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Supporting Information

Oxidation responsive polymers with a triggered degradation via arylboronate self-immolative

motifs on a polyphosphazene backbone

Aitziber Iturmendi†, Uwe Monkowius

‡, Ian Teasdale*

†

† Institute of Polymer Chemistry, Johannes Kepler University, Altenberger Straße 69, 4040 Linz,

Austria ‡ Institute of Inorganic Chemistry, Johannes Kepler University, Altenberger Straße 69, 4040 Linz,

Austria

*ian.teasdale@jku.at

Materials and methods

Syntheses were carried out under argon atmosphere in glovebox (MBRAUN) or under nitrogen using

standard Schlenk line techniques. All glassware was dried in an oven overnight at 120°C prior to use.

Solvents were purchased from Merck, VWR and Alfa Aesar and used without further purification.

Triethylamine (Et3N) was dried over molecular sieves and distilled prior to use. The polyetheramine

copolymer (PEO-PPO-NH2), sold under the trade name Jeffamine M-1000 and with a nominal

molecular weight of 1000 g mol-1

, was donated by Huntsman Performance Products (Netherlands)

and used as received. The monomer trichlorophosphoranimine (Cl3P=N-SiMe3) was synthetized

according to literature procedure as described previously1. All other chemicals were purchased from

Sigma Aldrich, TCI chemicals and Fluorochem and used without further purification.

1H NMR spectroscopy was recorded on a Bruker 300 MHz spectrometer using D2O, DMSO-d6 or

acetone-d6 as an internal reference. 31

P NMR (121 MHz) experiments were carried out using 85%

phosphoric acid as an external standard. Gel permeation chromatography (GPC) was measured with

a Viscothek GPCmax instrument equipped with a PFG column from PSS (Mainz, Germany) (300 mm x

8 mm, 5 µm particle size). DMF containing 10 mM LiBr was used as the mobile phase at a flow rate of

0.75 ml min-1

at 60°C. The molecular weights were estimated using conventional calibration of the

refractive index detector versus polystyrene standards from PSS. Electrospray ionization mass

spectroscopy (ESI-MS) characterization was recorded on Agilent 1100 series HPLC with LC/MSD mass

detector in the negative ion mode. 10 mmol methanol was used as eluent at 0.7 mL min-1

. UV-Vis

spectra were carried out on a Perkin Elmer Lambda 25 UV/Vis spectrophotometer. A Malvern

Zetasizer Nano-ZS instrument (Malvern Instruments, UK) was used for dynamic light scattering (DLS)

measurements. A 4 mW standard laser was used at a 633 nm wavelength with the detector angle at

173°. The polymer was dissolved in deionized H2O (1 mg mL-1

) and filtered through a 0.2 µm nylon

filter and measured at 25°C.

Synthesis of Boc-gly-arylboronic acid pinacol ester

N-(tert-Butoxycarbonyl)glycine (Boc-Gly-OH) (0.90 g, 5.1 mmol) and 4-(dimethylamino)pyridine

(DMAP) (62.6 mg, 0.5 mmol) were dissolved in 40 mL CH2Cl2. Then, 4-

(hydroxymethyl)benzeneboronic acid pinacol ester (1.2 g, 5.1 mmol) dissolved in 15 mL CH2Cl2 was

added and stirred for 1 hour. N,N′-Dicyclohexylcarbodiimide (DCC) (1.06 g, 5.1 mmol) was dissolved

in 20 mL CH2Cl2 and transferred to the previous reaction which was cooled to 0°C. After stirring the

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reaction for 2 days at room temperature, precipitated urea was then filtered off. The filtrate was

extracted twice with 10% NH4Cl, twice with 5% NaHCO3, twice with saturated sodium chloride and

dried over MgSO4. The solvent was removed under vacuum and further dried to yield boc-gly-

arylboronic acid pinacol ester as a white viscous oil. Yield = 1.82 g (91%). 1H NMR (300 MHz, DMSO-

d6, δ): 1.29 (s, 12H), 1.38 (s, 9H), 3.74 (d, J = 6.2 Hz, 2H), 5.15 (s, 2H), 7.27 (br,s, 1H), 7.37 (d,

J = 8.1 Hz, 2H), 7.66 (d, J = 7.9 Hz, 2H).

