Interfacial Ring-Opening Polymerization (irop) of Amino Acid Derived N-thiocarboxyanhydrides Towards Well-defined Polypeptides

Transcription

1 Electronic Supplementary Information (ESI) Interfacial Ring-Opening Polymerization (irop) of Amino Acid Derived N-thiocarboxyanhydrides Towards Well-defined Polypeptides Jinbao Cao, David Siefker, Brandon A. Chan, Tianyi Yu, Lu Lu, Mirza A. Saputra, Frank R. Fronczek, Weiwei Xie, and Donghui Zhang* Department of Chemistry and Macromolecular Studies Group, Louisiana State University, Baton Rouge, Louisiana 70803, United States. Department of Mechanical & Industrial Engineering, Louisiana State University, Baton Rouge, LA 70803, United States Corresponds to: 1

2 Materials All chemicals were purchased from Sigma-Aldrich and used as received unless specified. L-glutamic acid γ-benzyl ester (H-Glu(OBz)-OH) and ε-n-carbobenzyloxy-l-lysine (H-Lys(Z)- OH) were purchased from AAPPTec, LLC and used as received. BLG-NCA was synthesized by using a published procedure. 1 All solvents are regular ACS grade solvents and were used directly in the reactions without any special drying or purification step unless specified. All reactions are conducted in open air unless otherwise noted. Instrumentation 1 H and 13 C NMR spectra were recorded on a Bruker AV-400 or AV-500 spectrometer. Chemical shifts in parts per million (ppm) were referenced relative to proton impurities or 13 C isotope of deuterated solvents (e.g., CDCl 3 ). SEC-DRI analyses were performed with an Agilent 1200 system equipped with three Phenomenex 5 μm, mm columns [100 Å, 1000 Å and Linear(2)], Wyatt DAWN EOS multi-angle light scattering (MALS) detector (GaAs 30 mw laser at λ=690 nm) and Wyatt Optilab rex differential refractive index (DRI) detector with a 690 nm light source. DMF containing 0.1 M LiBr was used as the eluent at a flow rate of 0.5 ml min -1. The temperature of the column and detector was 25 C. FTIR spectra were collected on a Bruker Alpha FT-IR spectrometer. Electrospray ionization mass spectroscopy (ESI MS) was conducted on an ESI TOF 6210 (Electrospray Time-of-Flight) mass spectrometer (Agilent Technologies). The sample solutions used in ESI MS analysis were prepared by dissolving the sample (5 mg) in chloroform (0.5 ml). The experiments were carried out in positive mode ionization. MALDI-TOF MS experiments was conducted on a Bruker UltrafleXtreme tandem time-of-flight (TOF) mass spectrometer. The instrument was calibrated with Peptide Calibration Standard II consisting of standard peptides Angiotensin I, Angiotensin II, Substance P, Bombesin, ACTH clip 1-17, ACTH clip 18-39, and Somatoratin 28 (Bruker Daltonics, Billerica, MA) prior to experiment. A saturated methanol solution of α-cyano-4-hydroxycinnamic acid was used as matrix. Samples were prepared by mixing a THF solution of polymers (5 mg/ml) with matrix at 1:1 volume ratio, which were then deposited onto a 384-well ground-steel sample plate using the dry droplet method. Experiments were done in positive reflector mode. The data analysis was performed with flexanalysis software. TGA experiments were carried out on a TA 2950 TGA under a nitrogen atmosphere with a heating rate of 10 C/min. The scanned temperature range was rt.-600 C. Data were analyzed with Thermal Advantage Software. HPLC analyses were conducted on Dionex Ultimate 3000 system equipped with Chiralcel column (OD- H 0.46 cm 25 cm). The elution solvent mixture consists of 85% hexanes and 15% isopropanol. The NTA samples for HPLC were prepared by dissolving NTA in 1:1 isopropanol/hexanes mixture to make a 2 mg/ml concentration solution. Prior to injection into the HPLC, the solutions were filtered through 0.22 µm PTFE filters. X-ray Crystallographic Analysis of Single Crystals of NTA Monomers Single crystals of BG-NTA (2% ee), Lys-NTA (2% ee) and Leu-NTA (100% ee) were used in X-ray crystallographic analysis. They were obtained by slow solvent evaporation from a chloroform solution of the monomers. Structures of BG-NTA were determined from data collected at T = 90K with MoKα radiation on a Bruker Kappa Apex-II diffractometer equipped with a Triumph focusing monochromator. The racemic form of BG-NTA, space group P2 1 /c, exhibited two enantiomeric molecules overlapped with :0.0491(14) populations. The 2

