SUPPRTING INFRMATIN Ring-pening Polymerization of N-Carboxyanhydrides Initiated by a Hydroxyl Group Špela Gradišar, Ema Žagar, and David Pahovnik* National Institute of Chemistry, Department of Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia Experimental Materials. Chemicals: β-benzyl-l-aspartate (BLA, 99%, Iris Biotech GmbH), γ-benzyl-lglutamate (BLG, 99%, Acros rganics), triphosgene (98%, Aldrich), calcium hydride (95%, Sigma-Aldrich), phosphorus pentoxide (P 2 5, 99%, Sigma-Aldrich), methanesulfonic acid (MSA, 99.5%, Sigma-Aldrich), and solvents: tetrahydrofuran (THF, 99.9%, anhydrous, Sigma-Aldrich), n-hexane (99%, Merck), chloroform (99%, anhydrous, Sigma-Aldrich), diethyl ether (99.7%, Merck), N,N-dimethylformamide (DMF, 99.8%, Merck) and trifluoroacetic acid (TFA, 99%, Aldrich) were used as received. N-Ethyldiisopropylamine (EDIPA, 98%, Sigma-Aldrich) and 3-phenyl-1-propanol (PPA, 98%, Aldrich) were dried over calcium hydride and distilled under vacuum. α-methoxy-ω-hydroxy poly(ethylene glycol) (PEG-H, M p = 1964 g mol -1, Iris Biotech GmbH) was dried over P 2 5 overnight, whereas hydroxyl-terminated polystyrene (PS-H, M n = 2200 g mol -1, D = 1.06, Polymer Source) was used as received. Methods. 1 H NMR spectra were recorded on a Varian Unity Inova 300 MHz instrument (Varian, Inc., USA). All measurements were carried out in DMS-d 6 with a few drops of TFA at room temperature in the pulse Fourier transform mode with both the relaxation delay and the acquisition time of 5 s. Tetramethylsilane (TMS, δ = 0) was used as an internal chemical-shift standard. Size-exclusion chromatography coupled to a multi-angle light-scattering photometer (SEC-MALS) measurements were performed using a Hawlett-Packard pump series 1100 coupled to a Dawn Heleos multi-angle light-scattering photometer with a GaAs linearly polarized laser (λ 0 = 661 nm) and to an ptilab rex interferometric refractometer (RI), operating at the same wavelength as the photometer (both instruments are from Wyatt Technology Corp., USA). The separations were carried out at 50 C using successively coupled MIXED-E and ligopore columns (Agilent) with a precolumn in 0.1 M solution of LiBr in N,N-dimethylacetamide (DMAc) at a flow rate of 0.5 ml min -1. The masses of the samples injected onto the column were typically 1.0 10-3 g, whereas the solution concentration was 1.0 10-2 g ml -1. For the data acquisition and evaluation Astra 5.3.4 software (Wyatt Technology Corp., USA) was utilized. S1
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TF MS) measurements were carried out on a Bruker UltrafleXtreme MALDI-TF mass spectrometer (Bruker Daltonik, Bremen, Germany). Samples were dissolved in HFIP (10 mg ml -1 ) and mixed with a solution of matrix, 2,5-dihydroxybenzoic acid in THF (30 mg ml -1 ), and sodium trifluoroacetate in THF (10 mg ml -1 ) in a volume ratio of 1:10:3. 0.4 µl of thus prepared solution was spotted on the target plate (dried-droplet method). The reflective positive ion mode was used to acquire the mass spectra of the samples. The calibration was done externally with the poly(methyl methacrylate) standards using the nearest neighbour positions. Synthesis of BLA NCA and BLG NCA Into a dried argon-purged flask BLA (5.00 g, 22.4 mmol) or BLG (5.00 g, 21.1 mmol) in dry THF (50 or 60 ml) was suspended. A solution of triphosgene (3.52 g, 11.9 mmol or 3.32 g, 11.2 mmol) in dry THF (15 ml) was then slowly added. The reaction mixture was stirred and heated up in an oil bath to 55 C for 75 min to obtain clear solution. The reaction mixture was concentrated to approximately 25% of its original volume by rotary evaporator and then n- hexane was added to precipitate the product. The white product was filtered, washed with n- hexane, and crystalized three times from THF/n-hexane. The resulting NCA monomers were dried under vacuum. Y (BLA NCA) = 90%. 