Supporting Information

Supporting Information Synthesis of End-functionalized Phosphate and Phosphonate- polypeptides by Ring- pening Polymerization of their Corresponding N-carboxyanhydride (NCA) Soumen Das, Mrityunjoy Kar and Sayam Sen Gupta* Materials and method: All chemicals were purchased from sigma-aldrich and used as received unless otherwise specified. All the solvents used were obtained from Merk India. Hexanes, DMF and acetonitrile were dried by conventional methods and stored in the glove box. THF was freshly distilled over sodium wire and ethyl acetate was freshly distilled from calcium hydride. FT-IR spectra were recorded on Perkin Elmer FT-IR spectrum GX instrument. 1 H NMR and 31 P NMR spectrum was obtained with Bruker spectrometer (200.13 MHz, 400.13 MHz). 31 P NMR shifts are reported in ppm relative to 85% H 3 P 4 at 0 ppm. 13 C NMR spectrum and DEPT were recorded on Bruker spectrometer (50.23 MHz) and reported relative signals according to deuterated solvent used. Size exclusion chromatography of the polymer was performed in VISKTEK TDA 305-040 TRIPLE DETECTR ARRAY refractive index (RI), viscometer (VISC), low angle light scattering (LALS), right angle light scattering (RALS) GPC/SEC MDULE. Separations were achieved by three columns (T6000M, GENERAL MIXED RG 300X7.8 MM) and one guard column (TGAURD, RG GUARD CL 10x4.6 MM), 0.025 M LiBr in DMF as the eluent at 60 C. GPC/LS samples were prepared at concentrations of 5 mg/ml. A constant flow rate of 1 ml/min was maintained, and the instrument was calibrated using PMMA standards. Circular Dichroism Measurements Solutions of polymers were filtered through 0.22 μm syringe filters. CD (180 250 nm) spectra of the phospho-polypeptides (0.25 to 1.0 mg/ml in acetonitrile or in 10 mm phosphate buffer ph 7.2) were recorded (JASC CD SPECTRPLARIMETER, Model J- 815) in a cuvette with a 1 mm path length. All the spectra were recorded for an average of three scans and the spectra were reported as a function of molar ellipticity [θ] versus wavelength. The molar ellipticity was calculated using the standard formula, [θ]= S1

(θ 100 Mw)/(C l), where θ= experimental ellipticity in milli degrees, Mw = average molecular weight, C= concentration in mg/ml, and l = path length in cm. The % α helicity was calculated by using the formula % α helicity= ( [θ] 222nm + 3000)/ 39000. Synthesis of N-Boc-L-Cystine: HC NHBoc S S CH NHBoc L-Cystine (10 g, 41.6 mmol) was dissolve in 9:1 water:tetrahydrofuran (100 ml). 6 M NaH in water was added drop wise until ph 10 was reached. Then di-tert-butyl dicarbonate (24.5 g, 112.3 mmol) was added drop wise into that solution. The reaction mixture was stirred for 24 hrs. Then the reaction mixture acidified by drop wise addition of 2 (N) HCl with stirring until the solution reached ph 2. The solids were extracted with ethyl acetate (3x200 ml) and the combined organic layer washed with ph 2 water (2x100 ml) followed by brine solution and dried over sodium sulphate. Solution was filtered and concentrated under reduced pressure to get a white solid compound. The white solids were washed with hexane for several times to get completely pure N-Boc-L-Cystine (98% yield). 1 H NMR (200.13 MHz, DMS-d 6 ): δ 1.37 (s, 18H), 2.80-2.92 (dd, J=13.39, 9.98, 2H), 3.07-3.16 (dd, J=13.52, 4.17, 2H), 4.08-4.22 (m, 2H); 13 C NMR (50.23 MHz, DMS-d 6 ): δ 28.21 (6C), 39.84 (2C), 52.74 (2C), 78.33 (2C), 155.43 (2C), 172.5 (2C) Synthesis of N-Boc-L-Cysteine: HC NHBoc 1 SH N-Boc-L-Cystine (10.0 g, 22.7 mmol) was dissolve in 200 ml of THF: H 2 (10:1) and triphenyl phosphine (6.54 g, 24.9mmol) was added. The reaction mixture stirred at room temperature for overnight. The THF was evaporated under reduced pressure, then water was added (100 ml) and the solution was made basic to ph 10 with 6 M NaH. The aqueous phase was extracted with ethyl acetate (4x100 ml) to remove excess PPh 3 and PPh 3. Then 2 (N) HCl was added drop wise with stirring until the solution reached ph 2, with the S2

