S1
Supporting Information
Synthesis of End-functionalized Phosphate and Phosphonate- polypeptides by Ring-
Opening 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. 1H
NMR and 31P NMR spectrum was obtained with Bruker spectrometer (200.13 MHz, 400.13
MHz). 31P NMR shifts are reported in ppm relative to 85% H3PO4 at 0 ppm. 13C 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 VISKOTEK TDA 305-040 TRIPLE DETECTOR ARRAY
refractive index (RI), viscometer (VISC), low angle light scattering (LALS), right angle light
scattering (RALS) GPC/SEC MODULE. Separations were achieved by three columns
(T6000M, GENERAL MIXED ORG 300X7.8 MM) and one guard column (TGAURD, ORG
GUARD COL 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 (JASCO CD SPECTROPOLARIMETER, 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]=
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(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:
L-Cystine (10 g, 41.6 mmol) was dissolve in 9:1 water:tetrahydrofuran (100 mL). 6 M NaOH
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).
1H NMR (200.13 MHz, DMSO-d6): 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); 13C NMR (50.23 MHz, DMSO-d6): 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:
N-Boc-L-Cystine (10.0 g, 22.7 mmol) was dissolve in 200 mL of THF: H2O (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 NaOH. The aqueous
phase was extracted with ethyl acetate (4x100 mL) to remove excess PPh3 and PPh3O. Then 2
(N) HCl was added drop wise with stirring until the solution reached pH 2, with the
NHBoc
HOOCS
S
NHBoc
COOH
NHBoc
HOOCSH
1
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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 Na2SO4. The solution was filtered and condensed to give colourless oily N-
Boc-L-Cysteine 1 in 97% yields.
1H NMR (200.13 MHz, DMSO-d6): δ 1.43 (s, 9H), 2.46-2.54 (t, 1H), 2.66-2.95 (m, 2H),
4.01-4.15 (m, 1H); 13C NMR (50.23 MHz, DMSO-d6): δ 28.14 (3C), 39.30, 56.10, 78.27,
155.37, 172.09
Synthesis of allyl diethylphosphate (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 ºC 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).
1H NMR (200.13 MHz, CDCl3): 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); 13C NMR (50.23 MHz, CDCl3): δ 16.74 (2C), 64.41
(2C), 68.41, 118.61, 133.24
Synthesis of allyl diethylphosphonate (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 ºC.
Then it was distilled under reduced pressure at temperature 85º-90 ºC to obtained allyl
diethylphosphonate 3 as a colourless liquid (90% yield).
PO
O
O
O2
PO
O
O
3
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1H NMR (200.13 MHz, CDCl3): 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); 13C NMR (50.23 MHz, CDCl3): δ 16.26 (2C), 31.95,
61.38 (2C), 119.33, 127.05
Synthesis of N-Boc-L-cysteine-diethylphosphate (4):
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
Na2SO4, 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).
1H NMR (200.13 MHz, CDCl3): 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); 13C
NMR (50.23 MHz, CDCl3): δ 15.93 (2C), 28.07 (3C), 28.28, 29.82, 52.99, 64.06 (2C), 65.9,
79.88, 155.22, 173.61; 31P NMR (400.13 MHz, CDCl3): δ -1.51
Synthesis of N-Boc-L-cysteine-diethylphosphonate (5):
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).
1H NMR (200.13 MHz, CDCl3): 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); 13C
NHBoc
HOOCS O
P
O O
O
4
NHBoc
HOOCS P
O
O
O
5
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NMR (50.23 MHz, CDCl3): 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; 31P NMR (400.13 MHz, CDCl3): δ 33.24
General procedure for the removal of Boc group:
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).
1H NMR (200.13 MHz, DMSO-d6): 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); 13C
NMR (50.23 MHz, DMSO-d6): δ 15.67 (2C), 24.74 , 27.27, 30.68, 51.44, 62.83 (2C), 66.98,
169.17
5a was prepared from 5 according to the above procedure and recovered as oil (96% yield).
1H NMR (200.13 MHz, DMSO-d6): 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); 13C
NMR (50.23 MHz, DMSO-d6): 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):
NH2
HOOCS O
P
O O
O
4a
HOOCS P
O
O
O
5a
NH2
S OP
O O
O
NHO
O
O 4b
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To H2N-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 ºC 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 CHCl3 and precipitated into 100 mL of dry hexane to get a white solid product.
