Stepwise PEG synthesis featuring deprotection and coupling in one pot

The stepwise synthesis of monodisperse polyethylene glycols (PEGs) and their derivatives usually involves using an acid-labile protecting group such as DMTr and coupling the two PEG moieties together under basic Williamson ether formation conditions. Using this approach, each elongation of PEG is achieved in three steps – deprotection, deprotonation and coupling – in two pots. Here, we report a more convenient approach for PEG synthesis featuring the use of a base-labile protecting group such as the phenethyl group. Using this approach, each elongation of PEG can be achieved in two steps – deprotection and coupling – in only one pot. The deprotonation step, and the isolation and purification of the intermediate product after deprotection using existing approaches are no longer needed when the one-pot approach is used. Because the stepwise PEG synthesis usually requires multiple PEG elongation cycles, the new PEG synthesis method is expected to significantly lower PEG synthesis cost.


Experimental Details
General information: All compounds from commercial sources were used as received unless noted otherwise. THF was distilled over Na/benzophenone under nitrogen. Compounds 3d [1], 3e [2], 3g [3], and 3j [3] were synthesized following reported procedure. All reactions were carried out under nitrogen using oven-dried glassware. Thin layer chromatography (TLC) was performed using Sigma-Aldrich TLC plates, silica gel 60F-254 over glass support, 250 μm thickness. 1 H and 13 C NMR spectra were obtained on a Varian UNITY INOVA spectrometer at 400 and 100 MHz, respectively. Chemical shifts (δ) were reported in reference to solvent peaks (residue CHCl3 at δ 7.24 ppm for 1 H and CDCl3 at δ 77.00 ppm for 13 C). HRMS was obtained on a Thermo HR-Orbitrap Elite Mass Spectrometer. LRMS was obtained on a Thermo Finnigan LCQ Advantage Ion Trap Mass Spectrometer.
Screening base-labile protecting groups for PEG synthesis -Testing if the groups in 3a-l can be removed under basic conditions: In an oven dried 25 mL flask, 3a-k or 3l (0.734 mmol, 1 equiv) was dissolved in THF (4 mL). The solution was cooled to −78 °C. KHMDS (1 M in THF, 1.468 mL, 1.468 mmol, 2 equiv) was added via a syringe. The reaction mixture was stirred while warming to 0 °C gradually. After 2 h, TLC analyses (see below) were carried out. All compounds 3a-l were found to be consumed. Thus, the base-labile protecting groups in them meet the criterion of being labile under basic conditions required for PEG synthesis. Compound 3a was also tested using the base t-BuOK/LDA and found consumed under the conditions [4,5].
Screening base-labile protecting groups for PEG synthesis -Testing stability of protecting groups under the basic Williamson ether formation conditions: Compounds DMTrO(PEG)4OTs (1) [6] and MeO(PEG)4OH (4) were dried over P2O5 in a desiccator under vacuum for 2 days. Compound 4 (41 mg, 0.201 mmol, 1 equiv) was dissolved in THF (200 µL) under nitrogen. The solution was cooled to −78 °C, and KHMDS (0.241 mL, 0.241 mmol, 1 M in THF, 1.2 equiv) was added dropwise via a syringe. After addition, the reaction flask was placed in an ice bath for ~30 min. The mixture was then cooled to −78 °C. The solution of 1 (195 mg, 0.301 mmol, 1.5 equiv) and 3a-k or 3l (0.301 mmol, 1.5 equiv) in THF (500 µL) was added via a cannula dropwise. The reaction mixture was warmed to rt gradually over ~3 h. After stirring at rt for ~30 min, the mixture was heated to 60 °C and stirred vigorously at the temperature for 24 h. TLC analyses (see below) were carried out to determine if the Williamson ether formation reaction could proceed to form product 5 without the consumption of compound 3a-k or 3l. All the compounds except 3h were found to be able to survive the basic Williamson ether formation reaction conditions. Thus, the base-labile protecting groups in them (except 3h) meet the criterion of being stable under basic coupling conditions required for PEG synthesis.
DMTrO(PEG)4O(CH2)2Ph (7): The suspension of NaH (60% in mineral oil, 716 mg, 17.9 mmol, 2.5 equiv.) in anhydrous DMF (25 mL) in a 2-neck round bottom flask under nitrogen was cooled on an ice bath. The solution of Ph(CH2)2OH (2.14 mL, 17.9 mmol, 2.5 equiv) in anhydrous DMF (15 mL) was added dropwise via a cannula over ~1 h. After addition, the reaction mixture was stirred at 0 °C for ~1 h. The ice bath was removed. This gave the solution of NaO(CH2)2Ph. Compound 1 (4.66 g; 7.17 mmol, 1 equiv), which had been dried over P2O5 under high vacuum overnight, was dissolved in anhydrous DMF (15 mL). The solution was added to the solution of S3 NaO(CH2)2Ph dropwise via a cannula. After addition, the mixture was stirred vigorously at 60 °C for 24 h. After cooling to rt, the reaction was quenched with EtOH. DMF was removed on a rotary evaporator under high vacuum. The residue was partitioned between EtOAc (250 mL) and 5% K2CO3 (100 mL). The organic phase was washed with 5% K2CO3 (100 mL × 3), dried over anhydrous Na2SO4, and filtered. The filtrate was evaporated to dryness under reduced pressure and further dried under high vacuum. The residue was purified with flash chromatography (SiO2, Et3N/hexanes 1:9) to give compound 7 (4.02 g, 96%) as a yellow oil: TLC Rf = 0.3 (SiO2, hexanes/EtOAc 3:1); 1  Ph(CH2)2O(PEG)4 (6): Compound 7 (2.17 g, 3.62 mmol, 1 equiv.) was dissolved in dry DCM (10 mL). To the solution was added TFA (433 µL, 3.62 mmol, 1 equiv). The reaction mixture was stirred vigorously. After ~5 min, TLC indicated that compound 7 was consumed. The reaction was quenched with solid NaOH and a small volume of water until pH ~9. The mixture was then partitioned between DCM (total about 200 mL) and brine (75 mL). The aqueous phase was washed with DCM (100 mL × 3). The combined organic phase was dried over anhydrous Na2SO4, and filtered. The filtrate was evaporated to dryness, and the residue was purified with flash chromatography (SiO2, EtOAc) to give compound 6 (568 mg, 77%) as a yellow oil: TLC Rf = 0.10 (SiO2, hexanes/EtOAc 1:3); 1 (2): The compound was synthesized using a reported procedure with modifications [7]. The solutions of 6 (9.22 g, 46.5 mmol, 1 equiv) in THF (50 mL) and NaOH powder (22.3 g, 557 mmol, 12 equiv) in water (50 mL) were combined and stirred at 0 °C for 5 min. The solution of TsCl (26.5 g, 139.5 mmol, 3 equiv) in THF (50 mL, note that it is important to keep the ratio of total THF and water at around 2:1 v/v) was added dropwise over 10 min while the reaction mixture was stirred at 0 °C. After addition, stirring was continued while the temperature was raised to rt gradually. The progress of the reaction was monitored by TLC, and complete reaction was observed within 24 h. The mixture was partitioned between 5% Na2CO3 (300 mL) and EtOAc (500 mL). The aqueous phase was extracted with EtOAc (200 mL × 3). The combined organic phase was dried over anhydrous Na2SO4 and filtered. Volatiles were removed under reduced pressure, and the residue was further dried under vacuum from an oil pump. Compound 2 (12.7 g, 60%) was obtained as a colorless oil after flash chromatography purification (SiO2, hexanes/EtOAc 1:0 to 2:1): TLC Rf = 0.30 (SiO2, hexanes/EtOAc 1:1); 1