3 structure of the S form of Lys-NTA, space group P2 1, was determined from data collected at T = 200 K with MoKα radiation on a Nonius KappaCCD diffractometer. A phase change with twinning occurs below about 180 K, so data were collected from a single crystal at a higher temperature. The asymmetric unit has two independent molecules, and no indication of the presence of the R enantiomer was evident. The absolute configuration was confirmed, with Flack parameter x = 0.12(9). The structure of Leu-NTA, space group P , was determined from data collected at T = 90 K with MoKα radiation on a Bruker Kappa Apex-II diffractometer. The asymmetric unit has two independent molecules, which form a hydrogen-bonded dimer. The absolute configuration was confirmed, with Flack parameter x = 0.13(7) and no indication of the presence of the R enantiomer was evident. CIFs have been deposited at the Cambridge Crystallographic Data Centre, CCDC and , respectively. X-ray Powder Diffraction (XRD) Analysis of NTA Monomers The XRD measurement of BG-NTA was performed on in-house PNAnalytical Empyrean instrument, using the characteristic X-ray of Cu target. The range of scattering angle covers from 4 up to 90. The correction to a fixed slit was done before the analysis of spectrum. The samples were ground to powder and uniformly distributed on a zero-background silicon wafer for the measurement. For the measurement, phase identification and the lattice parameters were refined by LeBail refinements using LHPM RIETICA. The background of the powder pattern was determined by 6-parameter polynomial function. The area was integrated numerically with Simpson s rules. The integrated observed pattern was subtracted with the integrated background and the calculated pattern. The calculated pattern was derived from the single crystal data, which was treated as a full contribution from the 100% crystalline sample. The remaining was considered as contribution from the non-crystalline phase. The crystalline percentage (crystallinity) was defined and calculated as the ratio of diffraction contribution from the non-crystalline phase to the contribution from 100% crystalline sample. Synthesis of S-Ethoxythiocarbonyl Mercaptoacetic Acid (XAA) The synthesis of XAA is modified from a published procedure. 2 NaOH (9.31 g, 23.3 mmol) was first dissolved in chilled DI water (233 ml), followed by addition of chloroacetic acid (22.02 g, mmol) to afford a clear solution. Potassium ethyl xanthogenate (37.36 g, mmol) was then added to the above solution. The mixture was allowed to stir at room temperature for one day, followed by acidification with 4 M HCl(aq.) to ph ~ 1. The resulting cloudy mixture was then extracted with chloroform (3 150 ml). The combined organic extract was dried over anhydrous MgSO 4, filtered, and concentrated under vacuum. Hexanes (500 ml) were then added to the oily residue with vigorous stirring to afford an off-white solid. The solid was collected by filtration and washed with hexanes and dried under vacuum to give the final product as a white solid (38.06 g, 91% yield). 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): 1.43 (t, 3H, CH 3 ), 3.97 (s, 2H, CH 2 ), 4.65 (q, 2H, CH 2 ). Synthesis of γ-benzyl-dl-glutamic Acid N-Thiocarboxyanhydrosulfide (BG-NTA) H-Glu(OBz)-OH (4.95 g, 20.9 mmol) and XAA (3.76 g, 20.9 mmol) were suspended in a saturated NaHCO 3 (aq.) solution (70 ml). The suspension was stirred vigorously for two days at room temperature to afford a clear solution. The clear solution was then acidified with 37% 3