formation of white precipitate. The white solids were extracted by ethyl acetate (3x100mL) and the combined organic phases were washed with ph 2 water (100 ml) followed by brine, and dried with Na 2 S 4. The solution was filtered and condensed to give colourless oily N- Boc-L-Cysteine 1 in 97% yields. 1 H NMR (200.13 MHz, DMS-d 6 ): δ 1.43 (s, 9H), 2.46-2.54 (t, 1H), 2.66-2.95 (m, 2H), 4.01-4.15 (m, 1H); 13 C NMR (50.23 MHz, DMS-d 6 ): δ 28.14 (3C), 39.30, 56.10, 78.27, 155.37, 172.09 Synthesis of allyl diethylphosphate (2): P 2 To a solution of diethyl chlorophosphate (2.0 g, 11.6 mmol) in dry THF (20 ml) was added a solution of allyl alcohol (0.66 g, 11.37 mmol) and triethyl amine (1.4 g, 13.92 mmol) in dry THF (20 ML) at 0 º C under nitrogen. The resulting mixture was stirred at room temperature for overnight. A precipitated white mass was filtered off and the filtrate was concentrated by evaporation of the solvent to obtained allyl diethylphosphate 2 as a transparent liquid (96% yield). 1 H NMR (200.13 MHz, CDCl 3 ): δ 1.19-1.26 (m, 6H), 3.99-4.08 (m, 4H), 4.38-4.46 (m, 2H), 5.11-5.30 (m, 2H), 5.74-5.93 (m, 1H); 13 C NMR (50.23 MHz, CDCl 3 ): δ 16.74 (2C), 64.41 (2C), 68.41, 118.61, 133.24 Synthesis of allyl diethylphosphonate (3): P 3 Triethyl phosphite (25 g, 150 mmol) and allyl bromide (27.22 g, 225 mmol) were taken into a 100 ml RB equipped with a condenser. The reaction mixture was heated for 20h at 150 º C. Then it was distilled under reduced pressure at temperature 85 º -90 º C to obtained allyl diethylphosphonate 3 as a colourless liquid (90% yield). S3

1 H NMR (200.13 MHz, CDCl 3 ): δ 1.14-1.21 (m, 6H), 2.39-2.54 (m, 2H), 3.89-4.03 (m, 4H), 5.02-5.12 (m, 4H), 5.54-5.78 (m, 1H); 13 C NMR (50.23 MHz, CDCl 3 ): δ 16.26 (2C), 31.95, 61.38 (2C), 119.33, 127.05 Synthesis of N-Boc-L-cysteine-diethylphosphate (4): NHBoc HC S 4 P N-Boc-Cysteine 1 (2.84 g, 12.87 mmol) and allyl diethylphosphate 2 (1.0 g, 5.15 mmol) dissolved in dry DMF (25 ml) in a 50 ml test tube with joint. 2, 2-dimethoxy-2-phenyl acetophenone (0.396 g, 1.54 mmol) was added and reaction mixture was vacuum purged and backfilled with nitrogen for three times. Then the test tube irradiated to 365 nm light for 1h. The yellowish coloured DMF solution added into 300 ml of water and extracted with ethyl acetate (3x100 ml). The ethyl acetate layers were washed with water then brine, dried over Na 2 S 4, filtered and condensed to oil. The crude product was purified by silica gel column chromatography using ethyl acetate-pet ether with1% acetic acid as the mobile phase to afford the colourless oily N-Boc-L-cysteine-diethylphosphate 4 (94% yield). 1 H NMR (200.13 MHz, CDCl 3 ): δ 1.22-1.29 (t, J=6.32, 6H), 1.35 (s, 9H), 1.78-1.91 (m, 2H), 2.54-2.61 (t, J=6.82, 2H), 2.82-3.02 (m, 2H), 3.97-4.11 (m, 6H), 4.39-4.48 (m, 1H); 13 C NMR (50.23 MHz, CDCl 3 ): δ 15.93 (2C), 28.07 (3C), 28.28, 29.82, 52.99, 64.06 (2C), 65.9, 79.88, 155.22, 173.61; 31 P NMR (400.13 MHz, CDCl 3 ): δ -1.51 Synthesis of N-Boc-L-cysteine-diethylphosphonate (5): HC NHBoc S 5 P N-Boc-L-cysteine-diethylphosphonate 5 was prepared from N-Boc-L-Cysteine 1 and allyl diethylphosphonate 3 according to the procedure for 4 and was recovered as colourless oil (82% yields). 1 H NMR (200.13 MHz, CDCl 3 ): δ 1.23-1.30 (t, J= 7.07, 6H), 1.39 (s, 9H), 1.70-1.87 (m, 4H), 2.55-2.61 (t, J=6.69, 2H), 2.85-3.05 (m, 2H), 3.99-4.13 (m, 4H), 4.43-4.49 (m, 1H); 13 C S4