1H NMR (200.13 MHz, CDCl3): 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); 13C NMR (50.23 MHz, CDCl3): δ 16.17
(2C), 29.04, 29.93, 33.53, 58.41, 64.51 (2C), 65.71, 152.00, 169.09; FT-IR (CHCl3) 1785 cm-
1 and 1860 cm-1 υCO (unsymmetrical stretching).
Figure 1. FT-IR Spectra of diethylphosphate-L-Cysteine-N-Carboxyanhydride in CHCl3,
shows two unsymmetrical infrared stretching at 1860 and 1785 cm-1.
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Synthesis of diethylphosphonate-L-Cysteine-N-Carboxyanhydride (5b):
Diethylphosphonate-L-Cysteine-N-Carboxyanhydride 5b was prepared from 5a according to
the procedure for 4b and recovered as colourless oil (65% yields).
1H NMR (200.13 MHz, CDCl3): 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); 13C NMR (50.23 MHz, CDCl3): δ 16.47
(2C), 22.00, 24.81, 32.88 (2C), 58.68, 62.19 (2C), 151.98, 169.11; FT-IR (CHCl3) 1785 cm-1
and 1860 cm-1 υCO (unsymmetrical stretching).
Synthesis of poly-diethylphosphate-L-Cysteine (4c, 4d and 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-NH2 (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).
S PO
O
ONH
O
O
O
5b
S
NHH
O
HN R
O
PO
O
O
n= 25, 404c, 4d
R= Propargyl
4e
R= N3-(CH2CH2O)6
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Polymer 4c. 1H NMR (400.13 MHz, CDCl3): 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); 31P NMR (400.13 MHz, CDCl3): δ -0.52
Polymer 4d. 1H NMR (400.13 MHz, CDCl3): 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); 31P NMR
(400.13 MHz, CDCl3): δ -0.55
Synthesis of poly-diethylphosphonate-L-Cysteine (5c, 5d and 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. 1H NMR (400.13 MHz, CDCl3): 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); 31P NMR (400.13 MHz,
CDCl3): δ 31.91
Polymer 5d. 1H NMR (400.13 MHz, CDCl3): 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); 31P NMR (400.13 MHz,
CDCl3): δ 31.96
Polymer 5e. 1H NMR (400.13 MHz, CDCl3): δ 1.30-1.33 (m, 6H), 1.87 (br, 4H), 2.65-3.28
(m, 4H), 3.62-3.65 (m, for -CH2CH2O unit in initiator), 4.08 (br, 5H)
S
NHH
O
HN RP
O
O
O
n= 25, 405c, 5d
R= Propargyl
5e
R= N3-(CH2CH2O)6
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Figure 2. 31P NMR shows that the broadening of phosphorous peak in phosphonate
polypeptide. Black one represents 31P 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 ºC under N2 atm for 24 hrs. The
reaction mixture was evaporated to dryness to get a deep brown residue. The residue was re-
dissolve in MeOH and dialyzed (using dialysis tubing MWCO of 2 KDa) against MeOH 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).
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Polymer 5f. 1H NMR (400.13 MHz, D2O): 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); 31P NMR (400.13 MHz,
D2O): δ 25.30
Polymer 5g. 1H NMR (400.13 MHz, D2O): 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); 31P NMR (400.13 MHz,
D2O): 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 0C and at room
temperature.
25_CYS_PHOSPHATE_DEPROTECTED_1H.ESP
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
DEUTERIUM OXIDE
4.7
5
4.6
14
.60
3.9
13
.89
3.3
13
.30
3.1
73
.14 3.0
22
.98 2.7
22
.68
2.6
5
2.0
1
1.8
81
.80
1.2
3
Figure 3. 1H NMR spectra (CDCl3) of deprotected 25-cysteine-Phosphate 4c.
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25_CYS_PHOSPHATE_DEPROTECTED_31P.ESP
14 12 10 8 6 4 2 0 -2 -4 -6 -8 -10 -12 -14Chemical Shift (ppm)
1.2
10
.90
Figure 4. 31P NMR spectra (D2O) 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 CuSO4, 5H2O (5 equivalent) and sodium ascorbate (5
equivalent) in a solvent mixture THF: MeOH: H2O (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 MeOH. 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).