4 concentrated HCl to ph ~ 3, followed by extraction with ethyl acetate (3 60 ml). The combined organic extract was dried with anhydrous MgSO 4, filtered, and concentrated under vacuum. The oily residue was then re-dissolved in ethyl acetate (70 ml) under nitrogen, followed by the addition of PCl 3 (2.2 ml, 25 mmol) at room temperature. The reaction mixture was stirred at room temperature for 20 h and then sequentially washed with a saturated NaHCO 3 (aq.) solution (100 ml), DI water (100 ml), and brine solution (100 ml). The organic phase was separated and dried over anhydrous MgSO 4, filtered, and concentrated under vacuum to afford a light yellow oil. The oil was dissolved in a minimum amount of THF and precipitated into excess hexanes with vigorous stirring to afford an off-white solid (3.24 g). The crude solid product was further purified by flash chromatography with ethyl acetate/hexanes (2:3, v/v) (R f = 0.33) off a silica gel (pore size 60 Å, particle size µm) column. A white solid (2.84 g, 49% yield) was collected via removal of the solvent under vacuum after the chromatography purification. HRMS-ESI (m/z): [M + H] + calculated for C 13 H 14 NO 4 S , found Melting point: 71.6 C 72.5 C. α 25 D = in CHCl 3 c = H NMR (400 MHz, CDCl 3 ) δ (ppm): 7.42 (s, 1H, NH), 7.34 (m, 5H, C 6 H 5 ), 5.12 (s, 2H, CH 2 ), 4.37 (t, 1H, CH), 2.55 (t, 2H, CH 2 ), (m, 2H, CH 2 ) 13 C NMR (400 MHz, CDCl 3 ) δ (ppm): , , , , , , , 67.02, 66.09, 29.46, Synthesis of ε-n-carbobenzyloxy-dl-lysine N-Thiocarboxyanhydrosulfide (Lys-NTA) NaOH (2.87 g, 71.8 mmol) was dissolved in chilled DI H 2 O (120 ml). H-Lys(Z)-OH (10.04 g, 35.8 mmol) and XAA (6.47 g, 35.9 mmol) were sequentially added to the above clear solution to afford a cloudy mixture. The reaction mixture was stirred vigorously for three days at room temperature followed by acidification with 4 M HCl(aq.) to ph ~ 3 and extraction with ethyl acetate (3 200 ml). The combined organic extract was dried over anhydrous MgSO 4, filtered, and concentrated under vacuum to ~120 ml, to which PCl 3 (3.7 ml, 42 mmol) was added under nitrogen. The reaction was allowed to stir at room temperature for 20 h and then sequentially washed with a saturated NaHCO 3 (aq.) solution (200 ml), DI water (100 ml) and brine solution (100 ml). The organic phase was separated, dried over anhydrous MgSO 4, filtered, and concentrated under vacuum to afford a light yellow oil. The oil was re-dissolved in minimal THF and precipitated into excess hexanes under vigorous stirring to afford a white solid (8.26 g, 25.7 mmol, 72% crude yield). A fraction of the crude solid (2.50 g, 7.76 mmol) was further purified by flash chromatography using diethyl ether/hexanes (10:1, v/v) (R f = 0.22 in 10:1 diethyl ether/hexanes) off a silica gel column. The purified product was collected as a white solid (2.08 g, 83% yield) via solvent evaporation under vacuum after the chromatography purification. HRMS-ESI (m/z): [M + H] + calculated for C 15 H 19 N 2 O 4 S , found Melting point: C C. α 25 D = in CHCl 3 c = H NMR (400 MHz, DMSO-d 6 ) δ (ppm): 9.32 (s, 1H, NH), 7.33 (m, 5H, C 6 H 5 ), 7.24 (t, 1H, NH), 5.01 (s, 2H, CH 2 ), 4.55 (t, 1H, CH), 3.00 (m, 2H, CH 2 ), 1.72 (m, 2H, CH 2 ), 1.40 (m, 4H, CH 2 CH 2 ). 13 C NMR (500 MHz, CDCl 3 ) δ (ppm): , , , , , , , 67.03, 66.87, 40.23, 31.94, 29.39, Synthesis of D,L-Methionine N-Thiocarboxyanhydrosulfide (Met-NTA) NaOH (6.78 g, 170 mmol) was dissolved in chilled DI H 2 O (250 ml). D,L-methionine (10.12 g, 67.8 mmol) and XAA (12.23 g, 67.8 mmol) were sequentially added to the above clear 4

