A new approach to bioconjugates for proteins and peptides ( pegylation ) utilising living radical polymerisation

Article abstract of DOI:10.1039/b407712a

The synthesis of protein-polymer bioconjugates is reported using N-succinimidyl ester functionalised polymers from transition metal mediated living radical polymerisation.

Full text of DOI:10.1039/b407712a

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A new approach to bioconjugates for proteins and peptides
‘‘pegylation’’) utilising living radical polymerisation{
(
Fran c¸ ois Lecolley, Lei Tao, Giuseppe Mantovani, Ian Durkin, Sylvie Lautru and David M. Haddleton*
Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: D.M.Haddleton@warwick.ac.uk; Fax: 144 24 7652 4112; Tel: 144 24 7652 3256
Received (in Cambridge, UK) 24th May 2004, Accepted 5th July 2004
First published as an Advance Article on the web 28th July 2004
The synthesis of protein–polymer bioconjugates is reported
using N-succinimidyl ester functionalised polymers from
transition metal mediated living radical polymerisation.
good yield by coupling of 2-bromo propionate and N-hydroxy-
succimide in the presence of DCC. Living radical polymerisation of
mPEGMA was carried out at 30 uC using Cu(I)Br/N-(ethyl)-2-
8
pyridylmethanimine as catalyst to give mono-functionalised
polymers with structure 3 and 4. Catalyst residues were removed
by ultrafiltration to yield white products.{ The presence of the
Protein bioconjugation has been an area of increasing interest since
the pioneering work by Abuchowski et al. Bioconjugation is the
1
1
covalent binding of a protein to a synthetic or natural polymer
chain to form a new macromolecule. Bioconjugation offers the
opportunity to substantially alter the pharmacokinetics and
pharmacodynamic properties of protein therapeutics. Poly(ethylene
glycol methyl ether) (mPEG), a non-toxic polymer, has been
N-succinimidyl ester moiety at the chain ends was confirmed by H
NMR analysis as a singlet at 2.8 ppm. The synthesis of pure mono-
functionalised polymers with zero contamination from a, v
disubstituted polymer is a significant asset of this synthetic route.
A disadvantage of conventional linear PEGylating reagents is the
presence of di-functionalised byproducts inherent to the ring-
opening polymerisation. These can result in unwanted crosslinking
between proteins during the conjugation. Gel permeation chro-
matography (GPC) showed that the molecular weight distribution
2
extensively used for conjugation, ‘‘pegylation’’. In most cases,
suitable functionalisation of the polymer is the first step in the con-
jugation. This process is the ‘‘activation’’ step and the conjugation
group is chosen to match the available groups on proteins. A
3
common route for protein conjugation is the reaction of an
appropriate terminally-functionalised polymer with the e-amino
group of lysine residues and the N-terminal amino group present in
the polypeptides. A well developed method of activation is the
transformation of the a-terminus of polymers with succinimidyl
esters, which leads to the formation of stable amido linkages with
was very narrow (M
w
/M
n
v 1.15) for all of the materials obtained.
This is essential as a polydisperse polymer reflects on the poly-
9
dispersity of the protein–polymer conjugates.
The reactivity of N-succinimidyl ester function in polymers 3 and
1
was evaluated by monitoring the rate of hydrolysis by online H
4
NMR experiments, whereby the reaction was carried out in an
NMR tube within the cavity of the spectrometer (Fig. 1) in various
deuterium oxide buffers. It was found that the hydrolytic stability
of the activated polymers was strongly dependent on the pH of the
medium. At mild acidic pH (pH ~ 6), the cleavage of the
succinimde group was almost negligible, whilst, as expected, under
basic conditions the hydrolysis of the functionalised polymers was
very fast (at pH ~ 9, t1/2(4) ~ 0.2 h). The hydrolytic stability
experiments also demonstrated that polymers initiated by 1 were
much more reactive towards water, and hence towards nucleophiles
such as amines, than polymers initiated by 2. This agrees with
4
proteins (Scheme 1). It is noted that while the chemistry for the
conjugation of polymers to proteins has been widely developed,
hitherto few polymer types have been used with mPEG remaining
popular due to its non-hazardous and non-immunogenic pro-
perties, as well as its ability to solubilise biological molecules.