CH2)2O(PEG)12O(CH2)2Ph (8):
Compound 2 (2.19 g, 4.83 mmol, 2.5 equiv.) was dried over P2O5 under vacuum in a desiccator overnight. A suspension of NaH (60% in mineral oil, 193 mg, 4.83 mmol, 2.5 equiv) in dry THF (5 mL) under nitrogen was cooled on an ice bath. The solution of (PEG)4 (333 µL, 1.93 mmol, 1 equiv) in dry THF (10 mL) was added via a cannula dropwise over ~20 min. After addition, the reaction was allowed to proceed for ~30 min. The ice bath was removed, and compound 2 in THF (10 mL) was added via a cannula dropwise over ~10 min. After addition, the mixture was stirred vigorously at 60 °C for 24 h. The reaction was quenched with EtOH. THF was removed under reduced pressure. The residue was partitioned between DCM (100 mL) and saturated NH4Cl (50 mL). The aqueous phase was washed with DCM (100 mL × 3). The combined organic phase was dried over anhydrous Na2SO4 and filtered. The filtrate was evaporated to dryness, and compound 8 was purified with flash chromatography (SiO2, EtOAc/MeOH 100:0 to 100:3) to give a colorless oil ( (9): Compounds 2 and 8 were dried over P2O5 in a desiccator under vacuum for 2 days. Compound 8 (1.3 g, 1.8 mmol, 1 equiv) was dissolved in dry THF (5 mL) under nitrogen. The solution was cooled to −78 °C, and KHMDS (4.6 mL, 1 M in THF, 2.5 equiv) was added dropwise via a syringe. After addition, the reaction flask was placed in an ice bath for ~3 h. TLC analysis indicated that both 8 and Ph(CH2)2O(PEG)12 were not in the reaction mixture. The mixture was then cooled to −78 °C for ~10 min, and the solution of 2 (3.8 g, 8.3 mmol, 4.5 equiv.) in THF (10 mL) was added dropwise via a cannula over ~10 min. The reaction mixture was allowed to warm up to room temperature gradually over a period of ~3 h. After stirring at room temperature for ~30 min, the mixture was heated to 60 °C and stirred vigorously at the temperature for 24 h. THF was removed under reduced pressure. The residue was partitioned between DCM (100 mL) and saturated NH4Cl (20 mL). The aqueous phase was washed with DCM (100 mL × 3). The combined organic phase was dried over anhydrous Na2SO4 and filtered. Flash chromatography (SiO2, EtOAc to DCM/Et2O/MeOH 100:8:4) gave compound 9 (1.765 g, 86%) as a yellow waxy solid: TLC Rf = 0.40 (SiO2, DCM/Et2O/MeOH 10:1:1); 1  Compound 9 was also synthesized using tBuOK/LDA instead of KHMDS as the base under otherwise identical conditions. Similar yields were obtained.