Caged Polyphosphazenes with a Visible&#X02010

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Photocleavable Polymers www.mrc-journal.de Coumarin-Caged Polyphosphazenes with a Visible-Light Driven On-Demand Degradation

Aitziber Iturmendi, Sabrina Theis, Dominik Maderegger, Uwe Monkowius, and Ian Teasdale*

desired response. Consequently, several Polymers that, upon photochemical activation with visible light, undergo photo-responsive polymers have been pre- rapid degradation to small molecules are described. Through functionaliza- pared that have great promise in diverse tion of a polyphosphazene backbone with pendant coumarin groups sensitive applications such as drug delivery, porous membranes, and film patterning.[4b,9] to light, polymers which are stable in the dark could be prepared. Upon irra- Macromolecules that undergo photo diation, cleavage of the coumarin moieties exposes carboxylic acid moieties chemical backbone degradation are also along the polymer backbone. The subsequent macromolecular photoacid is of significant interest.[10] Most reported found to catalyze the rapid hydrolytic degradation of the polyphosphazene photo-cleavable systems require ultraviolet backbone. Water-soluble and non-water-soluble polymers are reported, which (UV) light to achieve cleavage, which can due to their sensitivity toward light in the visible region could be significant be a drawback especially in biomedical environments due to low penetration and as photocleavable materials in biological applications. biocompatibility, hence a shift to longer wavelengths is required.[11] Coumarin and its analogs are widely employed in There is considerable demand for stimuli-responsive polymers various fields such as medicine, polymer science, cosmetics, that can undergo chemical or physical changes in response to and biology.[12] Coumarin derivatives can be readily synthesized triggers such as pH,[1] biomolecules,[2] temperature, oxidation,[3] and functionalized to red-shift the light absorption,[12] cage the and light.[4] Macromolecules which undergo triggered backbone desired molecule as protecting groups,[13] and/or to be incorpo- disassembly or degradation upon response to stimuli could be of rated on the polymer main chain as photoresponsive units.[14] particular importance for a range of future applications including It is also reported that coumarin photocages can be cleaved via sensory materials, lithography, and triggered release systems.[5] two-photon processes with near-infra-red irradiation.[15] In addi- One approach involves self-immolative polymers[6] which are tion, functionalized aliphatic polycarbonates,[16] photodegradable designed to undergo end-to-end disassembly upon a triggering hydrogels,[17] and drug delivery systems[18] based on coumarin event.[7] More recently, a small number of chain-shattering poly derivatives have been developed. Furthermore, the ability of some mers have been described, a term used to describe polymers coumarin polyesters to undergo chain scission or crosslinking with multiple responsive cleavage sites along the backbone.[3b,8] upon certain wavelength irradiation has been demonstrated.[14,19] Among the investigated stimuli, light is notably attractive Herein, we present a novel approach to photodegradable due to its ability to exert spatial and temporal control over the polymers based on coumarin-functionalized polyphosphazenes. Polyphosphazenes are unique due to their hydrolytically Dr. A. Iturmendi, D. Maderegger, Prof. I. Teasdale unstable backbone which can be tailored through the incorpo- Institute of Polymer Chemistry ration of different substituents.[20] Furthermore, the polyphosp- Johannes Kepler University Linz hazene degradation pathway is known to be acid-catalyzed,[21] a Altenberger Strasse 69, 4040 Linz, Austria E-mail: [email protected] process which can be intramolecular when acidic substituents [22] Dr. S. Theis are present on the polymer backbone. Hence we proposed Institute of Inorganic Chemistry that through functionalization of polyphosphazenes with a Johannes Kepler University Linz coumarin-caged amino acid as a pendant group along the back- Altenberger Strasse 69, 4040 Linz, Austria bone, the sensitivity of the polymers to hydrolysis would be Prof. U. Monkowius accelerated upon irradiation by effectively producing a macro- Linz School of Education molecular photoacid which could subsequently catalyze its own Johannes Kepler University Linz Altenberger Strasse 69, 4040 Linz, Austria degradation. The ORCID identification number(s) for the author(s) of this article First 7-(N,N-diethylamino)-4-(hydroxymethyl)coumarin 1 can be found under https://doi.org/10.1002/marc.201800377. (Figure S1, Supporting Information) was prepared according [15] © 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, to literature procedures. After reaction with N-(tert-butox- Weinheim. This is an open access article under the terms of the Creative ycarbonyl)glycine (Boc-gly-OH) and deprotection, this gave Commons Attribution License, which permits use, distribution and re- the coumarin-caged glycine 3 (Figures S2 and S3, Supporting production in any medium, provided the original work is properly cited. Information).[23] Coumarin 1 was chosen as caging group as it DOI: 10.1002/marc.201800377 is expected to be photochemically active in the visible region

