Synthesis of PEG‐Polycycloether Block Copolymers: Poloxamer Mimics Containing a Rigid Helical Block

Abstract Poloxamers are amphiphilic block copolymers consisting of poly(ethylene glycol) (PEG) and poly(propylene glycol) segments. Their self‐assembly and interfacial properties are tied to the relative hydrophilicity and hydrophobicity of each block and can therefore be adjusted by changing block lengths. Here, a series of PEG‐polycycloether block copolymers is synthesized that have the same structure as a poloxamer, but they encompass a rigid polycyclic backbone as the hydrophobic block. A variety of polymer structures are synthesized, for example diblock or triblock architectures, with/without olefinic units, atactic or isotactic backbone, and different block lengths. Due to their amphiphilicity, self‐assembly into spherical aggregates (diameters ranging from 64 to 132 nm) at low concentrations (critical aggregation concentration as low as 0.04 mg mL−1) is observed in water. Low surface tensions (as low as 26.7 mN m−1) are observed as well as the formation of stable high internal phase emulsions (HIPEs) irrespective of the oil/water ratio. This contrasts with the properties of the commonly used poloxamers P188 or P407 and illustrates the significance of the rigid polycycloether block. These new colloidal properties offer new prospects for applications in emulsion formulations for biomedicine, cosmetics, and the food industry.


Typical procedure for the synthesis of chloride end-functionalized poly(epoxybutene) -Cl-PEB30
PEB was synthesized according to a modified literature procedure. [32]TPPAlCl was prepared in a flame dried Schlenk tube by dissolving TPPH2 (1.35 g, 2.19 mmol, 1 equiv) in DCM (40 mL)   and adding a 1 M diethylaluminum chloride solution in hexane (1 equiv) slowly.After 3 h the volatiles were evaporated using vacuum and a nitrogen cooled trap.TPPAlCl was then dried overnight.Racemic or isotactic 3,4-epoxy-1-butene (4.20 g, 4.83 mL, 60.0 mmol, 30 equiv) was added the next day to the dried (TPP)AlCl and the resulting mixture was subsequently stirred at ambient temperature for 2 days.The mixture was quenched by adding 1 M aqueous HCl (2 mL) under stirring.The volatiles were removed, and the resulting polymer was dissolved in a 1:1 methanol/DCM mixture for purification by preparative SEC (Sephadex LH-20, methanol/DCM S4 1:1).The fractions were concentrated, and the polymer was dried under vacuum overnight to give a brown oil (atactic Cl-PEB30: 3.44 g, 82%).
Typical procedure for the reduction of poly(epoxybutene) chloride end-group leading to H-PEB30 Atactic or isotactic H-PEB30 was synthesized by dissolving the corresponding Cl-PEB30 (400 mg, 190 μmol, 1 equiv) in THF (4 mL), then slowly adding a LiAlH4 solution in THF (1 M, 2.0 mL, 2.0 mmol, 10 equiv).The mixture was stirred at ambient temperature for 48 h, quenched with methanol (1 mL) then concentrated under vacuum.DCM (10 mL) and a 10% aqueous Rochelle salt solution (10 mL) were added and the mixture was stirred for 4 h.The organic phase was then separated, washed three times with deionized water, dried over magnesium sulfate, and filtered.
The volatiles were removed under vacuum, and the resulting polymer was dissolved in a 1:1 methanol/DCM mixture for purification by preparative SEC (Sephadex LH-20, methanol/DCM 1:1).The fractions were concentrated, and the polymer was dried under vacuum overnight to give a brown oil (atactic H-PEB30: 324 mg, 81%).

Typical procedure for the hydroxy substitution of poly(epoxybutene) chloride end-group leading to HO-PEB30
Atactic or isotactic HO-PEB30 was synthesized by dissolving the corresponding Cl-PEB30 (500 mg, 240 μmol, 1 equiv) and silver nitrate (210 mg, 2.50 mmol, 10 equiv) in a mixture of ethanol (10 mL) and deionized water (2 mL).The reaction mixture was then refluxed for 48 h and subsequently concentrated under vacuum.The resulting polymer was dissolved in DCM (10 mL), and the resulting solution washed three times with water, dried over magnesium sulfate, and filtered.The volatiles were removed under vacuum, and the resulting polymer was dissolved in a 1:1 methanol/DCM mixture for purification by preparative SEC (Sephadex LH-20, methanol/DCM 1:1).The fractions were concentrated, and the polymer was dried under vacuum overnight to give a brown oil (atactic HO-PEB30: 430 mg, 86%).

