Synthesis, crystal structure, and coordination properties of a helical peptide having β-(3-pyridyl)-l-alanine and l-glutamic acid residues

Article abstract of DOI:10.1016/j.jorganchem.2006.03.051

A novel helical peptide containing β-(3-pyirdyl)-l-alanine (Pal) and l-glutamic acid (Glu) residues has been designed and successfully prepared as a model ligand of metalloenzyme active sites. The helical peptide, Boc-Leu-Aib-Glu-Leu-Leu-Pal-Aib-Leu-OEt (1) (Boc = tert-butoxycarbonyl, Aib = 2-aminoisobutylic acid) yields fine crystals as an acetnitrile solvate. The metal ion binding affinities of 1 were tested for CoCl₂ using UV/vis, CD, Raman, and ¹H NMR spectroscopies. The non-linear fitting calculations have revealed the 1:1 complex for CoCl₂ with the binding constant 3.6 (±0.7) × 10² M^-1.

Full text of DOI:10.1016/j.jorganchem.2006.03.051

Journal of Organometallic Chemistry 692 (2007) 79–87
www.elsevier.com/locate/jorganchem
Synthesis, crystal structure, and coordination properties of a helical
peptide having b-(3-pyridyl)-L-alanine and L-glutamic acid residues
a,
a
b
b
Hiroyuki Oku *, Yosuke Kimura , Mitsuo Ohama , Norikazu Ueyama ,
a
a
Keiichi Yamada , Ryoichi Katakai
a
Department of Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
b
Received 27 February 2006; received in revised form 20 March 2006; accepted 24 March 2006
Available online 3 September 2006
Abstract
A novel helical peptide containing b-(3-pyirdyl)-L-alanine (Pal) and L-glutamic acid (Glu) residues has been designed and successfully
prepared as a model ligand of metalloenzyme active sites. The helical peptide, Boc-Leu-Aib-Glu-Leu-Leu-Pal-Aib-Leu-OEt (1) (Boc =
tert-butoxycarbonyl, Aib = 2-aminoisobutylic acid) yields fine crystals as an acetnitrile solvate. The metal ion binding affinities of 1 were
tested for CoCl2 using UV/vis, CD, Raman, and 1H NMR spectroscopies. The non-linear fitting calculations have revealed the 1:1 com-
plex for CoCl2 with the binding constant 3.6 ( 0.7) · 102 Mꢀ1
ꢀ 2006 Elsevier B.V. All rights reserved.
.
Keywords: b-(3-pyridyl)-L-alanine; L-Glutamic acid; CoCl2; Peptide; Helix; Crystal structure; Circular dichroism spectroscopy; Resonance Raman
spectroscopy; Metalloenzyme; Active site
1. Introduction
tion geometry and the nature of the ligands [10]. Recent
topics of cobalt derivatives are found for ferritins, which
have been used for the nano-fabrication of cobalt-oxide
nanoparticles through the oxidation reaction at the dinu-
clear metal ion center [11–13].
Metalloenzymes such as ferritin and methane monooxy-
genase contain the coordination site and the recognition
site for Fe ions on helical sequences. For example, Glu61
and His57 are on the same helix as estimated coordination
ligands in a ferroxidase center of human mytochondrial
ferritin of heavy chain subunit [1]. In the case of methane
monooxygenase from Methylosinus trichosporium OB3b
Continuing the study of enzyme active site models [14–
16] and peptide crystals [17–21], we have planned to synthe-
size model complexes with a helical peptide to simulate
these enzyme active sites. As an N-ligand, we have used b-
(3-pyridyl)-L-alanine (Pal) instead of L-histidine (His). Pal
is a non-coded amino acid, and is often used as an analog
of His and phenylalanine (Phe) due to its structural similar-
ity [22,23]. The reason why we have chosen Pal residue is the
advantage for the peptide synthesis where the side chain of
Pal does not require any protecting group. On the contrary,
His requires an imidazole protection both on Boc- and
Fmoc-chemistry and has been known as one of ‘‘difficult
amino-acid residues’’. Thus, in this study, we have prepared
a helical sequence having Pal and Glu residues to study the
crystal structure and coordination reaction with CoCl2 as a
structural model of metalloenzyme active sites.
[2], two residues on the same helix, Glua243 and Hisa246
bind an iron atom of the Fe2O2 diamond-core at the hydro-
phobic oxidation cavity.
