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Supplementary data
A new approach to Bioconjugates for proteins and peptides (“Pegylation”) utilising Living Radical
Polymerisation
François Lecolley, Lei Tao, Giuseppe Mantovani, Ian Durkin, Sylvie Lautru and David M. Haddleton*
Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom. Fax: +44 24 7652
4112; Tel: +44 24 7652 3256; E-mail: D.M.Haddleton@warwick.ac.uk
Materials. Copper(I) bromide (Aldrich, 98%) was purified according to the method of Keller and Wycoff [1].
N-(ethyl)-2-pyridylmethanimine was prepared as described earlier [2]. 2-bromoisobutyryl bromide (Aldrich,
98%) was stored under nitrogen. Triethylamine (TEA) (Fischer, 99%) was stored over potassium hydroxide
pellets. Dichloromethane (DCM) was distilled over CaH2 and store under dry nitrogen. All other reagents and
solvents were obtained at the highest purity available from Aldrich Chemical Company and used without
further purification unless stated.
Analytical methods. All reactions were carried out using standard Schlenk techniques under an inert
atmosphere of oxygen-free nitrogen, unless otherwise stated. Rf values refer to analytical TLC performed using
pre-coated silica gel 60 F254 and developed in the solvent system indicated. Compounds were visualized by
use of UV light (254 nm) or a basic solution (10% K2CO3 in water) of KMnO4. Merck 60 (230 – 400 Mesh)
silica gel was used for column chromatography. Monomer conversions were determined by NMR by
monitoring the disappearance of the peaks corresponding to the vinyllic protons ( = 6.2 ppm and 5.6 ppm)
using the peak related to the C(O)OCH2 ( ~ 3.9 ppm) as a reference. The latter corresponds to both the
monomer and the polymer signals and therefore remains constant during the polymerisation. Mn(NMR) were
calculated by comparing the peak related to the succinimidyl ring ( = 2.7 ppm) to the peak corresponding to
the methyl ether ( = 3.2 ppm). NMR spectra were obtained on a Bruker DPX300 spectrometer. All chemical
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shifts are reported in ppm () relative to tetramethylsilane, referenced to the chemical shifts of residual solvent
resonances (1H and
13
C). The following abbreviations were used to explain the multiplicities: s=singlet,
d=doublet, q=quartet. Molecular mass distributions were determined by Size Exclusion Chromatography (SEC)
in a system fitted with a 5 mm guard column, two Polymer Labs mixed E columns, a differential refractive
index detector, and an auto sampler. The system was eluted with tetrahydrofuran at a rate of 1 ml/min. Toluene
(0.2% vol) was used as the flow marker. Poly(MMA) standards (106 – 200 g.mol-1) were used to calibrate the
SEC. The Mn reported in the Mn versus conversion plots are obtained from SEC data calibrated with
Poly(MMA) standards and are uncorrected. Those plots are reported to show that Mn increases linearly with the
conversion. Infrared absorption spectra were recorded on a Bruker VECTOR-22 FTIR spectrometer using a
Golden Gate diamond attenuated total reflection cell. Melting points are uncorrected. The yields are not
optimized. The HPLC system was fitted with a 5 mm guard column, a BioSep-SEC-S3000 column and a UV
detector continuously measuring the relative absorbance of the mobile phase at 215 nm. The system was eluted
with 0.1% v/v trifluoroacetic acid solution in water and acetonitrile (69/31 v/v) at a rate of 0.5 ml/min. SDSPAGE was conducted using a stacking polyacrylamide gel of 5% crosslinking and a resolving polyacrylamide
gel of 15% cross-linking and a running buffer consisting of 25 mM of TRIS base, 250 mM of glycine and 0.1%
of SDS at pH 8.7.
Preparation of initiator 1
N-hydroxysuccinimide-2-bromopropionate (1)
O
O
N
O
Br
O
1
N-Hydroxysuccinimide (4.51 g, 39.2 mmol) and 2-bromopropionic acid (2.90 ml, 32.7 mmol) were dissolved,
under dinitrogen atmosphere, in anhydrous DCM (1000 ml) in a 2000 ml round-bottomed flask, equipped with
a magnetic stirrer. The flask was cooled to 0°C and a solution of N,N'-Dicyclohexylcarbodiimide (6.70 g, 32.68
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mmol) in DCM (50 ml) was added dropwise. After stirring at room temperature overnight the reaction mixture
was filtered and the solvent removed under reduced pressure to give a yellow solid that was purified by flash
chromatography (CC, SiO2, Et2O, Rf (1) = 0.31). Obtained 7.20 g (28.9 mmol, 74%) of (1) as a white solid.