5 solution. The reaction mixture was stirred vigorously for two days at room temperature followed by acidification with 37% conc. HCl to ph ~ 3 and extraction with chloroform (2 150 ml). The combined organic extract was dried over anhydrous MgSO 4, filtered, and concentrated under vacuum to afford a yellow oil. The oil was then dissolved in chloroform (200 ml), to which PCl 3 (7.9 ml, 91 mmol) was added under nitrogen. The reaction was allowed to stir at room temperature for 20 h and then sequentially washed with a saturated NaHCO 3 (aq.) solution (200 ml), DI water (200 ml), and brine solution (200 ml). The organic phase was separated, dried over anhydrous MgSO 4, filtered, and concentrated under vacuum to afford a light yellow oil, which slowly crystallized to form light a yellow solid. The crude solid was recrystallized from CH 2 Cl 2 /hexanes to afford a white solid (8.11 g, 42.5 mmol, 63% yield). HRMS-ESI (m/z): [M + H] + calculated for C 6 H 9 NO 2 S , found Melting point: 61.0 C 62.2 C. 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): 7.48 (s, 1H, NH), 4.50 (m, 1H, CH), 2.67 (t, 2H, CH 2 ), (m, 2H, CH 2 ), 2.11 (s, 3H, CH 3 ). 13 C NMR (400 MHz, CDCl 3 ) δ (ppm): , , 65.93, 31.52, 29.50, Synthesis of L-Leucine N-Thiocarboxyanhydrosulfide (Leu-NTA) The synthetic of Leu-NTA is modified from a published procedure. 3 NaOH (1.72 g, 43.0 mmol) was first dissolved in DI water (72 ml) in an ice bath. L-Leucine (2.82 g, 21.5 mmol) and XAA (3.87 g, 21.5 mmol) were then added to the aqueous solution, which was stirred for two days to form a clear solution. The clear solution was worked up by acidification with 4 M HCl (aq.) to ph ~ 3. Ethyl acetate (3 200 ml) was used to extract the aqueous solution. The combined organic solution was then washed with brine, separated, and dried over anhydrous MgSO 4. Clear oil was afforded after the organic solution was filtered and concentrated under vacuum. The clear oil was used directly for the cyclization step by dissolving in THF (43 ml) together with imidazole (1.46 g, 21.5 mmol) in an ice bath. Nitrogen was purged through the solution for 10 min before PBr 3 (2.42 ml, 25.8 mmol) was added dropwise to the above solution. An ice cold mixture of ethyl acetate (100 ml) and saturated NaHCO 3 (aq.) solution (100 ml) was poured into the reaction after 10 min stirring. The organic layer was separated from the basic aqueous solution, which was then successively washed with cold 1M HCl (aq.) (50 ml), saturated NaHCO 3 (aq.) solution (50 ml), and brine (50 ml). The organic layer was concentrated to afford clear oil after being dried over anhydrous MgSO 4 and filtration. The crude oil was then recrystallized form ethyl acetate/hexanes to afford a needle-like solid (2.41 g, 65%). 1 H NMR (400 MHz, CDCl 3 ) δ (ppm): 7.13 (s, 1H, NH), 4.37 (m, 1H, CH), 1.79 (m, 2H, CH 2 ), 1.70 (m, 1H, CH), 1.00 (m, 6H, CH 3 ). Interfacial Ring-Opening Polymerization (irop) of NTAs A representative polymerization procedure is given as followed. BG-NTA (52.8 mg, mmol) was suspended in hexanes (0.87 ml) in open air. A measured volume of a stock solution of hexylamine in hexanes (74.7 µl, 1.58 µmol, 133 mm) was added to the above mixture. The polymerization was stirred at 50 C for 2 days to reach nearly quantitative conversion. The solvent was then removed under vacuum to afford the reaction mixture as a solid which was redissolved in DMF (TFA in the case of irop of Met-NTA and Leu-NTA). The final polymer product was precipitated by adding excess diethyl ether into the polymer solution, separated by filtration, and dried under vacuum to afford a white solid (36.2 mg, 91% yield). 5

6 Homogeneous Solution-Phase Ring-Opening Polymerization of NTAs A representative polymerization procedure is given as followed. BG-NTA (59.9 mg, mmol) was dissolved in anhydrous dioxane (0.40 ml) under nitrogen atmosphere in glovebox. A measured volume of a stock solution of hexylamine in hexanes (31.1 µl, 1.79 µmol, 57.5 mm) was added to the above solution. The polymerization was stirred at 50 C for 2 days before sampling a reaction aliquot for conversion analysis. The polymer was isolated by precipitation into diethyl ether, followed by filtration, diethyl ether wash and vacuum dry. Chain Extension Experiments via irop of Lys-NTA Lys-NTA (61.2 mg, mmol) was suspended in heptane (0.95 ml) in open air. A measured volume of a stock solution of hexylamine in hexanes (28.5 µl, 3.79 µmol, 133 mm) was added to the above mixture. The polymerization was stirred at 80 C for 48 h to allow a quantitative monomer conversion. The solvent was removed under vacuum to afford the reaction mixture as a solid. A measured amount of solid (17.2 mg) was taken for further SEC and 1 H NMR analysis. The remaining solid was re-suspended in a known volume of heptane (0.64 ml). A second batch of Lys-NTA monomer (41.2 mg, mmol) was added to the above suspension. The reaction suspension was again stirred at 80 C for two days to allow for quantitative monomer conversion. The solvent was then removed under vacuum and the solid residue was characterized by SEC and 1 H NMR methods. Solid-State Polymerization of BG-NTA BG-NTA (20.4 mg, mmol) was weighed into a vial in open air. A measured volume of a stock solution of hexylamine in hexanes (30.7 µl, 1.46 µmol, 47.6 mm) was added to the above mixture. The mixture was gently shaken to facilitate the mixing of the initiator solution with the solid monomer and allowed to react for ~ 2 min at room temperature. The volatile (i.e., hexane) was subsequently removed under vacuum. The vial containing the remaining solid mixture was placed in oil bath at 50 C for 1 h and allowed to cool down to room temperature. The solid mixture was dissolved in DMF (0.5 ml). Aliquots were then taken for conversion analysis by 1 H NMR spectroscopy and molecular weight analysis by SEC method. Kinetic study of irop of NTAs Six parallel reactions of irop of NTA ([M] 0 :[I] 0 = 80:1, in hexanes) were set up in separate vials and allowed to proceed at 50 C in an oil bath. At varying time intervals (e.g., 1, 2, 4 h, etc.), one vial was taken out of the oil bath and allowed to cool to room temperature. The solvent was then removed under vacuum and the resulting solid mixture was dissolved in DMF (TFA in the case of Leu-NTA) to form a homogeneous solution. Aliquots were then taken for conversion and molecular weight analysis by 1 H NMR and SEC method, respectively. The kinetic studies were repeated twice. 6