Nevertheless a few examples of conjugation have been reported
using polystyrene as the synthetic polymer to form amphiphilic
10
5
block copolymers, or super amphiphiles. In this report, we
described the synthesis of N-succinimidyl ester functionalised
initiators and their subsequent use in the transition metal mediated
living radical polymerization (TMM-LRP) of poly(ethylene glycol)
methyl ether methacrylate (mPEGMA). Living radical polymerisa-
tion provides an excellent method for the polymerisation of a
diverse range of vinyl monomers (acrylates, methacrylates and
the observations of Harris and coworkers for a-branched
N-succinimidyl esters-mPEG. Although this difference in
reactivity is often ascribed to a different steric hindrance on the
3,4
6
styrenes) in a controlled manner, with excellent tolerance towards
a variety of functional groups. Moreover this is a facile way to
incorporate an initiator site into a molecule leading to a wide range
of functionalised polymers.
7
The N-succinimidyl functional initiators were prepared as shown
in Scheme 2. N-Succinimidyl 2-bromo-2-methylpropionate 2 was
obtained in very high yield by acylation of N-hydroxysuccimide
with 2-bromopropionyl bromide, while an analogous synthetic
protocol applied to the synthesis of 1 yielded a quite complicated
mixture of products. Initiator 1 was subsequently prepared in very
Scheme 1 Coupling of proteins to N-succinimidyl ester functionalised
polymers.
Scheme 2 Reagents and conditions: (i) 2-Bromopropionic acid, dicyclo-
2 2
hexylcarbodiimide, CH Cl ; (ii) 2-bromo-2-methylpropionyl bromide,
{
Electronic supplementary information (ESI) available: Experimental
triethylamine, CH Cl
2
poly(ethylene glycol) methyl ether methacrylate, toluene 50% v/v;
[PEGMA]:[initiator]:[Cu(I)Br]:[ligand] ~ 5:1:1:2.
2
; (iii) Cu(I)Br, N-(ethyl)-2-pyridylmethanimine,
procedures on prepared compounds and characterisation. See http://
www.rsc.org/suppdata/cc/b4/b407712a/
2
0 2 6
C h e m . C o m m u n . , 2 0 0 4 , 2 0 2 6 – 2 0 2 7
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Fig. 3 SDS-PAGE for the conjugation of lysozyme with 3. (a) Protein
standards. (b) Lysozyme. (c) Protein–polymer bioconjugate.
polypeptides and other hydrophilic monomers polymerisable under
TMM-LRP conditions (acrylates, methacrylates and styrenes).
The authors would like to thank the University of Warwick (FL
and TL) and the Marie Curie Fellowship Network (GM MEIF-
CT-2003–501305 and SL) for funding, they also thank Dr Adam
Clarke for his precious help with the online H NMR experiments,
Dr Greg Challis and Warwick Effect Polymers Ltd. for useful
discussions.
Fig. 1 Kinetic plots for the hydrolysis of various Poly(mPEGMA)-NHS at
different pH and buffer ionic strength.
1
Notes and references
1
(a) A. Abuchowski, T. Van Es, N. C. Palczuk and F. F. Davis, J. Biol.
Chem., 1977, 252, 3578; (b) A. Abuchowski, J. R. McCoy, N. C. Palzuk,
T. Van Es and F. F. Davis, J. Biol. Chem., 1977, 252, 3582.
R. B. Greenwald, Y. H. Choe, J. McGuire and C. D. Conover, Adv.
Drug Deliv. Rev., 2003, 55, 217.
(a) S. Zalipsky, Bioconjugate Chem., 1995, 6, 150; (b) M. J. Roberts,
M. D. Bentley and J. M. Harris, Adv. Drug Deliv. Rev., 2002, 54, 459.
J. M. Harris and A. Kozlowski, US 5672662, 1997.
(a) J. M. Hannink, J. J. L. M. Cornelissen, J. A. Farrera, P. Foubert,
F. C. De Schryver, N. A. J. M. Sommerdijk and R. J. M. Nolte, Angew.
Chem., Int. Ed. Engl., 2001, 40, 4732; (b) M. J. Boerakker, J. M. Hannink,
P. H. H. Bomans, P. M. Frederik, R. J. M. Nolte, E. M. Meijer and
N. A. J. M. Sommerdijk, Angew. Chem., Int. Ed. Engl., 2002, 41, 4239;
2
3
4
5
(
c) M. L. Becker, J. Liu and K. L. Wooley, Chem. Commun., 2003, 180.