NMR (50.23 MHz, CDCl 3 ): δ 16.14 (2C), 21.98, 22.46, 25.27, 28.14 (3C), 34.06, 53.08, 62.01 (2C), 79.94, 155.25, 173.37; 31 P NMR (400.13 MHz, CDCl 3 ): δ 33.24 General procedure for the removal of Boc group: NH 2 HC S 4a P N-Boc-L-cysteine-diethylphosphate 4 (1.0 g, 2.4 mmol) was added into the HCl/THF mixture (4 M, 20 ml) and stirred for 4h at room temperature. The solvent was removed under reduced pressure to yield the product 4a as oil. The oily product was washed with ethyl acetate for several times and then dried at room temperature under vacuum for 24h (97% yields). 1 H NMR (200.13 MHz, DMS-d 6 ): δ 1.19-1.26 (m, 6H), 1.82-1.92 (m, 2H), 2.60-2.68 (t, J=7.2, 2H), 3.04-3.07 (d, J= 5.43, 2H), 3.93-4.08 (m, 6H), 4.12 (br, 1H), 8.64 (br, 3H); 13 C NMR (50.23 MHz, DMS-d 6 ): δ 15.67 (2C), 24.74, 27.27, 30.68, 51.44, 62.83 (2C), 66.98, 169.17 HC NH 2 S 5a P 5a was prepared from 5 according to the above procedure and recovered as oil (96% yield). 1 H NMR (200.13 MHz, DMS-d 6 ): δ 1.18-1.25 (m, 6H), 1.82-1.90 (m, 2H), 2.61-2.68 (t, J=6.44, 2H), 3.02-3.05 (d, J= 5.43, 2H), 3.89-4.04 (m, 4H), 4.10 (br, 1H), 8.66 (br, 3H); 13 C NMR (50.23 MHz, DMS-d 6 ): δ 16.38 (2C), 22.25 (2C), 25.16, 30.87, 51.93, 61.07 (2C), 169.49 Synthesis of diethylphosphate-l-cysteine-n-carboxyanhydride (4b): NH S 4b P S5

To H 2 N-L-Cysteine-phosphate 4a (0.5 g, 1.58 mmol) freshly distilled out THF was added under argon followed by a solution of triphosgene (0.234 g, 0.79 mmol) in dry THF (2 ml). Then the reaction mixture was stirred at 50 º C for 1.5h under argon. The solution was evaporated to dryness under reduced pressure to get yellowish oil. The crude product was purified by silica gel column chromatography with a gradient of freshly distilled 95% ethyl acetate in dry hexanes. Collect 12 different fractions (10 ml) from column and analyzed by TLC. Fractions containing pure NCA were combined and removal solvent under reduced pressure to give diethylphosphate-l-cysteine-nca 4b as colourless oil (68% yield). Diethylphosphate-L-Cysteine-NCA can be obtained as a solid by precipitation according to the following procedure. The oily product obtained from column was dissolved in 5 ml of dry CHCl 3 and precipitated into 100 ml of dry hexane to get a white solid product. 1 H NMR (200.13 MHz, CDCl 3 ): δ 1.31-1.38 (m, 6H), 1.90-2.02 (m, 2H), 2.63-3.14 (m, 4H), 4.03-4.22 (m, 6H), 4.53-4.58 (m, 1H), 8.12 (br, 1H); 13 C NMR (50.23 MHz, CDCl 3 ): δ 16.17 (2C), 29.04, 29.93, 33.53, 58.41, 64.51 (2C), 65.71, 152.00, 169.09; FT-IR (CHCl 3 ) 1785 cm - 1 and 1860 cm -1 υ C (unsymmetrical stretching). Figure 1. FT-IR Spectra of diethylphosphate-l-cysteine-n-carboxyanhydride in CHCl 3, shows two unsymmetrical infrared stretching at 1860 and 1785 cm -1. S6

Synthesis of diethylphosphonate-l-cysteine-n-carboxyanhydride (5b): NH S 5b P Diethylphosphonate-L-Cysteine-N-Carboxyanhydride 5b was prepared from 5a according to the procedure for 4b and recovered as colourless oil (65% yields). 1 H NMR (200.13 MHz, CDCl 3 ): δ 1.32-1.38 (m, 6H), 1.82-2.05 (m, 4H), 2.67-3.15 (m, 4H), 4.01-4.26 (m, 4H), 4.52-4.57 (m, 1H), 8.27 (br, 1H); 13 C NMR (50.23 MHz, CDCl 3 ): δ 16.47 (2C), 22.00, 24.81, 32.88 (2C), 58.68, 62.19 (2C), 151.98, 169.11; FT-IR (CHCl 3 ) 1785 cm -1 and 1860 cm -1 υ C (unsymmetrical stretching). Synthesis of poly-diethylphosphate-l-cysteine (4c, 4d and 4e): P HN R S H NH n= 25, 40 4c, 4d R= Propargyl R= N 3 -(CH 2 CH 2 ) 6 4e To a solution of diethylphosphate-l-cysteine NCA 4b in dry dioxane or DMF (100 mg/ml) was added with propargyl amine or azido-peg-nh 2 (0.5 M) as initiator inside the glove box. The reaction was stirred at room temperature and the reaction generally completed within 36 to 72 hrs. The progress of the reaction was monitored by FT-IR spectroscopy by comparing with the intensity of the initial NCA s anhydride stretching at 1785 cm -1 and 1860 cm -1. Aliquots were removed after completion of the reaction for GPC analysis. Reaction were removed from the glove box and precipitated into diethyl ether. Solids were collected by centrifugation and washed with ph 2.0 water (HCl) followed by DI water. The polymers were lypholized to yield white solids (87-92% yield). S7