Cobalt substitution have been widely reported for dinu-
clear iron proteins, such as hemerythrins [3], ribonucleotide
reductase [4], methane monooxygenases [5], ferritines [6,7],
and de novo enzymes [8,9]. Because Co(II) is very similar to
Fe(II) both in ionic radious and charge and has d–d
absorptions that can be a sensitive probe for the coordina-
,
*
Corresponding author. Tel./fax: +81 277 30 1343.
E-mail address: oku@chem.gunma-u.ac.jp (H. Oku).
0022-328X/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2006.03.051

80
H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
2. Experimental
2.1. General procedures
benzotriazole (HOBt) was used as an additive for coupling
efficiency and to prevent racemization. The progress of
each reaction was monitored by TLC with ninhydrin reac-
tion. All the products were purified by recrystallization or
column chromatography. The purity was confirmed by
1H NMR and TLC. The final deprotection reaction for –
OBzl group in the Glu residue was carried out by using
5% Pd–C/H2 in MeOH at room temperature. Physico-
chemical data of the intermediates and the final product
1 were described below.
Leucine and 1-ethyl-3-(3-dimethylaminopropyl) carbo-
diimide hydrochloride (EDCÆHCl) were obtained from
Nihon–Rikagaku–Yakuhin (Tokyo, Japan). Other
reagents for peptide synthesis were obtained from Peptide
Institute. Thin-layer chromatography (TLC) was carried
out by using Kieselgel 60F254 aluminum plates obtained
from Merck. Melting points were measured on a microan-
alytical melting point apparatus, YANACO MP-500P and
were uncorrected. Optical rotations were measured on a
JASCO DIP-1000 digital polarimeter (cell path
length = 10 cm) at 20 ꢁC. 1H NMR spectra were taken
on JEOL lambda-500 and JEOL AL-300 spectrometers
using CDCl3, dimethyl sulfoxide-d6 (DMSO-d6) and
CD3CN as solvents. UV/vis and CD spectra were recorded
on a SHIMADZU UVPC-3100 and on a JASCO J-720,
respectively.
2.2.2. Boc-Aib-Leu-OEt (2)
20
Yield 65%, m.p. = 120–121 ꢁC, ½aꢁD ¼ ꢀ19:7ꢂ (c = 0.1,
MeOH), Rf = 0.70 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 6.81 (d, 1H), 4.86 (s,
1H), 4.54 (m, 1H), 4.14 (q, 2H), 1.71–1.52 (m, 3H), 1.48
(s, 3H), 1.45 (s, 3H), 1.41 (s, 9H), 1.24 (t, 3H), 0.91 (d, 6H).
2.2.3. Boc-Pal-Aib-Leu-OEt (3)
20
Yield 45%, m.p. = 141–142 ꢁC, ½aꢁD ¼ ꢀ25:6ꢂ (c = 0.1,
MeOH), Rf = 0.45 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 8.50 (q, 1H), 8.45 (d,
1H), 7.57 (m, 1H), 7.24 (q, 1H), 6.80 (d, 1H), 6.60 (s,
1H), 5.09 (d, 1H), 4.54 (m, 1H), 4.25–4.13 (m, 3H), 3.10
(q, 2H), 1.70–1.57 (m, 3H), 1.54 (s, 3H), 1.47 (s, 3H),
1.42 (s, 9H), 1.27 (t, 3H), 0.95 (d, 6H).
2.2. Synthesis of peptide 1
2.2.1. Synthetic procedure
The optically active Pal amino acid was prepared by
hydrogenation of N-acetyl-b-(3-pyridyl)-a,b-dehydroala-
nine, which was obtained from N-acetyl-glycine and pyri-
dine-3-aldehyde, followed by acylase-catalyzed enzymatic
resolution [24–26]. Boc amino acids were prepared by stan-
dard procedure using di-tert-butyl dicarbonate, (Boc)2O
[27,28].