Synthesis of Polymer 1

Polymers were synthesized according to literature procedure for the phosphine-initiated

polymerization of trichlorophosphoranimine.2 The following procedure describes the procedure used

for the synthesis of polymer 1. All polymers were synthesized with the same ratio of monomer to

chlorinated phosphine (50/1). Briefly, triphenylphosphine (12.00 mg, 45.7 µmol, 1 eq.) and

hexachloroethane (C2Cl6) (11.9 mg, 50.3 µmol, 1.1 eq.) dissolved in 1 mL anhydrous CH2Cl2 were

stirred for 16 hours at room temperature. After this time Cl3P=N-SiMe3 (0.51 g, 2.3 mmol, 50 eq.),

also dissolved in 0.5 mL anhydrous CH2Cl2, was added and stirred for 24 hours. Macromolecular

substitution of polydichlorophosphazene was carried out as follows: 0.91 g (2.3 mmol, 1 eq.) of boc-

gly-arylboronic acid pinacol ester was deprotected in CF3CO2H /CH2Cl2 (1:3) for 3 hours. The solvent

was removed under vacuum and further dried by co-evaporation with toluene and chloroform to

obtain the glycinate arylboronic acid pinacol ester (Figure S2). In the glove box, it was re-dissolved in

15 mL anhydrous THF and an excess of Et3N was added to neutralize TFA residues. The solution of

polydichlorophosphazene (0.51 g, 2.3 mmol, 1 eq.) in anhydrous DCM was transferred to the solution

of glycinate arylboronic acid pinacol ester and stirred at room temperature for 16 h. Then an excess

of Jeffamine 1k (3.20 g, 3.2 mmol, 1.4 eq) dissolved in 15 mL anhydrous THF and Et3N (0.4 mL,

3.2 mmol, 1.4 eq) mixture was added to the partially substituted polymer. The solution was further

stirred at room temperature for 16 h. The suspension was filtered in order to remove the insoluble

hydrochloride salt and the solvent was concentrated under vacuum. The polymer was purified by

dialysis (12 kDa cut-off) in dry EtOH (over molecular sieves) for 5 days. The solvent was removed

under vacuum to yield a white wax. Yield = 0.79 g (26 %). 1H NMR (300 MHz, acetonitrile-d3, δ): 1.07

(br, 9H), 1.28 (br, 3H), 3.29 (s, 4H), 3.55 (s, 84H), 3.78 (br, 2H), 5.12 (br, 2H), 7.34 (br, 2H), 7.77 (br,

2H). 31

P NMR (121 MHz, acetonitrile-d3, δ): 1.03 ppm. GPC: Mn = 68 500 g mol-1

, Mw = 105 600 g mol-1

,

Mw/Mn = 1.5. DLS: d = 11.23 ± 0.44 nm.

Synthesis of Polymer 2

Following the same procedure as with polymer 1, 0.96 g (2.5 mmol, 1 eq.) of boc-gly-arylboronic acid

pinacol ester was deprotected and added to a solution of polydichlorophosphazene (0.55 g,

2.5 mmol, 1 eq.) in anhydrous THF and Et3N. The reaction mixture was stirred at room temperature

for 16 h in the glove box. Meanwhile an excess of glycine ethyl ester hydrochloride (0.62 g, 4.4 mmol,

1.8 eq.) in anhydrous THF (20 mL) and Et3N (1 mL) was stirred at room temperature for 24 h.

Et3NH+Cl

- was filtered and transferred to the partially substituted polymer. The solution was allowed

to react for further 16 h at room temperature. The suspension was filtered and the solvent was

concentrated under vacuum. The polymer was purified by three precipitations into H2O from THF and

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seven precipitations into n-heptane from DCM. Yield = 0.24 g (22%). 1H NMR (300 MHz, acetone-d6,

δ): 1.12 – 1.29 (br, 15H), 4.02 (br, 6H), 5.08 (br, 2H), 7.30 (br, 2H), 7.71 (br, 2H). 31

P NMR (121 MHz,

acetone-d6, δ): 2.03 ppm.

Synthesis of Polymer 3

Et3N (2.00 mL, 14.3 mmol) was added to a suspension of glycine ethyl ester hydrochloride (1.10 g,

7.9 mmol) in THF (70 mL) at room temperature. The reaction mixture was stirred for 24 h to form

glycine ethyl ester and the precipitated was removed by filtration. Polydichlorophosphazene (0.55 g,

2.5 mmol, 1 eq.) in anhydrous DCM was added to the filtrate and the reaction mixture was stirred at

room temperature for 24 h in the glove box. The suspension was filtered and the solvent was

concentrated under vacuum. The polymer was purified by three precipitations into H2O from THF and

four precipitations into n-heptane from DCM. Yield = 0.22 g (35%). 1H NMR (300 MHz, acetone-d6, δ):

1.27 (t, 3H), 3.89 (br, 2H), 4.14-4.21 (q, 2H). 31

P NMR (121 MHz, acetone-d6, δ): 3.01 ppm.

Degradation studies of polymer 1

Polymer 1 (15.05 mg) was dissolved in 968 µL of D2O and the solution was transferred to a NMR

tube. H2O2 was added to make 10 mmol solution and 1H and

31P NMR spectra were recorded at

various time points. As a control, 15.25 mg of polymer 1 was dissolved in 1013 µL of D2O, transferred

to a NMR tube and measured at the same time points. GPC studies were done in a similar way.