Polymer 4c. 1 H NMR (400.13 MHz, CDCl 3 ): δ 1.31-1.35 (m, 6H), 1.94-1.95 (m, 2H), 2.25 (br, 1H for alkyne proton in initiator), 2.57-2.74 (m, 2H), 2.93-3.29 (m, 2H), 4.1-4.17 (m, 7H); 31 P NMR (400.13 MHz, CDCl 3 ): δ -0.52 Polymer 4d. 1 H NMR (400.13 MHz, CDCl 3 ): δ 1.31-1.34 (m, 6H), 1.93-1.95 (m, 2H), 2.24 (br, 1H for alkyne proton in initiator), 2.57-3.29 (m, 4H), 4.08-4.15 (m, 7H); 31 P NMR (400.13 MHz, CDCl 3 ): δ -0.55 Synthesis of poly-diethylphosphonate-l-cysteine (5c, 5d and 5e): P S H NH HN R n= 25, 40 5c, 5d R= Propargyl R= N 3 -(CH 2 CH 2 ) 6 5e poly-diethylphosphonate-l-cysteine (5c, 5d and 5e) were prepared by according the procedure for 4c and recovered as white solid (87-92% yield). Polymer 5c. 1 H NMR (400.13 MHz, CDCl 3 ): δ 1.28-1.31 (m, 6H), 1.85 (br, 4H), 2.25 (br, 1H for alkyne proton in initiator), 2.51-3.27 (m, 4H), 4.05-4.12 (m, 5H); 31 P NMR (400.13 MHz, CDCl 3 ): δ 31.91 Polymer 5d. 1 H NMR (400.13 MHz, CDCl 3 ): δ 1.29-1.32 (m, 6H), 1.86 (br, 4H), 2.25 (br, 1H for alkyne proton in initiator), 2.52-3.30 (m, 4H), 4.06-4.13 (m, 5H); 31 P NMR (400.13 MHz, CDCl 3 ): δ 31.96 Polymer 5e. 1 H NMR (400.13 MHz, CDCl 3 ): δ 1.30-1.33 (m, 6H), 1.87 (br, 4H), 2.65-3.28 (m, 4H), 3.62-3.65 (m, for -CH 2 CH 2 unit in initiator), 4.08 (br, 5H) S8

Figure 2. 31 P NMR shows that the broadening of phosphorous peak in phosphonate polypeptide. Black one represents 31 P NMR of N-Boc-L-cysteine-diethylphosphonate and red one for poly-diethylphosphonate-l-cysteine. Deprotection of poly-diethylphosphonate-l-cysteine (5c and 5d): The deprotections were performing by in situ generation of iodotrimethylsilane. For example, polymer 5c (100 mg, 0.0127 mmol) was dissolved in dry acetonitrile (5 ml) and sodium iodide (533.6 mg, 3.56 mmol) and trimethylsilyl chloride (386.7 mg, 3.56 mmol) were added sequentially. Then the reaction mixture was stirred at 45 º C under N 2 atm for 24 hrs. The reaction mixture was evaporated to dryness to get a deep brown residue. The residue was redissolve in MeH and dialyzed (using dialysis tubing MWC of 2 KDa) against MeH for 24 hrs to remove all the organic impurities. Then it dialyzed against DI water for another 48 hrs, with water changes at least 5 times. Dialyzed polymer was lyophilized to get 5f as white solid (~85% yield). S9

Polymer 5f. 1 H NMR (400.13 MHz, D 2 ): δ 1.66-1.78 (br, 4H), 2.18 (br, 1H, for alkyne proton in initiator), 2.63 (br, 2H), 2.92-3.00 (br, 2H), 4.55 (br, 1H); 31 P NMR (400.13 MHz, D 2 ): δ 25.30 Polymer 5g. 1 H NMR (400.13 MHz, D 2 ): δ 1.80-1.94 (br, 4H), 2.17 (br, 1H, for alkyne proton in initiator), 2.66 (br, 2H), 2.94-3.02 (br, 2H), 4.58 (br, 2H); 31 P NMR (400.13 MHz, D 2 ): δ 25.37 Deprotection of poly-diethylphosphate-l-cysteine (4c and 4d): Polymers 4c and 4d were deprotected by following the procedure of 5c at 45 0 C and at room temperature. 25_CYS_PHSPHATE_DEPRTECTED_1H.ESP DEUTERIUM XIDE 4.61 4.60 3.91 3.89 3.31 3.30 3.17 3.14 3.02 2.98 2.72 2.68 2.65 2.01 1.88 1.80 1.23 4.75 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 3. 1 H NMR spectra (CDCl 3 ) of deprotected 25-cysteine-Phosphate 4c. S10