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40_Cysteine_Phosphonate_Clicked with BenZ_Azide.esp
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 0Chemical Shift (ppm)
253.965.00
7.43
7.27
4.09
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
Figure 5: Calculation of polymer molecular weight (Mn) 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: MeOH: H2O (2:
0.5: 0.25) was added alkyne fluorecein (2.0 mg, 3 eq), CuSO4 (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 MeOH. The resultant
polymer re-precipitated for couple of times by addition of diethyl ether to the methanolic
Ace
tone
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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 (λmax= 500 nm, ε= 90,000 M-1cm-1) in MeOH. 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.
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Figure 7. UV-VIS spectra of the fluorescein labelled polymer 5e solution (6.6 µM) in
MeOH.
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-NH2 in dioxane.
1H, 13C, DEPT and 31P NMR Spectra of monomers and polymers
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Figure 9: 1H NMR of N-Boc-L-Cystine (DMSO-d6)
NHBOC_CYSTINE_1H.ESP
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 0Chemical Shift (ppm)
18.002.102.212.15
DMSO-d6
4.2
24
.20
4.1
74
.16
4.1
34
.11
4.0
8
3.1
63
.14
3.0
93
.07
2.9
22
.87
2.8
52
.80 2.5
0
1.3
7
Figure 10: 13C NMR of N-Boc-L-Cystine (DMSO-d6)
NHBOC_CYSTINE_13CESP.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
DMSO-d6
17
2.5
0
15
5.4
3 78
.33
52
.74
39
.51
28
.21
NHBoc
HOOCS
S
NHBoc
COOH
NHBoc
HOOCS
S
NHBoc
COOH
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Figure 11: DEPT of N-Boc-L-Cystine (DMSO-d6)
NHBOC_CYSTINE_DEPTESP.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
53
.21
39
.84
28
.68
Figure 12: 13C NMR of N-Boc-L-Cysteine (DMSO-d6)
NHBOC_CYSTEINE_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
DMSO-d6
17
2.0
9
15
5.3
7 78
.27
56
.10
39
.51
28
.14
NHBoc
HOOCS
S
NHBoc
COOH
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Figure 13: DEPT of N-Boc-L-Cysteine (DMSO-d6)
NHBOC_CYSTEINE_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
56
.09
39
.30
28
.13
Figure 14: 1H NMR of allyl diethylphosphate (CDCl3)
ALKENE PHOSPHATE_1H.ESP
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 0Chemical Shift (ppm)
6.134.071.962.031.00
CHLOROFORM-d
7.2
7 5.8
85
.85
5.8
25
.79
5.7
75
.74
5.3
05
.29
5.2
15
.16
5.1
65
.11
4.4
6 4.4
54
.42
4.4
24
.41
4.3
84
.05
4.0
13
.97
3.9
3
1.2
6 1.2
61
.23 1
.22
1.1
9 1.1
9
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S18
Figure 15: 13C NMR of allyl diethylphosphate (CDCl3)
ALKENE PHOSPHATE_13C.ESP
170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
13
3.2
4
11
8.6
1
77
.00
68
.41
64
.41 1
6.7
4
Figure 16: DEPT of allyl diethylphosphate (CDCl3)
ALKENE PHOSPHATE_DEPT.ESP
170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
13
2.6
6
11
8.0
6
67
.96
63
.75
16
.18
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S19
Figure 17: 1H NMR of allyl diethylphosphonate (CDCl3)
C_ALKENE_PHOSPHONATE_1H.ESP
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 0Chemical Shift (ppm)
6.112.004.041.950.94
CHLOROFORM-d
7.2
7
5.7
45
.69
5.6
65
.62
5.5
75
.12
5.1
15
.10
5.0
95
.07 5.0
45
.03
5.0
2
4.0
34
.00
3.9
63
.92
3.8
9
2.5
42
.50
2.4
32
.39
1.2
11
.17
1.1
4
Figure 18: 13C NMR of allyl diethylphosphonate (CDCl3)
