Bio-inspired Polymers for Nanoscience Research

Byoung-Chul Lee, Michael D. Connolly and Ronald N. Zuckermann*

Biological Nanostructures Facility

The Molecular Foundry, Lawrence Berkeley National Laboratory
1 Cyclotron Rd., Berkeley, CA 94720, rnzuckermann@lbl.gov

ABSTRACT R2
O
R4
O
N N OH
Peptoids are a novel class of non-natural biopolymer HN N N
based on an N-substituted glycine backbone that are ideally O
R1 O R3 O R5
suited for nanomaterials research [1]. This bio-inspired
material has many unique properties that bridge the gap Figure 1. Peptoids are a novel class of bio-inspired
between proteins and bulk polymers [2]. Like proteins, materials.
they are a sequence-specific heteropolymer, capable of
folding into specific shapes and exhibiting potent biological proteins do [4]. By choosing oligomerization chemistry
activities; and like bulk polymers they are chemically and that is more robust than Nature’s polypeptide or nucleic
biologically stable and relatively cheap to make. Peptoids acid backbones, we should be able to construct folded
are efficiently assembled via automated solid-phase synthetic nanostructures that are much more stable. As we
synthesis from hundreds of chemically diverse building continue to understand the chemical and physical
blocks, allowing the rapid generation of huge combinatorial mechanisms of biopolymer function, we should be able to
libraries [3]. This provides a platform to discover import these structural features into completely synthetic,
nanostructured materials capable of protein-like molecular industrially useful materials.
recognition and function. Man-made bulk polymers can be highly robust, useful
materials. However, the methods used to produce them
Keywords: peptoids, biomimetic polymers, combinatorial lack the ability to precisely control the monomer sequence.
chemistry, self-assembly, helix bundle. Although techniques like atom transfer radical
polymerization (ATRP) allow unprecedented control of
1 BIOMIMETIC POLYMERS polymer composition [5], they can only make linear or
branched block copolymer-type structures. The self-
One of the fundamental challenges in nanoscience is to organization in man-made polymers is currently limited
develop methods that allow the synthesis of materials with mainly to simple forms of striation and layering -- lamellae,
precisely defined 3-dimensional structures. Such micelles, hexagonal and discotic phases, and bicontinuous
techniques would allow for the positioning of molecular structures.
moieties at defined distances and angles in space. This kind In recent years chemists have made great strides in the
of control over materials synthesis would be extremely synthesis of non-natural sequence-specific heteropolymers
powerful, as chemically reactive groups, chromophores, [2]. Like biopolymers, these materials can fold into defined
metals, nanocrystals, biologically active molecules, etc structures, but have the advantage that they can incorporate
could all be arranged in precise geometries relative to each a much wider range of chemical functionalities. Because
other. This will undoubtedly lead to new generations of each monomer is added one at a time, this class of materials
nanostructured materials with very sophisticated properties. must be made by solid-phase synthesis, where monomers
In order to build such nanostructured materials, we look are added iteratively. It is now possible to synthesize such
to Nature for inspiration. Natural nanostructures exhibit a materials in the length regime of small proteins [6] (~50
fantastic array of precisely defined shapes, and yet their monomers). Thus, the challenge facing nanoscientists is
underlying architecture is profoundly simple. Enzymes, now turning from synthesis to design.
receptors, antibodies, structural proteins, DNA and RNA
are all biopolymers based upon the predominantly linear 2 PEPTOID OLIGOMERS
repetition of a relatively small number of monomer
building blocks. The key is that the 20 amino acids or the 4 Peptoids are a class of non-natural biomimetic oligomer
nucleotides are arranged into specific sequences that based on an N-substituted glycine backbone [1] (Fig. 1) that
determine the information content, structure and function. combine many of the advantageous properties of bulk
Our group and others have focused on techniques to polymers with those of proteins. Peptoid oligomers are of
chemically synthesize sequence-specific heteropolymers particular interest for building defined nanostructured
that can not only position a chemically diverse set of materials because of their ease of synthesis [7] and their
monomers in a particular order, but can fold into specific chemical and biological stability. Peptoid oligomers are
secondary and tertiary structures the way nucleic acids and

28 NSTI-Nanotech 2007, www.nsti.org, ISBN 1420061836 Vol. 2, 2007

 O
Br O R' O
OH R' NH2
H Br N
N N H N
DIC, DMF DMSO
R R R
STEP 1 STEP 2

Figure 2. The submonomer method allows the rapid solid-phase synthesis of peptoids from cheap and readily
available starting materials. Hundreds of diverse side chains can be introduced from the corresponding primary amine.

resistant to degradation by many common proteases [8], for Despite the fact that the peptoid backbone is achiral,
example. Because the peptoid backbone has a similar side peptoid oligomers can be folded into helical secondary
chain spacing and polarity to peptides, it is not surprising structures [11-16] (Fig.4). This is accomplished by
that peptoids have been shown by several laboratories to incorporating bulky, chiral side-chains into the oligomer.
have a wide variety of potent biological activities [1]. These helical secondary structures are extremely stable to
We have developed a rapid and highly efficient method chemical denaturants and temperature [17]. The unusual
to synthesize peptoids [7]. The solid-phase submonomer stability of the helical structure may be a consequence of
method uses a two-step monomer addition cycle wherein the steric hindrance of backbone ø angle by the bulky chiral
each monomer is assembled from cheap and readily side-chains [11, 13].
available starting materials (Fig. 2). The overall coupling
efficiency for each cycle is typically in excess of 99%.
This allows us to make materials in the size regime of small
proteins and polymers (Fig. 3).