Melting point: 69-70°C.
1
H NMR (CDCl3)  (ppm) 1.96 (d, 3H, CH(CH3)Br, J = 6.78 Hz), 2.86 (s, 4H, Hcycl), 4.61 (q, 1H, CH(CH3)Br,
J= 7.03 Hz).
13
C{1H} NMR (CDCl3)  (ppm) 21.67 (1C, CH(CH3)Br) 25.74 (2C, Ccycl), 34.97 (1C,
CH(CH3)Br), 166.17 (1C, C=O), 168.69 (2C, Ccycl=O). IR (solid, ATR cell)  (cm-1) 1808, 1781 (Ccycl=O),
1729 (C=O). Mass spectroscopy (+EI, m/z) 248. Elem. Anal. Theoretical for C 7H8NO4Br: C, 33.62; H, 3.22; N,
5.60. Found: C, 33.47; H, 3.16; N, 5.46.
Preparation of initiator 2
N-hydroxysuccinimide-2-bromo-2-methylpropionate (2)
O
O
N
O
Br
O
2
N-Hydroxysuccinimide (11.86 g, 100.0 mmol) and triethylamine (28.1 ml, 200 mmol) were dissolved in DCM
(2000ml), under dinitrogen atmosphere, in a 2500 ml round-bottomed flask equipped with a magnetic stirrer.
The flask was cooled to 0°C and 2-bromo-2-methylpropionyl bromide (13.9 ml, 110 mmol) was added
dropwise. The mixture was stirred for 45 min at 0°C and allowed to reach room temperature. After 2 hours, the
reaction mixture was poured into an excess of cold water and extracted with diethyl ether (3 x 50 ml). The
organic layer was subsequently washed with a saturated aqueous solution of sodium carbonate (3 x 50 ml),
diluted HCl aqueous solution (pH= 4.5, 3 x 50 ml), and again with saturated aqueous solution of sodium
carbonate (3 x 50 ml). The organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent
removed under reduced pressure to afford (2) (22.7 g, 138 mmol, 86%) as a white solid. Melting point 72-74°C.
1
H NMR (CDCl3)  (ppm) 2.08 (s, 6H, C(CH3)2Br), 2.87 (s, 4H, Hcycl). 13C{1H} NMR (CDCl3)  (ppm) 26.03
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(2C, Ccycl), 31.09 (2C, C(CH3)2Br), 51.60 (1C, C(CH3)2Br), 167.89 (1C, C=O), 169.02 (2C, Ccycl=O). IR (solid,
ATR cell)  (cm-1) 1803, 1772 (Ccycl=O), 1728 (C=O). Mass spectroscopy (+EI, m/z) 266. Elem. Anal.
Theoretical for C8H10NO4Br: C, 36.39; H, 3.82; N, 5.30; Br, 30.26. Found: C, 36.35; H, 3.82; N, 5.03; Br, 30.17.
Preparation of polymer 3
Polymerisation procedures
O
O
O
O
N
Br
O
O
Cu(I)Br / toluene
O
+
N
O
Br
O
O m
N
n
O
m~9
O
O
O
m
N
Two polymerisations were carried out, each containing a different ratio monomer / initiator. In a typical
polymerization, a dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), N-succinimidyl 2bromopropionate (1) (0.569 g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling
between nitrogen and vacuum three times. To a second Schlenk tube was added PEGMA (10 ml, 22.74 x 10 -3
mol), N-(ethyl)-2-pyridylmethanimine (0.64 ml, 4.54 mmol) and toluene (10 ml). The mixture was immediately
subjected to five freeze-pump-thaw degassing cycles. This solution was then cannulated to the Schlenk tube
containing the initiator and Cu(I)Br. The resulting brown solution was stirred at 30°C (t=0). Samples were
removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular
weight analysis. Samples for molecular weight analysis were prepared by passing over an acidic alumina
column using THF as eluent to remove the copper complexes. Before analysis, the liquid was filtered with a
0.22 m hydrophobic filter.