25_CYS_PHSPHATE_DEPRTECTED_31P.ESP 1.21 0.90 14 12 10 8 6 4 2 0-2 -4-6 -8-10 -12-14 Figure 4. 31 P NMR spectra (D 2 ) of deprotected 25-cysteine-Phosphate 4d showing two phosphorus peaks. Calculation of molecular weight by end group analysis The alkyne terminated phosphopolypeptide 5d was reacted with excess amount of benzyl azide (5 equivalent) in presence of CuS 4, 5H 2 (5 equivalent) and sodium ascorbate (5 equivalent) in a solvent mixture THF: MeH: H 2 (2: 2: 0.1). The reaction was left for 24 hr under argon atmosphere. Then solvent was removed under reduced pressure and residue was redissolved in dichloromethane. It was then washed multiple times using dilute aqueous ammonia solution to remove copper salt. The dichloromethane was removed and the residue was re-dissolved in MeH. The resultant polymer re-precipitated for couple of times by addition of diethyl ether to the methanolic solution. The precipitated white polymer dried thoroughly and then went for NMR analysis (Figure 4). S11

40_Cysteine_Phosphonate_Clicked with BenZ_Azide.esp 7.27 7.43 4.09 Acetone 3.38 3.32 3.27 3.19 3.16 3.12 3.02 2.99 2.93 2.86 2.81 2.71 1.90 1.32 5.00 253.96 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 5: Calculation of polymer molecular weight (M n ) by using the characteristic proton peak of the aromatic moiety at 7.43 ppm in the 40-diethylphosphonate-L- Cysteine after clicked. Synthesis of fluorescein labelled Phosphonate-polypeptide: The alkyne labelled fluorescein was prepared according to literature report (Bioconjugate Chem. 2005, 16, 1536). To a solution of 10 mg (0.0013 mmol) of 5e in THF: MeH: H 2 (2: 0.5: 0.25) was added alkyne fluorecein (2.0 mg, 3 eq), CuS 4 (0.17 mg, 0.50 eq) and sodium ascorbate (1.0 eq) under nitrogen and the reaction mixture was stirred for 24 hrs. The completion of the reaction was monitored by the near dissaperance (more than 90-95%) of the azide stretching by FT-IR. Then, the solvent was removed under reduced pressure and the reaction mixture was dissolved in DCM. It was then washed multiple times using dilute aqueous ammonia solution to remove copper (I) salt and excess fluorecein alkyne. The dichloromethane was removed and the residue was re-dissolved in MeH. The resultant polymer re-precipitated for couple of times by addition of diethyl ether to the methanolic S12

solution. Fluorecein labelled polymer was thoroughly dried (6 mg) and its absorption spectra taken in UV-vis spectrophotometer. Figure 6. FT-IR spectra for azide functionalized polymer 5e (black) and the crude reaction mixture upon completion of the click reaction (red). Method for estimation of azide concentration into 5e: The fluorecein moiety was incorporated into 5e using Cu (I) catalyzed azide alkyne click chemistry. The concentration of the fluorescein labelled polymer 5e was calculated using the Mn value of 7,357 kda that was obtained from NMR. Since only one fluorescein moiety will be conjugated to the polymer if all the polymer chains have one azide group attached to its end, the concentration of fluoresceinin solution would be equal to the concentration of the polymer. The concentration of fluorescein in solutions of 5e was estimated from its absorption spectra (λ max = 500 nm, ε= 90,000 M -1 cm -1 ) in MeH. The percentage of azide group incorporated was estimated from the ratio of the experimentally calculated concentration from absorption spectra of fluorescein to the theoretical concentration calculated from Mn values of 5e. S13

Figure 7. UV-VIS spectra of the fluorescein labelled polymer 5e solution (6.6 µm) in MeH. Figure 8. Size exclusion chromatogram of synthesized polymers (A) 40-diethylphosphate-L- Cysteine, (B) 25-diethylphosphate-L-Cysteine, (C) 40-diethylphosphonate-L-Cysteine, (D) 25-diethylphosphonate-L-Cysteine initiated by propargyl amine in DMF and (E) 25- diethylphosphonate-l-cysteine (F) 15-diethylphosphonate-L-Cysteine initiated by azido- PEG-NH 2 in dioxane. 1 H, 13 C, DEPT and 31 P NMR Spectra of monomers and polymers S14

Figure 9: 1 H NMR of N-Boc-L-Cystine (DMS-d6) NHBC_CYSTINE_1H.ESP 1.37 HC NHBoc S S CH NHBoc DMS-d6 4.22 4.20 4.17 4.16 4.13 4.11 4.08 3.16 3.14 3.09 3.07 2.92 2.87 2.85 2.80 2.50 2.15 2.21 2.10 18.00 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 10: 13 C NMR of N-Boc-L-Cystine (DMS-d6) NHBC_CYSTINE_13CESP.ESP DMS-d6 HC NHBoc S S CH 39.51 172.50 155.43 78.33 52.74 NHBoc 28.21 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S15