The peptide 1 was prepared by stepwise elongation and
fragment condensation starting from L-leucine ethyl ester
hydrochloride (HClÆH-Leu-OEt). The scheme for the pep-
tide synthesis was shown in Fig. 1. The coupling reactions
were carried out using N,N0-dicyclohexylcarbodiimide
(DCC) or 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide
hydrochloride (EDCÆHCl) as a condensation reagent in
purified chloroform or DMF as a solvent. 1-Hydroxy-
2.2.4. Boc-Leu-Pal-Aib-Leu-OEt (4)
20
Yield 75%, m.p. = 77–78 ꢁC, ½aꢁD ¼ ꢀ34:1ꢂ (c = 0.1,
MeOH), Rf = 0.28 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 8.45 (q, 1H), 8.43 (d,
1H), 7.59 (m, 1H), 7.24 (q, 1H), 6.87 (d, 1H), 6.82 (s,
1H), 6.74 (d, 1H), 4.91 (d, 1H), 4.55 (m, 2H), 4.17 (q,
2H), 3.99 (m, 2H), 3.14 (t, 2H), 1.76–1.52 (m, 6H), 1.50
(s, 3H), 1.47 (s, 3H), 1.40 (s, 9H), 1.28 (t, 3H), 0.96–0.88
(m, 12H).
2.2.5. Boc-Leu-Leu-Pal-Aib-Leu-OEt (5)
20
Yield 72%, m.p. = 92–93 ꢁC, ½aꢁD ¼ ꢀ49:7ꢂ (c = 0.1,
MeOH), Rf = 0.30 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 8.45 (q, 1H), 8.38 (d,
1H), 7.58 (m, 1H), 7.21 (m, 2H), 6.96 (s, 1H), 6.84 (d,
1H), 6.54 (d, 1H), 4.98 (d, 1H), 4.67–4.58 (m, 2H), 4.15
(m, 3H), 4.03 (m, 1H), 3.46 (d, 1H), 2.88 (q, 1H), 1.75–
1.57 (m, 9H), 1.55 (s, 3H), 1.51 (s, 3H), 1.44 (s, 9H), 1.27
(t, 3H), 0.98–0.83 (m, 18H).
2.2.6. Boc-Glu(OBzl)-Leu-Leu-Pal-Aib-Leu-OEt (6)
20
Yield 70%, m.p. = 109–110 ꢁC, ½aꢁD ¼ ꢀ48:4ꢂ (c = 0.1,
MeOH), Rf = 0.44 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 8.34 (s, 2H), 7.58 (d,
1H), 7.36 (s, 5H), 7.24–7.13 (m, 3H), 7.02 (s, 1H), 6.92
(d, 1H), 6.70 (d, 1H), 6.23 (d, 1H), 5.15 (s, 2H), 4.76–
4.66 (m, 2H), 4.21 (m, 1H), 4.12–4.05 (m, 3H), 3.89 (m,
1H), 3.54 (d, 1H), 2.79 (t, 1H), 2.55 (m, 2H), 2.06 (m,
2H), 1.79–1.68 (m, 9H), 1.58 (s, 3H), 1.52 (s, 3H), 1.48 (s,
9H), 1.20 (t, 3H), 0.97–0.79 (m, 18H).
Fig. 1. Scheme for the synthesis of the peptide, 1.

H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
81
2.2.7. Boc-Leu-Aib-Glu(OBzl)-Leu-Leu-Pal-Aib-Leu-OEt
(7)
The carboxyl –OH was clearly found in a difference Fourier
map and refined with a riding model as well.
20
Yield 50%, m.p. = 171–173 ꢁC, ½aꢁD ¼ ꢀ6:0ꢂ (c = 0.1,
Crystal and refinement data for 1ÆCH3CN (C54H87N10-
MeOH), Rf = 0.41 (CHCl3–MeOH = 9:1 (=v/v)), 1H
NMR (300 MHz, CDCl3, 25 ꢁC) d 8.47 (s, 1H), 8.40 (d,
1H), 7.84 (d, 2H), 7.66 (d, 1H), 7.34–7.27 (m, 7H), 7.16
(m, 3H), 6.88 (d, 1H), 5.15 (s, 3H), 4.70–4.63 (m, 2H),
4.21–3.92 (m, 6H), 3.57 (d, 1H), 2.79 (t, 1H), 2.58 (m,
2H), 2.14 (m, 2H), 1.83–1.61 (m, 12H), 1.59 (s, 3H), 1.56
(s, 3H), 1.47 (s, 3H), 1.43 (s, 9H, CH), 1.41 (s, 3H), 1.20
(t, 3H), 0.99–0.80 (m, 24H).