7 Figure S1. HPLC trace of Leu-NTA (100% ee) obtained using PBr 3 acylation agent at 0 C. Figure S2. HPLC trace of BG-NTA (10% ee) obtained using PBr 3 acylation agent at 0 C (Relative percentage of the two peak areas: 55% (left), 45% (right)). Figure S3. HPLC trace of Lys-NTA (40% ee) obtained using PBr 3 acylation agent at 0 C (Relative percentage of the two peak areas: 70% (left), 30% (right)). Figure S4. HPLC trace of BG-NTA (2% ee) obtained using PCl 3 acylation agent at rt. (Relative percentage of the two peak areas: 51% (left), 49% (right)). 7

8 Figure S5. HPLC trace of Lys-NTA (2% ee) obtained using PCl 3 acylation agent at rt. (Relative percentage of the two peak areas: percentage 51% (left), 49% (right)). Figure S6. 1 H NMR spectrum of poly(methionine) polymer (PMET 41 ) obtained from irop of Met-NTA (TFA-d 1 ). The DP n was determined by the integration of methine proton (e) on the PMET backbone relative to that of the methyl protons (a) of hexylamide end-group using the equation e/(a/3). 8

9 Figure S7. 1 H NMR spectrum of poly(l-leucine) polymer (PLEU 38 ) obtained from irop of Leu- NTA (TFA-d 1 /CDCl 3 = 9:1 v:v). The DP n was determined by the integration of methine proton (g) on the PLEU backbone relative to that of the methylene protons (a) of hexylamide end-group using the equation g/(a/2). Figure S8. (A) Full and (B) expanded MALDI-TOF MS spectra of a low molecular weight poly(l-leucine) polymer (PLEU 20 ) obtained by irop of Leu-NTA together with (C) the chemical structure of PLEU showing the end-group structures as determined by the MS analysis. 9

10 (D) Comparison of selected experimental m/z with the calculated values based on the PLEU chemical structure shown in (C). Table S1. Polymerization conversion of BG-NTA, Lys-NTA and Leu-NTA in anhydrous 1,4- dioxane (DO) or DMF using different initiators. a Entry # Solvent Temperature NTA (ee) Initiator b [M] 0 :[I] 0 Conv. (%) c 1 DO r.t BG (2%) HA 50: DO r.t BG (2%) HA 80: DO r.t BG (2%) HA 120:1 5 4 DO r.t BG (2%) TEA 50: DO r.t BG (2%) HMDS 100: DO r.t BG (2%) Ni(BiPy) (COD) 100: DO 50 C BG (2%) TEA 50:1 7 8 DO 50 C BG (2%) HA 120: DO 50 C BG (2%) HMDS 120: DO 50 C BG (2%) Ni(BiPy) (COD) 120: DO r.t Leu (100%) HA 40: DMF r.t Leu (100%) HA 40: DO 50 C Leu (100%) HA 20: DO 50 C Leu (100%) HA 40: DO 50 C Leu (100%) HA 80: DMF 50 C Leu (100%) HA 80: DO r.t Lys (2%) HA 40: DMF r.t Lys (2%) HA 40: DO 80 C Lys (2%) HA 80: DMF 80 C Lys (2%) HA 80: DO 50 C Met (0%) HA 40: DO 50 C Met (0%) HA 80: DMF 50 C Met (0%) HA 80:1 3 a. All reactions were allowed to proceed under nitrogen in anhydrous solvents for 2 d at [M] 0 = 0.5 M except for Entry 4-6 which reacted for 1 d; b. HA = hexylamine, TEA = triethylamine, 10