Figure 11: DEPT of N-Boc-L-Cystine (DMS-d6) NHBC_CYSTINE_DEPTESP.ESP 28.68 HC NHBoc S S CH NHBoc 53.21 39.84 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 12: 13 C NMR of N-Boc-L-Cysteine (DMS-d6) NHBC_CYSTEINE_13C.ESP DMS-d6 172.09 155.37 78.27 56.10 28.14 39.51 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S16

Figure 13: DEPT of N-Boc-L-Cysteine (DMS-d6) NHBC_CYSTEINE_DEPT.ESP 28.13 56.09 39.30 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 14: 1 H NMR of allyl diethylphosphate (CDCl 3 ) ALKENE PHSPHATE_1H.ESP 1.23 1.22 7.27 CHLRFRM-d 5.88 5.85 5.82 5.79 5.77 5.74 5.30 5.29 5.21 5.16 5.16 5.11 4.46 4.45 4.42 4.42 4.41 4.38 4.05 4.01 3.93 3.97 1.26 1.26 1.19 1.19 1.00 2.03 1.96 4.07 6.13 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S17

Figure 15: 13 C NMR of allyl diethylphosphate (CDCl 3 ) ALKENE PHSPHATE_13C.ESP CHLRFRM-d 133.24 68.41 118.61 64.41 16.74 77.00 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 16: DEPT of allyl diethylphosphate (CDCl 3 ) ALKENE PHSPHATE_DEPT.ESP 132.66 16.18 67.96 63.75 118.06 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S18

Figure 17: 1 H NMR of allyl diethylphosphonate (CDCl 3 ) C_ALKENE_PHSPHNATE_1H.ESP 1.17 7.27 CHLRFRM-d 5.74 5.69 5.66 5.62 5.57 5.12 5.11 5.10 5.09 5.07 5.04 5.03 5.02 4.03 4.00 3.96 3.92 3.89 2.54 2.50 2.43 2.39 1.21 1.14 0.94 1.95 4.04 2.00 6.11 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 18: 13 C NMR of allyl diethylphosphonate (CDCl 3 ) C_ALKENE_PHSPHNATE_13C.ESP CHLRFRM-d 127.05 119.33 77.00 15.94 31.95 61.38 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S19

Figure 19: DEPT of allyl diethylphosphonate (CDCl 3 ) C_ALKENE_PHSPHNATE_DEPT.ESP 16.45 127.45 32.46 119.85 61.95 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 20: 1 H NMR of N-Boc-L-cysteine-diethylphosphate (CDCl 3 ) NHBC_CYSTEINE_PHSPHATE_1H.ESP 1.35 7.27 CHLRFRM-d 4.08 4.04 4.48 4.45 4.41 4.39 4.11 4.00 3.97 3.02 3.00 2.95 2.92 2.82 2.61 2.58 2.54 1.91 1.88 1.85 1.82 1.78 1.25 AcH 1.22 0.81 6.00 2.19 1.91 1.98 9.00 6.18 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S20

Figure 21: 13 C NMR of N-Boc-L-cysteine-diethylphosphate (CDCl 3 ) NHBC_CYSTEINE_PHSPHATE_13C.ESP 28.07 AcH 173.61 155.22 79.98 77.00 CHLRFRM-d 65.90 64.06 52.99 34.11 29.82 28.28 15.93 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 22: DEPT of N-Boc-L-cysteine-diethylphosphate (CDCl 3 ) NHBC_CYSTEINE_PHSPHATE_DEPT.ESP 53.14 16.10 AcH AcH 28.23 28.43 29.96 34.25 66.17 64.34 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S21

Figure 23: 31 P NMR of N-Boc-L-cysteine-diethylphosphate (CDCl 3 ) NHBC_CYSTEINE_PHSPHATE_31P.ESP -1.51 50 45 40 35 30 25 20 15 10 5 0-5 -10-15 -20-25 -30-35 -40 Figure 24: 1 H NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl 3 ) NHBC_CYSTEINE_PHSPHNATE_1H.ESP 2.02 1.39 7.27 CHLRFRM-d 4.49 4.45 4.43 4.13 4.10 4.06 4.02 3.99 3.03 2.98 2.96 2.94 2.93 2.85 2.61 2.58 2.55 1.85 1.80 1.78 1.74 1.23 AcH 1.27 0.75 3.91 2.08 1.84 3.96 9.01 6.06 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S22

Figure 25: 13 C NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl 3 ) NHBC_CYSTEINE_PHSPHNATE_13C.ESP 28.14 CHLRFRM-d 173.37 155.25 79.94 77.00 62.01 53.08 34.06 25.27 22.46 21.98 16.14 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 26: DEPT of N-Boc-L-cysteine-diethylphosphonate (CDCl 3 ) NHBC_CYSTEINE_PHSPHNATE_DEPT.ESP 53.20 28.26 AcH 16.39 AcH AcH 22.17 22.56 25.37 34.17 62.19 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S23