C_ALKENE_PHOSPHONATE_13C.ESP
160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
12
7.0
5 11
9.3
3
77
.00
61
.38
31
.95
15
.94
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S20
Figure 19: DEPT of allyl diethylphosphonate (CDCl3)
C_ALKENE_PHOSPHONATE_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
12
7.4
5
11
9.8
5
61
.95
32
.46
16
.45
Figure 20: 1H NMR of N-Boc-L-cysteine-diethylphosphate (CDCl3)
NHBOC_CYSTEINE_PHOSPHATE_1H.ESP
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 0Chemical Shift (ppm)
6.189.001.981.912.196.000.81
CHLOROFORM-d
7.2
7
4.4
84
.45
4.4
14
.39 4.1
14
.08
4.0
44
.00
3.9
7
3.0
23
.00
2.9
52
.92
2.8
22
.61 2
.58
2.5
4
1.9
1 1.8
81
.85
1.8
21
.78
1.3
51
.25
1.2
2
AcO
H
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S21
Figure 21: 13C NMR of N-Boc-L-cysteine-diethylphosphate (CDCl3)
NHBOC_CYSTEINE_PHOSPHATE_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
17
3.6
1
15
5.2
2
79
.98
77
.00
65
.90
64
.06
52
.99
34
.11
29
.82
28
.28
28
.07
15
.93
Figure 22: DEPT of N-Boc-L-cysteine-diethylphosphate (CDCl3)
NHBOC_CYSTEINE_PHOSPHATE_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
66
.17
64
.34
53
.14
34
.25
29
.96
28
.43
28
.23
16
.10
AcO
H
AcO
H
AcO
H
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S22
Figure 23: 31P NMR of N-Boc-L-cysteine-diethylphosphate (CDCl3)
NHBOC_CYSTEINE_PHOSPHATE_31P.ESP
50 45 40 35 30 25 20 15 10 5 0 -5 -10 -15 -20 -25 -30 -35 -40Chemical Shift (ppm)
-1.5
1
Figure 24: 1H NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl3)
NHBOC_CYSTEINE_PHOSPHONATE_1H.ESP
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 0Chemical Shift (ppm)
6.069.013.961.842.083.910.75
CHLOROFORM-d
7.2
7
4.4
94
.45
4.4
34
.13
4.1
0 4.0
64
.02
3.9
9
3.0
32
.98
2.9
62
.94
2.9
32
.85
2.6
1 2.5
82
.55
2.0
21
.85 1
.80
1.7
81
.74
1.3
91
.27
1.2
3
AcO
H
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S23
Figure 25: 13C NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl3)
NHBOC_CYSTEINE_PHOSPHONATE_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
17
3.3
7
15
5.2
5
79
.94
77
.00
62
.01
53
.08
34
.06
28
.14
25
.27
22
.46
21
.98
16
.14
Figure 26: DEPT of N-Boc-L-cysteine-diethylphosphonate (CDCl3)
NHBOC_CYSTEINE_PHOSPHONATE_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
62
.19
53
.20
34
.17
28
.26
25
.37
22
.56
22
.17
16
.39
AcO
H A
cOH
A
cOH
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S24
Figure 27: 31P NMR of N-Boc-L-cysteine-diethylphosphonate (CDCl3)
NHBOC_CYSTEINE_PHOSPHONATE_31P.ESP
56 48 40 32 24 16 8 0 -8 -16 -24 -32 -40Chemical Shift (ppm)
33
.24
Figure 28: 1H NMR of NH2-L-cysteine-diethylphosphate (DMSO-d6)
CYSTEINE_PHOSPHATE_DEPROTECTED_1H.ESP
9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
6.002.041.911.896.120.922.59
DMSO-d6
8.6
4
4.1
24
.08
4.0
44
.01
4.0
03
.96
3.9
3 3.0
73
.04 2
.64
2.6
02
.50
1.9
21
.90
1.8
9 1.8
61
.82
1.2
61
.23
1.1
9
TH
F
TH
F
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S25
Figure 29: 13C NMR of NH2-L-cysteine-diethylphosphate (DMSO-d6)
CYSTEINE_PHOSPHATE_DEPROTECTED_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
DMSO-d6
16
9.1
7
66
.64
62
.83
51
.44
39
.51
30
.68
27
.27
24
.74 15
.67
Figure 30: DEPT of NH2-L-cysteine-diethylphosphate (DMSO-d6)
CYSTEINE_PHOSPHATE_DEPROTECTED_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
66
.98
63
.30
51
.77
31
.01
27
.60
25
.10
15
.89