11 HMDS = hexamethyldisilazane, Ni(BiPy)(COD) = Ni(2,2 -bipyridyl)(1, 5-cyclooctadiene); c. determined by 1 H NMR analysis of reaction aliquots. Figure S9. (A) Full and (B) expanded MALDI-TOF MS spectra of a low molecular weight PBG polymer obtained by solution-phase polymerization of BG-NTA in dioxane initiated with hexylamine at room temperature ([M] 0 :[I] 0 =80:1, [M] 0 =0.5 M, 30% conversion, 2 d). (C) The PBG chemical structures where the end-groups are determined by the MS analysis. (D) Comparison of selected experimental m/z with the calculated values based on the PBG chemical structure shown in (C). 11

12 Figure S10. (A) Full and (B) expanded MALDI-TOF MS spectra of a low molecular weight PBG polymer obtained by solution-phase polymerization of BG-NTA in dioxane initiated with hexylamine at 50 C ([M] 0 :[I] 0 =80:1, [M] 0 =0.5 M, 35% conversion, 2 d). (C) The PBG chemical structure where the end-groups are determined by the MS analysis. (D) Comparison of selected experimental m/z with the calculated values based on the PBG chemical structure shown in (C). 12

13 Normalized DRI response Normalized DRI signal [M] 0 :[I] 0 = 40 [M] 0 :[I] 0 = 80 [M] 0 :[I] 0 = 120 [M] 0 :[I] 0 = Elution time (mins) Figure S11. Representative SEC chromatograms of PBG polymers obtained by irop of BG- NTA (2% ee) at 50 C in hexanes suspension ([M] 0 :[I] 0 =40:1, 80:1, 120:1 and 150:1) % 33% 49% 65% 77% 99% Elution time (mins) Figure S12. Representative SEC chromatograms of PBG polymers obtained from the irop of BG-NTA (2% ee) at different conversions (Conditions: [M] 0 :[I] 0 = 80 :1, 50 C, hexanes suspension) 13

14 Figure S13. (A) Full and (B) expanded MALDI-TOF MS spectra of a low molecular weight PBG polymer obtained by irop of BG-NTA (2% ee) together with (C) the chemical structures of PBG polymers showing the end-group structures as determined by the MS analysis. (D) A comparison of selected experimental m/z with the calculated values based on the PBG polymer structure in (C). 14

15 Normalized DRI Signal [M] 0 :[I] 0 = 80 [M] 0 :[I] 0 = 150 [M] 0 :[I] 0 = Elution time (mins) Figure S14. Representative SEC chromatograms of PLYS polymers obtained by irop of Lys- NTA (2% ee) at 80 C in heptane suspension ([M] 0 :[I] 0 = 80:1, 150:1 and 200:1) 15

16 Figure S15. (A) Full and (B) expanded MALDI-TOF MS spectra of a low molecular weight PLYS 7 polymer obtained by irop of Lys-NTA in heptane initiated with hexylamine at 80 C. (C) The PLYS chemical structure where the end-groups are determined by the MS analysis. (D) Comparison of selected experimental m/z with the calculated values based on the PLYS chemical structure shown in (C). The minor specie (P2) was formed via free radical induced side chain fragmentation during ionization process. The mechanism of their formation has been previously reported. 4,5 16

17 Figure S16. (A) Full and (B) expanded MALDI-TOF MS spectra of polymet 40 polymer obtained by irop of Met-NTA in hexanes initiated with hexylamine at 50 C. (C) The polymet chemical structure where the end-groups are determined by the MS analysis. (D) Comparison of selected experimental m/z with the calculated values based on the polymet chemical structure shown in (C). 17

18 Normalized DRI Response PLYS after chain extension PLYS prior to chain extension Elution time (mins) Figure S17. SEC chromatograms of PLYS polymers obtained from the chain extension experiment by irop of Lys-NTA (2% ee) in heptane at 80 C. The red and black curves respectively correspond to the PLYS polymers obtained from the first ([M] 0 :[I] 0 =50:1) and second irop of Lys-NTA ([M] 0 :[I] 0 =57:1) that were conducted sequentially. Each step of the polymerization was allowed to reach quantitative conversion. Table S2. Chain extension study of irop of Lys-NTA in heptane at 80 C Entry [M] 0 :[I] 0 M n (Theo.) (kg/mol) M n (Exp.) a. (kg/mol) PDI a. Conversion b. 1 st batch 50: nd batch 57: a. determined by SEC-DRI-MALS analysis (dn/dc = ml/g for PLYS in 0.1 M LiBr/DMF at 25 C); b. determined by 1 H NMR spectroscopy. (%) 18