(a) M. Kamigaito, A. Tsuyoshi and M. Sawamoto, Chem. Rev., 2001,
01, 3689; (b) K. Matyjaszewski and J. Xia, Chem. Rev., 2001, 101, 2921.
6
1
Fig. 2 SEC HPLC traces following the reaction of N-succinimidyl esters
functionalised Poly(mPEGMA) (M ~ 6400 g mol , M /M ~ 1.11)
n w n
with lysozyme.
21
7 (a) D. M. Haddleton, C. Waterson, P. J. Derrick, C. B. Jasieczek and
A. J. Shooter, Chem. Commun., 1997, 683; (b) D. M. Haddleton,
A. M. Heming, D. Kukulj, D. J. Duncalf and A. J. Shooter,
Macromolecules, 1998, 31, 5201; (c) A. Marsh, A. Kahn,
D. M. Haddleton and M. J. Hannon, Macromolecules, 1999, 32,
8725; (d) A. Marsh, A. Kahn, M. Garcia and D. M. Haddleton, Chem.
Commun., 2000, 2083.
electrophilic centre, electronic contributions provided by the
a-substituents cannot be ruled out.
Lysozyme was used as a model protein for conjugation experi-
1
1
8
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,
ments to both polymers in anhydrous DMSO in the presence of
triethylamine. Size-exclusion HPLC was used to monitor the
evolution of the conjugate (Fig. 2). In the case of polymer 4, no
reaction was observed after 24 h. Further analysis by Sodium
Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
confirmed the absence of any conjugates. For polymer 3, the
reaction was fast and the HPLC trace showed the complete
disappearance of the lysozyme after 6 h. SDS-PAGE analysis
confirmed the absence of the lysozyme starting material and
revealed that an approximate number of 6 to 7 polymeric chains
Macromolecules, 1999, 32, 2110; 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.
9 F. M. Veronese, Biomaterials, 2001, 22, 405.
10 (a) G. W. Cline and S. B. Hanna, J. Am. Chem. Soc., 1987, 109, 3087;
(b) G. W. Cline and S. B. Hanna, J. Org. Chem., 1988, 53, 3583.
1
1 Low molecular weight succinimidyl ester terminated poly(mPEGMA)
prepared from initiator 1 (M
21
n w n
~ 6400 g mol , M /M ~ 1.11)
(89.5 mg, 14 mmol) and lysozyme (10 mg, 0.7 mmol) were dissolved in
1
2
were conjugated (Fig. 3). It is noted that lysozyme has 6 lysine
groups in addition to the terminal amine.
10 ml of anhydrous DMSO and 0.5 ml of anhydrous TEA and stirred at
ambient temperature under nitrogen. Samples were taken periodically
and analyzed by SEC-HPLC. The HPLC system was fitted with a 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
In summary, transition metal mediated living radical polymerisa-
tion has been successfully employed for the synthesis of two
different types of N-succinimidyl ester functionalised poly-
(mPEGMA) with different reactivities depending on the type of
2
1
acetonitrile (69/31 v/v) at a rate of 0.5 ml min . The crude product was
analysed by SDS-PAGE (polyacrylamide resolving gel cross-linking:
initiator used. This versatile synthetic approach provides a novel
route to protein–synthetic polymer bioconjugates using living
radical polymerization. This strategy has been applied using
lysozyme as a model protein and poly(mPEGMA) as the synthetic
polymer, it looks to be very general and will be extended to other
15%, running buffer: 25 mM TRIS base, 250 mM glycine, 0.1% SDS,
pH ~ 8.7).
12 M. O. Glocker, C. Borcher, W. Fiedler, D. Suckau and M. Przybylski,
Bioconjugate Chem., 1994, 5, 583.
C h e m . C o m m u n . , 2 0 0 4 , 2 0 2 6 – 2 0 2 7
2 0 2 7