Polymer 1 (15.04 mg) was dissolved in deionized water (968 µL) and H2O2 was added to make

10 mmol solution. 253 µL aliquots were taken at various time points and water was evaporated.

1.5 mL of DMF (containing 10 mM LiBr) was added and filtered before injection. The same procedure

was carried out in absence of H2O2 (15.01 mg of polymer 1 dissolved in 1013 µL of deionized water).

Degradation studies of polymer 2

Polymer 2 (15.07 mg) was dissolved in acetone-d6 (600 µL) and the solution was transferred to a

NMR tube. After addition of H2O2 to make 10 mmol solution, 1H and

31P NMR spectra were recorded

at different time points. As a control, 16.04 mg of polymer 2 was dissolved in acetone-d6 and

transferred to a NMR tube. Then 50 µL of D2O was added and 1H and

31P NMR were measured at

same time points.

Degradation studies of polymer 3

Polymer 3 (15.91 mg) was dissolved in acetone-d6 (600 µL) and the solution was transferred to a

NMR tube. After addition of H2O2 (to make 100 mM solution), 1H and

31P NMR spectra were recorded

at various time points.

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References

1. Wang, B.; Rivard, E.; Manners, I., A new high-yield synthesis of Cl3P=NSiMe3, a monomeric

precursor for the controlled preparation of high molecular weight polyphosphazenese. Inorg. Chem.

2002, 41 (7), 1690-1691.

2. Wilfert, S.; Henke, H.; Schoefberger, W.; Brüggemann, O.; Teasdale, I., Chain-End-

Functionalized Polyphosphazenes via a One-Pot Phosphine-Mediated Living Polymerization.

Macromol. Rapid Commun. 2014, 35 (12), 1135-1141.

3. Linhardt, A.; König, M.; Schöfberger, W.; Brüggemann, O.; Andrianov, A.; Teasdale, I.,

Biodegradable Polyphosphazene Based Peptide-Polymer Hybrids. Polymers 2016, 8 (4), 161.

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Figure S1. 1

H NMR spectrum of boc-gly-arylboronic acid pinacol ester in DMSO (*).

Figure S2. 1

H NMR spectrum of glycinate arylboronic acid pinacol ester in DMSO (*).

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Figure S3. (a)

1H NMR and (b)

31P NMR spectrum of polymer 1 in acetonitrile-d3 (*). Due to the larger

number of protons from the Jeffamine compared to the boronic acid ester group, there is some

uncertainity in the integration. Partial hydrolysis of the labile pinacol ester can be assumed because

of the purification method.

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Figure S4. UV-Vis spectra of 4-(hydroxymethyl)benzeneboronic acid pinacol ester (0.36 mg mL

-1, line

a), polymer 1 (0.65 mg mL-1

, line b) and polymer 2 (0.41 mg mL-1

, line c) in EtOH.

Figure S5. GPC chromatograph of polymer 1 with DMF as eluent containing 10 mM LiBr.

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Figure S6. Molecular size distribution by intensity (a) and volume (b) of polymer 1 in deionized H2O

with a concentration of 1 mg mL-1

at 25°C. Molecular size distribution by intensity shows a bimodal

distribution due to some agglomeration of the amphiphilic polymers.3

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Figure S7. 31

P NMR spectrum of polymer 1 in D2O (absence of H2O2). The polymer remains stable for

several weeks in aqueous solution.

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Figure S8. (a)

1H NMR and (b)

31P NMR spectrum of polymer 2 in acetone-d6 (*).

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Figure S9. 1H NMR spectrum of polymer 2 in acetone-d6 (*) before H2O2 addition and after 4h in

presence of H2O2. The oxidation of arylboronic acid pinacol ester to phenol is complete in 4 h

releasing the boronic pinacol ester which is hydrolyzed to boric acid and pinacol.

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Figure S10. Entire 1H NMR spectra

of Fig 4b.

1H NMR tracking of the self-immolation pathway of

polymer 2 in 10 mM acetone solution of H2O2.

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Figure S11. 31

P NMR spectrum of polymer 2 (a) in 10 mM acetone solution of H2O2. After 30 days the

polymer fully degrades to phosphates; (b) in acetone/water solution without H2O2. In the same time-

frame (30 days) no significant sign of phosphate is observed.

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Figure S12. ESI-MS of the H2O2-induced degradation products of Polymer 2.

Figure S13. 1H NMR tracking of polymer 2 in acetone/water solution. In absence of H2O2 arylboronic

acid pinacol ester cannot be oxidized, but it can be partially hydrolyzed releasing the pinacol.

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Figure S14. (a)

1H NMR and (b)

31P NMR spectrum of polymer 3 in acetone-d6 (*).

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Figure S15.

31P NMR spectrum of polymer 3 in 100 mM acetone solution of H2O2. Even at higher

concentration of H2O2 no relevant degradation is observed after 21 days.