Dendronized protein polymers: Synthesis and self-assembly of monodisperse cylindrical macromolecules

Article abstract of DOI:10.1021/ja046965q

Monodisperse dendronized protein polymers (DPPs), cylindrical dendrimers containing protein core, can be efficiently produced through a combined modular biosynthetic strategy. These DPP materials possess predictable size, shape, and solubility. In organic

Full text of DOI:10.1021/ja046965q

Published on Web 07/24/2004
Dendronized Protein Polymers: Synthesis and Self-Assembly of
Monodisperse Cylindrical Macromolecules
Michael A. Zhuravel,†,‡ Nicolynn E. Davis,† SonBinh T. Nguyen,‡ and Ilya Koltover*,†
Materials Science and Engineering and Chemistry Departments, Northwestern UniVersity, EVanston, Illinois 60208
Received May 23, 2004; E-mail: ilya@northwestern.edu
Self-assembly of ordered molecular patterns is a powerful route
towards new materials. Success of this synthetic strategy depends
on the availability of well-defined molecular building blocks with
nanoscale dimensions. Dendrimers, highly branched macromol-
ecules consisting of dendritic wedges (dendrons) attached to a small
core, are one type of such nanoscale building blocks.1 Although
most dendrimers have spherical architectures, recently the prepara-
tion of dendrimers with rodlike, cylindrical shapes has been
described. Percec2 and Schluter3 have shown that the polymerization
of dendronized monomers can give rise to dendritic polymers with
elongated shapes, while Tomalia demonstrated that rod-shaped
dendrimers can be prepared by stepwise growth of dendritic side
chains from a polymeric initiator backbone.4 Dendrimers prepared
by these pioneering strategies possess well-defined diameters (D)
and rigid shapes, but have a statistical distribution of lengths (L).
New synthetic approaches are needed to prepare molecules with
defined L and D that could self-assemble into nanostructures having
long-range order controlled by the exact molecular dimensions.
Current synthetic methods do not allow for the generation of
large polymeric molecules with strictly defined lengths. However,
there are several natural examples of monodisperse macromolecules,
such as rod-shaped viruses5 and biosynthetic polypeptides,6 that
self-assemble into structures with order directly templated by their
well-defined lengths. Motivated by these observations, we have
developed a synthetic approach that combines tools of biology and
chemistry to prepare cylindrical dendrimers with controlled dimen-
sions (Figure 1a). A monodisperse, R-helical polypeptide backbone
is expressed in a bacterial host (Escherichia coli) from engineered
DNA template (gene), thereby defining the length and overall rod
shape of the target cylindrical macromolecule. Synthetic dendrons
are then grafted to the reactive amino acid side chains along the
peptide backbone, covering it with a dendritic shell whose thickness
is controlled by the generation and chemical structure of the
dendrons. The resulting dendronized protein polymer (DPP) has
length L and diameter D defined by the biological and chemical
synthetic components, respectively. These DPPs self-assemble to
form highly ordered liquid crystalline (LC) structures unique to
molecules with absolutely defined shape and size.
Genetically engineered poly-L-glutamic acid was used as the DPP
backbone in the first experimental realization of our synthetic
scheme. We hypothesized that grafting dendritic wedges to every
glutamic acid residue of the homopeptide would maximize the steric
stabilization of the DPP shape and test the efficiency of the dendron-
grafting chemistry. Synthetic genes encoding polyglutamic acids
glun with three discrete lengths n ) 58, 76, and 94 were cloned,
transformed into E. coli cells, and expressed to yield the target-
purified monodisperse polypeptides.
Figure 1. (a) Modular synthesis of cylindrical DPPs. Dendritic wedges A
are grafted to the n reactive amino acid side chains (red) along a
monodisperse R-helical peptide backbone (green), generating a cylindrical
molecule with defined nanoscale length L and diameter D. For clarity, only
four of the grafted dendrons are shown here. (b) 1H NMR spectra of DPPs
with glu76 cores and four different dendritic shells. Note the increase in the
intensity of the dendritic shell resonances relative to that of the peptide
backbone with increasing dendron size.
conformation of the peptide core by forcing intramolecular hydrogen
bonding and produce DPPs that are soluble in organic solvents.
Frechet-type7 poly(benzyl ether) dendrons with an aldehyde function
at the focal point were converted to tosylhydrazones, which afforded
the corresponding diazo-functionalized dendrons (A, Figure 1a)
upon treatment with an aqueous base. Reaction of excess A with
the carboxylic acid side chains of the glun cores in DMSO efficiently
positioned the wedge-shaped benzyl ether dendrons around the
rodlike helical peptide core to complete the DPP cylinder. The large
molecular weight difference between monodisperse DPPs (25-81
kDa, depending on peptide length and dendron type) and unreacted
dendron byproducts (0.1-1 kDa), allowed us to purify DPPs in a
single step using size-exclusion chromatography. Purified DPPs
were isolated as white solids and were completely soluble in the
same common organic solvents as monomeric dendrons, indicating
