Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions

We first describe the synthesis and purification of PEG-MM B from 3 kDa O-(2-aminoethyl)polyethylene glycol (PEG-NH₂) and norbornene-N-hydroxysuccinimidyl (NHS) ester. The former compound can be purchased from Sigma Aldrich Inc., or prepared via anionic polymerization according to literature procedures^22,23. The latter compound can be prepared in two steps according to a published procedure²¹. Next we describe a synthesis of catalyst A from commercially available Grubbs 2^nd generation catalyst. We then demonstrate the use of this complex for brush-first BASP synthesis. This experiment details the procedure for making BASPs with N = 10, 15, and 20 from a BI with DP = 10. All reactions were performed in a fume hood using standard scintillation vials.

CAUTION: Always wear gloves, a lab-coat, and lab glasses, and follow common laboratory safety practices when working with hazardous chemicals. Any organic solvent must be handled in a fume hood. Solids can be weighed out on a balance outside the fume hood. Chemicals should not come into contact with skin, eyes, or mouth. It is strongly recommended to read the MSDS for every solvent and solid used in this procedure before beginning.

1. Preparation of PEG-MM B

1. Add PEG-NH₂ (300 mg, 0.0001 mol, 1.0 eq) to a 40 ml scintillation vial equipped with a stir bar.
2. Dissolve the PEG-NH₂ in 3 ml of anhydrous N,N-dimethylformamide (DMF).
3. Add 36 mg of norbornene-NHS ester (0.000105 mol, 1.05 eq)²¹.
4. Cap the vial and stir the reaction mixture overnight at room temperature.
5. Remove the stir bar and add diethyl ether to the reaction solution to precipitate the PEG-MM B.
6. Filter the white fluffy precipitate and wash extensively with diethyl ether. Alternatively, transfer the suspension to a 50 m centrifuge tube, centrifuge at 4,000 rpm for 5 min at room temperature, and then decant the supernatant. Add fresh diethyl ether, centrifuge, and decant again. We recommend repeating this procedure 3x for a total of 5x.
7. Dry the precipitate under vacuum for 24 hr to remove residual diethyl ether.

2. Purification of PEG-MM

In our previous report, the PEG-MM B was prepared from commercially available PEG-NH₂ and was used for BASP synthesis without further purification after drying (i.e., after step 1.7). In this study, we vary the PEG-NH₂ source (commercial versus homemade), and we compare BASP formation results before and after more rigorous preparative high performance liquid chromatography (prep-HPLC) MM purification. In the remainder of this study, the dried MM obtained after step 1.7 is referred to as B1. Prep-HPLC was used to purify B1 to give B2. An analogous prep-HPLC purified MM synthesized in our laboratory via anionic polymerization is referred to as B3. Prep-HPLC was performed using a Beckmann Coulter HPLC (127p solvent module and 166p detector module) with a 1-ml sample loop and an Agilent Zorbax 300SB-C18 PrepHT reverse-phase column at room temperature.

1. Set-up HPLC with solvent A: deionized water (Millipore purification system, 18.2 Ω) with 1% acetic acid; solvent B: acetonitrile.
2. Prime pumps and equilibrate column with 95% A and 5% B.
3. Dissolve PEG-MM in acetonitrile or MeOH (150 mg/ml).
4. Filter through a 13 mm 0.45 µm Nylon syringe filter.
5. Set HPLC method:
   – Flow rate: 20 ml/min
   – 0-1 min: linear gradient to 10% B and 90%A
   – 1-10 min: linear gradient to 90% B and 10% A
   – 10-13 min: switch to initial conditions (5% B and 95%) and re-equilibrate column
   – Set UV detector to detect absorbance at 256 nm
6. Load 0.8 ml of sample onto the sample loop.
7. Inject sample.
8. Collect the major absorbance peak (under the conditions specified, the product elutes between 5-7 min).
9. Repeat as necessary. Combine pure fractions together in a round-bottom flask.
10. Remove all solvent via rotary evaporation.
11. Redissolve the product in dichloromethane and add sodium sulfate. Gently shake or stir the flask periodically for ≅ 1 hr.
12. Filter the mixture using a fritted glass filter.
13. Concentrate via rotary evaporation. Dry under vacuum overnight.
14. The PEG-MM can be characterized by ¹H-NMR in CD₂Cl₂ (15-20 mg/0.7 ml CD₂Cl₂, 500 MHz or higher is recommended with over 128 scans and relaxation delay, d1 = 2.0 sec), and MALDI-TOF using positive ionization mode and 2-(4-hydroxyphenylazo)benzoic acid as the MALDI matrix.
15. The PEG-MM can be stored for months in a scintillation vial at 4 °C.

