Designed amino acid ATRP initiators for the synthesis of biohybrid materials

Article abstract of DOI:10.1021/ja0772546

A synthetic strategy to prepare peptide-polymer conjugates with precise sites of attachment is described. Amino acids modified with atom transfer radical polymerization (ATRP) initiators for the polymerization of styrenes and hacrylates were prepared. Fmoc-4-(1-chloroethyl)-phenylalanine (5) was synthesized in four steps from Fmoc-tyrosine. HATU-mediated amidation with glycine-OMe resulted in dipeptide (6). The initiator was effective for Cu(I)/bipyridine mediated bulk polymerization of styrene. Kinetic studies indicated a controlled polymerization, with high conversion (97%), and a polydispersity index (PDI) of 1.25. Fmoc-O-(2-bromoisobutyryl)-serine tert-butyl ester (10) was synthesized from Fmoc-Ser(OTrt)-OH in three steps. This initiator was employed in the ATRP of 2-hydroxyethyl methacrylate (HEMA), and kinetic studies indicated a controlled polymerization. Different monomer to initiator ratios resulted in poly-(HEMA) of different molecular weights and narrow PDIs (1.14-1.25). Conversions were between 70 and 99%. HEMA modified with N-acetyl-D-glucosamine (GlcNAc) was also polymerized to 84% conversion and the resulting PDI was 1.19. The t-butyl ester protecting group of 10 was removed, and the resulting amino acid (11) was incorporated into VM(H)VVQTK by standard solid-phase peptide synthesis. Polymerization resulted in the glycopolymer-peptide conjugate in 93% conversion and a PDI of 1.14.

Full text of DOI:10.1021/ja0772546

Published on Web 12/28/2007
Designed Amino Acid ATRP Initiators for the Synthesis of
Biohybrid Materials
Rebecca M. Broyer, Grace M. Quaker, and Heather D. Maynard*
Department of Chemistry and Biochemistry and the California NanoSystems Institute, UniVersity
of California, 607 Charles E. Young DriVe East, Los Angeles, California 90096-1569
Received September 19, 2007; E-mail: maynard@chem.ucla.edu
Abstract: A synthetic strategy to prepare peptide-polymer conjugates with precise sites of attachment is
described. Amino acids modified with atom transfer radical polymerization (ATRP) initiators for the
polymerization of styrenes and methacrylates were prepared. Fmoc-4-(1-chloroethyl)-phenylalanine (5) was
synthesized in four steps from Fmoc-tyrosine. HATU-mediated amidation with glycine-OMe resulted in
dipeptide (6). The initiator was effective for Cu(I)/bipyridine mediated bulk polymerization of styrene. Kinetic
studies indicated a controlled polymerization, with high conversion (97%), and a polydispersity index (PDI)
of 1.25. Fmoc-O-(2-bromoisobutyryl)-serine tert-butyl ester (10) was synthesized from Fmoc-Ser(OTrt)-
OH in three steps. This initiator was employed in the ATRP of 2-hydroxyethyl methacrylate (HEMA), and
kinetic studies indicated a controlled polymerization. Different monomer to initiator ratios resulted in poly-
(HEMA) of different molecular weights and narrow PDIs (1.14-1.25). Conversions were between 70 and
99%. HEMA modified with N-acetyl-D-glucosamine (GlcNAc) was also polymerized to 84% conversion and
the resulting PDI was 1.19. The t-butyl ester protecting group of 10 was removed, and the resulting amino
acid (11) was incorporated into VM(11)VVQTK by standard solid-phase peptide synthesis. Polymerization
resulted in the glycopolymer-peptide conjugate in 93% conversion and a PDI of 1.14.
Introduction
jugates can self-organize, driven by the interaction of the peptide
or protein moieties.5,8,9 These types of systems are especially
Polymer conjugates with peptides and proteins have been
widely used for applications in the areas of medicine, biotech-
nology, and materials science. These biohybrids make up an
important class of polymer therapeutics.1,2 In this context,
covalent attachment of synthetic polymers to proteins or peptides
offers a number of advantages. For example, the larger size
facilitates longevity by reducing metabolic elimination, and the
polymer reduces immunogenicity. Another important group of
bioconjugates are “smart” polymer conjugates that respond to
external stimuli such as changes in temperature or exposure to
light.3,4 Typically, upon stimulation, these “smart” polymers
undergo a hydrophobic collapse and phase separation, transfer-
ring this property to the attached biomolecule. This technology
has been used for enzyme switching, microfluidics, and affinity
separations. Polymer conjugates can undergo self-assembly in
aqueous solution driven by the hydrophobicity of the polymer
chain; a property that is useful in the design of bioactive
nanomaterials.5 “Giant amphiphiles” consisting of a protein or
enzyme polar head group and a nonpolar polystyrene tail have
been shown to form bioactive nanostructures.6,7 Further, con-
useful to form hydrogels for drug delivery and tissue engineer-
ing.
