Interrupted CuAAC Ligation: An Efficient Approach to Fluorescence Labeled Three-Armed Mikto Star Polymers

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1 Supporting Information Interrupted CuAAC Ligation: An Effiient Approah to Fluoresene Labeled Three-Armed Mikto Star Polymers Janin T. Offenloh, a,b Hatie Mutlu,* a,b and Christopher Barner-Kowollik* a,b, a Maromoleular Arhitetures, Institut für Tehnishe Chemie und Polymerhemie, Karlsruhe Institute of Tehnology (KIT), Engesserstr. 18, Karlsruhe, Germany, b Soft Matter Synthesis Laboratory, Institut für Biologishe Grenzflähen, Karlsruhe Institute of Tehnology (KIT), Hermann-von-Helmholtz-Platz 1, Karlsruhe, Germany. Shool of Chemistry, Physis and Mehanial Engineering, Queensland University of Tehnology (QUT), 2 George Street, QLD 4000, Brisbane, Australia hatie.mutlu@kit.edu, hristopher.barner-kowollik@kit.edu, hristopherbarnerkowollik@qut.edu.au Content A. Experimental proedures 1. Materials S2 2. Synthesis of the parent polymers S2 3. Typial proedure for the blok opolymer formation S4 4. Radial thiol-ene addition to the blok opolymer S4 B. Measurements and analytial methods 1. Nulear magneti resonane (NMR) spetrosopy S5 2. Size exlusion hromatography (SEC) S5 3. Dynami light sattering (DLS) S5 4. Ultaviolet-visible (UV-Vis) spetrosopy S6 5. Fluoresene spetrosopy S6 6. Attenuated total refletane infrared spetrosopy (ATR-IR) S6 7. Differential sanning alometry (DSC) S6 C. Additional data and figures 1. Suggested mehani pathway of the CuAAC interrupted by azirines S7 2. Additional SEC data of the miktro-arm star polymers S H NMR spetra S11 4. ATR-IR data S12 5. DLS data S13 6. UV Vis and fluoresene data S15 7. DSC data S17 D. Referenes S18 S1

2 A. Experimental proedures A.1 Materials Unless otherwise stated, all hemials were used as reeived. 1-Aetyl pyrene (Aros, 97 %), aluminium oxide (neutral, Aros), anisole (Aros, 99 %, dry), opper(ii) bromide (Aros, 99+ %), opper(i) iodide (Sigma Aldrih, 98 %), diethyl ether (VWR, normapur), N,N-diisopropylamine (DIPEA, Aros, 99.5 %), dithiothreitol (DTT, AppliChem Panrea, moleular biology grade), N,N-dimethylhydrazine (Merk, 98.0 %), 4-dimethylaminopyridine (DMAP, Sigma Aldrih, 99 %), N,Ndimethylformamide (DMF, Aros, 99 %, dry), 1-dodeanethiol (Alfa Aesar, 98 %), ethyl aetate (VWR, normapur), 1-ethyl-3-(3-dimethylaminopropyl)arbodiimide hydrogenhloride (EDC HCl, Roth, 99 %), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, Sigma Aldrih, 97 %), hexane (VWR, normapur), hexylamine (Merk, 99 %), iodomethane (Alfa Aesar, 99 %, stabilized with opper), methanol (VWR, normapur), methanol (Aros, 99.9 %, dry), poly(ethylene glyol)methyl ether azide (PEG-N 3, Sigma Aldrih, M n = 2000 g mol -1 ), poly(ethylene glyol) methyl ether thiol (PEG-SH, Sigma Aldrih, M n = 2000 g mol -1 ), sodium azide (Sigma Aldrih, 99.5 %), sodium hydride (Sigma Aldrih, 95 %, dry), tetrahydrofurane (THF, Aros, 99.5 %, extra dry, stabilized), triethylamine (Merk, for synthesis), trifluoroaeti aid (Alfa Aesar, 99 %), toluene (VWR, normapur). Azobisisobutyronitrile (AIBN, Fluka, 98 %) was rerystallized from MeOH. 2- ((Dodeylsulfanyl)arbonothioyl)sulfanyl propanoi aid was obtained from Lanxess Deutshland GmbH. Styrene (Merk, >= 99%) and methyl metharylate (MMA, Sigma Aldrih, 99 %) were passed over a olumn of basi aluminium oxide prior to polymerization. Az-Py was synthesized aording to literature. 1 Prop-2-ynyl 2-bromo-2-methylpropanoate was synthesized aording to literature. 2 Resin- N 3 (1.8 mmol of N 3 funtionality per mg) was synthesized by stirring hloromethyl polystyrene (Resin ross-linked with 1% DVB, TCI) with sodium azide in DMF at ambient temperature overnight followed by filtration und drying. A.2. Synthesis of the parent polymers Synthesis of PMMA-alkyne Aording to literature, mg CuBr 2 (0.49 mmol, 0.5 Eq.), 10.0 g MMA (99.90 mmol, Eq.), µl HMTETA (230.0 mg, mmol, 1.0 Eq.), mg prop-2-ynyl 2-bromo-2- methylpropanoate (0.999 mmol, 1.0 Eq.) and ml dry anisole were plaed in a Shlenk flask and degassed via three freeze-pump-thaw yles. The polymerization was started by plaing the flask into an oil bath at 90 C and stopped after 25 min by opening the flask and exposing the atalyst to air. The opper was removed by passing the mixture though a olumn of neutral aluminium oxide. The polymer was obtained as a white powder (2.8 g) by preipitation into water and drying under high vauum. S2