O13): P212121 (#19, orthorhombic), a = 10.525(1) A,
˚
3
˚
˚
˚
b = 21.969(2) A, c = 27.091(3) A, V = 6263.9 (13) A ,
Z = 4, Dcalc = 1.513 g/cm3, measured reflections = 55699,
unique reflections = 6323 (Rint = 0.079), R1 = 0.069,
wR2 = 0.069, goodness of fit = 0.86.
3. Results
3.1. Peptide design and synthesis
2.2.8. Boc0-Leu1-Aib2-Glu3-Leu4- Leu5-Pal6-Aib7-Leu8-
OEt9 (1)
To make a helical structure, we have chosen Leu and
Aib residues, which is generally considered as helix-form-
ing units [17–19,31–33]. The a,a-disubstituted amino acid,
Aib, is a sterically hindered amino acid and was used as
a conformational constraint [34,35] and thus suitable for
the stable formation of helical structures. Leu and Aib res-
idues are also suitable to make a soluble helix to various
organic solvents [31]. Especially in a Leu based helix, the
whole molecule is completely covered with iso-butyl side
chains of Leu residues [17–19,31]. When designing our heli-
cal sequence, two metal ion-binding residues, Glu and Pal,
were inserted into the positions where both carboxyl- and
pyridyl-groups are facing each other in an ideal helical
rod, such as -Glu-Leu-Leu-Pal-. Based on these structural
considerations, we have prepared an 8-mer sequence,
Boc-Leu-Aib-Glu-Leu-Leu-Pal-Aib-Leu-OEt (1), accord-
ing to the synthetic scheme (Fig. 1). The amino- and the
carobxy-terminous are both capped with a Boc- and an
ethyl ester, respectively for the helix stabilization and the
solubility to organic solvents. The obtained product, 1 suc-
cessfully shows fine crystallinity and solubility as expected
by the molecular design.
20
Yield 98%, m.p. = 122–123 ꢁC, ½aꢁD ¼ ꢀ33:5ꢂ (c = 0.1,
MeOH), Rf = 0.13 (CHCl3–MeOH-CH3COOH = 25:5:1
(=v/v/v)). ESI-MS (m/z) calcd for C52H87N9O13:1045.6.
1
Found, 1046.8 ([M + H]+). H NMR (500 MHz, CD3CN,
25 ꢁC): for Boc1, d 1.44 (s, 9H); for Leu2, 5.89 (d,
3JNHCa = 4.6 Hz, 1H, NH), 3.90 (1H, CaH), 1.67 (2H,
CbH), 1.50 (1H, CcH), 0.95 (3H, CdH), 0.91 (3H, CdH);
for Aib3, 7.64 (s, 1H, NH), 1.42 (6H, CbH); for Glu4,
3
7.79 (d, JNHCa = 3.0 Hz, 1H, NH), 4.05 (1H, CaH), 2.08
(1H, CbH), 2.00 (1H, CbH), 2.45 (2H, CcH); for Leu5,
3
7.83 (d, JNHCa = 5.3 Hz, 1H, NH), 4.15 (1H, CaH), 1.83
(2H, CbH), 1.56 (1H, CcH), 0.97 (3H, CdH), 0.91 (3H,
3
CdH); for Leu6, 7.48 (d, JNHCa = 5.3 Hz, 1H, NH), 3.96
(1H, CaH), 1.57 (2H, CbH), 1.28 (1H, CcH), 0.83 (3H,
CdH), 0.75 (3H, CdH); for Pal7, 8.49, 7.73, 7.21, 8.39 (pyr-
idine ring of C2H, C4H, C5H, C6H, respectively), 7.15 (d,
3JNHCa = 9.1 Hz, 1H, NH), 4.48 (1H, CaH), 3.47 (1H,
CbH), 2.72 (1H, CbH), 1.50 (1H, CcH), 0.95 (3H, CdH),
0.91 (3H, CdH); for Aib8, 7.64 (s, 1H, NH), 1.42 (6H,
3
CbH); for Glu4, 7.79 (d, JNHCa = 3.0 Hz, 1H, NH), 4.05
(1H, CaH), 2.08 (1H, CbH), 2.00 (1H, CbH), 2.45 (2H,
3
CcH); for Leu9, 7.83 (d, JNHCa = 5.3 Hz, 1H, NH), 4.15
(1H, CaH), 1.83 (2H, CbH), 1.56 (1H, CcH), 0.97 (3H,
CdH), 0.91 (3H, CdH); for OEt9, 4.07 (2H, –CH2–), 1.17
(3H, –CH3).
