Synthesis of thiol-clickable and block copolypeptide brushes via nickel-mediated surface initiated polymerization of α-amino acid N-carboxyanhydrides (NCAs)

Article abstract of DOI:10.1039/c1cc11534k

We describe the synthesis of homo-, block, and clickable copolypeptide brushes from low surface area substrates using nickel-mediated surface-initiated polymerization of α-amino N-carboxyanhydrides.

Full text of DOI:10.1039/c1cc11534k

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Chemical Communications
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Volume 47 | Number 22 | 14 June 2011 | Pages 6173–6480
ISSN 1359-7345
COMMUNICATION
Derek L. Patton et al.
Synthesis of thiol-clickable and block copolypeptide brushes via nickel-mediated surface
initiated polymerization of α-amino acid N-carboxyanhydrides (NCAs)

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COMMUNICATION
Synthesis of thiol-clickable and block copolypeptide brushes via
nickel-mediated surface initiated polymerization of a-amino acid
N-carboxyanhydrides (NCAs)wz
Bradley J. Sparks,a Jacob G. Ray,a Daniel A. Savin,a Christopher M. Staffordb and
Derek L. Patton*a
Received 17th March 2011, Accepted 31st March 2011
DOI: 10.1039/c1cc11534k
We describe the synthesis of homo-, block, and clickable
copolypeptide brushes from low surface area substrates using
nickel-mediated surface-initiated polymerization of a-amino
N-carboxyanhydrides.
of a-amino acid N-carboxyanhydrides (NCAs) initiated from
an amine-functionalized substrate in either the solution or
vapor phase.7,8 Since monomers can readily diffuse to the
propagating chain end, the surface-initiated approach yields
higher grafting densities as has been demonstrated for a broad
range of homopolypeptide surfaces.9 In contrast, relatively few
examples of block copolypeptide (BCPP) surfaces have been
reported in the literature. Wieringa et al.10 first reported the
synthesis of BCPPs from primary amine-functionalized
substrates in solution. Wang and Chang11 improved upon
the synthesis of BCPPs under surface-initiated vapor polymer-
ization, which significantly reduces the occurrence of side
reactions yielding higher film thicknesses.
Peptide-functionalized thin films are of great interest to the
materials science community and have been explored for an
array of applications including biomedical,1 anti-fouling,2
inorganic/peptide hybrids3 and stimuli responsive materials.4
Polypeptide brushes are a unique class of surface-grafted
polymers in that they allow the incorporation of structural
motifs typical of those found in proteins, such as a-helices,
b-sheets, and random coils. Like their native counterparts in
proteins, these surface-grafted motifs can respond dynamically
to changes in their environment.5 This feature presents an
opportunity to control the brush structure, function, and
response at a level difficult to achieve with conventional
organic polymers, but requires the development of synthetic
methods that enable the preparation of structurally complex
peptide surfaces.
Transition metal-mediated polymerization of NCAs,
as reported by Deming et al.,12–15 allows the preparation
of well-defined polypeptides including end-functionalized,
sequenced and BCPPs. Witte and Menzel16 utilized Ni(0)
initiators to polymerize NCAs from high surface area
polystyrene resins; however, no studies have been reported
on Ni-mediated polymerization of polypeptides from low
surface area substrates (i.e. silicon wafer). Due to the low
concentration of initiator on the surface, polymerization
behaviour can vastly differ from that of high surface area
polymerizations. Furthermore, the flat substrate represents a
model substrate configuration for biosensor platforms and
other thin film applications.
Polypeptide brushes have been synthesized using two
different methods.6 The ‘‘grafting to’’ approach involves a
one-step reaction of reactive end-groups of pre-synthesized
polypeptides with complementary reactive moieties on a surface.
The adsorption and reaction of preformed polypeptides with
surfaces quickly becomes diffusion-limited resulting in low
grafting densities. The ‘‘grafting from’’, or surface-initiated
approach, typically occurs via a ring-opening polymerization
In this communication, we describe the synthesis of homo,
block, and clickable copolypeptides via nickel-mediated
surface-initiated polymerization from low surface area
substrates. As examples, we prepare blocked combinations
of poly(N-carbobenzyloxy-L-lysine), poly(g-benzyl-L-glutamate)
and poly(S-tert-butylmercapto-L-cysteine) brushes from the
respective NCA monomers. Additionally, we show that the
cysteine-containing constituent lends itself well to a post-
polymerization modification via an efficient thiol-Michael click
reaction with maleimides resulting in a highly modular
approach to functional polypeptide surfaces.
a School of Polymers and High Performance Materials,
The University of Southern Mississippi, 118 College Drive # 10076,
Hattiesburg, MS 39406, USA. E-mail: derek.patton@usm.edu;
Tel: +1 601-266-4229
b Polymers Division, National Institute of Standards and Technology,
Gaithersburg, Maryland 20899, USA
w Electronic supplementary information (ESI) available: Synthetic
details and characterization parameters. See DOI: 10.1039/c1cc11534k
z Official contribution of the National Institute of Standards and
Technology; not subject to copyright in the US. Equipment, instruments,
and materials are identified in the paper in order to adequately specify
the experimental details. Such identification does not imply
recommendation by NIST, nor does it imply the materials are
necessarily the best available for the purpose.
As shown in Fig. 1, alloc-L-leucine-3-aminopropyltriethoxy-
silane, synthesized from the reaction of alloc-L-leucine-N-
hydroxysuccinimide with 3-aminopropyltriethoxysilane (see
ESIw), was used to covalently modify silicon oxide substrates
c
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Chem. Commun., 2011, 47, 6245–6247 6245