3 SEC: M n = 4000 g mol -1, Ð = 1.1 Synthesis of PS-SH 0.5 g DOPAT (1.4 mmol, 1.0 Eq.), 8.5 mg DMAP (69.3 µmol, 0.05 Eq.) and 562 µl dry MeOH (444.4 mg, 13.9 mmol, 10.0 Eq.) were dissolved in 5 ml dry THF and ooled to 0 C mg EDC HCl (1.7 mmol, 1.2 Eq.) were added and the mixture was left at ambient temperature overnight. After removal of the solvent, the residue was dissolved in ethyl aetate, washed with sat. NaHCO 3 solution, 1H HCl and brine, and dried over MgSO 4. After removal of the solvent, DOPAT-OMe was obtained as orange oil (498 mg, 1.3 mmol, 97 %) mg AIBN (92.6 µmol, 0.1 Eq.) and mg DOPAT-OMe (0.9 mmol, 1.0 Eq.) were dissolved in 9.6 g styrene (9.2 mmol, 10.0 Eq.) in a round-bottom flask. After purging for 30 min with argon, the flask was plaed in a 60 C oil bath for 17 h. The polymerization was stopped via ooling with liquid nitrogen and opening the flask to air. 2.2 g of a slight yellow polymer was obtained via two fold preipitation into ie-old MeOH. SEC: M n = 2700 g mol -1, Ð = 1.1 For the transformation of the RAFT-endgroup into thiol funtionality, 150 mg PS was dissolved in 1.5 ml dry DMF µl hexylamine (7.9 mg, 78.3 µmol, 1.2 Eq.) was added, and to prevent oxidation to disulfides, further 5.0 mg of DTT was added to the mixture. After stirring at ambient temperature overnight, the polymer was preipitated into ie-old MeOH and obtained as white powder. SEC: M n = 2600 g mol -1, Ð = 1.1 Redution to PEG-SH To remove the PEG-SS-PEG, 0.4 g of the purhased produt (PEG-SH) was dissolved in 2 ml dry DMF mg DTT (0.6 mmol, 3.0 Eq.) and 0.01 ml triethylamine as atalyst was added. The mixture was stirred at ambient temperature for three days. The polymer was obtained by two fold preipitation in ie-old diethyl ether. SEC: M n = 2600 g mol -1, Ð = 1.1 S3