3.2. Crystal structure of 1
Single crystals of 1 suitable for X-ray crystallography
was successfully obtained from a CH3CN solution as
shown in Fig. 2a. A solution of CH3OH or CHCl3 only
yielded colorless powders not suitable for the diffraction
study. Fig. 2b and c show the stereo drawing of 1 and a
packing diagram, respectively. The crystal structure has
revealed that a pyridyl- and a carboxyl-group are located
on the same side of the helical rod. Therefore this sequence
is suitable for the metal ion chelation on the same helix.
The torsion angles of main and side chains are listed in
Table 1. The / and w torsional angles of the helical resi-
dues from Leu1 to Leu5 have shown similar pairs of angles
except an Aib2 residue ranging from ꢀ61.3 to ꢀ65.6ꢁ and
ꢀ19.9 to ꢀ 26.3ꢁ, respectively. In the Aib2 residue, the /
and w values (ꢀ54.8ꢁ, ꢀ23.0ꢁ) very close to the helical part
of Leu1–Leu5. As a whole, the backbone torsion angles for
residues from Leu1 to Leu5 lie in the right handed 310 helix
region and not a-Helix in a Ramachandran plot [36–39].
For the side chains of Leu residues, the torsion angles of
2.3. X-ray crystallography
To obtain single crystals suitable for diffraction analysis,
for example, 10 mg of 1 was dissolved in 0.5 mL of warm
acetnitrile and completely dissolved, resulting in fine plate-
let crystals of 1ÆCH3CN after 3 h and kept for 12 h at
15 ꢁC. X-ray diffraction data were collected on a Rigaku
R-AXIS RAPID imaging plate area detector with mono-
˚
chromated Cu Ka radiation, k = 1.5418 A at ꢀ100 ꢁC.
We have successfully solved the structure of 1ÆCH3CN by
a direct method for macromolecular crystals, Sir2002
[29]. An empirical absorption collection program, DIFABS
[30] was applied which resulted in transmission factors
ranging from 0.76 to 1.07. Non-hydrogen atoms were
refined with anisotropic displacement tensors. H-atoms
except at the carboxyl group –OH of Glu3 were located
in calculated positions and refined with a riding model.

82
H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
Fig. 2. (a) A polarized light photograph of a crystal of 1, about 3 mm on a side. (b) Stereo drawing of 1 with 50% thermal ellipsoids. All hydrogen atoms
except NHs are omitted for clarity. (c) A packing diagram of 1. Intermolecular (purple) and intramolecular (green) hydrogen bonds are indicated as dotted
lines (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).
Leu4 and Leu5, which are located in the middle of the helix,
have suggested a stable orientation, gꢀ(t,gꢀ) [40–43]. The
rest of Leu residues, the chain head Leu1 and the chain
end Leu8 adopt the other stable form, t(g+,t). Similar con-
formational tendencies for the Leu side chain have been
observed for an 11-residue helix, Boc-Leu-Leu-Ala-(Leu-
Leu-Lac)3-Leu-Leu-OEt [17]. In this helix, the chain head
and the chain end Leu adopt the same t(g+,t) conforma-
tion. The rest of Leu residues show gꢀ(t,gꢀ) as observed
in the middle of the helix of 1. Therefore these pairs of v1
and v2 parameters are probably favourable for the L-leu-
cine based right-handed helices.
The intramolecular and intermolecular hydrogen bonds
are listed in Table 2. A total of seven intramolecular hydro-

H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
83
Lac5 followed by the type I b-turn from Leu4 to Aib7, and
ends with the other reverse turn from Glu3 to Leu8. The
shape of the whole molecule resembles a hydrophobic
rod with an end-cap. Complete covering of the polar pep-
tide main chain with hydrophobic alkyl side chains clearly
contribute to form a stable helical structure to hide amide
moieties in the main-chain and thus soluble to various
organic solvents.