The broad range of successful applications has increased the
demand for efficient methods to prepare synthetic biological
hybrids. Controlled radical polymerizations (CRPs), such as
atom transfer radical polymerization (ATRP),10,11 reversible
addition-fragmentation chain transfer (RAFT) polymerization,12-14
and nitroxide mediated radical polymerization (NMRP),15 have
emerged as powerful tools to make peptide and protein
conjugates. Important for resultant conjugate properties is that
(1) the binding site be defined on the protein and (2) the polymer
chain exhibit a narrow polydispersity.16 This has been achieved
using CRPs by three main approaches.17,18 First, polymers have
been synthesized from functionalized initiators or chain-transfer
agents with narrow molecular weight distributions and the direct
(8) Klok, H. A. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 1-17.
(9) Kopecek, J. Eur. J. Pharm. Sci. 2003, 20, 1-16.
(10) Matyjaszewski, K.; Xia, J. H. Chem. ReV. 2001, 101, 2921-2990.
(11) Kamigaito, M.; Ando, T.; Sawamoto, M. Chem. ReV. 2001, 101, 3689-
3745.
(1) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347-360.
(2) Duncan, R. Nat. ReV. Cancer 2006, 6, 688-701.
(3) Hoffman, A. S.; Stayton, P. S. Macromol. Symp. 2004, 207, 139-151.
(4) Hoffman, A. S. Clin. Chem. 2000, 46, 1478-1486.
(5) Elemans, J. A. A. W.; Rowan, A. E.; Nolte, R. J. M. J. Mater. Chem.
2003, 13, 2661-2670.
(12) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58, 379-410.
(13) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A: Polym. Chem. 2005,
43, 5347-5393.
(14) Barner, L.; Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C. Macromol.
Rapid Commun. 2007, 28, 539-559.
(15) Hawker, C. J.; Bosman, A. W.; Harth, E. Chem. ReV. 2001, 101, 3661-
3688.
(16) Kochendoerfer, G. G. Curr. Opin. Cell Biol. 2005, 9, 555-560.
(17) Heredia, K. L.; Maynard, H. D. Org. Biomol. Chem. 2007, 5, 45-53.
(18) Nicolas, J. M., G.; Haddleton, D. M. Macromol. Rapid Commun. 2007,
28, 1038-1111.
(6) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. J. Am. Chem. Soc. 2002, 124,
4224-4225.
(7) 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.
2002, 41, 4239-4241.
9
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J. AM. CHEM. SOC. 2008, 130, 1041-1047
1041

A R T I C L E S
Broyer et al.
Scheme 1. Incorporation of Amino Acid Initiator for Precise Biohybrid Synthesisa
a Polymer modification occurs at the desired residue only, without having to rely on that amino acid being distinct within the sequence.
ability to bind to specific amino acid side chains.19 Second,
polymers have been prepared by polymerizing from modified
proteins and peptides.20,21-27 The third route involves side-chain
polymers that have been produced by polymerizing peptide
monomers or by post-polymerization conjugations.28 Herein, we
report a method to prepare peptide-polymer conjugates whereby
the polymer is grown from a predetermined amino acid side
chain of a peptide.
There are several examples of peptide-polymer conjugates
produced by polymerizing from the biomolecule. Becker, Liu,
and Wooley demonstrated that this method was amenable to
resin-bound peptides.21 The N-terminus of the protein trans-
duction domain (PTD) of the HIV-TAT protein was modified
with a NMRP initiator. This resin-bound peptide initiator was
successful in generating block copolymers. The same group later
utilized this technique to prepare poly(acrylic acid)-block-
polystyrene from the antimicrobial peptide tritrpticin using both
NMRP and ATRP.24 The resulting conjugates formed well-
defined micelles and were shown to have enhanced anti-
microbial activity compared to the free peptide. ATRP has also
been employed for the polymerization of 2-hydroxyethyl
methacrylate (HEMA) from a resin-supported 2-bromopropio-
nyl-functionalized sequence of the protein fibronectin, GRGDS
(Gly-Arg-Gly-Asp-Ser).25 The bioconjugate exhibited cell adhe-
sion properties, indicating that the peptide was still active. Using
solid-phase peptide synthesis (SPPS), Bo¨rner and co-workers
prepared oligopeptide GDGFD functionalized at the N-terminus
with a 2-bromopropionate or a dithioester group for ATRP22
and RAFT,23 respectively. Cleavage of the macroinitiators from
the solid support followed by controlled polymerization of
n-butyl acrylate resulted in well-defined bioconjugates. Biesalski
and co-workers demonstrated that cyclic peptides modified with
initiators at the lysine residues self-assembled into peptide
nanotubes, orienting the initiating groups toward the outer
surface of the nanotube.26,29 The ensembles were utilized to
polymerize N-isopropylacrylamide. Van Hest synthesized ABA
triblock copolymers with a peptide as the center block.30 This
was accomplished by selective removal of serine protecting
groups on the resin bound peptide, followed by modification
with 2-bromoisobutyric acid and polymerization in solution from
the cleaved peptide. More recently, a valine based haloamide
initiator was used to polymerize tert-butyl acrylate and chain
extended with styrene in order to create a diblock copolymer
with amino acid functionality.27 All of these methods, while
effective to prepare biohybrids, involve modifying an existing
peptide either at the N or C terminus or at reactive side-chains
with the appropriate initiating group.