4 A.3. Typial proedure for the blok opolymer formation The following proedure is a variation of the proess reported in referene mg PMMA-alkyne (1.1 Eq.), 22.7 mg PEG-N 3 (1.0 Eq.), 4.0 mg Az-Py (17.3 µmol, 1.2 Eq.), 3.4 µl DIPEA (2.6 mg, 19.8 µmol, 1.4 Eq.) and 0.3 mg CuI (1.6 µmol, 0.1 Eq.) were dissolved in 1 ml dry DCM under argon atmosphere. After stirring at 0 C, the mixture was left at ambient temperature overnight. After 24 h, 0.6 mg resin- N 3 was added to the mixture to remove the exess of PMMA-alkyne. After further 24 h, the opper atalyst was removed via passing through a olumn of neutral aluminium oxide. The blok opolymer was obtained by preipitation into ie-old hexane. SEC: M n = 6500 g mol -1, Ð = 1.1 A.4. Radial thiol-ene addition to the blok opolymer 20.0 mg (1.0 Eq.), 1.0 Eq. of thiol (9.1 mg PS-SH, 8.0 mg PEG-SH or 0.8 mg 1- dodeanethiol) and 1.0 mg AIBN (6.0 µmol, 1.5 Eq.) were dissolved in 1 ml 1,4-dioxane under an argon atmosphere. Subsequently, the flask was plaed in a 60 C oil bath for 29 h. The modified blok opolymer was reeived via preipitation in ie-old hexane and drying under vauum. For PS-SH: SEC: M n = 6600 g mol -1, Ð = 1.2 For PEG-SH: SEC: M n = 6300 g mol -1, Ð = 1.2 For 1-dodeanethiol: SEC: M n = 7700 g mol -1, Ð = 1.1 S4

5 B. Measurements and analytial methods B.1. Nulear magneti resonane (NMR) spetrosopy NMR measurements were performed on a Bruker AM 400 spetrometer ( 1 H: 400 MHz) for hydrogen. The δ-sale was referened to the respetive solvent signal of hloroform-d 1 whih was employed as deuterated solvent. Abbreviations used in the desription of the materials synthesis inlude singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin), and multiplet (m). B.2. Size exlusion hromatography (SEC) The apparent number average molar mass (M n) and the molar mass distribution [Ð (polydispersity index) = M w/m n] of the polymers were determined via SEC measurements, whih were performed on a TOSOH Eo-SEC HLC-8320 GPC System, omprising an autosampler, a SDV 5 μm bead-size guard olumn (50 8 mm, PSS) followed by three SDV 5 μm olumns ( mm, subsequently 100 Å, 1000 Å and 10 5 Å pore size, PSS), and Waters 2487 dual wavelength absorbane detetor (analysis at 254 nm) in series with a refrative index detetor using tetrahydrofuran (THF) as the eluent at 30 C with a flow rate of 1 ml min -1. The SEC system was alibrated using linear polystyrene standards ranging from 266 to g mol -1. Calulation of the moleular weight proeeded via the Mark-Houwink-Sakurada (MHS) parameters for polystyrene (PS) in THF at 30 C, i.e., K = ml g -1, α = and for poly(methyl metharylate) in THF at 30 C, K = ml g -1, α = For poly(ethylene glyol), SEC alulations were arried out relative to a PMMA alibration. B.3. Dynami light sattering (DLS) The apparent hydrodynami diameters (Dh,app) were determined at 25 C by means of a dynami light sattering (DLS) analysis using a Zetasizer Nano ZS light sattering apparatus (Malvern Instruments, UK) equipped with He-Ne laser (at a wavelength of 633 nm, 4 mw). The Nano ZS instrument inorporates a non-invasive baksattering (NIBS) opti with a detetion angle of 173. The polymer solutions were prepared in aetonitrile, and were subsequently filtered into quartz uvettes. For mielles formation, µ-peg was dispersed in water while for -Dode, the nanopreipitation method with THF and water was employed. The prepared samples were stabilized prior to DLS analysis at the hosen temperature. All values of the apparent hydrodynami diameter for the mikto-arm polymers and mielles were averaged over tripliate measurements (11 runs/measurement), and were automatially provided by the instrument using a umulative analysis in ombination with the autoorrelation funtions (see below). S5

6 B.4. Ultraviolet-visible (UV-Vis) spetrosopy The UV-Vis spetra were reorded on a Cary 100 UV-Visible Spetrophotometer (Agilent Tehnologies, USA) equipped with a tungsten halogen light soure (190 to 900 nm, auray +/- 2 nm) and a R928 PMT detetor. Spetra were reorded in DCM at 20 C with a onentration of mmol ml -1 and olleted between 200 and 800 nm. Samples were baseline orreted with respet to the pure solvent. B.5. Fluoresene spetrosopy Fluoresene spetra were measured on a Varian Cary Elipse Fluoresene Spetrometer using quartz uvettes loaded with 400 µl of samples. An exitation wavelength of 344 nm was used and the emission was reorded from 355 to 800 nm. All spetra were reorded in DCM ( = mmol ml -1 ) at 20 C. B.6. Attenuated total refletane infrared spetrosopy (ATR-IR) All IR measurements were performed on a Bruker Alpha ATR-IR Spetrometer with a range of 400 to 4000 m -1 at ambient temperature. B.7. Differential sanning alometry (DSC) Differential sanning alorimetry studies were arried out with a Mettler-Toledo STAR system under nitrogen atmosphere using a sample mass of 5 mg. The glass transition temperature T g is reported as the midpoint of the step hange in the heat apaity and the melting point is reported as the minimum of the endothermi peak at a heating rate of 20 K min -1 starting from -75 C up to a temperature of 250 C of the seond heating san. S6