Table 1
Torsion angles (ꢁ) for Boc0-Leu1-Aib2-Glu3-Leu4-Leu5-Pal6-Aib7-Leu8-
OEt9 (1)
Residues Angles
/
w
x
v1
v2
Boc0
Leu1
ꢀ173.3(5)
ꢀ63.7(7) ꢀ26.3(8)
ꢀ54.8(7) ꢀ23.0(8)
176.8(5) ꢀ167.1(6)
59.4(8)
179.2(6)
Aib2
Glu3
Leu4
179.7(5)
ꢀ61.3(7) ꢀ19.9(8) ꢀ176.5(5)
76.1(7)
ꢀ76.0(7)
171.7(5)
ꢀ61.5(9)
ꢀ179.0(6)
ꢀ68(1)
ꢀ65.6(7) ꢀ20.9(8)
174.7(5)
3.3. Metal ion binding studies of 1 with CoCl2
Leu5
ꢀ62.0(8) ꢀ22.6(9) ꢀ178.9(5)
ꢀ77.3(8)
ꢀ71.4(7)
173.2(7)
3.3.1. Visible spectra
Pal6
Aib7
Leu8
ꢀ102.4(7)
ꢀ50.6(7)
1.8(8) ꢀ173.0(5)
41.0(7) 170.7(5)
ꢀ71.3(7) ꢀ32.4(9) ꢀ176.0(6) ꢀ171.8(7)
We have monitored the binding properties between 1
and CoCl2 by using a spectral change of visible spectra
(Fig. 3a). In this titration study, the condensed solution
of 1 was gradually added into the 0.1 mM of CoCl2 solu-
tion in CH3CN at 20 ꢁC. Upon addition CoCl2, the inten-
sity of the absorption maximum of at 570, 614, and
684 nm, assignable to d–d transitions of Co(II) ion,
decreased in intensity through many isosbestic points at
552, 578, 600, 632, and 662 nm. After the addition of
enough ligand peptide 1, new maxima found at 589 and
675 nm.
Plots of the decrease in the absorbance at 684 nm will
reach slowly to the saturation when the excess amount of
peptide is added to the solution as shown in Fig. 3b. The
non-linear fitting analysis [49] applied for these data points
has indicated a 1:1 complexation with the formation con-
stant (K) of 3.6 ( 0.7) · 102 Mꢀ1 [50]. The Job plot showed
a maximum value for the absorbance at 684 nm when the
mole fraction of 1 reached 0.5, thereby supporting a 1:1
binding between the peptide and CoCl2 (Fig. 4). Interest-
ingly, a kink was observed for the Job plot at 0.7 mole frac-
tion. This suggests the existence of smaller amount of a 3:2
or 2:1 complex for 1 and CoCl2. In the case of FeCl2 spe-
cies, a 3:2 association was dominantly formed in the same
reaction system.
ꢀ61(1)
ꢀ176.9(7)
The torsion angles for rotation about bonds of the peptide backbone (/,
w, and x) and about bonds of the amino acid side chains (v1, v2) are
described in Ref. [30].
gen bonds are found for carbonyl oxygens and amide NHs
and dotted as purple lines shown in Fig. 2c. Four of these
interactions are of (i, i + 3) types that form a 310 helical
structure from Leu1 to Leu5. The bond length of Nꢃ ꢃ ꢃO
are well corresponded to the reported data for 310-helices
in native proteins [44,45] and in synthetic peptides contain-
ing Aib residues [46,47]. Other intramolecular interactions
are found for the residues of (Glu3, Leu8) and (Leu4, Aib7)
that form a turn structure from Glu3 to OEt9. The hydro-
gen bond between Leu4 C@O and Aib7 NH is a typical type
I b-turn [48] where the pair of (/, w) for Leu5 and Pal6 are
(ꢀ62.0ꢁ, ꢀ22.6ꢁ) and (ꢀ102.4ꢁ, 1.8ꢁ), respectively.
For intermolecular interactions, two hydrogen bonds of
NHꢃ ꢃ ꢃO@C connect the peptide helices into infinite chains
along c-axis. In Fig. 2c, three molecular columns are shown
in this unit cell. The green dotted lines indicate these inter-
molecular hydrogen bonds. The connecting residues are
found at the head to tail positions, Leu1 to Pal6 and Aib2
toAib7. Interestingly the columns are connected each other
by an intermolecular hydrogen bond connecting the side
chains of Glu3 carboxyl- and Pal6 pyridyl-group, –OHꢃ ꢃ ꢃN.