1 H NMR (DMS-d 6 ): δ = 2.91 (dd, J 1 = 17.8 Hz, J 2 = 4.3 Hz, 1H, CH 2a ), 3.08 (dd, J 1 = 17.8 Hz, J 2 = 4.3 Hz, 1H, CH 2b ), 4.70 (m, 1H, CH), 5.14 (s, 2H, benzyl CH 2 ), 7.37 (m, 5H, aryl CH), 9.00 (s, 1H, NH). Y (BLG NCA) = 85%. 1 H NMR (DMS-d 6 ): δ = 2.02 (m, 2H, CH 2 ), 2.49 (m, 2H, CH 2 ), 4.48 (ddd, J 1 = 7.8 Hz, J 2 = 5.5 Hz, J 3 = 1.1 Hz, 1H, CH), 5.11 (s, 2H, benzyl CH 2 ), 7.37 (m, 5H, aryl CH), 9.11 (s, 1H, NH). General procedure for RP of BLA NCA and BLG NCA All polymerizations were carried out with a freshly prepared batch of the NCA monomer in a flame-dried Schlenk flask under an argon atmosphere. S2
Synthesis of poly(β-benzyl-l-aspartate) (PBLA) H N Ph H n Ph Sample A: The BLA NCA (0.250 g, 1.00 mmol) was suspended in dry chloroform (10 ml) in an argon atmosphere. Then, the methanesulfonic acid (MSA, 7.8 µl, 0.12 mmol) and 3- phenyl-1-propanol (PPA, 5.5 µl, 0.04 mmol), corresponding to a monomer/initiator mole ratio of 25/1, were added to the suspension which was then heated in an oil bath to 40 C and stirred for 24 h under argon atmosphere, when full conversion of the hydroxyl groups to ester bonds was achieved as determined by 1 H NMR analysis. After complete initiation, the reaction mixture was cooled down in an ice bath to 0 C and the propagation was started by the addition of N-ethyldiisopropylamine (EDIPA, 17.2 µl, 0.10 mmol). After 24 hours, 40% conversion of NCA monomer was achieved as determined by 1 H NMR. After 2 days, the reaction mixture became clear and full conversion of the monomer was detected. Reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (8000 rpm, 1 min) and dried under vacuum. The white powder was then dissolved in DMF and precipitated into water, washed with water several times and dried under vacuum for 24 h. Sample C was synthesized and isolated under the same experimental conditions as the Sample A, except the monomer/initiator mole ratio used was 40/1, whereas the initiator/acid/base mole ratio of 1/3/2.5 was the same as for the synthesis of Sample A. Figure S1: 1 H NMR spectrum of PBLA (Sample A in Table 1). S3
Figure S2: 1 H NMR spectrum of PBLA (Sample C in Table 1). Figure S3: Top: MALDI-TF mass spectrum and its enlarged region with denoted measured monoisotopic signals for the PBLA homopolypeptide (Sample C in Table 1), which was prepared by RP of BLA NCA initiated with PPA. Bottom: The proposed PBLA structures and their calculated exact masses ionized with sodium ion [M+Na] +. S4
Figure S4: SEC-MALS chromatograms of PBLA (Sample A (red) and Sample C (black) in Table 1); solid curves: refractive index detector responses, dotted curves: light-scattering detector responses at 90 angle, squares: molar mass as a function of elution volume. Synthesis of poly(γ-benzyl-l-glutmate) (PBLG) Ph H N H n Ph Sample B: The BLG NCA (0.250 g, 0.95 mmol) was dissolved in dry chloroform (5 ml) in an argon atmosphere. Then, the methanesulfonic acid (MSA, 7.4 µl, 0.11 mmol) and 3- phenyl-1-propanol (PPA, 5.2 µl, 0.04 mmol) were added to the solution. After the solution was stirred for 24 h at 40 C, it was cooled down to 0 C. Propagation was started by the addition of N-ethyldiisopropylamine (EDIPA, 16.3 µl, 0.10 mmol) and the reaction mixture was stirred for 24 h, when full conversion was achieved. Afterwards, the reaction mixture was poured into diethyl ether to precipitate the product. The precipitate was isolated by centrifugation (8000 rpm, 1 min) and dried under vacuum. The white powder was then dissolved in DMF and precipitated into water, washed several times with water and dried under vacuum for 24 h. Sample D was synthesized and isolated under the same experimental conditions as the Sample B, except the monomer/initiator mole ratio used was 40/1, whereas initiator/acid/base mole ratio of 1/3/2.5 was the same as for the synthesis of Sample B. S5