(19) For some examples, see: (a) Bontempo, D.; Heredia, K. L.; Fish, B. A.;
Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372-15373. (b) Qi, K.;
Ma, Q. G.; Remsen, E. E.; Clark, C. G.; Wooley, K. L. J. Am. Chem. Soc.
2004, 126, 6599-6607. (c) Bontempo, D.; Li, R. C.; Ly, T.; Brubaker, C.
E.; Maynard, H. D. Chem. Commun. 2005, 4702-4704. (d) Mantovani,
G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.; Cornelissen, J. J.;
Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966-2973. (e) Sen Gupta, S.;
Raja, K. S.; Kaltgrad, E.; Strable, E.; Finn, M. G. Chem. Commun. 2005,
4315-4317. (f) Va´zquez-Dorbatt, V.; Maynard, H. D. Biomacromolecules
2006, 7, 2297-2302. (g) Hong, C. Y.; Pan, C. Y. Macromolecules 2006,
39, 3517-3524. (h) Liu, J.; Bulmus, V.; Barner-Kowollik, C.; Stenzel, M.
H.; Davis, T. P. Macromol. Rapid Commun. 2007, 28, 305-314. (i) Heredia,
K. L.; Tolstyka, Z. P.; Maynard, H. D. Macromolecules 2007, 40, 4772-
4779.
We were interested developing an approach that would allow
us to modify precisely one amino acid in a peptide or protein
without having to rely on that amino acid occurring infrequently.
For example, we desired a strategy that would enable us to
modify one serine in a peptide or protein that contains many
serines, threonines, and tyrosines. Such a strategy would allow
us to construct precise bioconjugates for finely tailored applica-
tions. One of the many possible uses of this approach would
be to synthesize peptides or proteins modified at a controlled
number of sites with glycopolymers to form conjugates that
better mimic glycosylation patterns found in nature.31 We
envisioned that by designing an unnatural amino acid containing
an ATRP initiator at the side chain, incorporating it into a
peptide, and polymerizing from the biomolecule, we would be
able to achieve such specific modification (Scheme 1). To our
knowledge this tactic has not yet been undertaken. We targeted
two classes of initiators, one modified with a 1-chloroethyl-
(20) For examples from proteins, see: (a) Bontempo, D.; Maynard, H. D. J.
Am. Chem. Soc. 2005, 127, 6508-6509. (b) Lele, B. S.; Murata, H.;
Matyjaszewski, K.; Russell, A. J. Biomacromolecules 2005, 6, 3380-3387.
(c) Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.;
Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 16955-16960. (d) Zeng,
Q.; Li, T.; Cash, B.; Li, S.; Xie, F.; Wang, Q.; Chem. Commun. 2007,
1453 - 1455. (e) Boyer, C. B.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel,
M. H.; Barner-Kowlik, C. J. Am. Chem. Soc. 2007, 129, 7145-7154.
(21) Becker, M. L.; Liu, J. Q.; Wooley, K. L. Chem. Comm. 2003, 180-181.
(22) Rettig, H.; Krause, E.; Bo¨rner, H. G. Macromol. Rapid Commun. 2004,
25, 1251-1256.
(23) ten Cate, M. G. J.; Rettig, H.; Bernhardt, K.; Bo¨rner, H. G. Macromolecules
2005, 38, 10643-10649.
(24) Becker, M. L.; Liu, J. Q.; Wooley, K. L. Biomacromolecules 2005, 6, 220-
228.
(25) Mei, Y.; Beers, K. L.; Byrd, H. C. M.; Vanderhart, D. L.; Washburn, N.
R. J. Am. Chem. Soc. 2004, 126, 3472-3476.
(26) Couet, J.; Biesalski, M. Macromolecules 2006, 39, 7258-7268.
(27) Venkataraman, S.; Wooley, K. L. Macromolecules 2006, 39, 9661-9664.
(28) For some examples see: (a) Godwin, A.; Hartenstein, M.; Muller, A. H.
E.; Brocchini, S. Angew. Chem., Int. Ed. 2001, 40, 594-597. (b) Ayres,
L.; Vos, M. R. J.; Adams, P. J. H. M.; Shklyarevskiy, I. O.; van Hest, J.
