Nanometer-sized amino acids for the synthesis of nanometer-scale water-soluble molecular rods of precise length

Article abstract of DOI:10.1021/ja072648i

This paper introduces the unnatural amino acid Abc^2K as a nanometer-length building block for the creation of water-soluble molecular rods of exceptional size. Abc^2K is a water-soluble variant of the unnatural amino acid 4′-amino-[1,

Full text of DOI:10.1021/ja072648i

Published on Web 05/16/2007
Nanometer-Sized Amino Acids for the Synthesis of Nanometer-Scale
Water-Soluble Molecular Rods of Precise Length
Chris M. Gothard, Nosheen A. Rao, and James S. Nowick*
Department of Chemistry, UniVersity of California, IrVine, IrVine, California 92697-2025
Received April 16, 2007; E-mail: jsnowick@uci.edu
Chart 1. Molecular Model of Abc2K Decamer H-(Abc2K
)10-OH
This paper introduces the unnatural amino acid Abc2K as a
nanometer-length building block for the creation of water-soluble
molecular rods in nanometer unit lengths up to at least ten
nanometers. Over the past two decades, molecular rods have been
developed that can be assembled in precise constitution and length
from individual building blocks.1-4 Many of these oligomers, such
as staffanes and oligo(p-phenylene ethynylene)s, require carbon-
carbon bond formation for their synthesis and are designed to be
soluble in organic solvents.1 Nucleic acids provide one attractive
approach to water-soluble oligomers and have been used as rodlike
components in a variety of architectures.2 Peptides and peptide
derivatives provide a particularly attractive approach, both because
of their biological compatibility and relevance and because of their
ease of assembly through widely available peptide synthesis
technologies.3 Here, we introduce water-soluble rodlike peptides
of exceptional size that are constructed from a 1.0-nm long θ-amino
acid. These θ-peptides are easy to assemble and purify with standard
peptide synthesis and purification technologies and can be combined
with biologically relevant peptides.
Scheme 1. Synthesis of Fmoc-Abc2K(Boc)-OH (1)
of the methyl ether groups with aqueous HBr in AcOH provides
4-bromo-2,5-dihydroxybenzoic acid (4). Protection of the acid group
as the phenacyl ester generates ester 5. Introduction of the Boc-3-
aminopropoxy “K(Boc)” side chains by Williamson ether synthesis
forms diether 6. Suzuki cross-coupling with 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)aniline (6a) using 3-5 mol % PdCl2(dppf)‚
CH2Cl2 gives the biphenyl derivative H-Abc2K(Boc)-OCH2COPh (7).
Fmoc-protection of the amino group of 7 affords Fmoc-Abc2K(Boc)
-
OCH2COPh (8). Finally, removal of the phenacyl group with Zn
in aqueous AcOH provides Fmoc-Abc2K(Boc)-OH (1). Apart from
the modest yield of the Grignard reaction, these reactions afford
76-95% yields and easily allow the preparation of 1 in ca. five-
gram batches.
We designed Abc2K as a water-soluble variant of the unnatural
amino acid 4′-amino-[1,1′-biphenyl]-4-carboxylic acid (Abc) with
lysinelike propyloxyammonium side chains at the 2- and 5-posi-
tions.5 The amino acid Abc2K is readily prepared as building block
1, with Fmoc protection of the aniline main chain and Boc
protection of the side chains, to be compatible with standard Fmoc-
based solid-phase synthesis. Molecular modeling studies of Abc2K
oligomers show only minor effects from torsional and bending
motions and support a model in which the oligomers are relatively
straight and rigid. Monte Carlo-stochastic dynamics (MC/SD)
studies of the decamer H-(Abc2K)10-OH in MacroModel with the
AMBER* force field and GB/SA water solvation at 300 K show
an average length of 10.1 nm with 0.5 nm standard deviation. Only
if unstable cis-amide linkages are introduced does substantial
bending of the rods occur. Chart 1 provides a molecular model of
the decamer H-(Abc2K)10-OH.
Fmoc-Abc2K(Boc)-OH (1) can be used like an ordinary Fmoc-
protected amino acid in solid-phase peptide synthesis. Oligomers
up to and including the decamer (9a-i) were easily prepared on
Rink amide resin and purified by reverse-phase HPLC (RP-HPLC).
Coupling reactions were typically performed with 3 equiv of 1 and
12 hr coupling times. Either HCTU and 2,4,6-collidine or diiso-
propylcarbodiimide and HOAt were used as coupling agents, with
the former eventually being found preferable. The oligomers have
good water solubility and are readily characterized by standard
1
analytical techniques, such as RP-HPLC, ESI-MS, and H NMR
spectroscopy in D2O solution.
Figure 1 illustrates these data for hexamer 9f. The H NMR
1
spectra of the oligomers are sharp at low concentrations but broaden
The Fmoc-Abc2K(Boc)-OH building block (1) is efficiently pre-
pared on a multigram scale from commercially available 1,4-
dibromo-2,5-dimethoxybenzene (2). Generation of a Grignard
