A R T I C L E S
Heredia et al.
Preparation of polymer bioconjugates is generally achieved
by reaction of preformed semitelechelic polymers with specific
amino acid residues. Lysine side chains are frequently targeted
with amine-reactive end-groups such as activated esters, iso-
cyanates, or through reductive amination.19-21 However, a
protein may contain several internal lysine residues in addition
to the N-terminal amine. Therefore, this approach is often
nonspecific, and the resultant protein-polymer conjugate is
heterogeneous in the number and placement of the polymer
chains. Heterogeneity in the structure reflects on the biological
properties of the conjugate and often results in decreased protein
activity.22 Hence, creating well-defined adducts is important,
and site-specific modification of the protein is a better approach
to prepare such biomolecules. Site-specific modification of
proteins is also important for directed immobilization onto
chains on the protein can be precisely determined. In addition,
if a protein lacks free thiols for conjugation, genetic engineering
can incorporate cysteine residues in specific positions, for
example away from the active site, such that a polymer can be
1,33
attached without hindering protein activity. We have recently
demonstrated that atom transfer radical polymerization (ATRP)
can be used to prepare pyridyl disulfide semitelechelic poly(2-
hydroxyethyl methacrylate) that without any postpolymerization
modification conjugates to bovine serum albumin (BSA) via a
34
reversible disulfide bond. Maleimide end-functionalized poly-
(PEG methacrylate) has also been prepared by ATRP and
conjugated to BSA and to glutathione through covalent C-S
35
attachment of the polymer. Other reactive groups which are
36
often employed for thiol conjugation include vinyl sulfone and
37
iodoacetamide.
6
surfaces and ensures that biorecognition sites are accessible.
Each of these approaches to prepare protein-polymer con-
jugates attaches a preformed polymer to the protein. Most often
an excess of polymer is used, which must be removed from the
conjugate; this can be difficult to achieve if the polymer and
protein are similar in size. In addition, it can be difficult to
determine the number and location of the polymer chains in
the final conjugate, particularly when multiple attachment sites
are possible. Recently, we reported an alternative way to prepare
In addition, for “smart” polymer switches, placement of the
polymer chain near the protein or enzyme active site is critical
for reversible activity control.16 Self-assembly of enzymes and
proteins modified with hydrophobic chains also requires well-
defined conjugates.2
3,24
Various creative methods have been explored to obtain site-
specific bioconjugates. Examples include oxime formation by
25
reaction of ketone-modified tyrosine residues or lysine resi-
38
protein-polymer conjugates that circumvents these issues. The
approach involves first modifying the protein with initiation sites
for polymerization and then polymerizing from the protein
macroinitiator to form the bioconjugate in situ. Polymers had
previously been grafted from proteins by randomly generating
2
2,26
dues
with aminooxy end-functionalized PEGs. Cofactor
27
reconstitution between an enzyme and polymer has also proven
to be an efficient strategy. Polymers with ligands for protein
binding sites are used; for example, biotinylated polymers that
2
4,28-31
bind to streptavidin or avidin have been synthesized.
3
9-43
radicals on amino acid side chains,
although in these
Furthermore, Griffith et al. have made use of affinity interactions
examples, the number and sites of polymerization could not be
controlled. However, by first modifying the protein, we showed
that the polymerization is initiated from specific domains, and
the resultant locations of polymer conjugation can be predeter-
mined. Specifically, the protein streptavidin (SAv) was modified
with a biotinylated initiator and used for the formation of the
by the preparation of protein-polymer conjugates consisting
2+
of polymers with side chain Ni complexes and a polyhistidine-
tagged growth factor.32
Cysteine residues are frequently targeted for site-specific
conjugation by exploiting thiol chemistry. Generally, proteins
contain very few, if any, cysteines that do not participate in
disulfide bonds. Therefore, by making available and then
targeting free cysteines the number and placement of polymer
3
8
conjugate in situ. The initiation sites were defined by place-
ment of the modified biotins, and conjugate formation was
efficient. This methodology has the advantage that compared
to the traditional approach, purification is simplified: (1)
unreacted small molecules such as residual monomer are readily
removed by simple dialysis or chromatography; and (2) deter-
mination of the number and placement of initiation sites, and
therefore resulting polymer, is readily achieved by mass
spectrometry techniques. Because the macroinitiator was formed
by the interaction of functionalized biotin, the identity of the
protein in this example was limited to those that bind the ligand,
namely streptavidin, avidin, and recombinant derivatives such
as NeutrAvidin.
