Design, synthesis, and complementary recognition of β-hairpin peptides stabilized by artificial DNA base-pairing amino acids

Article abstract of DOI:10.1039/b924239b

A novel DNA base-pairing β-hairpin peptide involving intramolecular hydrogen bonds for induction of β-hairpin secondary structure and intermolecular complementary hydrogen bonds between thymine and diaminopyridine for molecular recognition has been synthe

Full text of DOI:10.1039/b924239b

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Design, synthesis, and complementary recognition of b-hairpin peptides
stabilized by artificial DNA base-pairing amino acidsw
Katsuhiro Isozaki*a and Kazushi Mikiab
Received (in Cambridge, UK) 18th November 2009, Accepted 13th February 2010
First published as an Advance Article on the web 8th March 2010
DOI: 10.1039/b924239b
A novel DNA base-pairing b-hairpin peptide involving intra-
molecular hydrogen bonds for induction of b-hairpin secondary
structure and intermolecular complementary hydrogen bonds
between thymine and diaminopyridine for molecular recognition
has been synthesized.
bonds between adenine and thymine, and between guanine
and cytosine.7
Amino acid Taa was synthesized from commercially available
5-iodouracil in a three-step chemical transformation involving
alkylation with a haloalkyl ester at the 1-position under
basic conditions, a Sonogashira reaction with a di-Boc-protected
alkynylamine, and subsequent hydrogenation with o-nitroben-
zenesulfonylhydrazide (NBSH),8 as shown in Scheme 1.
Finally, the protected amino acid (Boc)2TaaOEt (4) was
hydrolyzed to afford 5, which was then incorporated into
peptide sequences.
b-Hairpins1–4 have a particular fascination and tremendous
potential as biologically and supramolecularly important
fragments because of their key role in DNA repair2a and
replication2b upon their molecular recognition of, and selective
binding to, DNAs. Much effort has been devoted to the
synthesis of stabilized b-hairpins by linking peptide side-chains
via disulfide bonds,3 complexation to metal atoms,4 or
incorporating hairpin-favorable sequences bearing proline
residues.5 Recently, de novo design has extensively focused
on the development of functional b-hairpin peptides containing
non-natural amino acids as hairpin-inducers.6 These amino
acids stabilize the b-hairpin secondary structure by geometrical
restriction with piperazinone,6a ferrocene,6b,d ornithine,6c
dibenzofuran,6e,h bipyridine,6f and azobenzene6g backbones.
The resulting artificial b-hairpin peptides have been found to
show novel functionalities, such as redox response,6b gelation,6e
electron-transfer,6f and photoisomerization properties.6g
However, there has been no report to date concerning the
molecular recognition abilities of these hairpin-inducers. The
rational incorporation of molecular recognition abilities into
hairpin-inducers could lead to the development of novel
bioorganic supramolecular assemblies between b-hairpins
and DNAs or organic guests such as DNA bases or RNAs.
Herein we report the DNA base-pairing amino acid Taa, which
shows molecular recognition properties based on complementary
hydrogen bonds, as the next generation of b-hairpin-inducers.
The DNA base-pairing amino acid Taa, which contains a
thymine backbone, was designed to have two properties—
intramolecular hydrogen-bonding properties between terminal
amino and carboxyl groups for the induction of b-hairpin
secondary structures, and molecular-recognition abilities
based on complementary hydrogen bonds similar to those
found in DNA bases, which show characteristic molecular-
recognition properties based on complementary hydrogen
Sequential peptide-coupling reactions were carried out in
solution using the peptide fragments Boc-AlaPheVal-OH and
H-AlaPheVal-OMe, which were obtained by deprotection of
Boc-AlaPheVal-OMe (8), and 1-ethyl-3-(3-dimethylamino
propyl)carbodiimide hydrochloride (EDCl) as coupling agents to
afford the heptapeptide Boc-Ala-PheValTaaAlaPheVal-OMe
(7). Interestingly, the second coupling reaction between
Boc-AlaPheVal-OH and H-TaaAlaPheVal-OMe, obtained by
deprotection of 6 with TFA, did not proceed in CH2Cl2
solution, although coupling occurred smoothly in DMF
solution. This observation suggests the presence of a strong
intramolecular hydrogen bond between the deprotected amine
N–H and amide CQO in non-polar media. Peptide 7 was
expected to form a stabilized b-hairpin secondary structure
based on the strong intramolecular hydrogen bonds.