Figure S5: 1 H NMR spectrum of PBLG (Sample B in Table 1). Figure S6: 1 H NMR spectrum of PBLG (Sample D in Table 1). S6
Figure S7: Top: MALDI-TF mass spectrum and its enlarged region with denoted measured monoisotopic signals for the PBLG homopolypeptide (Sample B in Table 1), which was prepared by RP of BLA NCA initiated with PPA. Bottom: The proposed PBLA structures for two main populations and their calculated exact masses ionized with sodium ion [M+Na] +. Figure S8: Top: MALDI-TF mass spectrum and its enlarged region with denoted measured monoisotopic signals for the PBLG homopolypeptide (Sample D in Table 1), which was prepared by RP of BLA NCA initiated with PPA. Bottom: The proposed PBLA structures for two main populations and their calculated exact masses ionized with sodium ion [M+Na] +. S7
Figure S9: SEC-MALS chromatograms of PBLG (Sample B (red) and Sample D (black) in Table 1); solid curves: refractive index detector responses, dotted curves: light-scattering detector responses at 90 angle, squares: molar mass as a function of elution volume. Synthesis of polypeptide-based hybrid block copolymers Block copolymers containing polypeptide blocks (PEG-b-PBLA and PS-b-PBLA) were synthesized by ring-opening polymerization of BLA NCA using hydroxyl-terminated poly(ethylene glycol) (PEG-H) or polystyrene (PS-H) as a macroinitiator. Both block copolymers were synthesized in a similar manner by using the corresponding macroinitiators. Synthesis of poly(ethylene glycol)-b-poly(β-benzyl-l-aspartate) (PEG-b-PBLA) n H N Ph m H Sample E: The BLA NCA (0.400 g, 1.61 mmol) was suspended in dry chloroform (10 ml) in an argon atmosphere. Then, PEG-H (M p = 1964 g mol -1, 78.8 mg, 0.04 mmol) and methanesulfonic acid (MSA, 7.8 µl, 0.12 mmol) were added to the suspension. Initiation was completed after stirring the reaction mixture at 40 C for 48 h. Then, the reaction mixture was cooled down to 0 C. Propagation was started by the addition of N-ethyldiisopropylamine (EDIPA, 17.2 µl, 0.10 mmol), and it was completed after 24 h. The product was precipitated in diethyl ether, washed with diethyl ether several times and dried under vacuum for 24 h. S8
Figure S10: 1 H NMR spectrum of PEG-b-PBLA block copolymer (Sample E in Table 1). Synthesis of poly(styrene)-b-poly(β-benzyl-l-aspartate) (PS-b-PBLA) n Ph N H m H Sample F: The BLA NCA (0.400 g, 1.61 mmol) was suspended in dry chloroform (10 ml) in an argon atmosphere. Then, PS-H (M n = 2200 g mol -1, 88.3 mg, 0.04 mmol) and methanesulfonic acid (MSA, 7.8 µl, 0.12 mmol) were added to the suspension. Initiation of PS-H was completed after stirring the reaction mixture at 40 C for 24 h. Afterwards, the reaction mixture was cooled down to 0 C. Propagation was started by the addition of N- ethyldiisopropylamine (EDIPA, 17.2 µl, 0.10 mmol), and it was completed after 24 h. The product was precipitated in diethyl ether and dried in vacuum. Then, it was dissolved in DMF and precipitated into water, washed several times with water and dried under vacuum for 24 h. S9
Figure S11: 1 H NMR spectrum of PS-b-PBLA block copolymer (Sample F in Table 1). S10