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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 13C). 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. SDS-

PAGE 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)

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.

1H 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). 13C{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 C7H8NO4Br: 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)

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.

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1H NMR (CDCl3) (ppm) 2.08 (s, 6H, C(CH3)2Br), 2.87 (s, 4H, Hcycl). 13C{1H} NMR (CDCl3) (ppm) 26.03

(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

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 2-

bromopropionate (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

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when PEG-containing initiators were employed in the polymerization of MMA.3

Table1. Kinetic data for the polymerisation of PEGMA initiated by 1 in toluene solution (50% v/v) at 30°C

([PEGMA]0/[CuBr]0/[(1)]0/[ligand]0 = 5 (A) or 10 (B) /1/1/2).

Experiment Time

(h)

Conversion

(%)

ln([M]0/[M]) Mn, theo

(g.mol-1)

Mn, SEC

(g.mol-1)

Mw/Mn

(A) 1 10.8 0.115 280 2480 1.05

Initiator 2 19.9 0.221 520 2980 1.05

Efficiency 3 25.8 0.298 680 2820 1.13

41% 4 27.7 0.520 730 2930 1.15

5 47.6 0.645 1250 3990 1.17

6 57.7 0.860 1510 4010 1.18

7 74.8 1.251 1960 4400 1.19

8 82.8 1.760 2170 5170 1.12

kp[Pol*] = 0.144 h-1

(B) 2 4.5 0.046 230 3040 1.07

Initiator 4 17.3 0.190 870 3710 1.30

Efficiency 6 30.2 0.359 1510 4480 1.13

55% 8 40.2 0.514 2010 4950 1.11

10 49.3 0.679 2470 5530 1.10

21 92.3 2.564 4620 7980 1.09

kp[Pol*] = 0.083 h-1

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Figure 1. Kinetic plot 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.

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.

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3.9105

3.1997

2.6595

(ppm)2.62.72.82.93.03.13.23.33.43.53.63.73.83.94.04.14.2

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

In a typical polymerization, a dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), N-

succinimidyl 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)-2-

pyridylmethanimine (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

HaHb

Hc

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column using THF as eluent to remove the copper complexes. Before analysis, the liquid was filtered with a

0.22m 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

Time(h)

Conversion(%)

ln([M]0/[M]) Mn, theo

(g.mol-1)Mn, SEC

(g.mol-1)Mw/Mn

Toluene 1 8.9 0.0933 450 2350 1.10Propyl ligand 2 18.4 0.204 920 2860 1.26

3 27.1 0.316 1360 3100 1.204 34.7 0.4259 1740 3600 1.1317 80.8 1.6510 4050 5670 1.06

kp[Pol*] = 0.096 h-1

Anisole 2 17.0 0.1861 850 2670 1.11Ethyl ligand 4 26.8 0.3116 1340 3460 1.12

6 34.8 0.4277 1740 4260 1.1122 81.4 1.6809 4080 6350 1.06

kp[Pol*] = 0.076 h-1

Anisole 2 9.8 0.1030 490 2070 1.10Propyl ligand 4 16.8 0.1837 842 2480 1.12

6 28.7 0.3378 1440 2870 1.1327 83.4 1.7985 4180 6280 1.06

kp[Pol*] = 0.069 h-1

Figure 4. First order kinetic plot for the LRP of PEGMA initiated by (2),

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[PEGMA]0/[CuBr]0/[(2)]0/[Propyl Ligand]0 = 10/1/1/2 in toluene (50% v/v) at 30°C.

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.

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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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

Experiment Kp[Pol*]a

(h-1)Mn, theo

b

(g.mol-1)Mn, exp

c

(g.mol-1)Mw/Mn

Toluene / Propyl ligand 0.096 4000 6200 1.05Anisole / Ethyl ligand 0.076 4100 6400 1.06Anisole / Ethyl ligand 0.069 4200 6300 1.06

a Kp[Pol*] = rate constant of propagation. b 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).

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

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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

intervals of time.

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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Figure 9. Kinetic plot for the hydrolysis of polymer (4) at different buffer concetrations.

Coupling reactions with lysozyme using polymer 3General 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.

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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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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 Reaction time: 20 hours

(a) (b) (c) (a) (b) (c)

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.

lysozyme lysozyme

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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..