TH
F
TH
F
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S26
Figure 31: 1H NMR of NH2-L-cysteine-diethylphosphonate (DMSO-d6)
CYSTEINE_PHOSPHONATE_DEPROTECTED_1H.ESP
9 8 7 6 5 4 3 2 1 0Chemical Shift (ppm)
6.002.101.881.813.960.942.72
DMSO-d6
8.6
6
4.1
04
.04
4.0
03
.97
3.9
33
.89
3.0
53
.02
2.6
82
.64
2.6
12
.50
1.9
01
.86
1.8
41
.82
1.2
51
.21
1.1
8
Figure 32: 13C NMR of NH2-L-cysteine-diethylphosphonate (DMSO-d6)
CYSTEINE_PHOSPHONATE_DEPROTECTED_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
DMSO-d6
16
9.4
9
61
.07
51
.93
39
.51
30
.87
25
.16
22
.25
16
.38
TH
F
TH
F
TH
F
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S27
Figure 33: DEPT of NH2-L-cysteine-diethylphosphonate (DMSO-d6)
CYSTEINE_PHOSPHONATE_DEPROTECTED_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
60
.88
51
.85
30
.79
25
.09
22
.18
16
.21
Figure 34: 1H NMR of diethylphosphate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHATE_NCA_1H.ESP
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 0Chemical Shift (ppm)
6.111.963.996.050.960.92
CHLOROFORM-d
8.1
2
7.2
7 4.5
8 4.5
54
.53
4.1
94
.14
4.1
14
.07
4.0
3
3.1
43
.12 3
.07
3.0
52
.95
2.9
2 2.7
32
.70
2.6
72
.63
2.0
21
.99 1
.96
1.9
31
.90
1.3
81
.34
1.3
1
TH
F
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S28
Figure 35: 13C NMR of diethylphosphate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHATE_NCA_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
16
9.0
9
15
2.0
0
77
.00
65
.71
64
.51 5
8.4
1
33
.53
29
.04
16
.17
Figure 36: DEPT of diethylphosphate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHATE_NCA_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
65
.67
64
.47
58
.49
33
.58
30
.09
29
.11
16
.24
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S29
Figure 37: 1H NMR of diethylphosphonate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHONATE_NCA_1H.ESP
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 0Chemical Shift (ppm)
6.014.034.094.100.930.81
CHLOROFORM-d
8.2
7
7.2
7 4.5
7 4.5
44
.52
4.2
2
4.1
9 4.1
7 4.1
5 4.1
34
.10
4.0
74
.03
4.0
1
3.1
53
.13
3.0
73
.06
2.9
52
.92
2.8
8 2.8
52
.82
2.7
62
.73
1.9
6 1.9
41
.90
1.8
71
.85
1.8
21
.38
1.3
51
.32
Figure 38: 13C NMR of diethylphosphonate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHONATE_NCA_13C.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
CHLOROFORM-d
16
9.1
1
15
1.9
8
77
.00
62
.19
58
.68
32
.88
24
.81
22
.00
16
.47
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S30
Figure 39: DEPT of diethylphosphonate-L-Cysteine-N-Carboxyanhydride (CDCl3)
CYSTEINE_PHOSPHONATE_NCA_DEPT.ESP
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0Chemical Shift (ppm)
62
.23
58
.72
32
.88
24
.83
22
.02
16
.39
Figure 40: 1H NMR of 25-diethylphosphate-L-Cysteine (CDCl3)
25_CYSTEINE_PHOSPHATE_1H.ESP
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 0Chemical Shift (ppm)
154.8249.831.0057.0052.13173.84
CHLOROFORM-d
7.2
7
4.1
7 4.1
54
.12
4.1
14
.10
4.1
03
.29
3.2
73
.12
3.1
03
.09
3.0
22
.70
2.6
8 2.6
72
.65
2.6
32
.61
2.5
71
.95
1.9
4
1.3
51
.33
1.3
1
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S31
Figure 41: 31P NMR of 25-diethylphosphate-L-Cysteine (CDCl3)
25_CYSTEINE_PHOSPHATE_31P.ESP
48 40 32 24 16 8 0 -8 -16 -24 -32 -40 -48Chemical Shift (ppm)
-0.5
2
Figure 42: 1H NMR of 40-diethylphosphate-L-Cysteine (CDCl3)
40_CYSTEINE_PHOSPHATE_1H.ESP
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 0Chemical Shift (ppm)
