Communications
DOI: 10.1002/anie.200702247
RNA Recognition
Macrocyclic Helix-Threading Peptides for Targeting RNA**
Malathy Krishnamurthy, Kristina Simon, Anita M. Orendt, and Peter A. Beal*
The design and synthesis of RNA-binding small molecules
with the ability to bind with high affinity and selectivity to
specific intracellular targets is an important step in the
development of new tools for the study of RNA function and
new therapeutics that target RNA.[1–3] Unfortunately, syn-
thetic molecules capable of selective binding to predeter-
mined RNA targets are elusive. Our laboratory has developed
helix-threading peptides (HTPs) that target certain duplex
RNA structures selectively by threading intercalation.[4–9]
These molecules have a heterocyclic core that directs them
to sites predisposed to intercalation in RNA.[5] Peptide
functional groups attached to the heterocycle extend into
the dissimilar RNA duplex grooves.[6] The peptide appen-
dages provide stabilizing interactions in the grooves of the
target RNA. However, these short, linear peptides can adopt
multiple, energetically similar conformations. Only a subset of
these conformations allow for specific amino acid–nucleotide
interactions. Thus, entropic losses upon binding can erode the
beneficial effects of groove contacts. To overcome this, we are
currently exploring different approaches to restrict the
conformational flexibility of HTPs. Macrocyclic peptides are
an important class of low-molecular-weight ligands for
biological receptors and new methods for their synthesis
have been reported recently.[10] The cyclic peptide scaffold
offers several advantages over the linear counterpart. The
limited conformational flexibility of a cyclic scaffold enables
orientation of different functional groups on the peptide
backbone into distinct positions in space, thereby providing
rigid recognition surfaces for receptors. In addition, cyclic
peptides often have increased cell permeability compared
with their linear counterparts.[11] Herein, we describe the
generation of macrocyclic HTPs through ring-closing meta-
thesis and the RNA-binding properties of these new mole-
cules.
Efficiency and functional group tolerance have made ring-
closing metathesis (RCM) a popular approach for cyclizing
peptides.[12] RCM has been used effectively to generate
peptides with medium to large ring sizes.[13–16] To apply this
method to HTPs, we chose to append an allyl group to the 2-
phenylquinoline core of these molecules.[8,9] If such a struc-
ture were introduced at the N terminus of a peptide that also
contained an olefin-bearing C-terminal residue, the meta-
thesis reaction would generate a macrocyclic HTP. We
anticipated that this would significantly limit conformational
flexibility in the peptide backbone given the near planarity of
the atoms of the 2-phenylquinoline that are included in the
macrocycle. The requisite carboxylic acid was generated
through a short, efficient synthesis (Scheme 1). Displacement
of a thiomethyl group from the known Meldrumꢀs acid
derivative 1[17,18] by using methyl anthranilate, followed by
thermal cyclization, formed the appropriately substituted
quinolone 2 in 64% yield (Scheme 1).[19] Chlorination at the
C4 atom preceded radical bromination at the benzylic
position to give compound 3.[20] This compound was then
subjected to the conditions of a Stille coupling by using vinyl
tributyltin to form the p-allyl substituent on the 2-phenyl ring
of 4.[21] Amination with Boc-protected 4-aminobenzylamine
in the presence of tin tetrachloride[20] followed by hydrolysis
of the methyl ester provided acid 5 in good overall yield.
Macrocyclic HTPs were prepared on a Rink amide resin
by using standard procedures for assembling peptides with 9-
fluorenylmethoxycarbonyl (Fmoc)-protected amino acids
(Scheme 2). Linear peptide synthesis was initiated by cou-
pling commercially available Fmoc-protected allylglycine to
the support followed by coupling the additional a-amino
acids. The last residue added was 2-(p-allylphenyl)quinoline
acid 5. The resin-bound bis-olefin linear peptides were
subjected to ring-closing metathesis conditions in dichloro-
ethane at 608C for 30 h by using the Hoveyda–Grubbs
second-generation ruthenium catalyst (Scheme 2).[13] HPLC
purification afforded cyclic HTPs 6 and 7, which differ in the
N!C sequence of the a-amino acids present in the macro-
cycle. We measured vicinal coupling constants for the major
product isolated for each peptide. The high J values observed
(15.6 Hz for 6 and 15.3 Hz for 7) are indicative of trans
stereochemistry for the newly formed double bond. An
energy-minimized model for HTP 6 containing the trans
double bond is shown in Scheme 2 (see the Supporting
Information for details).
[*] M. Krishnamurthy, Prof. P. A. Beal
Department of Chemistry
University of California, Davis
One Shields Ave, Davis, CA 95616 (USA)
Fax: (+1)530-752-8995
E-mail: beal@chem.ucdavis.edu
M. Krishnamurthy, K. Simon, A. M. Orendt
Department of Chemistry
University of Utah
315 South 1400 East, Salt Lake City, UT 84112 (USA)
A. M. Orendt
Center for High Performance Computing
University of Utah
315 South 1400 East, Salt Lake City, UT 84112 (USA)
[**] P.A.B. would like to acknowledge support from the National
Institutes of Health (USA) (GM057214). The computational
resources for this project have been provided by the National
Institutes of Health (Grant number NCRR 1 S10 RR17214-01) on the
Arches Metacluster, administered by the University of Utah Center
for High Performance Computing.
Supporting information for this article (including experimental
or from the author.
Ribonuclease footprinting was used to analyze the RNA
binding properties for newly synthesized macrocyclic HTPs
7044
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 7044 –7047