C. M. Macromolecules 2003, 36, 5967-5973. (c) Schilli, C. M.; Muller,
A. H. E.; Rizzardo, E.; Thang, S. H.; Chong, Y. K. AdV. Control./LiV.
Rad. Polym. 2003, 854, 603-618. (d) Griffith, B. R.; Allen, B. L.;
Rapraeger, A. C.; Kiessling, L. L. J. Am. Chem. Soc. 2004, 126, 1608-
1609. (e) Mori, H.; Iwaya, H.; Nagai, A.; Endo, T. Chem. Commun. 2005,
4872-7874. (f) Mori, H.; Sutoh, K.; Endo, T. Macromolecules 2005, 38,
9055-9065. (g) Mori, H.; Matsuyama, M.; Sutoh, K.; Endo, T. Macro-
molecules 2006, 39, 4351-4360. (h) Hwang, J.; Li, R. C.; Maynard, H. D.
J. Controlled Release 2007, 122, 279-286.
(29) Couet, J.; Jeyaprakash, J. D.; Samuel, S.; Kopyshev, A.; Santer, S.; Biesalski,
M. Angew. Chem., Int. Ed. 2005, 44, 3297-3301.
(30) Ayres, L.; Hans, P.; Adams, J.; Lowik, D.; van Hest, J. C. M. J. Polym.
Sci., Part A: Polym. Chem. 2005, 43, 6355-6366.
(31) Dwek, R. A. Chem. ReV. 1996, 96, 683-720.
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Initiators for the Synthesis of Biohybrid Materials
A R T I C L E S
Scheme 2. Synthesis of N-Fmoc-4-(1-chloroethyl)-phenylalanine Initiator (5)
Scheme 3. Synthesis of Dipeptide and Polymerization of Styrene
Table 1. Results of ATRP of Styrene
phenyl group designed for the ATRP of styrenes and one
modified with a 2-bromoisobutyrate group for the ATRP of
methacrylates. In designing derivatives of phenylalanine and
serine, respectively, we chose a 9-fluorenylmethoxycarbonyl
(Fmoc) protection route that would conveniently allow for solid-
phase peptide synthesis of the resulting amino acid initiators.
Amino acid and peptide synthesis and polymerization of styrene,
HEMA, and a â-linked N-acetyl-D-glucosamine (O-GlcNAc)
modified monomer is reported. In addition, a single site
modification of a peptide containing more than one amino acid
with alcohol functionality is described.
bipy
T
conv
(%)a
Mn
PDIb
b
initiator
[M]o/[I]o
(eq.)
(
°
C)
(theory)
Mn
6
6
6
5
100:1
100:1
100:1
100:1
3
2
3
3
110
110
130
130
92
54
97
95
9600
5600
10 100
9900
10 100
11 800
12 600c
13 300
1.40
1.30
1.25
1.27
a Determined from the 1H NMR spectra. b Determined by GPC in THF
against polystyrene standards prior to precipitation. c After purification by
precipitation, the Mn of this polymer was 13 500 and the PDI 1.23.
dipeptide initiator 6 (Scheme 3). The coupling reaction was
surveyed using a variety of conditions, and the most effective
involved using excess N,N′-diisopropylethylamine (DIPEA) (3
equiv) along with 2-(1H-9 azobenzotriazole-1-yl)-1,1,3,3-tet-
ramethylammonium hexafluorophosphate (HATU) as a coupling
agent to give the amide product in 44% yield.36 The origin of
the relatively low yield of the coupling reaction was likely due
to the reactivity of the chloroethyl-phenyl side chain. To increase
the yields and to be able synthesize longer sequences, we
envision direct incorporation of the free acid of vinyl precursor
4 during solid-phase synthesis followed by concurrent instal-
lation of the halogen and cleavage from the resin. Not only
would such a strategy avoid side reactions, particularly during
the Fmoc deprotection steps of the synthesis, it would produce
the desired peptide macroinitiator in one fewer step.
