Design and Synthesis of N-Maleimido-Functionalized Polymers
A R T I C L E S
an estimated business worldwide of $4.6 billion, opening the
way for other inhaled protein therapeutics.8
pH and below, this functionality is reported to react ap-
proximately 1000 times faster with thiols than with amines.
There has been a preliminary report on the use of a modified
pyridyl sulfide with 2-hydroxy ethyl methacrylate which allowed
direct conjugation of the polymer to cysteine residues of bovine
serum albumin.16
Early work in PEGylation concentrated on lysine-specific
conjugation, usually leading to statistical multisite attachment.
However, the use of a single site attachment leading to one
polymer conjugate per protein has obvious advantages. This can
be achieved via the reaction of free cysteine residues in proteins,
which is an excellent approach for site-specific modification.
Where necessary, free cysteine units can be introduced to
appropriate positions of polypeptide surfaces either by reduction
of disulfide bridges or by genetic engineering modification.5 In
this latter case, PEGylation of the cysteine muteins can afford
homogeneously modified mono-PEGylated species that can be
readily purified and retain high biological activity. The terminal
reactive chain-ends used for these purposes include maleimide,
vinyl sulfone, iodoacetamide, and orthopyridyl disulfide units.3
The macromolecular structure of the polymer conjugate has
also proven to be crucial for the properties of the corresponding
bioconjugates. Previous reports indicate, for example, that the
use of branched PEG in place of a linear chain can, in some
cases, improve a number of properties, such as resistance to
proteolysis, to the action of antibodies and resulted in a lower
immunogenicity, due to the so-called “umbrella-like” shape of
these polymers.9,10 The molecular weight and molecular weight
distribution (MWD) of the polymer conjugates are also impor-
tant parameters as the MWD of the polymers is reflected in the
polydispersity of the peptide-polymer conjugate.5 We envisaged
that the concept of PEGylation could be extended to many
functionalized hydrophilic water-soluble polymers obtainable
by controlled radical polymerization, featuring a narrow mo-
lecular weight distribution and containing appropriate R-terminal
reactivity.11-13 Transition-metal-mediated living radical polym-
erization (TMM-LRP), often called atom transfer radical po-
lymerization (ATRP), is a technique that allows a great control
over the molecular weight distribution and the architecture of
polymers. Moreover, it is very tolerant of a variety of functional
groups and protic solvents, including water.14,15 The extreme
versatility of this living polymerization process allows tailoring
of both the molecular weight and the macromolecular structure,
the latter, a factor that can also influence the selectivity of the
conjugation reaction. In the present work, we focused our
attention toward poly(methacrylates) containing a maleimide
terminus. The choice of this reactive chain-end is due to the
high reactivity and selectivity of the maleimide toward the
cysteine residues present at the protein surface. Indeed, at neutral
In the present work, we report the synthesis and characteriza-
tion of new R-maleimide-functional polymers and their conjuga-
tion with a model peptide and the carrier protein BSA.
Experimental Section
All reactions were carried out using standard Schlenk techniques
under an inert atmosphere of oxygen-free nitrogen, unless otherwise
stated. Copper(I) bromide (Aldrich, 98%) was purified according to
the method of Keller and Wycoff.17 N-(Ethyl)-2-pyridylmethanimine,
N-(n-propyl)-2-pyridylmethanimine,18 and (2,2-dimethyl-1,3-dioxolan-
4-yl)methyl methacrylate19 were prepared as described earlier and stored
at 0 °C under a dinitrogen atmosphere. All other reagents and solvents
were obtained at the highest purity available from Aldrich Chemical
Co. and used without further purification unless stated.
4,10-Dioxatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (1): Maleic an-
hydride (30.0 g, 306 mmol) was suspended in 150 mL of toluene and
the mixture warmed to 80 °C. Furan (33.4 mL, 459 mmol) was added
via syringe and the turbid solution stirred for 6 h. The mixture was
then cooled to ambient temperature and the stirring stopped. After 1 h,
the resulting white crystals were collected by filtration and washed
with 2 × 30 mL of petroleum ether. Obtained was 44.4 g (267 mmol,
87% yield) of 1 as small white needless. Mp 124-127 °C (dec). IR
(neat): ν˜ ) 1857, 1780, 1309, 1282, 1211, 1145, 1083, 1019, 947,
920, 902, 877, 847, 800, 732, 690, 674, 633, 575 cm-1 1H NMR
.