NOTE: the molecular weights of the polymers are substantially higher than expected. This could be ascribed to
an initiating efficiency of about 40-55 % under our experimental conditions. The initiating efficiency seemed to
be related to the intrinsic nature of the monomer / initiator system and did not change remarkably by varying
parameters such as solvent, temperature or catalyst concentration. An analogous behaviour had been observed
when PEG-containing initiators were employed in the polymerization of MMA.3
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Table1. Kinetic data for the polymerisation of PEGMA initiated by 1 in toluene solution (50% v/v) at 30°C
Experiment
(A)
Initiator
Efficiency
41%
(B)
Initiator
Efficiency
55%
([PEGMA]0/[CuBr]0/[(1)]0/[ligand]0 = 5 (A) or 10 (B) /1/1/2).
Time
Conversion
ln([M]0/[M])
Mn, theo
Mn, SEC
(h)
(%)
(g.mol-1)
(g.mol-1)
1
10.8
0.115
280
2480
2
19.9
0.221
520
2980
3
25.8
0.298
680
2820
4
27.7
0.520
730
2930
5
47.6
0.645
1250
3990
6
57.7
0.860
1510
4010
7
74.8
1.251
1960
4400
8
82.8
1.760
2170
5170
kp[Pol*] = 0.144 h-1
2
4.5
0.046
230
3040
4
17.3
0.190
870
3710
6
30.2
0.359
1510
4480
8
40.2
0.514
2010
4950
10
49.3
0.679
2470
5530
21
92.3
2.564
4620
7980
kp[Pol*] = 0.083 h-1
Mw/Mn
1.05
1.05
1.13
1.15
1.17
1.18
1.19
1.12
1.07
1.30
1.13
1.11
1.10
1.09
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3.0
o
[M]:[I] 5:1 @ 30 C
[M]:[I] 10:1
2.5
ln ([M]0/[M])
2.0
1.5
1.0
0.5
0.0
0
5
10
15
20
25
time / h
Figure 1. Kinetic plot for the LRP of PEGMA initiated by (1),
1.3
1.2
1.1
1.0
8000
Mw / Mn
[PEGMA]0/[CuBr]0/[(1)]0/[L]0 = 5 or 10/1/1/2 in toluene (50% v/v) at 30°C.
o
[M]:[I] 5:1 @ 30 C
[M]:[I] 10:1
7000
Mn / g.mol
-1
6000
5000
4000
3000
2000
1000
0
0
10
20
30
40
50
60
70
80
90
100
Conversion / %
Figure 2. Evolution of the molecular weight distribution and polydispersity for the LRP of PEGMA initiated by (1),
[PEGMA]0/[CuBr]0/[(1)]0/[L]0 = 5 or 10/1/1/2 in toluene (50% v/v) at 30°C.
Ha
Ha
Ha
Ha
O
O
N
Br
O
n
Hc
Hb
O
O
O
O
Hb
O p
Hc
Hc
2.6595
3.1997
3.9105
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Hc
Hb
Ha
4.2
4.1
4.0
3.9
3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
3.0
2.9
2.8
2.7
2.6
(ppm)
Figure 3. Selected region (2.7-4.3 ppm) of the 1H NMR spectrum of polymer (3) (Mn = 6400 g.mol-1, Mw/Mn = 1.09).
Preparation of polymer 4
Polymerisation procedure
O
O
O
O
N
Br
O
O
Cu(I)Br / toluene
O
+
N
O
Br
O
O m
N
m~9
n
O
O
O
O
m
N
In a typical polymerization, a dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), Nsuccinimidyl 2-bromo-2-methylpropionate (2) (0.601 g, 2.27 mmol) and a magnetic follower prior to being
deoxygenated by cycling between nitrogen and vacuum three times. To a second Schlenk tube was added
PEGMA (10 ml, 22.74 mmol), N-(ethyl)-2-pyridylmethanimine (0.64 ml, 4.54 mmol) or N-(n-propyl)-2pyridylmethanimine (0.71 ml, 4.54 mmol) and toluene or anisole (10 ml). The mixture was immediately
subjected to five freeze-pump-thaw degassing cycles. This solution was then cannulated to the Schlenk tube
containing the initiator and Cu(I)B. The resulting brown solution was stirred at 30°C (t=0). Samples were
removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular
weight analysis. Samples for molecular weight analysis were prepared by passing over an acidic alumina
column using THF as eluent to remove the copper complexes. Before analysis, the liquid was filtered with a
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0.22m hydrophobic filter.