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Fig. 1 Synthetic route for the preparation of homo- and block
copolypeptide brushes via nickel-mediated surface-initiated polymer-
ization of a-amino acid N-carboxyanhydrides (NCAs).
Fig. 2 XPS survey (left) and corresponding high resolution Ni2p
spectra (right) for (a) tethered Ni-initiator complex, (b) poly(N-carbo-
benzyloxy-L-lysine), (c) poly(g-benzyl-L-glutamate), (d) poly(N-carbo-
benzyloxy-L-lysine-block-S-tert-butylmercapto-L-cysteine), and (e)
poly(g-benzyl-L-glutamate-block-S-tert-butylmercapto-L-cysteine).
with a thin layer of alloc-amide groups. Successful modification
of the surface was confirmed by ellipsometry and X-ray
photoelectron spectroscopy (XPS). The mean thickness of
the dry silane-modified layer was 1.7 nm ꢀ 0.6 nm as measured
by ellipsometry (std. dev. represents variability from repeat
experiments, which is taken as the standard uncertainty
of the measurement). The XPS survey scan of the alloc-amide
functionalized surface indicated the expected chemical
composition with observed bonding energies at 99 eV (Si2p),
285 eV (C1s), 399 eV (N1s), and 532 eV (O1s) (see ESIw
for XPS spectrum). The alloc-amide substrates were then
complexed with bis(cyclooctadiene) nickel(0) (Ni(COD)2)
and 1,10-phenanthroline in dry dimethylformamide (DMF)
to create a surface tethered Ni(0) initiator complex from which
surface-initiated polymerization (SIP) of NCAs could be
facilitated. After washing the substrates extensively with
DMF to remove excess/unbound Ni species, the Ni complexation
on the surface was confirmed by XPS. As shown in Fig. 2a, the
survey and high resolution Ni2p spectra indicate the presence
of nickel with binding energy observed at 855 eV (Ni2p).
The active Ni-initiator substrates were then submerged in a
0.26 mol Lꢁ1 NCA monomer solution in dry DMF for 24 h.
After polymerization, Soxhlet extraction in THF was used to
remove any physisorbed polymer from the polypeptide brush.
For the purpose of this communication, optimization of
experimental parameters such as monomer concentration,
initiator grafting density, temperature, ligand, etc. were not
explored. These parameters will be fully investigated in a
forthcoming publication. After extraction, the poly(lysine)
and poly(benzyl glutamate) peptide brushes yielded thicknesses
of 4.2 nm ꢀ 1.7 nm and 3.6 nm ꢀ 1.8 nm, respectively. The
low film thickness observed is likely a direct result of the low
monomer concentrations employed in this study. The polypeptide
surfaces were characterized by XPS where the retention of the
Ni end-group would indicate a degree of controlled polymer-
ization from the surface. As shown in high resolution Ni2p
spectra in Fig. 2b and c, peaks corresponding to the Ni2p
binding energies were observed albeit in reduced intensity
compared to the Ni-initiator in Fig. 2a. This is expected as
we are now probing the entire film thickness, as indicated
by the increase in the C1s and N1s intensities in the survey
spectra associated with the peptide backbone. An additional
indication of a controlled polymerization process was demon-
strated through the continued growth of the homopolypeptides
and formation of block copolypeptides. BCPPs were
synthesized by subsequently submerging the homopolymer
substrates prepared as previously described in a 0.26 mol Lꢁ1
NCA–S-tert-butylmercapto-L-cysteine monomer solution.
After 24 h reaction time and subsequent washing, the
poly(cysteine) block segment thicknesses were measured at
4.4 nm ꢀ 1.5 nm by ellipsometry. The equivalent thickness