reagent from 2 followed by reaction with dry ice and acidic aqueous
workup affords 4-bromo-2,5-dimethoxybenzoic acid (3). Cleavage
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10.1021/ja072648i CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
well-behaved hybrid peptides. To demonstrate the ease of synthe-
sizing and handling peptides containing both R-amino acids and
Abc2K, we synthesized peptide 11, which contains two RGD units
separated by a two-nanometer spacer composed of two Abc2K units.
RP-HPLC analysis showed the crude peptide to be 79% pure, and
purification proceeded smoothly by standard RP-HPLC techniques.
The peptide is water soluble and easy to characterize by ESI-MS
and 1H NMR spectroscopy. We anticipate that the size, water
solubility, and ease of incorporation into peptides will make Abc2K
attractive as a rigid linker in various biologically relevant
applications.1f,3c,9
Figure 1. Characterization data for the Abc2K hexamer H-(Abc2K)6-NH2
(9f): analytical RP-HPLC, ESI-MS, and H NMR spectroscopy.
1
In conclusion, Abc2K oligomers constitute a new class of water-
soluble molecular rods that can be synthesized in precise and
defined lengths up to at least 10 nm in 1 nm increments. The Fmoc-
Adc2K(Boc)-OH building block is compatible with standard solid-
phase peptide synthesis techniques, and the resulting peptides are
water soluble and compatible with analytical and purification
techniques that are standard for peptides. The ease of assembly
and handling of the Abc2K nanometer-scale molecular rods should
permit researchers who are not highly skilled in organic chemistry
to use these new architectural elements in areas such as nanotech-
nology, materials science, and cell biology.
Figure 2. FRET study of peptides 10a-j: (a) fluorescence emission spectra
of 1.75 µM solutions of the peptides in 50% aqueous DMSO solution with
335 nm excitation; (b) fluorescence intensity at λmax
.
at higher concentrations, suggesting that little or no aggregation
occurs at tenth-millimolar concentrations but that self-association
occurs at higher concentrations.
Acknowledgment. We thank the NIH (Grant GM-49076) and
ACS-PRF (Grant 38986-AC1) for financial support. C.M.G. thanks
the UCI Institute for Brain Aging and Dementia for training grant
support (Grant NIA-5T32AG00096). N.A.R. thanks the Pakistan
Higher Education Commission for fellowship support.
Fluorescence resonance energy transfer (FRET) studies support
a model in which the Abc2K oligomers behave as rigid rods with a
length of 1.0 nm per monomer unit (Figure 2).3aef,6 For these studies,
we designed a homologous series of oligomers containing the FRET
donor and acceptor groups EDANS and Dabcyl linked to the amino
acids Glu and Lys and separated by 1-10 Abc2K units [10a-j,
Ac-Lys-Lys-Glu(EDANS)-(Abc2K)n-Lys(Dabcyl)-Lys-Lys-NH2, n
) 1-10].7 We incorporated flanking Lys residues to enhance
solubility and reduce aggregation, which proved to be problematic
with the hydrophobic EDANS and Dabcyl groups.8 FRET studies
were performed in 50% aqueous DMSO (to minimize aggregation
of the longer oligomers) with 335 nm excitation and 400-600 nm
detection. The oligomers exhibit increasing fluorescence with
increasing length through the 8-mer 10h, with the largest increases
occurring between the 4-mer (10d), 5-mer (10e), and 6-mer (10f).
The 9-mer (10i) and 10-mer (10j) exhibit slight decreases and blue-
shifting of the fluorescence.
Supporting Information Available: Synthetic procedures, NMR,
ESI-MS, HPLC data, and other experimental details. This material is
available free of charge via the Internet at http://pubs.acs.org.
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The observed dependence of fluorescence on oligomer length is
reasonably consistent with that predicted by the Fo¨rster equation
6
[energy transfer efficiency ) 1/(1 + R6/R0 )].6 For the EDANS-
Dabcyl pair, the Fo¨rster radius (R0) is 3.3 nm. If each Abc2K unit
provides 1 nm separation (R) between the EDANS and Dabcyl
groups, relatively little fluorescence is predicted for the 1- and
2-mers, substantially increasing fluorescence is predicted for the
3-, 4-, and 5-mers, and nearly complete fluorescence is predicted
for the 5- to 10-mers. The discrepancy between the predicted and
observed fluorescence is consistent with the flexibility of the Glu
and Lys tethers (ca. 1 nm) and is comparable to variations observed
in a related system with a polyproline spacer.3f The slight decreasing
fluorescence and blue-shifting of the 9-mer and 10-mer may reflect
slight aggregation of these compounds.
The Abc2K amino acid can be combined with other amino acids
using standard Fmoc-based peptide synthesis techniques to give
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