We next investigated the ATRP of styrene utilizing the
dipeptide as an initiator (Scheme 3). Styrene was polymerized
in bulk at 110 °C using 100 equiv to initiator 6, CuCl and 2,2′-
bipyridine (bipy) as the catalyst (6:CuCl:bipy ) 1:1:3). The
resulting polymer was synthesized in 92% conversion, with a
number-average molecular weight (Mn) of 10 100 Da and a
polydispersity index (PDI) of 1.40 (Table 1). Maintaining a 110
°C reaction temperature, while reducing the equivalents of bipy
from 3 to 2 resulted in a polymer with a narrower molecular
Results and Discussion
Synthesis of Polystyrene-Peptide Conjugates. We started
with a commercially available tyrosine to prepare an artificial
amino acid containing the requisite phenylethyl halide moiety
for the polymerization of styrene (Scheme 2). Chemical
manipulation of Boc-protected tyrosine to 4-hydroxymethyl-L-
phenylalanine via a key aryl triflate derivative had been reported
previously by Morera et al.32 We adopted a similar intermediate
to prepare N-Fmoc-4-(1-chloroethyl)-phenylalanine (5). Com-
mercially available Fmoc-L-tyrosine was protected as the tert-
butyl ester.33 Subsequent treatment with triflic anhydride34 using
2.5 equiv of pyridine gave the triflate intermediate 3 in 64%
yield. This compound was then subjected to Stille coupling
conditions,35 using tetrakis(triphenylphosphine) palladium(0)
(Pd(PPh3)4) and dry lithium chloride to introduce a vinyl group
on the aromatic ring (4) in 70% yield. Simultaneous chlorine
installation and deprotection of the ester protecting group was
achieved by treating 4 with HCl in acetic acid to give the
1-chloroethyl derivative 5 in 60% yield. The free acid end of 5
was coupled to glycine methyl ester hydrochloride to form
(32) Morera, E.; Ortar, G.; Varani, A. Synth. Commun. 1998, 28, 4279-4285.
(33) Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. J. Org. Chem.
2003, 68, 6795-6798.
(34) Shieh, W. C.; Carlson, J. A. J. Org. Chem. 1992, 57, 379-381.
(35) Echavarren, A. M.; Stille, J. K. J. Am. Chem. Soc. 1987, 109, 5478-5486.
(36) Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem. 1998,
63, 9678-9683.
9
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A R T I C L E S
Broyer et al.
by both 1H NMR spectroscopy and GPC. The semilogarithmic
kinetic plot was linear and the polymer Mn increased linearly
with conversion (Figure 1). The PDI decreased throughout the
reaction, similar to what had been observed for the ATRP of
styrene utilizing unfunctionalized initiator, 1-phenylethyl chlo-
ride.37 These results indicated a controlled polymerization from
the dipeptide initiator.
To ensure that the biohybrid material had been synthesized,
polymer 7 was purified by precipitation into methanol and
1
subjected to analysis by H NMR spectroscopy. The peaks
corresponding to the peptide end group were clearly visible in
the spectrum (see Supporting Information). In particular, peaks
corresponding to the Fmoc hydrogens of the initiator (at 7.76,
7.55, and 7.38 ppm) were observed, providing good evidence
that this moiety did not thermally deprotect during the polym-
erization. These peaks also enabled the estimation of molecular
weight from the 1H NMR spectrum. The result was 13 900 Da,
which was in excellent agreement with the molecular weight
observed by GPC after precipitation of the polymer (13 500 Da).
This data additionally suggested that the peptide was conjugated
to the chain.
In a study of various carboxylic acid-containing initiators,
Matyjaszewski found that unprotected carboxylic acids had low
efficiency in the ATRP of styrene.38 Haddleton also found that
addition of high concentrations of carboxylic acids to the ATRP
of methyl methacrylate was deleterious to the polymerization.39
However, Matyjaszewski also found that initiators with halide
groups remote from the carboxylic acid moiety gave reasonable
efficiencies for the polymerization of styrene.38 Thus, although
5 contained a free acid, we decided to explore this initiator in
the ATRP of styrene (Scheme 4) employing the optimized
conditions found for initiator 6. Styrene (100 equiv) was
polymerized in bulk at 130 °C (5:CuCl:bipy ) 1:1:3). The
resulting polymer was synthesized with high conversion (95%),
a Mn of 13 300 Da and a PDI of 1.27. This result indicated that
5 could effectively produce well-defined polystyrene, which
suggested that the chloride was remote enough to avoid
complications with the free acid moiety. Purity of the initiator
was important as polymerizations that used impure samples
showed remarkably increased polydispersities (data not shown).
Figure 1. Kinetic plot (top) determined by 1H NMR and plot of the
evolution of Mn with conversion (bottom) determined by GPC in THF for
the bulk polymerization of styrene 6:[bipy]:[styrene] ) 1:3:100 at 130 °C.