247.2680.101.00170.99282.46
CHLOROFORM-d
7.2
7
4.1
54
.14
4.1
24
.11
4.0
94
.09
4.0
8
3.2
93
.27
3.1
02
.72
2.7
02
.68
2.6
62
.65
2.6
42
.62
2.6
02
.24
1.9
51
.94
1.9
3
1.3
41
.33
1.3
1
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S32
Figure 43: 31P NMR of 40-diethylphosphate-L-Cysteine (CDCl3)
40_CYSTEINE_PHOSPHATE_31P.ESP
48 40 32 24 16 8 0 -8 -16 -24 -32 -40 -48Chemical Shift (ppm)
-0.5
5
Figure 44: 1H NMR of 25-diethylphosphonate-L-Cysteine (CDCl3)
25_CYSTEINE_PHOSPHONATE_1H.ESP
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 0Chemical Shift (ppm)
171.64111.171.00110.01122.42
CHLOROFORM-d
7.2
7
4.1
2
4.0
84
.08
4.0
64
.05
3.2
73
.26
3.0
73
.06
3.0
02
.75
2.7
22
.70 2
.63
2.5
62
.54
2.5
32
.51
2.2
51
.85
1.3
11
.30
1.2
8
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S33
Figure 45: Calculation of polymer molecular weight (Mn) 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_PHOSPHONATE_1H.ESP
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 1.0Chemical Shift (ppm)
171.64111.171.00110.01
2.7
72
.75 2
.74
2.7
2 2.7
02
.69
2.6
3
2.5
62
.54
2.5
32
.51
2.2
5
1.8
5
1.3
11
.30
1.2
8
Figure 46: 31P NMR of 25-diethylphosphonate-L-Cysteine (CDCl3)
25_CYSTEINE_PHOSPHONATE_31P.ESP
56 48 40 32 24 16 8 0 -8 -16 -24 -32 -40 -48Chemical Shift (ppm)
31
.92
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S34
Figure 47: 1H NMR of 40-diethylphosphonate-L-Cysteine (CDCl3)
40_CYSTEINE_PHOSPHONATE_1H.ESP
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 0Chemical Shift (ppm)
282.10178.861.00180.97207.22
CHLOROFORM-d
7.2
7
4.1
3
4.0
94
.08 4.0
74
.06
3.2
8
3.1
23
.11
3.0
83
.07
3.0
12
.73
2.7
12
.64
2.5
72
.55
2.5
32
.52
2.2
61
.86
1.3
21
.31
1.2
9
Figure 48: Calculation of polymer molecular weight (Mn) 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_PHOSPHONATE_1H.ESP
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 1.1Chemical Shift (ppm)
282.10178.861.00180.97
2.7
6 2.7
42
.73 2.7
12
.69
2.6
4
2.5
72
.55
2.5
32
.52
2.2
6
1.8
6
1.3
21
.31
1.2
9
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S35
Figure 49: 31P NMR of 40-diethylphosphonate-L-Cysteine (CDCl3)
40_CYSTEINE_PHOSPHONATE_31P.ESP
56 48 40 32 24 16 8 0 -8 -16 -24 -32 -40 -48Chemical Shift (ppm)
31
.96
Figure 50: 1H NMR of 25-diethylphosphonate-L-Cysteine initiated by N3PEG6NH2
(CDCl3)
25_CYSTEINE_PHOSPHONATE_N3PEG_1H.ESP
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 0Chemical Shift (ppm)
155.88101.11104.4922.00115.59
CHLOROFORM-d
7.2
7
4.0
94
.08
3.6
53
.65
3.6
43
.62
3.2
8
3.1
23
.07
3.0
12
.81
2.6
5
1.8
7
1.3
31
.31
1.3
0
S
NHH
O
HN RP
O
O
O
n= 25
5e N3-(CH2CH2O)6R=
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S36
Figure 51: 1H NMR of 25-phosphonate-L-Cysteine (D2O)
25_CYS_PHOSPHONATE_DEPROTECTED_1H.ESP
4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)
DEUTERIUM OXIDE4
.75
4.5
5
3.0
02
.92 2
.63
2.1
8
1.7
8
1.6
6
Figure 52: 31P NMR of 25-phosphonate-L-Cysteine (D2O)
25_CYS_PHOSPHONATE_DEPROTECTED_31P.ESP
80 70 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 -60 -70Chemical Shift (ppm)
25
.30
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013
S37
Figure 53: 31P NMR of 40-phosphonate-L-Cysteine (D2O)
40_CYS_PHOSPHONATE_DEPROTECTED_31P.ESP
80 70 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 -60 -70Chemical Shift (ppm)
25
.37
Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013