7 C. Additional data and figures C.1. Suggested mehani pathway of the CuAAC interrupted by azirines A B Sheme S1. Copper(I)-atalyzed Azide-Alkyne Cyloaddition interrupted by 2H-azirines. A: reation overview. B: Suggested mehanisti pathway. 4 S7

8 C.2. Additional SEC data of the mikto-arm star polymers I norm / a.u. + PS-SH starting reation mixture -PS results after 29 h reation time retention time / min Figure S1. Comparative SEC traes (THF, RI) of the employed and PS-SH reation mixture along the resulting µ-ps, whih was obtained after radial thiol-ene reation at 60 C for 29 h. + PEG-SH staring reation mixture -PEG results after 29 h reation time I norm / a.u retention time / min Figure S2. Comparative SEC traes (THF, RI) of the employed and PEG-SH reation mixture along the resulting µ-peg, whih was obtained after radial thiol-ene reation at 60 C for 29 h. S8

9 A -Dode I norm / a.u retention time / min B -Dode I / a.u retention time / min Figure S3. SEC (THF, A: RI detetion, B: UV detetion) results of the modifiation proess of with 1-dodeanethiol. S9

10 A PEG-SH -PEG I / a.u. I norm / a.u retention time / min B PEG-SH -PEG retention time / min Figure S4. SEC (THF, A: RI detetion, B: UV detetion) results of the modifiation proess of with PEG-SH. S10

11 C.3. 1 H NMR spetra b a Py a+b b+ d e Py a+b b / ppm Figure S5. 1 H NMR (400 MHz, CDCl 3) of before (top) and after the radial thiol-ene addition of 1-dodeanethiol (bottom). Py a b a+b b+ g d f e e f d e g i f DTT DTT m l Py a+b b / ppm Figure S6. 1 H NMR (400 MHz, CDCl 3) spetra of the blok opolymer (top), PEG-SH (middle) and the resulting mikto-arm star polymer µ-peg (bottom). S11

12 C.4. ATR-IR data C=O PS-SH C-H arom. C=C C-H arom. C-S -PS -Dode wavenumber / m -1 CH 2, long hain Figure S7. ATR-IR spetra of, PS-SH and the mikto-arm polymers, µ-ps and -Dode. Charateristi vibrations for the aromati C-H are detetable at 2920 m -1 and 697 m -1 for the C-H streth and for the C-H out-of-plane bending, respetively. Furthermore, at 627 m -1 a signal for the C-S streth is visible. However, due to weak absorbane of thiols, no peak for the S-H streth between m -1 is present. The IR spetrum of µ-ps represents a ombination of the polymer bloks, PMMA, PEG and PS, while for -Dode a signal at 726 m -1, indiating a C-H rok of long hain alkanes appears. S12

13 C.5. DLS data A 5 C 25 C 45 C D 5 C 25 C 45 C Correlation Coeffiient I number / % -Dode diameter / nm 5 C 25 C 45 C I volume / % -Dode diameter / nm 5 C 25 C 45 C Correlation Coeffiient I volume / % I number / % -PEG diameter / nm B E 5 C 25 C 45 C -PEG diameter / nm C F 5 C 25 C 45 C E E7 Time / s Time / s Figure S8. Temperature dependent DLS studies of -Dode and µ-peg in water at a onentration of 2 mg ml -1. S13

14 A I number / % g ml g ml g ml g ml g ml g ml -1 D I number / % g ml g ml g ml g ml g ml g ml g ml -1 -Dode -PEG diameter / nm diameter / nm B I volume / % g ml g ml g ml g ml g ml g ml -1 E I volume / % g ml g ml g ml g ml g ml g ml -1 -Dode -PEG diameter / nm diameter / nm C Correlation Coeffiient g ml g ml g ml g ml g ml g ml -1 F Correlation Coeffiient g ml g ml g ml g ml g ml g ml g ml E E7 Time / s Time / s Figure S9. Conentration dependent DLS studies of µ-peg and -Dode in water at 25 C. S14