Figure 1. a) Photocleavage reaction of the coumarin-caged glycine 3. b) Change of the emission spectra of 3 in MeOH/H2O (4:1) solution upon irradiation with HBO lamp (100 W, cut-off filter at 395 nm, conc. 10−7 mol L−1). (— before irradiation; …5 min irradiation; ¨10 min irradiation; •15 min 1 −1 1 irradiation; -- 25 min irradiation). c) H NMR analysis of the photocleavage of 3 in MeOD/D2O (4:1) solution (conc. 0.082 mol L ). Entire H NMR spectra are shown in Figure S5, Supporting Information.

due to the electron-donating diethylamino group.[24] The chlorine atoms were substituted with either Jeffamine M-1000, kinetics of the elementary photochemical reaction of 3 has been a polyether monoamine, or glycine ethyl ester to obtain pol- investigated recently by flash-photolysis[23] but not under steady ymer P1 and polymer P2, respectively. illumination. Hence, we first investigated the photo-cleavage Polymer P1 was synthetized using Jeffamine M-1000 as the reaction of compound 3 upon irradiation (100 W, ≥395 nm) second substituent to give a water-soluble polymer. M-1000 in MeOH/H2O (4:1) solution. UV–vis spectroscopy (Figure S4, was also chosen to ensure that the hybrid polymer remained Supporting Information) showed a decrease in absorption hydrolytically stable in the timeframe of the photochemical intensity and shifted to lower wavelength (from 382 to 377 nm) reactions[21] and hence to be able to induce a photochemical after irradiation, indicative of the decaging of the glycine moiety degradation without significant interference from undesired (Figure 1a). Interestingly, an increase in intensity and slight hydrolytic degradation. The polymer was purified by dialysis hypsochromic shift (from 478 to 473 nm) was observed in the in the dark and characterized by 1H and 31P NMR spectros- emission spectra upon irradiation (Figure 1b). This ceased after copy (Figure S6, Supporting Information), size exclusion chro- approximately 25 min irradiation, indicating completion of the matography (SEC) in DMF containing 10 mm LiBr (Figure S7, 1 −1 photosolvolysis reaction. H NMR spectroscopy (Figure 1c) also Supporting Information, Mn,GPC = 149 000 g mol , and confirmed the nature of the photocleavage reaction, albeit with Mw/Mn = 1.03, measured using multidetector calibration) and a lower reaction rate due to the higher concentration required dynamic light scattering (DLS) (Figure S8, Supporting Informa- −7 −1 −1 −1 (10 mol L vs 0.082 mol L ). tion, d = 12.25 nm ± 0.38 nm in H2O at 1 mg mL ). According Our approach was to extend this photodecaging phenom- to 1H and 31P NMR spectroscopy complete backbone substi- enon to “cage” a hydrolytically unstable glycine-substituted tution in a ratio of nearly 34:66 (coumarin derivative:M-1000) phosphazene moiety through incorporation of the coumarin- could be observed with no additional peaks in the 31P NMR caged glycine onto a polyphosphazene backbone. Polydichlo- corresponding to partially substituted phosphorous atoms. rophosphazene was first prepared via phosphine-mediated UV–vis spectroscopy (Figure S9, Supporting Information) polymerization of trichlorophosphoranimine[25] (Scheme 1, showed the loading to be approximately 14 wt%, which corre- see Supporting Information for detailed procedure). First, the sponds roughly to 35:65 ratio, coumarin derivative to M-1000 coumarin-cage 3 was added in order to substitute the majority substituents. of chlorine atoms. For related amino acid esters, it is known The polymer was irradiated (100 W, ≥395 nm) in aqueous that monosubstitution at the phosphorus atom is strongly solution to investigate its photochemical properties. The photo- favored due to the higher reactivity of Cl2PN in comparison reaction was followed by UV–vis (Figure 2a) and fluorescence to ClRPN,[26] therefore, we expect a distribution of the cou- spectroscopy (Figure 2b). In the UV–vis spectra, one prominent marin groups along the backbone. Thereafter, the remaining long wavelength absorption band with a maximum at 386 nm