Table 2. Kinetic data for the polymerisation of PEGMA initiated by 1 in toluene or anisole solution (50% v/v)
at 30°C ([PEGMA]0/[CuBr]0/[(2)]0/[ligand]0 = 10/1/1/2).
Solvent /
Ligand
Toluene
Propyl ligand
Time
(h)
1
2
3
4
17
Anisole
Ethyl ligand
2
4
6
22
Anisole
Propyl ligand
2
4
6
27
Conversion ln([M]0/[M])
(%)
8.9
0.0933
18.4
0.204
27.1
0.316
34.7
0.4259
80.8
1.6510
kp[Pol*] = 0.096 h-1
17.0
0.1861
26.8
0.3116
34.8
0.4277
81.4
1.6809
kp[Pol*] = 0.076 h-1
9.8
0.1030
16.8
0.1837
28.7
0.3378
83.4
1.7985
kp[Pol*] = 0.069 h-1
Mn, theo
Mn, SEC
(g.mol-1) (g.mol-1)
450
2350
920
2860
1360
3100
1740
3600
4050
5670
Mw/Mn
1.10
1.26
1.20
1.13
1.06
850
1340
1740
4080
2670
3460
4260
6350
1.11
1.12
1.11
1.06
490
842
1440
4180
2070
2480
2870
6280
1.10
1.12
1.13
1.06
1.8
1.6
1.4
ln([M]0/[M])
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
8
10
12
14
16
18
time / h
Figure 4. First order kinetic plot for the LRP of PEGMA initiated by (2),
[PEGMA]0/[CuBr]0/[(2)]0/[Propyl Ligand]0 = 10/1/1/2 in toluene (50% v/v) at 30°C.
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1.4
1.3
1.1
Mw/Mn
1.2
1.0
7000
6000
Mn / g.mol
-1
5000
4000
3000
2000
1000
0
0
10
20
30
40
50
60
70
80
90
100
Conversion / %
Figure 5. Evolution of the molecular weight distribution and polydispersity for the LRP of PEGMA initiated by
(2), [PEGMA]0/[CuBr]0/[(2)]0/[ethyl Ligand]0 = 10/1/1/2 in toluene at 30°C.
2.0
Ethyl ligand
Propyl ligand
1.8
1.6
1.4
ln([M]0/[M])
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
5
10
15
20
25
30
time / h
Figure 6. First order kinetic plot for the LRP of PEGMA initiated by (2),
[PEGMA]0/[CuBr]0/[(2)]0/[L]0 = 10/1/1/2 in anisole (50% v/v) at 30°C.
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1.2
Mw/Mn
1.1
1.0
7000
Ethyl ligand
Propyl ligand
6000
Mn / g.mol
-1
5000
4000
3000
2000
1000
0
0
10
20
30
40
50
60
70
80
90
100
Conversion / %
Figure 7. Evolution of the molecular weight distribution and polydispersity for the LRP of PEGMA initiated by
(2), [PEGMA]0/[CuBr]0/[(2)]/[L]0 = 10/1/1/2 in anisole (33% v/v) at 30°C.
Table 3. Characterisation of Poly(PEGMA) prepared by LRP
Kp[Pol*]a
Mn, theo b
Mn, exp c
Mw/Mn
-1
-1
-1
(h )
(g.mol )
(g.mol )
Toluene / Propyl ligand
0.096
4000
6200
1.05
Anisole / Ethyl ligand
0.076
4100
6400
1.06
Anisole / Ethyl ligand
0.069
4200
6300
1.06
a
b
Kp[Pol*] = rate constant of propagation. Mn, theo = ([M]0 / [I]0 x M.W.mPEGMA x Conv.)
/ 100 + M.W.(2) . c determined by SEC calibrated with Poly(MMA) standards – THF
(stabilised with topanol).
Experiment
Hydrolytic stability of N-succinimidyl terminated Poly(PEGMA) 3
A set of two on line 1H NMR experiments was carried out, each using a different buffer. Polymer 3 (Mn= 6400
g/mol, PDI= 1.09) (50 mg, 7.81 mol) were introduced in an NMR tube and dissolved in 0.5 ml of the
appropriate phosphate buffer (pH = 8, C=100 mM or 200 mM). NMR spectra were recorded regularly at regular
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intervals of time.