19 Figure S18. (A) Plot of conversion versus time ( ) and the non-linear fitting (R 2 =0.994) of the data ( ) for the irop of Leu-NTA. Fitting equation: conversion = 1-exp(-k obs *t), k obs = 0.33 ± 0.02 h -1 (n=2) (B) Plot of ln([m] 0 :[M]) versus time ( ) and the linear fitting (R 2 =0.995) of the data ( ) for the irop of Leu-NTA. Fitting equation: [ln([m] 0 :[M]) = k obs t, k obs =0.31±0.02 h -1 (n=2) (Polymerization condition: [M] 0 = 0.2 M, [I] 0 = 2.5 mm, [M] 0 :[I] 0 =80:1, in hexanes at 50 C in air). (C) 19

20 Figure S19. (A) Plot of conversion versus time ( ) for irop of BG-NTA and the non-linear fitting (R 2 =0.982) of the data ( ); fitting equation: conversion = 1-exp(-k obs *t), k obs = 0.20 ± 0.01 h -1 (n=2). (B) Plot of ln([m] 0 :[M]) versus time ( ) for irop of BG-NTA and the linear fitting (R 2 =0.980) of the data ( ); fitting equation: ln([m] 0 :[M]) = k obs t, k obs =0.19 ± 0.01 h -1 (n=2) (Polymerization condition: [M] 0 = 0.2 M, [I] 0 = 2.5 mm, [M] 0 :[I] 0 =80:1, in hexanes at 50 C in air). (C) Plots of [M] 0 -[M] versus time ( ) for irop of BG-NTA and the linear fitting (R 2 =0.968) of the data ( ); fitting equation: [M] 0 -[M] = k obs t, k obs =0.014 ± 0.01 M h -1 (n=2). The irop of BG-NTA is more adequately fitted with the first-order kinetic rate law than the zero-order rate law. The irop polymerization kinetic is expected to be more complex due to the contribution of monomer dissolution kinetics and polymerization kinetics. Table S3. The effect of a continuous N 2 flow on the polymerization conversion of BG-NTA and Lys-NTA in DMF using hexylamine initiator. a Entry # N 2 flow NTA (ee) [M] 0 :[I] 0 Conversion (%) b 1 No BG (2%) 80: Yes BG (2%) 80: No Lys (2%) 80:1 6 4 Yes Lys (2%) 80:1 47 a. All reactions were conducted at room temperature in anhydrous DMF for 2 d using hexylamine initiators at [M] 0 = 0.2 M with or without a continuous flow of N 2 (flow rate = ~250 ml/min); b. determined by 1 H NMR analysis of reaction aliquots. 20

21 Figure S20. A). ESI-MS spectrum of the reaction product from Leu-NTA and hexylamine in 1:1 molar ratio in 50 C hexanes ([M] 0 =0.2 M, 3 h). B) The PLEU chemical structure where the endgroups are determined by the MS ESI analysis. C) Comparison of selected experimental m/z with the calculated values based on the PLEU chemical structure shown in B). Figure S21.The corresponding 13 C{ 1 H} NMR spectra of the reaction product from (1) Leu-NTA and hexylamine in 1:1 molar ratio in 50 C dioxane ([M] 0 =0.2 M, 3 h), (2) after purging the stoichiometric reaction product in dioxane with nitrogen for 1 h and (3) from the same stoichiometric reaction in 50 C hexane ([M] 0 =0.2 M, 3 h). Reaction aliquots were directly dissolved in CDCl 3 for NMR analysis. 21

22 Figure S22. A). ESI-MS spectrum of the reaction product from Leu-NTA and hexylamine in 1:1 molar ratio in 50 C dioxane ([M] 0 =0.5 M, 3 h). B) The PLEU chemical structure where the endgroups are determined by the MS ESI analysis. C) Comparison of selected experimental m/z with the calculated values based on the PLEU chemical structure shown in B). 22

23 Figure S23. (A) Full and (B) expanded MALDI-TOF MS spectra of the PLEU polymer obtained by the solution polymerization of LEU-NTA in 50 C dioxane using hexylamine initiator ([M] 0 :[I] 0 =120:1, [M] 0 =0.5 M, 30% conversion, 2 d), (C) the expanded MS spectrum showing the experimental isotope pattern, (D) the simulated MS isotope pattern of the species P1 (n=13) and P2 (n=12) in a 10:1 ratio, (E) the simulated MS isotope pattern of the species P1 (n=13) alone, (F) the simulated MS isotope pattern of the species P2 (n=12) alone, (G) the PLEU 23