Scheme 1. Synthesis of Polymer P1 and Polymer P2 bearing the coumarin derivative as a caging group of glycine. Reagents and conditions: i) CH2Cl2, n = 50, and 18 h for polymer P1, and bulk polymerization (solvent free), n = 400, and 96 h for polymer P2; ii) 0.73 equivalents of coumarin-caged glycine 3, THF, Et3N, rt, 24 h for polymer P1 and 48 h for polymer P2; iii) excess of R (Jeffamine M-1000 and 24 h for polymer P1, and glycine ethyl ester and 48 h for polymer P2), THF, Et3N, rt.

could be observed, which corresponds to the absorption of cou- of the photocleavage in the polymers was also clearly observed marin. The maximum of the emission band was approximately (Figure S12, Supporting Information). 500 nm. Aqueous solutions of the polymer showed no changes Figure 3a shows the proposed degradation mechanism during 24 h in the dark (Figure S10, Supporting Information). induced by the photoreaction, in which cleavage of the cou- However, under irradiation with visible light, the sample changed marin cage exposes the free acid groups of the glycine moieties color (from yellow to orange) and significant changes in the UV– along the polyphosphazene backbone. Thus, once the protecting vis and fluorescence spectra could be observed. The intensity of group was cleaved, the polymer undergoes a chain-shattering the absorption band at 386 nm gradually decreased and shifted type of degradation.[8a,28] This process was further investigated to 381 nm. Four isosbestic points at 457, 359, 266, and 236 nm by 31P NMR spectroscopy in which significant depletion of the are visible indicative for a clean photo-reaction. The fluorescence polyphosphazene backbone peak was observed upon polymer spectra showed both an increase of the emission band and a irradiation (90 min irradiation at 0.164 mg mL−1 concentration hypsochromic shift from 508 to 499 nm. This behavior is compa- in H2O and stored for 3 days in the dark, Figure S13a, Sup- rable to the small-molecule studies on compound 3. porting Information). After a longer period, further degradation Since the photo-cleavage was deemed to be completed after accompanied by the appearance of the sharp peak associated 90 min irradiation in H2O, a further sample was irradiated at with phosphate formation could also be observed. The polymer the same concentration (0.16 mg mL−1) and analyzed by SEC kept in the dark even for 7 days did not show any signs of deg- (Figure 2e). Meanwhile, an identical sample held for 24 h radation (Figure S13b, Supporting Information). Furthermore, (Figure 2f) and even 7 days (Figure S11a, Supporting Informa- 1H NMR spectroscopy revealed the successful cleavage of the tion) without irradiation showed no signs of degradation. This caging group upon irradiation with the appearance of sharp confirmed the destabilizing effect of glycine after decaging ren- peaks corresponding to the coumarin group while no changes dering the polymer hydrolytically unstable. were observed in the dark sample (Figure S14, Supporting As the degradation upon irradiation was significant but Information). incomplete, the polymer was kept in the dark for a further Amino acid ester-substituted polyphosphazenes are known period (Figure S11b, Supporting Information). However, total as biodegradable polymers.[20a,29] Therefore, polymer P2 was degradation of the polymer could not be achieved even keeping synthesized using glycine ethyl ester as second substituent. it for 7 days in aqueous solution after irradiation. Since Jef- After purification by dialysis in the dark, polymer P2 was suc- famine M-1000 (34:66 ratio) was used in excess, parts of the cessfully obtained as it could be confirmed by 1H and 31P NMR polymer might consist of blocks constituted only by the M-1000 spectroscopy (Figure S15, Supporting Information), and SEC −1 substituent which are relatively stable against hydrolytic deg- (Mn,GPC = 249 000 g mol , and Mw/Mn = 1.8, measured using radation.[21] It is known that some polymers containing cou- multidetector calibration in DMAc containing 57.6 mm LiBr marin derivatives can undergo 2 + 2 photodimerization of and 0.1 m acetic acid). A ratio of approximately 33:66 coumarin the 3,4-double bond forming a cyclobutane ring.[27] For the derivative to glycine ethyl ester could be confirmed by 1H NMR small-molecule caged glycine 3, we could not identify any with complete backbone substitution observed in the 31P NMR photodimerization product. Furthermore, the predominance spectroscopy.