3. Preparation of Catalyst A

1. Add Grubbs 2^nd generation catalyst (500 mg, 0.589 mmol) to a 20 ml vial equipped with a stir bar.
2. Add pyridine (approximately 0.474 ml, 5.89 mmol, 10 eq) to the vial. The solution color should immediately change from red to green. Allow the reaction to stir until all of the red color has disappeared and the solution has become viscous (15-30 min).
3. Fill the reaction vial with cold pentane to precipitate complex A.
4. Filter the suspension to collect the green precipitate (catalyst A). Wash 4x with 15 ml of cold pentane.
5. Dry the green solid under vacuum overnight.
6. Complex A can be stored for months at room temperature in a benchtop desiccator without significant loss of activity. For extra precaution, we typically store the complex in a -20 °C freezer inside a glovebox. For convenience, we pre-weigh known amounts of A into 4 ml scintillation vials immediately after drying (step 3.5). We then store these vials in the glovebox freezer. When ready to run a ROMP reaction, we simply take one vial out of the glovebox and use as described below (step 4.4).

4. Preparation of Stock Solution of Living Brush Polymer (BI) with DP = 10

1. In a 3 ml vial with a gas-tight screw cap equipped with a stir bar, weigh out 65 mg (0.020 mmol, 10 eq) of MM B. This amount corresponds to 20 mg of MM for each of the 3 different sizes of BASPs, and 5 mg leftover for GPC analysis of the BI. Use a spatula to add the MM directly to the bottom of the vial. Try to prevent material from adhering to the sides of the vial as this scenario could lead to MM contamination in the final BASP product.
2. Dissolve the MM B in 158 µl of THF. Immediately cap the vial after adding THF to avoid solvent evaporation. Note: The final concentration of MM during the polymerization should be 0.05 M. If 158 µl of THF is added here, then 243 µl of catalyst solution, step 4.4, will be added to give 401 µl of THF total, which corresponds to [MM] = 0.05 M. The amount of solvent during this step can be varied, as long as the amount of solvent during step 4.4 is also varied to give [MM] 0.05. We have found that polymerizations carried out with [MM] < 0.05 sometimes do not proceed to complete conversion.
3. Let the solution stir until all of the MM is dissolved. Heat lightly if necessary. Avoid splattering the viscous solution onto the sides or the cap of the vial.
4. Next, add a known amount (2.8 mg for this example) of catalyst A to a 3 ml vial (or obtain a vial with pre-weighed catalyst A). Add anhydrous THF (466 µl in this example) to give a 6 mg/ml catalyst solution. Cap the vial immediately. Allow the catalyst to completely dissolve; gently shake the vial if needed. This catalyst solution should be used immediately for ROMP. Note 1: The catalyst solution should be a forest green color. If it is black, or greenish-brown, then it has likely decomposed, and it will probably not yield satisfactory ROMP results. If decomposition occurs, we suggest preparing fresh catalyst (according to section 3 above), or using freshly distilled THF. Note 2: The amount of THF added to A is chosen to ensure that the final [MM] is ~0.05. This amount can be adjusted, as long as compensatory adjustments are made to the MM solution in step 4.2.
5. Add 243 µl (1.46 mg; 1 eq to B) of the catalyst solution quickly (not dropwise) to the stirring MM solution. Try to keep the needle tip just above the MM solution when adding to the vial. Avoid splashing the reaction mixture onto the sides and cap of the vial, as splashing can lead to residual MM and BI impurity in the final BASP.
6. Cap the vial immediately and let the reaction mixture stir for 15 min to form the brush macroinitiator (BI).