complete sequestration of the polar peptide cores within the
dendritic jackets.8 To expand the range of similar dendritic
architectures, we synthesized first-generation dendrons with two
(D) and three (T) peripheral benzyls, with dendron sizes increasing
in the order D0 < D1 < T < D2.
1
We chose to conjugate the glun peptides with hydrophobic
dendrons since complete dendronizatition would stabilize R-helical
The H NMR spectra of DPPs dissolved in CDCl3 (Figure 1b)
show the expected R, â, and γ proton signals of the glutamic acid
residues, as well as the benzyl (Bn), aryl (Ar), and phenyl (Ph)
proton signals of dendron side chains. Remarkably, comparison of
† Materials Science and Engineering Department.
‡ Chemistry Department.
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J. AM. CHEM. SOC. 2004, 126, 9882-9883
10.1021/ja046965q CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
The observed long-range ordered self-assembly of DPP materials
is driven by excluded volume interactions between rigid DPP
cylinders.10 In dry DPP films, where a ) D, SAXD scans show
the expected increase of cylinder diameters D with increasing size
of the dendron side chains (Figure 2c). Remarkably, the measured
D values agree very well with dimensional estimates that are based
on simple geometrical computer models of helical peptide back-
bones surrounded by dendritic side chains (12.9 and 29 Å for
glun-D0 and -D2, respectively). Comparison of SAXD scans of the
three glun-D1 molecules dissolved in m-cresol at the same concen-
tration (Figure 2d), shows that the periodicity of DPP stacks
increases with L. Knowing the amount of solvent in each sample,
we estimate that the thicknesses of DPP layers are 8.7, 11.4, and
14.1 nm, identical to the expected lengths L of the respective DPP
molecules.
In conclusion, we have developed a combined biosynthetic route
toward a new class of cylindrical dendrimers with predictable size,
shape, and solubility. Our DPP materials self-assemble into highly
ordered LC phases with ordering length-scales (a, d) controlled by
the dimensions of the DPP molecules. The modular nature of our
biosynthetic strategy can be used to expand the range of accessible
DPP interactions and shapes through the variation of peptide
sequences, dendron functionality, and dendron architectures. These
studies, as well as complete investigations of the unique liquid
crystalline phases formed in DPP solutions, are currently in
progress.
Figure 2. (a) SAXD pattern of glu76-D1 DPP dissolved in m-cresol (40 wt
% solvent) showing lamellar (along the z-axis) and hexagonal (along the
xy-plane) reflections. (b) Schematic illustration of the self-assembled
structure of DPP solutions. (c) SAXD data of dry films prepared from DPPs
with glu76 cores and varying dendron shells showing hexagonal packing
peaks. (d) SAXD data of glun-D1 samples at constant 40 wt % m-cresol
concentrations showing lamellar stacking peaks.
Acknowledgment. This work was supported by the NSF (Grants
DMR-0134874, -0094347, and -0076097) and the ACS-PRF (Grant
37669-G7). X-ray diffraction experiments were carried out at the
DND-CAT beam line of the Advance Photon Source (ANL),
supported by the DOE Contract No. W-31-102-Eng-38.
the integrated signal intensities of the backbone and side-chain
protons reveals essentially quantitative dendron-conjugation for all
synthesized DPPs, regardless of the size of the dendritic wedges
and despite the high density (1.5 Å/residue) of reactive glutamic
acid side chains. MALDI-TOF analyses also confirmed >95%
derivatization of the carboxylic acid groups. Importantly, the success
of our synthetic approach does not depend on 100% efficiency in
the grafting reaction: the L and D of the DPPs are fixed even if a
few of the glutamic acid side chains remain unreacted, as long as
the peptide is mostly covered by the dendrons.
The monodispersity and the R-helical conformation of the glun
peptide backbones establish the three discrete lengths L ) n × 1.5
Å ) 8.7, 11.4, and 14.1 nm for the synthesized DPPs, with four
diameters corresponding to the different types of dendron side
chains.9 The self-assembly of the DPPs was investigated in m-cresol.
Concentrated DPP/m-cresol solutions exhibit optical anisotropy
(birefringence) that increases with the DPP concentrations, indicat-
ing formation of ordered LC structures. Small-angle X-ray diffrac-
tion (SAXD) patterns of DPP/m-cresol solutions, sealed between
mica windows and oriented by gentle shearing, show two orthogonal
sets of sharp diffraction peaks (Figure 2a). Reflections with ratios
of 1:x3:x4 along the xy-plane indicate hexagonal packing of DPP
cylinders with well-defined spacing a between the cylinder axis.
Along the z-axis, reflections with ratios 1:2:3:4 reveal a layered
structure with periodicity d . a. The diffraction pattern is consistent
with the structure shown in Figure 2b: cylindrical DPPs form
periodically stacked layers with thickness equal to L, and hexagonal
packing of molecules within each layer; solvent occupies the space
between the molecules in each layer (a > D) and between the layers
(d > L).
Supporting Information Available: Experimental details (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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poly(γ-benzyl L-glutamic acid). They required addition of CF3COOH (ca.
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