(a)

(b)

(c)
Figure 4. View down the axis of a model of a
15mer peptoid helix.

Amphiphilic peptoid helices can be packed together to

Figure 3. Length series of a peptoid synthesis as form helical bundles, a significant step toward synthesizing
monitored by analytical HPLC during the synthesis
of a peptoid 50 mer via the submonomer method. a completely artificial protein [18]. Recently, a single-
(a) 10mer, (b) 38mer, (c) 50mer. chain multi-helical compact protein-like nanostructure was
synthesized by linking individual helical units together [6].
A distinguishing feature of peptoids is that we can The ability to synthesize robust helices, displaying a
incorporate literally hundreds of different side chain wide variety of chemical functionalities that can self
moieties into a specific sequence of defined polymer length. assemble into ordered structures offers us a unique platform
Since the side chain functionality is introduced via a to create novel functional nanostructured materials.
primary amine submonomer, we can use a tremendous
number of commercially available amines directly as 3 COMBINATORIAL DISCOVERY OF
building blocks [3]. In addition, we have developed a NANOMATERIALS
variety of methods to incorporate many kinds of polar,
reactive and heterocyclic functionalities into peptoids [9]. Ultimately, we aim to create stable nanostructures with
This allows rapid synthesis of biomimetic oligomers with protein-like functions from non-natural polymers. But
far more chemical diversity than natural peptides, which many challenges remain before we can rationally design a
vastly increases the probability of discovering novel sequence that will fold into a predictable defined tertiary
oligomers with the desired activity. Methods to incorporate structure. Despite decades of study, the rules that govern
chemoselective ligation functionalities in high yield have the kinetics and thermodynamics of folding polymer chains
also been developed [10] which allow peptoids to be readily into stable tertiary structures are still not fully understood.
incorporated into devices. Thus, alternative methods are needed to circumvent this
problem. The way Nature solves the problem is by a

NSTI-Nanotech 2007, www.nsti.org, ISBN 1420061836 Vol. 2, 2007 29

process of sequence evolution: biopolymers sequences are
varied by mutation and the fittest mutant survives on to the
next generation. This iterative process is repeated over and
over, yielding sequences with optimal function. Because
peptoid synthesis itself is not a major limitation, we can
also apply this iterative biomimetic discovery process by
using combinatorial library discovery methods. We make
very large combinatorial libraries of structured peptoid
oligomers and screen them directly for function.
Combinatorial discovery techniques generate large
numbers of compounds in parallel. Much in the way that
the immune system is a library of billions of antibodies that
can recognize foreign molecules, combinatorial libraries
can yield compounds with high binding affinities to targets
and/or potent biological activities [19].
We have used combinatorial chemistry techniques to
make large libraries of peptoid helices, and have screened Figure 5. Custom robotic combinatorial peptoid
them for their ability to self-assemble into compact helical synthesizer capable of fully automated mix & split
bundles [18]. We have linked these helices together to library synthesis.
form a single-chain helical bundle structure [6]. We expect
that this folded structure can serve as the basis to build In order to study each individual oligomer as a separate
functional folded protein-like nanostructures. By compound, it can be advantageous to array out individual
randomizing certain portions of this and related folded resin beads (each containing a unique compound) from the
structures, we aim to select individual sequences capable of synthesis mixture into a multi-well plate. We have
specific functions. developed a bead-arraying instrument that can array out
1000 beads per day into multi-well plates. Peptoids can
3.1 Automated Tools for Synthesis and then be tested for activity as individual compounds. Assays
Screening can be performed while the compound is attached to the
bead, or the compounds can be cleaved and screened in
In order to screen large libraries of peptoids for new solution.
functional structures, we have designed and custom-built One key property of peptoids that is essential to the
several key combinatorial synthesis technologies. The success of our library approach is that peptoid oligomers
high-throughput synthesis and evaluation of thousands of can be sequenced by tandem electrospray mass
individual peptoids is made possible by robotic parallel spectrometry. Thus, once a peptoid-bead is identified from
synthesis, “mix & split” synthesis and single-bead array a library that has the desired activity, the peptoid’s identity
technology. can be unambiguously determined by single-bead
Since the submonomer method [7] requires the iterative sequencing. Since the peptoid backbone fragments along
addition of monomers, a very large number of reagent the amide bond in the mass spectrometer, a ladder of ions is
addition and resin washing steps are required. Automation obtained from which the monomer sequence can be
of this process is essential to produce peptoid oligomers in determined (Fig. 6). We have developed synthesis linkers
a timely and reproducible fashion [20]. We have designed
and built our own custom robotic synthesizers. Each
instrument consists of 30 - 40 fritted reaction vessels, and
allows the parallel synthesis of peptoid oligomers (Fig. 5).
The automated two-step monomer addition cycle takes < 30
minutes, so that the synthesis of a series of 20mers can be
synthesized overnight.
In addition to automation of the chemistry, the robotic
synthesizer is also capable of performing resin mixing and
resin splitting operations required by the “mix & split”
combinatorial synthesis method [20]. This allows a very
large number of different sequences to be synthesized
simultaneously in such a fashion that each resin bead in the
combinatorial mixture contains a single compound [21]. Figure 6. Peptoids can be rapidly sequenced by
This means that we can generate tens of thousands of tandem electrospray mass spectrometry.
Fragmentation at each backbone amide yields a
peptoid oligomers in a single robot run. ladder of ions that can be easily interpreted.

30 NSTI-Nanotech 2007, www.nsti.org, ISBN 1420061836 Vol. 2, 2007

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