(400.03 MHz, CDCl3, 298 K): δ ) 3.17 (s, 2H, CH), 5.45 (t, J ) 1.0
Hz, 2H, CHO), 6.57 (t, J ) 1.0 Hz, 2H, CHvinyl). 13C{1H} NMR (100.59
MHz, CDCl3, 298 K): δ ) 48.85 (2C, CH), 82.35 (2H, CHO), 137.12
(2C, CHvinyl), 170.04 (2C, CO). Anal. Calcd for C8H6O4: C, 57.84; H,
3.64. Found: C, 57.74; H, 3.68. Mass spectrometry (+EI) m/z (%):
167 [MH+] (<1), 121 (7), 98 (22), 94 (13), 68 (100).
4-(2-Hydroxyethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-
dione (2): The anhydride 1 (2.00 g, 12.0 mmol) was suspended in
MeOH (50 mL) and the mixture cooled to 0 °C. A solution of
ethanolamine (0.72 mL, 12.0 mmol) in 20 mL of MeOH was added
dropwise (10 min), and the resulting solution was stirred for 5 min at
0 °C, then 30 min at ambient temperature, and finally refluxed for 4 h.
After cooling the mixture to ambient temperature, the solvent was
removed under reduced pressure, and the white residue was dissolved
in 150 mL of CH2Cl2 and washed with 3 × 100 mL of water. The
organic layer was dried over MgSO4 and filtered. Removal of the
solvent under reduced pressure furnished an off-white residue that was
purified by flash chromatography (CC, SiO2, 100% ethyl acetate, Rf
(2) ) 0.26) to give 2 (1.04 g, 5.00 mmol, 42% yield) as a white solid.
Mp 139-141 °C (dec). IR (neat): ν˜ ) 3472, 1681, 1435, 1405, 1335,
1269, 1168, 1100, 1053, 1013, 959, 916, 875, 850, 807, 722, 705, 654
cm-1. 1H NMR (400.03 MHz, CDCl3, 298 K): δ ) 1.90 (bs, 1H, OH),
2.90 (s, 2H, CH), 3.69-3.72 (m, 2H, NCH2), 3.76-3.78 (m, 2H,
OCH2), 5.28 (t, J ) 0.9 Hz, 2H, CH), 6.52 (t, J ) 0.9 Hz, 2H, CHvinyl).
13C{1H} NMR (100.59 MHz, CDCl3, 298 K): δ ) 41.77 (1C, NCH2),
47.50 (2C, CH), 60.18 (1C, OCH2), 81.04 (2C, CHO), 136.60 (2C,
CHvinyl), 176.97 (2C, CO). Anal. Calcd for C10H11NO4: C, 57.41; H,
5.30; N, 6.70. Found: C, 57.16; H, 5.37; N, 6.62. Mass spectrometry
(8) Powell, K. Nat. Biotechnol. 2004, 22, 1195-1196.
(9) Veronese, F. M.; Monfardini, C.; Caliceti, P.; Schiavon, O.; Scrawen, M.
D.; Beer, D. J. Controlled Release 1996, 40, 199-209.
(10) Veronese, F. M.; Caliceti, P.; Schiavon, O. J. Bioact. Compat. Polym. 1997,
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2004, 126, 13220-13221.
(13) Few examples of hybrid materials formed by (poly)peptides and polymers
other than PEG have been reported: (a) Shimoboji, T.; Ding, Z.; Stayton,
P. S.; Hoffman, A. S. Bioconjugate Chem. 2001, 12, 314-319. (b) Ranucci,
E.; Spagnoli, G.; Sartore, L.; Ferruti, P.; Caliceti, P.; Schiavon, O.; Veronese,
F. M. Macromol. Chem. Phys. 1994, 195, 3469-3479. (c) Caliceti, P.;
Schiavon, O.; Morpurgo, M.; Veronese, F. M.; Sartore, L.; Ranucci, E.;
Ferruti, P. J. Bioact. Compat. Polym. 1995, 10, 103-120. (d) Hannink, J.
M.; Cornelissen, J. J. L. M.; Farrera, J. A.; Foubert, P.; De Schryver, F.
C.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Angew. Chem., Int. Ed. 2001,
40, 4732-4734. (e) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. J. Am.
Chem. Soc. 2002, 124, 4224-4225.
(16) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem.
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(17) Keller, R. N.; Wycoff, H. D. Inorg. Synth. 1946, 1-4.
(18) Haddleton, D. M.; Crossman, M. C.; Dana, B. H.; Duncalf, D. J.; Heming,
A. M.; Kukulj, D.; Shooter, A. J. Macromolecules 1999, 32, 2110-2119.
(19) Perrier, S.; Armes, S. P.; Wang, X. S.; Malet, F.; Haddleton, D. M. J.
Polym. Sci., Part A: Polym. Chem. 2001, 39, 1696-1707.
(14) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. ReV. 2001, 101, 3689-
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(15) Matyjaszewski, K.; Xia, J. Chem. ReV. 2001, 101, 2921-2990.
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