The X-ray structural analysis of 1 is summarized as fol-
lows: the octapeptide begins with the 310-helix from Leu1 to
The Job plot result has suggested the tiny existence of a
peptide:metal = 3:2 or 2:1 complex. However, it seems
curious why the titration result was successfully analyzed
based on a 1:1 complex formation. Actually the non-linear
fitting analysis based on 3:2 complexes has also resulted the
Table 2
Intramolecular and intermolecular hydrogen bond geometries (A, ꢁ) for Boc -Leu -Aib -Glu -Leu -Leu -Pal -Aib -Leu -OEt (1)
0
1
2
3
4
5
6
7
8
9
˚
Acceptor C@O
Donor NH
Oꢃ ꢃ ꢃN
Hꢃ ꢃ ꢃO
Nꢃ ꢃ ꢃHꢃ ꢃ ꢃO
Type (assignment)
O102 (Boc0)
N131 (Pal3)
3.006(6)
2.974(6)
2.930(6)
3.035(6)
3.402(6)
3.090(6)
2.928(6)
3.032(6)
2.696(7)
2.075
2.042
2.048
2.175
2.472
2.161
1.994
2.175
1.489
166.0
166.5
153.7
149.9
166.1
165.7
167.1
152.6
158.5
i, i + 3 (310 helix)
i, i + 3 (310 helix)
i, i + 3 (310 helix)
i, i + 3 (310 helix)
i, i + 5 (reverse turn)
i, i + 3 (type I b-turn)
Head to tail
O111 (Leu1)
N141 (Leu4)
O121 (Aib2)
N151 (Leu5)
O131 (Glu3)
N161 (Pal6)
O131 (Glu3)
N181 (Leu8)
O141 (Leu4)
N171 (Aib7)
O161 (Pal6)
N111 (Leu1)
O171 (Aib7)
N121 (Aib2)
Head to tail
Side chain to side chain
N166 (Pyridyl group of Pal6)
O136 (Carboxyl group of Glu3)
˚
Hydrogen atoms were placed at idealized positions with N–H = 0.95 A.

84
H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
Fig. 4. Job plot for 1 and CoCl2 in CN3CN, monitored at Co(II)
absorption at 684 nm ([Peptide] + [CoCl2] = 0.10 mM at 20 ꢁC).
5 mm NMR gastight tube, followed by the 0.25, 0.50,
1.0, 2.0 equivalent of CoCl2 in each spectral measurement.
As shown in Fig. 6b, the signals of the pyridyl group CHs
(7.0–8.5 ppm), Pal6 CbH2 (2.7 and 3.4 ppm) and Glu3
CcH2 (2.45 ppm) are disappeared in the presence of only
0.25 equivalent of CoCl2. The disappearance has indicated
the coordination to both side chains of Glu3 and Pal6 res-
idues. The resonance signals due to amide NHs which
appeared in the range of 5.5–8.0 ppm, became weak and
broad upon addition of the metal chloride. Those
broaden signals are gradually shifted low field by increas-
ing the amount of CoCl2. Similarly the signals due to
Pal6, Leu1, Leu4, Leu5, Leu8, and Glu3 CaHs, which
appeared at 3.5–4.6 ppm changed to broad multiplets.
Fig. 3. (a) Absorption spectral changes for CoCl2 with the titration of 1 in
CH3CN ([CoCl2] = 0.10 mM, at 20 ꢁC). (b) Absorption changes at
684 nm. Data were analyzed by non-linear curve fitting analysis,
K = [CoCl2-peptide]/[CoCl2][peptide]. The calculated results are
DAbs1 = 0.0238 ( 0.0020) and K = 3.6 ( 0.7) · 102 Mꢀ1
.
formation constant (K) of 4.6 ( 0.5) · 102 Mꢀ2/3 [50],
although the 3:2 species is only dominant at the peptide-
rich environment.
3.3.2. Circular dichroism spectra
Circular dichroism spectra are shown in Fig. 5 for the
model peptide 1 in the absence (solid line) and the presence
of metal ion, CoCl2 (dotted lines) in CN3CN at 20 ꢁC
([peptide] = 1.0 mg/mL). The CD spectrum of 1 exhibited
a positive maximum at 192nm and a negative maximum
at 206 nm, indicating the formation of helical structure.
Only tiny negative shoulder was observed at 222 nm, which
is typically observed for an a-helical structure [51–53]. By
the addition of CoCl2 species to the solution of 1, the con-
tinuous spectral change in intensity are observed for both
CD bands at 191 and 206 nm. This intensity changing of
the spectra suggested the slight conformational stabiliza-
tion of the peptide, probably the increasing the helical
structure among the total conformational ensemble, upon
interaction with CoCl2.