Figure 27: 31 P NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl 3 ) NHBC_CYSTEINE_PHSPHNATE_31P.ESP 33.24 56 48 40 32 24 16 8 0-8 -16-24 -32-40 Figure 28: 1 H NMR of NH 2 -L-cysteine-diethylphosphate (DMS-d6) CYSTEINE_PHSPHATE_DEPRTECTED_1H.ESP 1.23 8.64 4.12 4.08 4.04 4.01 4.00 3.93 3.96 THF 3.07 3.04 2.64 2.60 2.50 THF DMS-d6 1.92 1.90 1.89 1.86 1.82 1.26 1.19 2.59 0.92 6.12 1.89 1.91 2.04 6.00 9 8 7 6 5 4 3 2 1 0 S24

Figure 29: 13 C NMR of NH 2 -L-cysteine-diethylphosphate (DMS-d6) CYSTEINE_PHSPHATE_DEPRTECTED_13C.ESP DMS-d6 39.51 169.17 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 66.64 62.83 Figure 30: DEPT of NH 2 -L-cysteine-diethylphosphate (DMS-d6) CYSTEINE_PHSPHATE_DEPRTECTED_DEPT.ESP 66.98 51.44 51.77 THF THF 30.68 27.27 24.74 31.01 25.10 15.67 15.89 27.60 63.30 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S25

Figure 31: 1 H NMR of NH 2 -L-cysteine-diethylphosphonate (DMS-d6) CYSTEINE_PHSPHNATE_DEPRTECTED_1H.ESP 1.21 8.66 3.97 4.10 4.04 4.00 3.93 3.89 1.25 1.18 3.05 3.02 2.68 2.64 2.61 2.50 DMS-d6 1.90 1.86 1.84 1.82 2.72 0.94 3.96 1.81 1.88 2.10 6.00 9 8 7 6 5 4 3 2 1 0 Figure 32: 13 C NMR of NH 2 -L-cysteine-diethylphosphonate (DMS-d6) CYSTEINE_PHSPHNATE_DEPRTECTED_13C.ESP DMS-d6 169.49 61.07 51.93 THF 30.87 25.16 22.25 16.38 39.51 THF THF 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S26

Figure 33: DEPT of NH 2 -L-cysteine-diethylphosphonate (DMS-d6) CYSTEINE_PHSPHNATE_DEPRTECTED_DEPT.ESP 16.21 51.85 22.18 25.09 30.79 60.88 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 34: 1 H NMR of diethylphosphate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHATE_NCA_1H.ESP 4.19 1.34 4.07 8.12 7.27 4.14 4.11 CHLRFRM-d 4.58 4.55 4.53 1.31 1.38 THF 4.03 3.14 3.12 3.07 3.05 2.95 2.92 2.73 2.70 2.67 2.63 2.02 1.99 1.96 1.90 1.93 0.92 0.96 6.05 3.99 1.96 6.11 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S27

Figure 35: 13 C NMR of diethylphosphate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHATE_NCA_13C.ESP CHLRFRM-d 169.09 152.00 65.71 64.51 58.41 33.53 29.04 16.17 77.00 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 36: DEPT of diethylphosphate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHATE_NCA_DEPT.ESP 58.49 16.24 30.09 65.67 64.47 29.11 33.58 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S28

Figure 37: 1 H NMR of diethylphosphonate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHNATE_NCA_1H.ESP 1.35 8.27 7.27 CHLRFRM-d 4.57 4.54 4.52 4.19 4.17 4.15 4.13 4.07 4.10 4.22 4.03 4.01 3.07 3.06 2.95 2.92 3.15 3.13 2.88 2.85 2.82 2.76 2.73 1.96 1.94 1.85 1.90 1.82 1.87 1.32 1.38 0.81 0.93 4.10 4.09 4.03 6.01 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 38: 13 C NMR of diethylphosphonate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHNATE_NCA_13C.ESP CHLRFRM-d 169.11 151.98 62.19 58.68 32.88 24.81 22.00 16.47 77.00 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 S29

Figure 39: DEPT of diethylphosphonate-l-cysteine-n-carboxyanhydride (CDCl 3 ) CYSTEINE_PHSPHNATE_NCA_DEPT.ESP 32.88 62.23 24.83 22.02 16.39 58.72 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 40: 1 H NMR of 25-diethylphosphate-L-Cysteine (CDCl 3 ) 25_CYSTEINE_PHSPHATE_1H.ESP 1.33 7.27 CHLRFRM-d 4.17 4.15 4.12 4.11 4.10 4.10 3.29 3.27 3.12 3.10 3.09 3.02 2.70 2.68 2.67 2.65 2.63 2.61 2.57 1.95 1.94 1.35 1.31 173.84 52.13 57.00 1.00 49.83 154.82 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S30

