Decelerated chirality interconversion of an optically inactive 3 10-helical peptide by metal chelation

Article abstract of DOI:10.1039/b803080d

A dynamically optically inactive 3₁₀-helical peptide possessing metal chelating ability between the side-chains at the i and i + 3th residues was synthesized, and its metal complexes were shown to stabilize the peptide structure and the helical sense. The Royal Society of Chemistry.

Full text of DOI:10.1039/b803080d

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Decelerated chirality interconversion of an optically inactive 310-helical
peptide by metal chelationw
Naoki Ousaka,*a Norihiko Tani,b Ryo Sekiyab and Reiko Kuroda*ab
Received (in Cambridge, UK) 21st February 2008, Accepted 19th March 2008
First published as an Advance Article on the web 21st April 2008
DOI: 10.1039/b803080d
A dynamically optically inactive 310-helical peptide possessing
metal chelating ability between the side-chains at the i and i +
3th residues was synthesized, and its metal complexes were
shown to stabilize the peptide structure and the helical sense.
Api residue possessing functional group at the side-chain is
also known to promote the helical structure.8,12 The peptide 1
was shown to adopt the 310-helical structure in the crystalline
and solution states.7d A careful modeling study based on the
crystal structure of 1 indicates that pyridine-3-carboxylic acid
(PyCO2H) is one of the most suitable and relatively small
metal coordinating moieties for the metal chelation between
the side-chains of Api residues at i and i + 3 positions which
are located on the same face of the 310-helix. Thus, ligand
peptide Cbz-[Aib2-Api(PyCO)]2-Aib2-OMe (2) (Scheme 1) was
synthesized from 17d by coupling with PyCO2H after removal
of the protecting Boc groups.w
An intramolecular side-chain metal ligation is a useful method
for stabilizing b-sheet, turn and helical structures in short
peptides.1 Metal ligation of an a-helix was achieved by co-
ordination to two amino acid side-chains spanning i and i + 4
positions located on one face of the helix.2,3 Similar attempts
to stabilize a-helical peptides have been demonstrated by using
side-chain constraints such as salt-,4a disulfide-,4b,c lactam-,4d
hydrazone-bridges,4e hydrogen bond surrogate,4f and hydro-
carbon crosslinks.4g The stabilization of the helical structure
may enhance biological activities and protease resistance
in vitro or in vivo.4f,g The side-chain constraint on residues i
and i + 3 in the 310-helix5 (a relatively common structural
motif in protein helices6) has also been investigated by using
covalent tether7 and salt-bridge formation.8 Fairlie and his co-
workers have demonstrated that His–X–X–His motif binds to
a transition metal ion and the resulting complex adopts some
turn structures.3d Oku and his co-workers have also demon-
strated that a helical structure is stabilized by complexation
with Co(II) ion.9 However, a well-defined 310-helical peptide
with metal ligation to the side-chains has rarely been demon-
strated.
A solution structure of 2 was investigated by 1H NMR
technique, and additionally by computational analysis. 1H
NMR titration experiments in a CD3CN solution were per-
formed upon addition of DMSO-d6 (strong hydrogen bond
accepting solvent) to determine the helix type (310- or a-helix).
The plots of NH chemical shifts against DMSO-d6 percentage
showed that two NH protons shifted substantially to lower
field on increasing DMSO-d6 ratio (shown in red in Fig. 1a),
whereas the remaining six NH protons stayed almost constant
(shown in black in Fig. 1a) due to intramolecular hydrogen
bonding. One of the two solvent exposed NH protons located
at higher field was assigned to the urethane N(1)H, and the
other to N(2)H.13 The structures of 2 optimized by semi-
empirical MO calculation (PM6 method14 in MOPAC2007)
with the four possible starting orientations of side-chain amide
carbonyl groups (types I–IV) afforded a typical 310-helical
conformation; the averaged |f|/|c| dihedral angles fell in the
range of |61| B |63|/|22| B |24|1 for all the four structures.z
Only the lowest energy structure (type II) is shown in
Fig. 2. The other optimized structures are shown in Fig. S2
(see ESIw).