1
3.3.3. H NMR spectra
Changes in the 1H NMR spectrum of 1 before and
after the addition of CoCl2 in CD3CN at 20 ꢁC are shown
in Fig. 6. The sample solution of 1 was prepared in a
Fig. 5. CD spectral changes of 1 by the addition of CoCl2 in CH3CN
([1] = 1.0 mM, at 20 ꢁC).

H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
85
3.3.4. Raman spectra
Fig. 7a and b show the Raman spectra of the concen-
trated oil from a CH3CN solution containing equiva-
lent mol of 1 and CoCl2 with 632.8 and 514.5 nm laser
excitation, respectively. The absorption spectra of this
reaction mixture have absorption maxima at around
570–690 nm that are due to d–d transition bands of
Co(II) as shown in Fig. 3. Therefore the wavenumbers
at 632.8 and 514.5 nm are located on the middle region
and an absorption edge of the d–d spectra, respectively.
With the 632.8 nm laser, the Raman spectra have shown
the enhancements at 311, 283, and 208 cmꢀ1. These
bands can be assignable to the mas(Co–Cl), ms(Co–Cl),
and m(Co–N) stretchings [54,55], respectively, if the com-
plex have a tetrahedral coordination core of CoIICl2NO.
Significant band was not observed for m(Co–O) stretching
that probably dose not show strong enhancement in res-
onance Raman effect due to the mostly ionic and scarcely
covalent character of the carboxylato coordination. Rela-
tively weak but significant bands were observed at 1451
and 1036 cmꢀ1 that are associated to the carboxylate
and pyridine coordinations to the Co(II) atom. In the
case of 514.5 nm excitation, only weak shoulders were
observed in the region, 350–200 cmꢀ1. This is clearly
due to the weak visible absorption of the d–d transition
bands associated with mainly metal d-orbitals and cova-
lent contribution from ligand p-orbitals. On the contrary,
numerous bands were observed in the range 600–
1500 cmꢀ1. Among them, the band at 1451, 1420,
1036 cmꢀ1 are assignable to carboxylate, amide III, pyr-
idyl group, respectively.
Fig. 6. 1H NMR spectral changes of 1 by the titration of CoCl2 in CD3CN
([1] = 3.7 mM, at 20 ꢁC). The spectra of (a) CH signals and (b) amide NHs
and pyridyl groups are shown.
These broaden spectra are indications of the slow
exchange rate between the free species of 1 and CoCl2
and the complex at 20 ꢁC.
Fig. 7. Raman spectra for the in situ formed peptide complex between 1
and CoCl2 (concentrated from CH3CN solution, [1] = [CoCl2] = 2 mM)
with 514.5 and 632.8 nm laser excitation.

86
H. Oku et al. / Journal of Organometallic Chemistry 692 (2007) 79–87
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4. Conclusion
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17276.
We have described the preparation of a helical sequence,
1, which contains Pal and Glu residues by solution-phase
synthesis. The successful analysis by X-ray diffraction has
revealed that the peptide have 310-helical structure in solid
state, because of its short sequence and the containing two
Aib residues. The titration study of 1 has shown relatively
weak chelating ability with CoCl2 as observed in 1H NMR
and visible spectra, although the Raman spectra have
strongly suggested the peptide coordination with a Co(II)
atom. We are now extending our study to histidine peptides
and other metal ions such as FeCl2 toward the total chem-
ical syntheses of metallo-enzyme active sites by combining
X-ray crystallographic analysis.
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5. Deposited material
Crystallographic data for the structure reported in this
paper have been deposited with the Cambridge Crystallo-
graphic Data Centre as CCDC 299275. Copies can be
obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ (Fax: international +44(0)
1223/336-033; E-mail: deposit@ccdc.com.ac.uk). Tables
of crystallographic data, atomic parameters and geometric
parameters are available as Supplementary data.
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61 (2005) o3864–o3866.
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Acknowledgements
HO thanks Grant-in-Aid for Scientific Research on Pri-
ority Areas (No. 14078101 and 16033211, Reaction Con-
trol of Dynamic Complexes) from Ministry of Education
Culture, Sports, Science and Technology, Japan.
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