5. Formation of BASPs

1. Add 3.6±0.1 mg (6.18 µmol, 10 eq to the amount of BI to be transferred in step 5.2), 5.5 ± 0.1 mg (9.28µmol,15 eq to the amount of BI to be transferred in step 5.2), and 7.3±0.1 mg (12.4µmol,20 eq to the amount of BI to be transferred in step 5.2) of crosslinker C to three separate 3 ml vials equipped with stir bars. Try to weigh the crosslinker directly onto the bottom of the vial to prevent material from adhering to the sides of the vial. Note: Crosslinker C is not highly soluble in THF. For this reason, the solid is used directly in this step. In cases where the crosslinker is soluble, then a concentrated stock solution of crosslinker can be made and various amounts of this solution can be transferred to vials. Again, the concentration of the final polymerizations should be >0.05 M; if solvent is added to the crosslinker then a compensatory reduction of solvent should be made elsewhere.
2. Add 123 µl (0.618 µmol) of the BI solution to each of the three vials containing C. Try to keep the needle tip just above the solid crosslinker when adding to the vial. Add the BI solution all at once rather than dropwise.
3. Cap the vials and stir the reactions at RT for until completion. With this specific MM and crosslinker combination, the reaction is complete in ~4 hr; continued stirring for up to 24 hr has no discernible effect on BASP growth. Monitor by GPC to ensure complete conversion of BI.
4. Quench the reactions by adding one drop of ethyl vinyl ether to the remaining BI solution and each of the N = 10, 15, and 20 BASP reaction mixtures. Stir for 10 min to ensure complete quenching.

6. GPC Sample Preparation

The GPC-MALLS results were obtained on an Agilent 1260 LC system equipped with a Shodex GPC KD-806M column, a Wyatt Dawn Heleos-II MALLS detector, and a Wyatt Optilab t-rEX refractive index detector at room temperature. DMF with 0.025 M LiBr at a flow rate of 1.0 ml/min was used as the eluent. Results were analyzed using Astra 6 software provided by Wyatt.

1. Using a new glass pipette for each reaction vial, dip the pipette tip into the reaction solution to draw up a small sample of the reaction. Wash down the inside of the pipette with 250 µl of 0.025 M LiBr in DMF to give a final concentration of roughly 3 mg/ml.
2. Filter the diluted sample through a 0.45 µm polytetrafluoroethylene filter before depositing the sample into a GPC vial.
3. Set-up GPC-MALLS runs and analyze the results once the runs are completed.

List of Abbreviations:

A: Grubbs 3^rd generation bis-pyridine catalyst

B: poly(ethylene glycol) (PEG) macromonomer (MM)

B1: PEG MM prepared using commercially available (Aldrich) PEG-NH₂ and used without HPLC purification.

B2: PEG MM prepared using commercially available (Aldrich) PEG-NH₂ and used after HPLC purification.

B3: PEG MM prepared using newly synthesized PEG-NH₂ and used after HPLC purification.

BASP: brush-arm star polymer

BI: living brush initiator

C: photodegradable crosslinker

Ð: molar mass dispersity index

DMF: N,N-dimethylformamide

DP: number average degree polymerization

GPC: gel permeation chromatography

Prep-HPLC: preparative high performance liquid chromatography

MALLS: multi-angle laser light scattering

MM: macromonomer

MW: molecular weight

M_w: weight average molar mass

N: number of crosslinker equivalents (ratio of C à A)

NHS: N-hydroxysuccinimidyl

PEG: polyethylene glycol

PEG-MM: norbornene-PEG macromonomer (also referred to as compound B)

ROMP: ring-opening metathesis polymerization

THF: tetrahydrofuran