24 chemical structure where the end-groups are determined by the MS analysis, and (H) the comparison of selected experimental m/z with the calculated values based on the PLEU chemical structure shown in (G). (* indicates PLEU polymers whose end-group structures are unknown. Note: the presence of urea-terminated PLEU species is particularly notable in the high MW region). Scheme S1. Proposed mechanism for the primary amine-initiated ROP of NTA. Scheme S2. Proposed termination reaction that occurs in polar solvent (e.g., dioxane or DMF) during the polymerization of amino-acid derived NTA (e.g., Leu NTA, Lys-NTA) 24

25 Figure S24. Lamellar packing of BG-NTA monomers as revealed by the X-ray crystallographic analysis of the single crystal of the monomer. (Note: BG-NTA side chains and the S enantiomer were not shown for an unobstructed view of the packing structure. The inter-lamellar distance along the a-axis is Å). Figure S25. In plane (bc plane) packing of BG-NTA monomers as revealed by the X-ray crystallographic analysis of the single crystal of the monomer. (Note: BG-NTA side chains and the S enantiomer were not shown for an unobstructed view of the packing structure. The green dash lines represent the distance between NH and C5 carbonyl (N to C distance). Unit for distance is Å). 25

26 Figure S26. Observed and calculated powder X-ray diffraction (XRD) pattern of the BG-NTA solid monomers. The crystallinity of the BG-NTA is calculated to be 95(3)%. Table S4. Conversions obtained from irop and solid-state ROP of BG-NTA (2% ee) and Lys- NTA (2% ee) using hexylamine initiators (50 C for BG-NTA, 80 C for Lys-NTA). Conversion in 1 h (%) NTA [M] 0 :[I] 0 irop solid-state ROP BG-NTA Lys-NTA

27 Normalized DRI signal solid-state ROP irop Elution time (mins) Figure S27. SEC chromatograms of PBG obtained from the solid-state ROP and irop of BG- NTA (2% ee) using hexylamine initiators at 50 C for 2 d ([M] 0 :[I] 0 =50:1) (Results: solid state, M n = 9.8 kg/mol, PDI = 1.95, 93% conversion in 2 d, theoretical M n = 10.5 kg/mol; irop, M n = 10.4 kg/mol, PDI = 1.20, 98% conversion in 2 d, theoretical M n = 10.8 kg/mol). Figure S28. (A) FTIR spectra of BLG-NCA and (B) BG-NTA (2% ee) solids that have been stored in open air for varying periods of time to determine their relative shelf-lives. (For both monomers, a new band at ~1545 cm -1 appears upon storage for varying periods of time. The peak corresponds to the amide II band, indicating formation of oligomeric peptides). 27

28 Weight (%) Deriv. Weight (%/ o C) Weight (%) Deriv. Weight (%/ o C) Table S5. A comparison of BLG-NCA and BG-NTA (2% ee) shelf-lives at different conditions Stored in air Stored in desiccator BLG-NCA <11 d ~ 1 m BG-NTA ~ 2 m >5 m Figure S29. FT-IR spectrum of BG-NTA (2% ee) in the solid state. The peaks at 1717 cm -1 and 1680 cm -1 correspond to the [O=C(5)] and [O=C(2)] bands respectively. The O=C(5) carbonyl stretching frequency of BG-NTA (1717 cm -1 ) is lower than that of the BLG-NCA (1842 cm -1 ), consistent with the carbonyl [O=C(5)] of BG-NTA being less electrophilic than that of BLG- NCA. A) o C B) o C o C o C o C Temperature ( o C) Temperature ( o C) Figure S30. Thermogravimetric analysis (TGA) (black curve) and the first derivative (blue curve) of TGA plot for BG-NTA (2% ee) (A) and BLG-NCA (B). 0 28

29 Figure S31. 1 H NMR spectrum of BG-NTA (2% ee) in CDCl 3 Figure S C{ 1 H} NMR spectrum of BG-NTA (2% ee) in CDCl 3 29

30 Figure S33. 1 H NMR spectrum of Lys-NTA (2% ee) in DMSO-d 6 Figure S C{ 1 H} NMR spectrum of Lys-NTA (2% ee) in CDCl 3 30

31 Figure S35. 1 H NMR spectrum of Leu-NTA in CDCl 3 Figure S36. 1 H NMR spectrum of Met-NTA in CDCl 3 31

32 Figure S C{ 1 H} NMR spectrum of Met-NTA in CDCl 3 Figure S38. Representative 1 H NMR spectrum of PBG polymer obtained from the irop of BG- NTA (2% ee) (CDCl 3 :TFA-d 1 =95:5, v/v). 32

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