The secondary structure of peptide 7 was identified by
spectroscopic measurements in CHCl3 solution. IR measurements
were used to clarify the intramolecular hydrogen bonds in
peptide 7 (Fig. S1w). Thus, the amide CQO vibration of
peptide 7 was observed at a low wavenumber of 1643 cmꢀ1
,
a typical value for b-sheet peptides.6a,d,e,h,9 In contrast,
non-hydrogen-bonded CQO vibrations were observed for
protected Taa (4) and 8, which are structural components of
peptide 7, at 1693 and 1670 cmꢀ1. The amide N–H stretching
vibration of peptide 7 was also found at lower wavenumbers
(3290 cmꢀ1) than those for 4 (3417 cmꢀ1) and 8 (3425 cmꢀ1).
These IR spectra suggested the b-hairpin structure of peptide
7. 2-D NMR measurements were carried out to identify the
solution structure of peptide 7. ROESY measurements showed
interstrand crosspeaks between peptide backbones or peptide
side chains (Fig. S2w), which directly clarify the b-hairpin
folding structure. These results confirmed the formation of a
b-hairpin secondary structure of peptide 7 based on intra-
molecular hydrogen bonds in CHCl3.
a Organic Nanomaterials Center, National Institute for Materials
Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.
E-mail: ISOZAKI.Katsuhiro@nims.go.jp; Fax: +81-29-860-4718;
Tel: +81-29-860-4718
The stability of the b-hairpin structure was analyzed by CD
measurements. The CD spectra of peptide 7 in CHCl3 show
the characteristic induction CD (ICD), which corresponds
to the absorption band of the achiral uracil ring, at around
b Graduate School of Pure and Applied Sciences, University of
Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan
w Electronic supplementary information (ESI) available. Experimental
section. See DOI: 10.1039/b924239b
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Fig. 1 CD and UV spectra of 4 (2.0 ꢂ 10ꢀ3 M, green), 8 (4.0 ꢂ 10ꢀ3 M,
black), DAP (2.0 ꢂ 10ꢀ3 M, orange), 7 (2.0 ꢂ 10ꢀ3 M, blue), and
7 : DAP = 1 : 1 (2.0 ꢂ 10ꢀ3 M, red) in CHCl3.
The molecular-recognition properties of peptide 7 were
examined using N,N0-diacetyl-2,6-diaminopyridine (DAP)
as a complementary hydrogen-bonding guest.11 1H NMR
titration experiments were carried out between thymine-
derivatives (T-derivative, 7 and 9) and DAP in CDCl3, with
the imide N–H proton chemical shifts of T-derivatives being
shifted downfield with increasing DAP content (Fig. S4w). The
titration curves were fit to a 1 : 1 binding isotherm (Fig. S5,
S6w),12,13 giving the association constants (Ka) between
T-derivatives 7 and 9 and DAP as 700 and 780 Mꢀ1, respectively
(Table 1). Despite the possibility of intra- or intermolecular
hydrogen bonding competition with the peptide backbone, the
association constant of peptide 7 is very similar to those for
structurally simpler T-derivative 9. Such a high association
constant and 1 : 1 complex formation between peptide 7 and
DAP is due to the induction of stable b-hairpin structure
which reduces the undesirable hydrogen bonding competition
with the peptide backbone, thus enabling the thymine moiety
to recognize the complementary DAP molecule.