Herein, we report the design, synthesis, and characterization
of a dynamically optically inactive 310-helical peptide7d,10
possessing a metal-chelating ability. The peptide-metal com-
plexes with a 25-membered metallocycle to stabilize the entire
peptide structure and decelerate helix-inversion of the peptide
which is composed of only achiral Ca-tetrasubstituted a-amino
acids.5,11
Variable temperature 1H NMR spectra of 2 (Fig. 3a)
showed that the relatively broad NH resonance at around
7.87 ppm at 20 1C becomes sharp at higher temperatures but
splits at lower temperatures. In contrast, protons neighboring
the N atom on the piperidine ring (around 4.4 ppm) are sharp
at lower temperatures but disappear at higher temperatures,
indicating that rotation of side-chain amide bonds is relatively
slow on the NMR time scale. These data suggest that the split
We chose the same octapeptide we studied previously, Cbz-
[Aib2-Api(Boc)]2-Aib2-OMe (Cbz
=
benzyloxycarbonyl;
Api(Boc) = 1-Boc-piperidine-4-amino-4-carboxylic acid; Boc
= tert-butoxycarbonyl; OMe = methoxy) (1),7d as a rigid
backbone to design the metal-ligating 310-helical peptide. The
high percentage of Aib residues in short peptides is known to
show a strong preference for the 310-helix,5,7–11 and the achiral
a Japan Science and Technology Agency, ERATO-SORST Kuroda
Chiromorphology Team, 4-7-6, Komaba, Meguro-ku, Tokyo, 153-
0041, Japan. E-mail: ousaka@chiromor2.erato.rcast.u-tokyo.ac.jp;
Fax: (+81) 3 5454 6601
b Department of Life Science, Graduate School of Arts and Sciences,
The University of Tokyo, 3-8-1, Komaba, Meguro-ku, Tokyo, 153-
8902, Japan. E-mail: ckuroda@mail.ecc.u-tokyo.ac.jp;
Fax: (+81) 3 5454 6600
w Electronic supplementary information (ESI) available: Experimental
details and additional spectroscopic data. See DOI: 10.1039/b803080d
Scheme 1
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Fig. 1 Solvent dependence on NH chemical shifts of peptides (a) 2
and (b) 3 in CD3CN/DMSO-d6 mixture at 20 1C in 500 MHz 1H NMR
spectra.
NH resonance at lower temperatures corresponds to the
side-chain amide orientations of Api residue.
We have studied the metal-chelation ability of 2. Pd(en)(O-
NO2)2 (en = ethylenediamine) (5) as the cis-blocked square-
planar Pd(II) complex was added to a CD3CN solution of 2. In
the 1H NMR spectrum of 1 : 0.5 (2 : 5) mixture at 20 1C (Fig.
S3w), two sets of resonances were clearly observed, one for 2
alone and the other for complex 2[Pd(en)]2+ (3) (Scheme 1).
After further addition of 0.5 equiv of 5 (total of 1 : 1 (2 : 5)
mixture), the resonances of 2 disappeared completely and only
one set of resonances was observed (Fig. S3w). Similarly, the
1H NMR spectrum of 2[Pt(en)]2+ (4) (Scheme 1) prepared
from 1 : 1 2 : Pd(en)(ONO2)2 stoichiometry showed a single set
of resonances (Fig. S4w), indicating that these peptide–metal
ions form a 1 : 1 complex. These 1 : 1 complexations are also
supported by electron spray ionization mass spectra of 3 and
4. As shown in Fig. 4, complex 3 exhibited an intense peak at
m/z value of 652.05 and a moderate peak at m/z value of
1367.07. These signals are assigned to the positively charged
species 3 and [3(ONO2)]+, respectively. Complex 4 gave a
similar spectrum.
Fig. 2 Side (left) and top (right) views of the right-handed structures
of 2 and 3 as energy-minimized by the semi-empirical MO calculation
(PM 6 method14).17 Hydrogen atoms are omitted for clarity. The
schematic representations indicate the side-chain orientations of amide
carbonyl.