−1 Figure 2. Changes of a) UV–vis absorption spectra and b) emission spectra of polymer P1 in H2O (0.167 mg mL ) upon irradiation with a HBO lamp (cut-off filter at 395 nm). Progress of c) absorbance intensity and d) emission intensity—each measured at the band maxima from spectra in (a) and −1 −1 (b)—versus time. SEC analysis of polymer P1 e) after irradiation in H2O (0.163 mg mL ) and f) in the dark in H2O (0.173 mg mL ) for 24 h (— before irradiation; -- 90 min irradiation; °24 h dark).

Irradiation was performed in acetonitrile:H2O solution analyzed by SEC (Figure 3c). Significant degradation could be (4:1) (Figure S16, Supporting Information) upon which observed after irradiation. Compared to polymer P1, the deg- the emission spectra showed a predominant increase espe- radation was more pronounced, presumably due to the better cially in the first irradiation minutes with an approximate leaving-group ability of the chosen cosubstituent. Mean- end after 45 min (Figure 3b). As the photocleavage seemed while, in the absence of light, only slight hydrolytic degrada- to have ceased after 45 min, a second sample was irradiated tion over 3 days could be observed (Figure S17, Supporting in the same solvent mixture (0.16 mg mL−1) for 45 min and Information).

Figure 3. a) Reaction scheme of the photochemical cleavage of the coumarin and of the rapid self-catalyzed degradation of the hydrolytically sensitive glycine-substituted polyphosphazene. b) Progress of emission intensity of polymer P2 versus time upon irradiation in acetonitrile: H2O (4:1) mixture. −1 c) SEC analysis of polymer P2 before and after irradiation in acetonitrile: H2O (4:1) mixture (0.16 mg mL ) (— before irradiation; -- 45 min irradiation). The design and synthesis of polyphosphazenes with a light- Acknowledgements driven, on-demand degradation mechanism have been described. The authors thank Wolfgang Gnong and Petra Gründlinger for their A coumarin-based photocage, which is sensitive to visible light, assistance. Prof. Oliver Brüggemann and Prof. Günther Knör are kindly was used to protect the carboxylic acid group of a glycine moiety acknowledged for generous access to laboratory facilities. The authors on a polyphosphazene backbone. Upon irradiation, the carbox- thank Melanie Kleindienst for contribution of artistic work. The NMR ylic acid was exposed which in turn catalyzed the polymer deg- experiments were performed in part at the Upper Austrian-South radation. In the dark, the polymers were hydrolytically stable Bohemian Research Infrastructure Center in Linz, co-financed by the in the investigated timeframe, that is, the degradation could European Union in the context of the project “RERI-uasb,” EFRE RU2- be initiated upon exposure to visible light. These results prove EU-124/100-2010 (ETC Austria-Czech Republic 2007–2013, project M00146). The authors acknowledge financial support of the Austrian the potential to use visible light to switch the hydrolytic stability Science Fund (FWF), P 27410-N28. of polyphosphazenes and hence trigger backbone degradation. While complete degradation was observed for P2, an incomplete degradation was observed for P1, indicating the importance of Conflict of Interest the nature of the secondary substituents on the overall polymer The authors declare no conflict of interest. stability. Hence, future work will aim to optimize the balance between the nature and loading of the photocage and further substituents with the aim of maintaining stability in the dark, Keywords while maintaining a complete on-demand degradation. The gen- on-demand degradation, photocages, photodegradable, eral proof-of-principle could also be extended to other photoc- polyphosphazenes, visible light ages to prepare polymers which respond to irradiation with yet longer wavelengths in the red region which would have a consid- Received: May 16, 2018 erable impact in biological applications. Revised: June 28, 2018 Published online: July 26, 2018

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