(
(
(
(
16) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G. H.; Harris,
J. M.; Hoffman, A. S. Nature 1995, 378, 472-474.
17) Ding, Z. L.; Long, C. J.; Hayashi, Y.; Bulmus, E. V.; Hoffman, A. S.;
Stayton, P. S. Bioconjugate Chem. 1999, 10, 395-400.
18) Pennadam, S. S.; Lavigne, M. D.; Dutta, C. F.; Firman, K.; Mernagh, D.;
Gorecki, D. C.; Alexander, C. J. Am. Chem. Soc. 2004, 126, 13208-13209.
19) Roberts, M. J.; Bentley, M. D.; Harris, J. M. AdV. Drug DeliVery ReV.
2
002, 54, 459-476.
(
20) Bailon, P.; Berthold, W. Pharm. Sci. Technol. Today 1998, 1, 352-356.
(
21) Tao, L.; Mantovani, G.; Lecolley, F.; Haddleton, D. M. J. Am. Chem. Soc.
2
004, 126, 13220-13221.
(
22) Kochendoerfer, G. G.; et al. Science 2003, 299, 884-887.
(
23) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. J. Am. Chem. Soc. 2002, 124,
4
224-4225.
(
24) 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.
(
25) Schlick, T. L.; Ding, Z. B.; Kovacs, E. W.; Francis, M. B. J. Am. Chem.
Soc. 2005, 127, 3718-3723.
(33) Rosendahl, M. S.; Doherty, D. H.; Smith, D. J.; Carlson, S. J.; Chlipala, E.
A.; Cox, G. N. Bioconjugate Chem. 2005, 16, 200-207.
(34) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem.
Soc. 2004, 126, 15372-15373.
(
26) Shao, H.; et al. J. Am. Chem. Soc. 2005, 127, 1350-1351.
27) Boerakker, M. J.; Hannink, J. M.; Bomans, P. H. H.; Frederik, P. M.; Nolte,
R. J. M.; Meijer, E. M.; Sommerdijk, N. A. J. M. Angew. Chem., Int. Ed.
(
(35) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.;
Cornelissen, J. J.; Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966-73.
(36) Khan, A.; Marsh, A. Synth. Commun. 2000, 30, 2599-2608.
(37) Kogan, T. P. Synth. Commun. 1992, 22, 2417-2424.
(38) Bontempo, D.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 6508-6509.
(39) Zhu, J. M.; Li, P. J. Polym. Sci., Part A: Polym. Chem. 2003, 41, 3346-
3353.
2
002, 41, 4239-4241.
(
(
(
(
(
28) Sun, X. L.; Faucher, K. M.; Houston, M.; Grande, D.; Chaikof, E. L. J.
Am. Chem. Soc. 2002, 124, 7258-7259.
29) Qi, K.; Ma, Q. G.; Remsen, E. E.; Clark, C. G.; Wooley, K. L. J. Am.
Chem. Soc. 2004, 126, 6599-6607.
30) Hou, S. J.; Sun, X. L.; Dong, C. M.; Chaikof, E. L. Bioconjugate Chem.
2
004, 15, 954-959.
(40) George, A.; Radhakrishnan, G.; Joseph, K. T. Polymer 1985, 26, 2064-
31) Bontempo, D.; Li, R. C.; Ly, T.; Brubaker, C. E.; Maynard, H. D. Chem.
Commun. 2005, 4702-4704.
2068.
(41) Chatterji, P. R. J. Appl. Polym. Sci. 1989, 37, 2203-2212.
(42) Dong, Q. Z.; Hsieh, Y. L. J. Appl. Polym. Sci. 2000, 77, 2543-2551.
(43) Imai, Y.; Iwakura, Y. J. Appl. Polym. Sci. 1967, 11, 1529 ff.
32) Griffith, B. R.; Allen, B. L.; Rapraeger, A. C.; Kiessling, L. L. J. Am.
Chem. Soc. 2004, 126, 1608-1609.
16956 J. AM. CHEM. SOC.
9
VOL. 127, NO. 48, 2005