60
Conversion / %
50
40
30
20
10
100 mM phosphate buffer (pH = 8)
200 mM phosphate buffer (pH = 8)
0
0
5
10
15
20
25
time / h
Figure 8. Kinetic plot for the hydrolysis of polymer (3) at different buffer concetrations.
Hydrolytic stability of N-succinimidyl terminated Poly(PEGMA) 4
A set of five on line 1H NMR experiments was carried out, each using a different buffer. N-succinimidyl
terminated Poly(PEGMA) 4 (Mn= 2700 g/mol, PDI= 1.12) (50 mg, 18.5 mol) were introduced in an NMR
tube and dissolved in 0.5 ml of the appropriate buffer (200 mM phosphate buffer (pH = 6 and pH = 8), 100 mM
phosphate buffer (pH = 8) or 200 mM borate buffer (pH = 9.2)). NMR spectra were recorded at regular
intervals of time.
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200 mM Borate (pH=9.2)
200 mM Phosphate (pH=8)
100 mM Phosphate (pH=8)
200 mM Phosphate (pH=6)
100
Conversion / %
80
60
40
20
0
0
50
100
150
200
250
300
350
400
450
500
550
Time / h
Figure 9. Kinetic plot for the hydrolysis of polymer (4) at different buffer concetrations.
Coupling reactions with lysozyme using polymer 3
General Procedure
Polymer (3) (Mn = 6400 g/mol, PDI= 1.11) (89.5 mg, 14.0 mol) and lysozyme (10 mg, 0.699 mol) was
dissolved in 10 ml of anhydrous DMSO and 0.5 ml of anhydrous TEA and stirred at room temperature under
nitrogen. Samples were taken periodically and analyzed by HPLC. The crude product was also analysed by
SDS-PAGE.
Lysozyme
Polymer
t=1h
t=2h
t=4h
t=6h
16
17
18
19
Retention Time / min
20
21
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Figure 10. HPLC traces for the reaction of polymer (3) (Mn=6400 g.mol-1, Mw/Mn =1.11) with Lysozyme
([polymer] / [lysozyme] 20:1)
Figure 11. SDS-PAGE for the conjugation of lysozyme with polymer (3) (Mn=6400 g.mol-1, Mw/Mn =1.11)
(20 equivalents).
Coupling reactions with lysozyme using polymers 4.
A set of three experiments was carried out, each containing a different ratio polymer / lysozyme. Each set of
experiments was left to react for either 4 hours or 20 hours. Polymer (4) (Mn = 2700 g/mol, PDI= 1.12) (41.6
mg, 15.4 mol) for a ratio 5/1, (83.2 mg, 30.8 mol) for a ratio 10/1 and (249.5 mg, 92.4 mol) for a ratio 30/1
and lysozyme (50 mg, 3.08 mol) was dissolved in 10 ml of 200 mM phosphate buffer (pH = 8) and stirred at
4°C hours under nitrogen. The reaction was followed by HPLC in the case of a ratio polymer / lysozyme 30/1.
In each case, the crude was purified in dialysis bag (Spectra/Por1, MWCO = 6-8000 g/mol) and analysed by
SDS-PAGE.
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Lysozyme
t=1h
t = 20 h
16
17
18
19
20
21
Retention Time / min
Figure 12. HPLC traces for the reaction of (4) (Mn=2700 g.mol-1, Mw/Mn =1.12) with lysozyme ([polymer] /
[lysozyme] 30:1)
Reaction time: 4 hours
(a)
(b)
(c)
lysozyme
Reaction time: 20 hours
(a)
(b)
(c)
lysozyme
Figure 13. SDS-PAGE for the conjugation of Poly(PEGMA)NHS with lysozyme at different reaction time and
different ratio polymer / protein (a) 5/1, (b) 10/1 and (c) 30/1.
References
1
Cu(I)Br was purified as necessary according to a method by Keller and Wycoff: R. N. Keller and H. D.
Wycoff, Inorg. Synth., 1947, 2, 1.
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2
Prepared by condensation of ethylamine with 2-pyridinecarboxaldehyde as decribed earlier: D. M.
Haddleton, M. C. Crossman, B. H. Dana, D. J. Duncalf, A. M. Heming, D. Kukulj and A. J. Shooter,
Macromolecules, 1999, 32, 2110.
3
S. Perrier and D. M. Haddleton, submitted to Eur. Polym. J..