Formation of a stable 1 : 1 complex between peptide 7 and
DAP was confirmed by IR and CD measurements. Thus, the
IR spectrum of a mixed solution of peptide 7 and DAP (1 : 1,
2.5 ꢂ 10ꢀ2 M, Fig. S1w) in CHCl3 shows amide CQO and
N–H vibrations shifted to lower wavenumbers (1643 and
3290 cmꢀ1, respectively). These values are similar to those
for peptide 7 alone, thereby confirming retention of the
b-hairpin structure of the 1 : 1 complex. The CD spectrum of
Scheme 1 Synthetic scheme of DNA base-pairing amino acid 4 and
subsequent peptide 7.
275 nm, together with a strong negative Cotton effect of the
chiral peptide chains below 230 nm (Fig. 1). Upon heating
the CHCl3 solution from 263 to 323 K, the ICD band was
red-shifted by about 30 nm (Fig. S3aw). This shift suggests
relaxation of the intramolecular hydrogen-bonding structure.
Surprisingly, the same CD pattern was also observed even
in highly polar trifluoroethanol (TFE), although the small
ICD value in this solvent suggests that the intramolecular
hydrogen bonds are much weaker (Fig. S3bw). Upon
increasing the temperature from 263 to 343 K in TFE,
the negative Cotton effect at around 220 nm corresponding
to the b-hairpin secondary structure was red-shifted and
became more negative.4,5g,6a,d,h,10 The CD pattern of
peptide 7 at higher temperature in TFE closely resembles the
CD pattern of 8 in CHCl3, which has a random conformation
with no strong intramolecular hydrogen bonds. These
results indicate the highly stabilized b-hairpin structure of
peptide 7.
Table 1 Association constants between T-derivatives and DAP
T-derivative R1
R2
Ka/Mꢀ1
7
9
BocAFVNH(CH2)3– –(CH2)3COAFVOMe 700
Me Hex 780
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Fig. 2 Most stable conformers of peptide 7 (a) and 1 : 1 complex of 7 and DAP (b) optimized at the B3LYP/6-31G* level. Side chains are shown as
lines for clarification. Red dotted line: intramolecular hydrogen bonds for inducing the b-hairpin, blue dotted lines: intermolecular hydrogen bonds
for DNA-like base pairing.
a solution of peptide 7 and DAP (1 : 1, 2.0 ꢂ 10ꢀ3 M, Fig. 1)
shows a positive ICD around 275 nm and a negative CD below
230 nm, similar to peptide 7. Surprisingly, a new ICD,
corresponding to the absorption band of DAP, is present at
around 290 nm. This result suggests that the 1 : 1 complex
is so stable that the chiral environment of the b-hairpin
structure binds to the non-covalently bonded DAP through
complementary hydrogen bonds.
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secondary structure of peptide 7 and its 1 : 1 complex. Thus,
conformational searches of peptide 7 by semi-empirical
calculations showed that the b-hairpin conformer is the most
stable (Fig. 2). Likewise, conformational searches of the 1 : 1
complex with restriction of the intermolecular hydrogen bonds
between the thymine moiety of 7 and DAP afforded the
b-hairpin structure as the most stable conformer.
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In conclusion, the artificial DNA base-pairing amino acid
T
aa successfully induces a b-hairpin secondary structure when
´
incorporated into peptide chains. The b-hairpin structure of
peptide 7 is so stable that the thymine moiety recognizes
the complementary DAP molecule through intermolecular
hydrogen bonds without steric repulsions and undesirable
intermolecular hydrogen bonds of peptide chains. This work
demonstrates the molecular-recognition properties of artificial
b-hairpin peptides containing DNA base-pairing amino acids,
which could lead to a novel bioorganic supramolecular
architecture with DNAs based on complementary
hydrogen bonds.
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This work was financially supported in part by a Grant-
in-Aid for Young Scientists (Start-up) from the Japan Society
for the Promotion of Science and a KANEKA Corporation
Award in Synthetic Organic Chemistry, Japan. The authors
gratefully acknowledge Prof. K. Ariga and Prof. M. Higuchi
for experimental assistance.
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13 GAs-Fit: A program that uses an evolutionary algorithm to solve
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 2947–2949 | 2949