc (0.928 g, 96% yield) as a pale yellow waxy powder. The completion of the hydrolysis was confirmed by 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) revealed the disappearance of ester proton peaks, shown at 1.28 and 4.32 ppm (—OCH2CH3)
Et-G1-NH2.2TFA Et-G1-NHBoc (2.42 g, 0.613 mmol) was dissolved in dichloromethane (15.33 ml) and stirred with TFA (2.36 ml, 30.7 mmol) for 1 hour at room temperature. Most of the volatiles were removed using a rotary evaporator to give ˜4.5 g of thick red extract. The crude product was stirred with 30 mL of diethyl ether to give a sticky powder and a slightly cloudy suspension. After decanting the liquid, the residue was stirred with 30 mL of diethyl ether. After decanting the solution, the pale white powder (waxy) was dried overnight in vacuo. The crude product was dissolved in 25 mL of dichloromethane and then washed with brine (20 mL), dried over Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to give Et-G1-NH2.2TFA (2.10 g, 86% yield). The completion of the deprotection was confirmed by the disappearance of -Boc group proton peak, shown at 1.44 ppm (s, 18H, —NHBoc).
CO2H-G1-Ethynyl HOBT (0.209 g, 1.362 mmol) was dried by azeotropic distillation using acetonitrile. To the residue was added a solution of 4-pentynoic acid (0.125 g, 1.277 mmol) in dichloromethane (20 ml). DCC (0.264 g, 1.277 mmol) was added and the mixture was stirred for 10 minutes to give a cloudy solution. A solution of Et-G1-NH2.2TFA (1.69 g, 0.426 mmol) with TEA (0.356 ml, 2.55 mmol) in dichloromethane (20 ml) was added. After stirring for 18 hours, the reaction mixture was filtered using a syringe filter and quenched with 0.1N HCl. All the organic volatiles were removed using a rotary evaporator and passed through an Amberlite column and then an ion-exchange column using DEAE Sepharose FF. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The organic phase was dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide Et-G1-Ethynyl.
Hydrolysis of Et-G1-Ethynyl The ethyl ester product was dissolved in water and the pH of the solution was adjusted to 13 using 0.5 N NaOH. After stirring overnight, the mixture was acidified to pH 3 and purified on an Amberlite column and an ion-exchange column using DEAE Sepharose FF to give 1.14 g (69% yield) of CO2H-G1-Ethynyl as the desired product. 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 2.03 (m, 2H, —CH2CH2CCH), 2.42 (t, 4H, —CH2CH2CCH), 2.53 (t, 4H, —CH2CH2CCH), 3.98-4.16 ppm (m, 4H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.62 ppm (q, 1H, —CH(CO2Et)NH—).
ii. Construction of Dendrimer via a Convergent Pathway
Et-G2-NH2.4HCl Et-G2-NHBoc (0.877 g, 0.076 mmol) was stirred with 20 mL of methanolic HCl (5 ml, 15.20 mmol) for 1 hour at room temperature. All the volatiles were removed under vacuum. The residue was dissolved in 30 mL of dichloromethane and washed with 25 mL of brine solution. The organic solution was dried over Na2SO4, filtered, concentrated, and dried in vacuo to give Et-G2-NH2.HCl (0.883 g, quantitative yield). 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (m, 3H, —OCH2CH3), 3.94-4.04 ppm (m, 12H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.17 ppm (m, 2H, —OCH2CH3). The completion of deprotection was confirmed by disappearance of t-Boc proton peak at 1.44 ppm (s, 36H, —NHBoc).
Et-G3-Ethynyl HOBT (0.051 g, 0.332 mmol) was dried by azeotropic distillation using 30 mL of acetonitrile. To the residue was added a solution of CO2H-G1-Ethynyl (1.133 g, 0.292 mmol) in dichloromethane (33 ml). DCC (0.060 g, 0.292 mmol) was added and the mixture was stirred for 2 hours at room temperature to give a cloudy solution. After addition of Et-G2-NH2HCl (0.75 g, 0.066 mmol) and TEA (0.074 ml, 0.532 mmol), the mixture was stirred for 16 hours at room temperature. The mixture was quenched with 6 mL of 0.1 N HCl. All the organic volatiles were removed using a rotary evaporator and the remaining aqueous solution was diluted with 15 mL of water. The resulting aqueous solution was passed through an Amberlite column and then an ion-exchange column using both DEAE Sepharose FF and SP Sepharose FF to remove excess acid dendron species and amino species resulting from incomplete reaction. The resulting aqueous solution was charged with NaCl (15% w/v) and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na2SO4, filtered, concentrated using a rotary evaporator, and dried in vacuo to provide pale yellow solids. Further purification was performed by stirring with 30 mL of diethyl ether for 30 minutes, filtering on a glass frit, and drying to give Et-G3-Ethynyl (1.221 g, 69% yield) as pale yellow crystalline material. Ion-exchange analysis on both DEAE and SP column revealed all neutral species. 1H NMR (Varian, 500 MHz, 10 mg/mL CDCl3) showed the usual backbone peak at 3.64 ppm (m, 4H, —(OCH2CH2)n—) and other major peaks at 1.28 ppm (m, 3H, —OCH2CH3), 2.03 (m, 2H, —CH2CH2CCH), 2.43 (t, 16H, —CH2CH2CCH), 2.53 (t, 16H, —CH2CH2CCH), 3.98-4.03 ppm (m, 28H two protons for each PEG, —NHC(═O)CH2—(OCH2CH2)n—), 4.17 ppm (m, 2H, —OCH2CH3), 4.40 ppm (q, 6H, —CH(CO—)NH—). 4.62 ppm (q, 1H, —CH(CO2Et)-NH—).
https://www.google.com/patents/US8383093
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