Scheme 4. Polymerization of Styrene from 5
weight distribution (PDI ) 1.30), although the conversion was
only 54%. The dipeptide initiator 6 contained an amide group
that may have competitively bound to the catalyst, requiring
additional bipy to maintain enough copper-bound ligand to
achieve high conversions. The reaction was then carried out at
higher temperatures according to the original conditions used
by Matyjaszewski.37 Styrene was polymerized in bulk at
130 °C using 100 equiv to initiator 6 and CuCl and bipy as the
catalyst (6:CuCl:bipy ) 1:1:3). In this case, the reaction
proceeded to high conversion (97%) and the polymer (7) had a
Mn of 12 600 Da and a narrow PDI of 1.25 (Table 1). Aliquots
removed periodically during the polymerization were analyzed
These results demonstrated that dipeptide initiator 6 with a
1-chloroethyl group was highly effective for the polymerization
of styrene. The ATRP amino acid was synthesized in a few
steps. The peptide initiator was produced, not by the typical
modification of the side chain, but rather by incorporation of
the initiation moiety during synthesis. The polymerizations were
Scheme 5. Synthesis of the Fmoc-O-(2-bromoisobutyryl)-serine Initiator
9
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Initiators for the Synthesis of Biohybrid Materials
A R T I C L E S
Table 2. Results of ATRP of Methacrylate Monomers from
Initiator 10
a
polymer
[M]0/[I]0
conva (%)
Mn (theory)
Mn
PDIb
polyHEMAc
polyHEMAc
polyHEMAc
12d
20
50
100
50
99
94
70
84
2500
6300
9100
4400
5900
11 800
15 400
1.14
1.22
1.25
1.19
14 000
a Determined by 1H NMR. b Determined by GPC in DMF (0.1 M LiBr)
against polymethyl methacrylate standards. c Polymerization conditions:
MeOH, 23 °C, [10]0:[CuBr]0:[bipy]0 ) 1:1:2. d Polymerization conditions:
MeOH:H2O (85:15), 23 °C,[10]0:[CuBr]0:[bipy]0 ) 1:1:2.
Figure 2. Kinetic plot (top) determined by 1H NMR and plot of the
evolution of Mn with conversion (bottom) determined by GPC in DMF
(0.1 M LiBr) for the polymerization of HEMA in methanol-d4 [10]:[bipy]:
[HEMA] ) 1:2:50 at 23 °C.
controlled and reached high conversions when three equivalents
of bipy were utilized in the reaction at 130 °C. The resulting
biohybrid materials were well-defined. Interestingly, amino acid
5 with a free acid could also be utilized as an initiator, suggesting
that the acid was far enough removed from the initiating species.
With these promising results, the scope of the types of polymers
accessible with this strategy was next explored.
Synthesis of Polymethacrylate-Amino Acid Conjugates.
We were also interested in synthesizing amino acid-based
initiators that would be optimal to polymerize methacrylate
monomers. Therefore, Fmoc protected serine derivative (10) was
designed because the 2-bromoisobutyrate moiety is a well-
known initiator for the ATRP of methacrylates.10,11 The
synthesis began by preparation of N-R-Fmoc-L-serine-tert-butyl
ester 9 in two steps following a previously reported procedure
(Scheme 5).33 The protected serine 9 was subsequently treated
with 2-bromoisobutyryl bromide in the presence of DIPEA to
afford the Fmoc-O-(2-bromoisobutyryl)-serine tert-butyl ester
initiator 10 in 66% yield.
Figure 3. Kinetic plot (top) determined by 1H NMR and plot of the
evolution of Mn with conversion (bottom) determined by GPC in DMF
(0.1 M LiBr) for the polymerization of the glycomonomer in methanol-d4:
D2O (85:15), [10]:[bipy]:[glycomonomer] ) 1:2:50 at 23 °C.
observed as the monomer to initiator ratio was increased (4400,
5900, and 11 800 Da, for 20, 50, and 100 equiv of monomer,
respectively). These molecular weights were estimated from the
1H NMR spectra (see Supporting Information) after extensive
purification of the samples. GPC was employed before and after
to ensure that fractionation did not occur during this purification
process. In all cases, the PDIs were narrow (1.14-1.25),
indicating that well-defined polymers were formed.
HEMA was selected for polymerization from amino acid 10
because polyHEMA is a biocompatible material known to form
hydrogels. It is widely used, for example, in the production of
contact lenses.40 Copper-mediated ATRP of HEMA (50 equiv)
was explored using a 1:1:2 ratio of 10:CuBr:bipy in methanol
at 23 °C. Progression of the polymerization was monitored by
1H NMR spectroscopy. The kinetic plot and Mn versus conver-
sion were both linear to 94% conversion (Figure 2), indicating
a controlled polymerization. The system was robust in that the
initial monomer-to-initiator ratios could be changed while
maintaining relatively high conversions. Conversions of 99%
and 70% were achieved for monomer to initiator ratios of 20:1
and 100:1, respectively (Table 2). Increasing Mn values were
With this success, next we investigated the polymerization
of a GlcNAc-modified HEMA. O-GlcNAc glycosylation is a
specific protein post-translational modification that covalently
attaches the sugar to a serine or threonine residue of proteins.
This type of post-translational modification has been detected
on transcription factors, protein kinases and cytoskeletal proteins
and is important in a range of diseases.41-43 In addition,
glycopolymers are known to mimic the bioactivities of natural
polysaccharides,44 yet are significantly easier to synthesize than
oligosaccharides. O-GlcNAc modified synthetic polymers at-
(40) Montheard, J. P.; Chatzopoulos, M.; Chappard, D. J. Macromol. Sci.-ReV.