due to slightly stronger hydrogen bonding. The theoretical
study of 3 also supports the 310-helical structure. The opti-
mized structure of 3 is shown in Fig. 2 (PM6 method14).z The
starting model was based on type I of 2, which has two amide
carbonyls in opposite orientations. The other orientations
(types II–IV) cannot make metal coordination without severe
strain. In fact, only single set of resonances is observed in the
NMR spectrum, although the side-chain amide bonds exhibit
slow rotation on the NMR time scale. This orientation was
also observed in the case of previously-reported covalent
system.7d The averaged |f|/|c| dihedral angles of optimized
structure, (|63|/|23|), are similar to the values of 2. These
results suggest that the backbone structures are almost
unchanged upon the metal ligation.y
The solution conformations of these complexes were deter-
mined by the same method used for 2. Only two NHs are
clearly exposed to solvent, indicating that 3 and 4 maintain the
310-helical structure (Fig. 1b, S5w). Some of the NHs exhibit
slight downfield shift compared to 2, however, similar ten-
dency was found in the case of the covalent system,7d probably
In the 1H NMR spectra of complexes 3 and 4, all the
protons of piperidine rings are very sharp and distributed over
Fig. 3 Variable temperature 1H NMR (500 MHz) spectra of 2 (a) and 3 (b) in CD3CN; [2] = 3.5 mM, [3] = 4.0 mM. Blue, green, and red circles
indicate protons of pyridine rings, piperidine rings, and amino groups (en), respectively. The full region spectra are provided in Fig. S6.w
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a wide chemical-shift range (Fig. 3b and S4w).z Positions and
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temperature changes relative to those of 2. Thus, the motion
of the piperidine rings and the rotation of side-chain amides
are constrained in 3 and 4. The protons of pyridine rings also
show similar trends and lower magnetic field shift due to the
coordination with the metal ion.z The resonances of the four
amino protons of ethylenediamine appear at different chemical
shifts due to the broken symmetry caused by complexation
with 2 (Fig. 3b, red circles). Remarkably, N-terminal methy-
lene protons of Cbz group (around 5.17 ppm) show diastereo-
topic splitting (Fig. 3b), although they are placed distant from
the metallocyclic region. The coalescence temperature of the
splitting resonance is above 70 1C (Fig. S6w). This observation
indicates that 3 and 4 exhibit slow helix-inversion.8 In other
words, deformation and re-formation of the hydrogen bonds
and reversal of f/c dihedral angles are necessary for the helix-
inversion, and the helix sense and the structure are stabilized
by the metal coordination.
In summary, we have designed and characterized the first
case of a well-defined 310-helical peptide possessing the metal
chelating site. The side-chain metal ligation of i and i + 3
residues not only stabilizes the structure but also decelerates
the helix-inversion in the optically inactive 310-helical peptide.
To the best of our knowledge, this is the stiffest helical
metallopeptide which is composed of achiral Ca-tetrasubsti-
tuted amino acids with side-chain metal ligation. Control of
the helical structure and its sense by metal-ligation is prefer-
able to covalent-bond formation in terms of by-product for-
mation in preparation. The Api(PyCO) residue possessing
high propensity for helix formation and metal coordination
may serve as a key residue for the de novo design of metal-
binding peptides.
8. T. S. Yokum, M. G. Bursavich, T. Gauthier, R. P. Hammer and
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Notes and references
14. J. J. P. Stewart, J. Mol. Model., 2007, 13, 1173.
15. For examples, see: (a) H. Jiang, J.-M. Le
´
Chem. Soc., 2003, 125, 3448; (b) C. Dolain, J.-M. Le
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z The starting conformations of 2 and 3 are modeled based on the
crystal structure of 17d without changing the backbone dihedral angles.
y Single crystals of these peptides suitable for X-ray diffraction
analysis could not be obtained.
ger and I. Huc, J. Am.
ger,
N. Delsuc, H. Gornitzka and I. Huc, Proc. Natl. Acad. Sci.
U. S. A., 2005, 102, 16146.
z The protons of piperidine rings, pyridine rings, and en are assigned
by 2D COSY spectra after the DMSO-d6 titration experiments
(Fig. S7).w TOCSY measurements for peptides 2 and 3 were performed
(Fig. S8).w
8 The artificial helical molecules composed of achiral non-biological
backbone sometimes exhibit slow helix-inversion,15 and even retain the
helix sense for a long period of time.16 However, in the case of
biological backbone, only one example has been reported.7d
16. For examples, see: (a) E. Yashima, K. Maeda and Y. Okamoto,
Nature, 1999, 399, 449; (b) H.-Z. Tang, P. D. Boyle and
B. M. Novak, J. Am. Chem. Soc., 2005, 127, 2136;
(c) T. Hasegawa, Y. Furusho, H. Katagiri and E. Yashima,
Angew. Chem., Int. Ed., 2007, 46, 5885.
17. The molecular graphics were drawn with: M. A. Thompson,
ArgusLab 4.0.1; Planaria Software LLC: Seattle, WA, 2004
(http://www.arguslab.com).
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This journal is The Royal Society of Chemistry 2008
2896 | Chem. Commun., 2008, 2894–2896