Typical procedure for the synthesis of polycycloether -PCE30
Isotactic or atactic PCE30 was synthesized according to a modified literature procedure. [32]In a large round-bottomed flask, atactic or isotactic H-PEB30 or HO-PEB30 (600 mg, 8.57 mmol) was stirred for 15 min at 84 °C in DCE (38 mL).Then, second-generation Grubbs catalyst (178 mg, 210 μmol, 2.5 mol%) in DCE was added slowly under argon.After 5 days, the reaction mixture was cooled to ambient temperature, treated with 100 equiv of DMSO and stirred for 1 h.The volatiles were removed under vacuum, and the residue was purified using preparative SEC (Sephadex LH-20, 3:1 methanol/DCM).The volatiles were removed under vacuum, and the resulting PCE was dried overnight (atactic H-PCE30: 398 mg, 83%).

Typical procedure for the synthesis of polycycloether-block-poly(ethylene glycol) -PCE30-b-PEG100
In a flame-dried Schlenk tube, atactic or isotactic H-PCE30 (300 mg, 0.14 mmol, 1 equiv) was added and dried overnight under vacuum.Sodium hydride was added (36 mg, 1.5 mmol, 10 equiv), dry THF was introduced (3 mL), then the mixture was stirred at 50 °C for 3 h.The mixture was cooled down to ambient temperature then ethylene oxide in THF (2.5 M, 6.0 mL, 15 mmol, 100 equiv) was added and the mixture was stirred at 50 °C for 2 days.An aqueous sodium hydroxide solution was then added (1 M, 1.0 mL) and the mixture was stirred overnight at ambient temperature.The mixture was finally quenched with an aqueous HCl solution (1 M, 1.0 mL) then the volatiles were removed under vacuum.The resulting polymer was dissolved in deionized water and dialyzed (1.0 kDa MWCO) against deionized water over two days.The resulting solution was freeze-dried and PCE30-b-PEG100 was obtained as a viscous brown oil (atactic PCE30-b-PEG100: 522 mg, 57% yield).

Typical procedure for the synthesis of saturated polycycloether-block-poly(ethylene glycol) -SPCE30-b-PEG100
Atactic or isotactic SPCE30-b-PEG100 was synthesized by adding the corresponding PCE30-b-PEG100 (58 mg) to a 25 mL round-bottom flask.Under an argon atmosphere, methanol was added (2 mL) followed by Pd/C (10% wt, 12 mg).Hydrogen was then introduced to the flask.The mixture was stirred at ambient temperature for 24 h.The reaction mixture was then filtered, concentrated under vacuum, and the resulting oil solubilized in water, followed by dialysis (1.5 kDa MWCO) against water for 24 h.The aqueous solution was freeze-dried and SPCE30-b-PEG100 was obtained as a viscous yellow oil (atactic SPCE30-b-PEG100: 42 mg, 76%).