Macromol. Chem. Phys. 1992, C32, 1-34.
(41) Whelan, S. A.; Hart, G. W. Circ. Res. 2003, 93, 1047-1058.
(42) Khidekel, N.; Ficarro, S. B.; Peters, E. C.; Hsieh-Wilson, L. C. P. Natl.
Acad. Sci. U.S.A. 2004, 101, 13132-13137.
(37) Wang, J. S.; Matyjaszewski, K. J. Am. Chem. Soc. 1995, 117, 5614-5615.
(38) Zhang, X.; Matyjaszewski, K. Macromolecules 1999, 32, 7349-7353.
(39) Haddleton, D. M.; Heming, A. M.; Kukulj, D.; Duncalf, D. J.; Shooter, A.
J. Macromolecules 1998, 31, 2016-2018.
(43) Slawson, C.; Hart, G. W. Curr. Opin. Struct. Biol. 2003, 13, 631-636.
(44) Ladmiral, V.; Melia, E.; Haddleton, D. M. Eur. Polym. J. 2004, 40, 431-
449.
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J. AM. CHEM. SOC. VOL. 130, NO. 3, 2008 1045

A R T I C L E S
Broyer et al.
Scheme 6. ATRP of O-GlcNAc Glycomonomer from Serine Initiator 10
Scheme 7. Polymerization from VM(11)VVQ(Trt)T(tBu)K(Mca)G Peptide
tached to peptides or proteins may mimic biological glycoso-
lation, particularly if the attachment can be made site specifi-
cally.45 It was for these reasons that the polymerization of a
GlcNAc-modified HEMA from initiator 10 was explored.
ATRP of the glycomonomer was carried out using initiator
10 with CuBr and bipy ([monomer]0:[initiator]0:[CuBr]0:[bipy]0
) 50:1:1:2). Initially, ATRP was explored using methanol as
the solvent. However, high molecular weights of the glyco-
polymer were insoluble in methanol, making it difficult to obtain
reproducible results. Therefore, methanol and water mixtures
were investigated, with the idea that water would help solubilize
the growing polymer. Indeed, it was found that the polymeri-
zation was homogeneous in MeOH:H20 (85:15, [M]0 ) 0.9 M)
and afforded polymer 12 in high conversion (84%) in 80 min.
Kinetic analysis indicated slight curvature in the kinetic plot;
yet the Mn versus conversion plot was linear and the PDIs were
narrow throughout the polymerization (Figure 3). The Mn after
Deprotection of the tert-butyl ester initiator 10 with 20%
trifluoroacetic acid (TFA) in dichlormethane (CH2Cl2) for 1 h
gave the acid form (11) in 90% yield (Scheme 5). Glycine
functionalized chlorotrityl resin was selected for the peptide
synthesis, as the resulting peptide could be cleaved from the
resin using relatively mild conditions (acetic acid:trifluoroet-
hanol:CH2Cl2; 2:2:6) leaving the amino acid side chain protect-
ing groups intact. At the C-terminal residue, the lysine was
replaced with a lysine derivative modified at the epsilon amine
with a (7-methoxycoumarin-4-yl)acetyl (Mca) group, so that the
end group could be monitored by fluorescence measurements.
The Mca group is known to fluoresce at 405 nm when stimulated
at 340 nm.46 Synthesis of VM(11)VVQ(Trt)T(tBu)K(Mca) was
achieved from glycine modified chlorotrityl resin, employing
Fmoc-based solid-phase peptide synthesis. The peptide was
purified by reverse phase preparatory HPLC prior to use.
ATRP from the protected peptide was conducted as for 10
using MeOH:H2O (85:15), 23 °C, [M]0:[peptide]0:[CuBr]0:
[bipy]0 ) 20:1:1:2, and [M]0 ) 0.9 M (Scheme 7). Similarly,
the polymerization reached 93% conversion to polymer in 80
min. Polymerization from the peptide resulted in a polymer with
a PDI of 1.14, demonstrating that a well-defined conjugate was
obtained. The Mn of the polymer was determined by GPC to
be 12 200 Da. Conjugate formation was confirmed using a
fluorometer after extensive purification to remove any unat-
tached peptide. Upon stimulation at 340 nm, fluorescence was
observed at 405 nm, as expected for the modified lysine Mca
derivative. This provided good evidence that the peptide was
indeed attached to the polymer and that the biohybrid was
formed.
1
extensive purification of the polymer was 15 400 Da by H
NMR spectroscopy. Importantly, the PDI was remarkably
narrow at 1.19, indicating that well-defined glycopolymers were
accessible.