Figure 41: 31 P NMR of 25-diethylphosphate-L-Cysteine (CDCl 3 ) 25_CYSTEINE_PHSPHATE_31P.ESP -0.52 48 40 32 24 16 8 0-8 -16-24 -32-40 -48 Figure 42: 1 H NMR of 40-diethylphosphate-L-Cysteine (CDCl 3 ) 40_CYSTEINE_PHSPHATE_1H.ESP 1.33 7.27 CHLRFRM-d 4.15 4.14 4.12 4.11 4.09 4.09 4.08 3.29 3.27 3.10 2.72 2.70 2.68 2.66 2.65 2.64 2.62 2.24 2.60 1.95 1.94 1.93 1.34 1.31 282.46 170.99 1.0080.10 247.26 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S31

Figure 43: 31 P NMR of 40-diethylphosphate-L-Cysteine (CDCl 3 ) 40_CYSTEINE_PHSPHATE_31P.ESP -0.55 48 40 32 24 16 8 0-8 -16-24 -32-40 -48 Figure 44: 1 H NMR of 25-diethylphosphonate-L-Cysteine (CDCl 3 ) 25_CYSTEINE_PHSPHNATE_1H.ESP 1.30 CHLRFRM-d 7.27 4.08 4.08 4.06 4.05 4.12 1.85 3.27 3.26 3.07 3.06 3.00 2.75 2.72 2.70 2.56 2.63 2.54 2.53 2.51 2.25 1.28 1.31 122.42 110.01 1.00 111.17 171.64 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S32

Figure 45: Calculation of polymer molecular weight (M n ) by using the characteristic proton peak of the initiator (alkyne C C-H) at 2.25 ppm in the 25-diethylphosphonate- L-Cysteine. 25_CYSTEINE_PHSPHNATE_1H.ESP 2.25 2.51 2.77 2.63 1.85 2.75 2.74 2.72 2.70 2.69 2.53 1.31 1.30 1.28 2.56 2.54 110.01 1.00 111.17 171.64 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 Figure 46: 31 P NMR of 25-diethylphosphonate-L-Cysteine (CDCl 3 ) 25_CYSTEINE_PHSPHNATE_31P.ESP 31.92 56 48 40 32 24 16 8 0-8 -16-24 -32-40 -48 S33

Figure 47: 1 H NMR of 40-diethylphosphonate-L-Cysteine (CDCl 3 ) 40_CYSTEINE_PHSPHNATE_1H.ESP 1.31 CHLRFRM-d 4.09 4.08 4.07 7.27 4.13 3.28 4.06 1.86 3.12 3.11 3.08 3.07 3.01 2.73 2.71 2.64 2.57 2.55 2.53 2.52 2.26 1.29 1.32 207.22 180.97 1.00 178.86 282.10 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 48: Calculation of polymer molecular weight (M n ) by using the characteristic proton peak of the initiator (alkyne C C-H) at 2.25 ppm in the 40-diethylphosphonate- L-Cysteine. 40_CYSTEINE_PHSPHNATE_1H.ESP 2.26 2.52 2.53 2.64 2.76 2.74 2.73 2.71 2.69 1.86 2.57 2.55 1.32 1.31 1.29 180.97 1.00 178.86 282.10 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 S34

Figure 49: 31 P NMR of 40-diethylphosphonate-L-Cysteine (CDCl 3 ) 40_CYSTEINE_PHSPHNATE_31P.ESP 31.96 56 48 40 32 24 16 8 0-8 -16-24 -32-40 -48 Figure 50: 1 H NMR of 25-diethylphosphonate-L-Cysteine initiated by N 3 PEG 6 NH 2 (CDCl 3 ) 25_CYSTEINE_PHSPHNATE_N3PEG_1H.ESP 1.31 P S H NH HN R n= 25 5e R= N 3 -(CH 2 CH 2 ) 6 CHLRFRM-d 7.27 3.62 3.28 3.65 3.65 3.64 3.12 3.07 3.01 2.81 2.65 1.87 4.09 4.08 1.33 1.30 115.59 22.00 104.49 101.11 155.88 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 S35

Figure 51: 1 H NMR of 25-phosphonate-L-Cysteine (D 2 ) 25_CYS_PHSPHNATE_DEPRTECTED_1H.ESP DEUTERIUM XIDE 4.55 3.00 2.92 2.63 2.18 1.78 1.66 4.75 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Figure 52: 31 P NMR of 25-phosphonate-L-Cysteine (D 2 ) 25_CYS_PHSPHNATE_DEPRTECTED_31P.ESP 25.30 80 70 60 50 40 30 20 10 0-10 -20-30 -40-50 -60-70 S36

Figure 53: 31 P NMR of 40-phosphonate-L-Cysteine (D 2 ) 40_CYS_PHSPHNATE_DEPRTECTED_31P.ESP 25.37 80 70 60 50 40 30 20 10 0-10 -20-30 -40-50 -60-70 S37