Emulsion preparation
To prepare an emulsion, the typical procedure consisted of dissolving the PEG-polycycloether block copolymer in water, adding the oil, keeping a total volume of 4 mL, then using IKA Ultra-Turrax T18 homogenizer for 4 minutes at 13,500 rpm.Toluene, n-dodecane and rapeseed oil were investigated as oil types.Blo9ck copolymer concentration ranged from 0.01% to 0.5%.Oil/water ratios ranged from 5% to 90%.The nature of the emulsion (O/W versus W/O) was determined by measuring the conductivity of the emulsion as well as doing a drop test; that is, putting a drop of the emulsion either in pure oil or pure water (See Supp.Info.Video S1). were performed on a JEOL 1200 EX TEM running at 80 kV, images were captured using a Cantega 2K X 2K camera and Olympus ITEM Software.To prepare negative stained samples: suspension droplets (5 μL) were placed on top of the surface of carbon coated 400 mesh copper grids that were previously glow discharged using a Quorum Q150T ES High vacuum system.Samples were left for 5 min to allow attachment, then grids were floated sample side down three times for 30 s each onto distilled water droplets before negative staining with 2% aqueous uranyl acetate for 5 min then allowed to air dry before imaging.High-resolution mass spectrometry (HRMS) was S8 performed on a Bruker microTOFq High Resolution Mass Spectrometer using an Electrospray (ESI) ion source coupled to a time-of-flight (ToF) analyzer.Multi-angle dynamic light scattering (MADLS) measurements were performed using an Anton Paar Litesizer 500 using forward scattering (15°), side scattering (90°) and back scattering (175°).The light source was a semiconductor laser diode at 40 mW, 658 nm.All experiments were performed three times and the average size distribution was calculated.Emulsion droplets were analyzed using a Leica DM750 binocular microscope without any dilution.Size distribution was collected using ImageJ software.
Enantiopure butadiene monoxide optical rotation was measured using an Autopol III Polarimeter, with a mean value obtained through 5 measurements.Surface tension values were measured by using an Ossila contact angle goniometer.Block copolymer solutions in water were prepared with a fixed concentration of 0.5 wt% and analyzed at ambient temperature.For each solution, shape analysis was performed on three drops, using 5 different frames for each drop, the surface tension value was then averaged (See Figure S6 and Table S4).Hydrophilic-Lipophilic Balance (HLB) values were calculated using Chemaxon HLB predictor through the MarvinSketch software, using Griffin's method.Circular dichroism studies were performed on a Chirascan circular dichroism spectrometer.Temperature was kept at 20 °C, spectra were recorded in triplicates from 180 to 260 nm, with an acquisition time of 1.0 s and a wavelength step increase of 1 nm.The sample concentration was kept at 0.1 mg/mL.The critical aggregation concentration (CAC) of each block copolymer was measured by fluorescence spectroscopy using pyrene as a fluorescence probe as described in the literature. [47]Measurements were made on a Horiba Duetta Bio fluorescence and absorbance spectrometer.A blank consisting of deionized water was used before each experiment.
Block copolymer solutions of different concentrations were prepared in water, before adding 12 μL of a 0.5 M pyrene solution in ethanol.Both blank and sample absorption spectra were recorded before each experiment to account for the Inner Filter Effect.Excitation wavelength was fixed at detector binning was 0.5 nm (1 pixel).Excitation and emission bandpass were 5 nm.Peak intensity was measured at 372 nm (I1) and 384 nm (I3).CAC was determined by plotting the I3/I1 ratio against the block copolymer concentration and reading the intersection of the regression trendlines at low and high concentrations.

Statistical Analysis
When dynamic light scattering was used, the standard deviation of the intensity-weighted diameter size was calculated using Anton Paar Kalliope particle sizing software.
The root-mean-square (RMS) error associated with surface tension values were calculated using Ossila contact angle goniometer software.An RMS error greater than 1 between the detected edge data and the polynomial fit is considered poor.
The standard deviation associated with the size distribution of emulsion droplets was calculated using ImageJ software, with a sample size of 100 droplets.

HLB values
Table S3.Hydrophilic-Lipophilic Balance (HLB) of the polymer and block copolymer synthesized, calculated through the Griffin method with the ChemSketch software.Table S4.Surface tension values at 20 °C of the different synthesized block copolymer as well as the calculated root mean squared error (RMSE).

Characterization 1 H
NMR and DOSY spectra were recorded on either a Bruker AVI DPX-400 or a Bruker DPX-400 (400 MHz) instrument.The chemical shifts are expressed in parts per million (ppm) referenced to TMS.D2O or CDCl3 was used as solvent.Size-exclusion chromatography (SEC) was conducted in THF at 35 °C using a column system with an Agilent PL Gel Guard Column (5 µm) and an Agilent PL Gel Mixed-D Column (5 µm) as well as an Agilent Infinity1260 II RID and calibration with poly(styrene) standards.Transmission electron microscopy (TEM) experiments

Figure S2 .
Figure S2.Size-exclusion chromatography traces of a) the homopolymers, b) diblock and c) triblock copolymers measured in THF.

Figure S6 .Figure S7 .
Figure S6.Surface tension measurement of a) PEG50-iPCE30-PEG50 b) PEG50-aSPCE30-PEG50 c) aPCE30-PEG100 d) iSPCE30-PEG100 e) iPCE30-PEG200 f) aPCE30-PEG200 g) iPCE30-PEG100 h) PEG50-aPCE30-PEG50 i) aSPCE30-PEG100 and j) water.The green line corresponds to the detected edge of the droplet, the blue line corresponds to the simulated fit used to calculate the surface tension.The difference between the simulated edge and the detected edge is the root mean squared error (RMSE).The instrument was calibrated on k) a 10.00 mm sphere.

Figure S8 .
Figure S8.Emulsion pictures of different PEG-PCE block copolymers at 0.5 wt.% concentration and a 65:35 n-dodecane/water ratio after a) 1 day and b) 4 months.

Figure S15 .
Figure S15.Size of poloxamers P188, P407 and P123 self-assemblies in water, at 20 °C and at a concentration of 5 mg/mL.Measured by dynamic light scattering, using back-scattering (175°) and intensity weighting.

Table S1 .
m/z values of Cl-PEB15, HO-PEB15 and H-PEB15 adducts.The adducts in bold are shown in Figure S1.