With these promising results, 10 was deprotected and
incorporated into a model peptide, VMSVVQTK, using solid-
phase peptide synthesis. This particular peptide was chosen
because the sequence is known to be O-GlcNAc modified in
the human cellular factor (HCF) protein, an abundant chromatin-
associated factor involved in cell proliferation and transcriptional
regulation.42 In addition, this peptide contains two different
alcohol groups (the serine and threonine side chains) making it
an interesting model system for site-specific modification.
(45) Vazquez-Dorbatt, V.; Maynard, H. D. Biomacromolecules 2006, 7, 2297-
(46) Lauer-Fields, J. L.; Broder, T.; Sritharan, T.; Chung, L.; Nagase, H.; Fields,
2302.
G. B. Biochemistry 2001, 40, 5795-5803.
9
1046 J. AM. CHEM. SOC. VOL. 130, NO. 3, 2008

Initiators for the Synthesis of Biohybrid Materials
A R T I C L E S
These results demonstrated that a serine-derived initiator was
highly effective for the polymerization of methacrylate-based
monomers. The amino acid initiator was prepared in a few steps
and was efficient in the synthesis of two important biopoly-
mers: polyHEMA and an O-GlcNAc side chain glycopolymer.
This amino acid was readily incorporated into a biologically
relevant peptide, and the corresponding glycopolymer-biohybrid
material was formed directly. Importantly, all polymers exhibited
narrow molecular weight distributions, demonstrating that well-
defined polymers were obtained utilizing this strategy.
should enable us to mimic glycosylation patterns found in nature,
whereby a particular amino acid is modified with a saccharide
or polysaccharide. In addition, this may result in glycopooly-
mer-peptide conjugates with unique bioactivities. We also
anticipate that other monomers will be readily polymerized. In
addition, we expect that polymerization from peptides supported
on a solid resin should be possible. Thus far, we demonstrated
utilizing solid-phase synthesis to incorporate an artificial amino
acid into a short peptide. This same approach may then be
extended to the incorporation of the amino acid initiator into
longer peptides or proteins by known native chemical ligation
technologies. Another potential use of these amino acids is
incorporation of the deprotected versions into proteins by
recombinant methods.47-51 These and other applications of this
method to produce peptide or protein conjugates with unprec-
edented control over the placement of the polymer chain are
currently being pursued.
Conclusions
Herein, a straightforward methodology to prepare peptide-
polymer biohybrid materials was described. Artificial amino
acids with side-chain ATRP initiators were designed and
synthesized. Use of these amino acids to prepare well-defined
polystyrene, polyHEMA, and glycopolymer biohybrid materials
was demonstrated. Previous technologies were limited to
modifying pre-existing peptides making it difficult to conjugate
to one particular amino acid in a peptide that contained many
of the residue. This methodology is unique in that it provides a
means to polymerize from a peptide at any point along the
sequence. Unlike use of selective deprotection group strategies
and subsequent modification, this approach should result in
quantitative incorporation of the initiation moiety. A variety of
applications may be envisioned. For example, biohybrid materi-
als composed of polystyrene are known to produce self-
assembled nanostructures that are bioactive.5 Until now, these
have been synthesized by conjugation of a preformed polymer
chain. The approach described here should allow the peptide
to be modified at a precise site, potentially resulting in increased
bioactivity or unique self-assembling properties of the conjugate.
In addition, polyHEMA is a well-known hydrogel material, and
formation of this polymer from peptides could be utilized to
produce modified gels for a variety of biomaterial applications.
Glycopolymers, including GlcNAc-modified materials, are
useful as mimetics of natural polysaccharides, and are signifi-
cantly more straightforward to synthesize compared to oligosac-
charides.44 We expect to be able to modify a wide variety of
peptides at one particular amino acid with a glycopolymer. This
Acknowledgment. This work was supported by the NSF
(CHE-0416359) and the ACS PRF through a Type G Grant.
H.D.M. is also grateful to Amgen (New Faculty Award) and
the Alfred P. Sloan Foundation Research Fellowship for funding.
G.M.Q. thanks the NIH funded Chemistry Biology Training
Program for a predoctoral fellowship. The authors appreciate
Dr. Jeremy Collette for mass spectrometry of the VM(11)-
VVQTKG peptide, Dr. James Tsay and Sam On Ho for help
with the fluorescence measurements, and Zachary Tolstyka for
assistance formatting some of the figures.
Supporting Information Available: Experimental details, 1H
NMR spectra of polymers. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA0772546
(47) Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 19-24.
(48) Mendel, D.; Cornish, V. W.; Schultz, P. G. Annu. ReV. Biophys. Biomol.
Struct. 1995, 24, 435-462.
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(51) Noren, C. J.; Anthonycahill, S. J.; Griffith, M. C.; Schultz, P. G. Science
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