measured for each chain extension suggests similar end-group
retention and blocking efficiency from both lower blocks.
A more detailed XPS study will be necessary to quantify
chain-end retention/blocking efficiency. Further supporting
the presence of the poly(cysteine) block in the Lys-b-Cys and
BGlu-b-Cys copolymers, XPS survey spectra (Fig. 2d and e)
showed a peak corresponding to the S2s binding energy at
229 eV (high resolution S2p available in the ESIw). Notably,
the Ni end-group is still observed in the Ni2p high
resolution spectra suggesting the possibility of preparing
short sequenced, multiblock polypeptide brushes using this
approach.
c
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in ellipsometric thickness was also observed consistent with an
increase in molecular mass of the maleimide functionalized
brush. Although the maleimide used in the current study was
convenient for XPS analysis, the modification of the pendant
thiol can easily be extended to other ‘‘enes’’ carrying
appendages useful for a broad range of applications, i.e.,
bioconjugation. This versatility allows for a simple and
effective way to realize polypeptide surfaces that are otherwise
unattainable by direct polymerization.
In summary, we have demonstrated a method to synthesize
homo-, block, and clickable copolypeptides via nickel-mediated
surface-initiated polymerization from low surface area substrates.
XPS showed that the Ni species remains attached to the end-
group of the brush, which leads us to believe that the Ni-complex
continues to play a mediating role during chain extension to form
block copolypeptides. Furthermore, we utilized the highly efficient
thiol-Michael reaction to prepare functional polypeptide surfaces
from thiol-containing poly(cysteine) brushes. Future work will
focus on the kinetics of brush growth, initiation efficiency, and
expanding the utility of thiol-clickable peptide surfaces.
This work was supported in part by NSF-PFI (Award
#0917730) and NSF CAREER (DMR-1056817). J.R.
acknowledges fellowship support from the NSF GK-12
Molecules to Muscles Program at USM (Award #0947944).
Notes and references
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Fig. 3 (a) Synthetic scheme for thiol-Michael modification of a
deprotected poly(cysteine) brush with a fluorine-labelled maleimide.
(b) XPS data indicate a successful modification as shown by the
observation of a peak at 689.5 eV in the high resolution F1s spectrum.
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particularly thiol-click reactions19–21 are a powerful approach
for engineering functional surfaces in a modular fashion.
Thiol-ene reactions, more specifically the thiol-Michael addition,
are of particular interest because of rapid kinetics, amiable
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mercapto-L-cysteine as previously described. Subsequently,
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thiols. These moieties were used as reactive handles for further
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employed to functionalize the cysteine brush with a fluorine-
labelled maleimide. The maleimide was dissolved in a dry THF
solution and the deprotected peptide substrate was submerged
in the maleimide solution for 24 h. The brush functionalization
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the pendant maleimide as indicated by the peak at 689.5 eV in
the high resolution F1s spectrum (Fig. 3b). A 3.2 nm increase
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c
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