15 C.6. UV Vis and fluoresene Data A absorbane / a.u. PMMA-alkyne PEG-N 3 Pyrene wavelength / nm B fluoresene emission / a.u. PMMA-alkyne PEG-N wavelength / nm Figure S10. UV Vis spetra (A) and fluoresene spetra (B, λ ex. = 344 nm) of PMMA-alkyne, PEG- N 3 and in DCM. Investigation of the optial properties via UV Vis and fluoresene spetrosopy reveals the typial pyrene absorbane and fluoresene emission for the blok opolymer. In the ase of the parent polymers, no pyrene an be deteted sine any typial fluoresene and absorbane an be deteted. The photograph on the right side depits the NMR tubes with the blok opolymer (left) and with the polymer mixture (right) dissolved in CDCl 3 ( = 40 mg ml -1 ), whih are exited at 344 nm. S15

16 A -Dode D -Dode absorbane / a.u. PS-SH -PS fluoresene emission / a.u. absorbane / a.u wavelength / nm wavelength / nm B E fluoresene emission / a.u. PS-SH -PS wavelength / nm wavelength / nm C absorbane / a.u. PEG-SH -PEG F fluoresene emission / a.u. PEG-SH -PEG wavelength / nm wavelength / nm Figure S11. UV Vis spetra (A-C) and fluoresene spetra (D-E, λ ex. = 344 nm) of the parent polymers, the blok opolymer and the three-armed mikto armed polymers in DCM. The fat that the UV Vis spetrum of PS-SH displays no remaining absorbane at 320 nm for the trithioarbonate group meaning that the aminolysis of the polystyrene prepared by RAFT polymerization was suessful. In addition, the UV Vis spetra of the modified blok opolymers do not indiate sharp absorption bands as for the pyrene in omparison to the blok opolymer itself. In other words, the addition of the polymer bloks at as a shield for the hromophore leading to a rougher absorption bands. The same phenomenon an be seen in the fluoresene spetra: The additional polymer bloks lead to a lower fluoresene emission due to shielding of the pyrene moiety. S16

17 C.7. DSC data Heat Flow / mw T g (PS) PMMA-alkyne -PS T g (PMMA) T g (PMMA) T / C Figure S12. DSC results of PMMA-alkyne, and µ-ps obtained with a heating rate of 20 K min -1. A glass transition temperature of 102 C for the starting PMMA blok is determined. In the ase of, no peak for melting of the PEG phase in the seond heating san urve is visible due to diffiulties of PEG to rystallize in an environment with 60 wt% or more PMMA. 6 In addition, the glass transition temperature of the PMMA blok may derease as the effet of the soft PEG blok, hene a T g of lose to 50 o C in the blue urve is assoiated with the PMMA blok. The three-armed mikto star polymer µ-ps exhibits harateristis of PS and PMMA by a glass transition temperature at 104 C and 156 C, respetively. S17

18 D. Referenes (1) Mueller, J. O.; Shmidt, F. G.; Blino, J. P.; Barner-Kowollik, C. Visible-Light-Indued Clik Chemistry. Angew. Chemie - Int. Ed. 2015, 54, 1 6. (2) Murtezi, E.; Yagi, Y. Simultaneous Photoindued ATRP and CuAAC Reations for the Synthesis of Blok Copolymers. Maromol. Rapid Commun. 2014, 35, (3) Kwak, Y.; Matyjaszewski, K. ARGET ATRP of Methyl Metharylate in the Presene of Nitrogen-Based Ligands as Reduing Agents. Polym. Int. 2009, 58, (4) Zhou, W.; Zhang, M.; Li, H.; Chen, W. One-Pot Three-Component Synthesis of Enamine- Funtionalized 1,2,3-Triazoles via Cu-Catalyti Azide Alkyne Clik (CuAAC) and Cu Catalyzed Vinyl Nitrene Transfer Sequene. Org. Lett. 2017, 19, (5) Brandrup, J.; Immergut, E. H.; Grulke, E. A. Polymer Handbook, 4th ed.; John Wiley Sons: New York, (6) Chen, N.; Yan, L. T.; Xie, X. M. Interplay between Crystallization and Phase Separation in PS- B-PMMA/PEO Blends: The Effet of Confinement. Maromoleules 2013, 46, S18