Novel short-chain analogues of somatostatin as ligands for Cu(II) ions. Role of the metal ion binding on the spatial structure of the ligand

Article abstract of DOI:10.1016/j.jinorgbio.2012.08.023

In this paper we present the studies on coordination abilities of two short-chain analogues of somatostatin with free N-terminal and protected amino group towards copper (II) ions. The octreotide is the most popular analogue of the somatostatin (peptide h

Full text of DOI:10.1016/j.jinorgbio.2012.08.023

Journal of Inorganic Biochemistry 117 (2012) 10–17
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Novel short-chain analogues of somatostatin as ligands for Cu(II) ions. Role of the
metal ion binding on the spatial structure of the ligand
Aleksandra Marciniak a, Marek Cebrat b, Żaneta Czyżnikowska a, Justyna Brasuń a,
⁎
a
Department of Inorganic Chemistry, Wroclaw Medical University, Szewska 38, 50‐139 Wroclaw, Poland
b
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14, 50‐383, Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2012
Received in revised form 27 August 2012
Accepted 31 August 2012
Available online 7 September 2012
In this paper we present the studies on coordination abilities of two short-chain analogues of somatostatin
with free N-terminal and protected amino group towards copper (II) ions. The octreotide is the most popular
analogue of the somatostatin (peptide hormone) used in medicine. Somatostatin analogues are used in diag-
nosis and treatment of the neuroendocrine tumors. Both analyzed analogues are characterized by the pres-
ence of two His instead of Cys residues in characteristic fragment of native peptide. We characterize
coordination abilities of the ligands using potentiometric and spectroscopic methods. His-analogues of so-
matostatin are effective ligands for copper (II) ions. Both peptides are able to form the complexes with the
cyclic structure.
Keywords:
Somatostatin
Copper complexes
Histidine
© 2012 Elsevier Inc. All rights reserved.
Potentiometric measurements
Spectroscopic studies
Coordination
1. Introduction
The somatostatin analogues are used in scintigraphy, targeted
chemo- and radiotherapy. The presence of somatostatin receptors in
Somatostatin (SST) is a peptide hormone existing in the human
body which takes part in the regulation of endocrine system and af-
fects neurotransmission and cell proliferation via interaction with so-
matostatin receptors and inhibition of numerous secondary hormones
[1].
The native somatostatin exists in two forms which differ in length
of the peptide chain. The first one is formed by 14 while the second
one by 28 amino acid residues, respectively [1]. Nevertheless, both
peptides have the same cyclic motif in their structures. This motif is
formed by the disulfide bridge between Cys and Cys residues. The
characteristic feature of the SST peptides is also the presence of the
–PheTrpLysThr-sequence [1] (Scheme 1a). The native SST hormone
has a short half-life (about 2 min) in the body. The analogues with
longer half-lives are used in medicine and they are very promising
tool in therapy and diagnosis.
tumor cells causes that somatostatin analogues could be used in visu-
alization and detection of tumors, in control of hormonal hyper-
secretion and tumor growth [4]. In the future new analogues of
somatostatin with high affinity for different somatostatin receptors
should be developed. The study of various radionuclides is also desir-
able [5].
The previous studies on coordination abilities of His-analogues
of vasopressin and oxitocine have shown that insertion of two His
amino acid residues instead of Cys causes huge increase of efficacy
in metal ion coordination. Moreover, both analogues form the cyclic
complex with the {NH2, N−, Nim(macrochelate)} binding mode in the
physiological range of pH [6]. These results allow us to perform
studies on binding abilities of His-analogues of somatostatin with
copper (II) ion. The copper was chosen due to the fact that its iso-
tope, 64Cu, is an attractive radionuclide for PET imaging and radio-
therapy [7,8]. Previous studies show that 64Cu-TETA-OCT could be
used as a PET imaging agent for patients with neuroendocrine tu-
mors [9].
The octreotide (OCT) is the most popular analogue of the somato-
statin used in medicine, e.g. the [111In-DTPA]-octerotide was used for
the scintigraphy of the neuroendocrine tumors [2]. This peptide is char-
acterized by the DPhe-c (Cys-Phe-DTrp-Lys-Thr-Cys)-Thr-ol sequence
[3].
In this paper we present studies performed for two short-chain an-
alogues of somatostatin with free and protected N-terminal amino
group (Scheme 1b,c). Both analogues are characterized by the pres-
ence of two His instead of Cys residues in characteristic fragment of
native peptide. The replacement of both Cys by His residues causes
loss of the cyclic motif in the peptide structure and also insertion of
effective donor atoms for the metal ions, especially for copper. The
⁎
Corresponding author. Fax: +48 71 784 03 36.
E-mail address: justyna.brasun@am.wroc.pl (J. Brasuń).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.08.023

A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
11
of the lyophilized peptides was >97% by analytical HPLC (Thermo
Separation Product; column: Vydac Protein RP C18, 250 Å 4.6 mm,
5 μm; linear gradient 0–100% B in 60 min, solvent A — 0.1% TFA in
water, solvent B — 0.1% TFA in 80% acetonitrile:water solution, UV de-
tection at 220 nm). Chemical identity of the ligands was confirmed by
ESI-MS on a Bruker micrOTOF-Q mass spectrometer. Analytical data
of the synthesized peptides are given in Table 1.
2.2. Potentiometric measurements
Potentiometric measurements were carried out using Molspin
pH-meter system with a Mettler Toledo InLab 422 semimicro com-
bined electrode at 25 °C calibrated in hydrogen ion concentration
using HCl [10]. The ligand concentration was 8×10−4
M and
pH-metric titrations were performed in 0.1 M KCl using sample vol-
umes of 1.2 mL. Alkali was added by using a 0.25 mL micrometer sy-
ringe. Stability constants and stoichiometry of the complexes were
calculated from titration curves using SUPERQUAD program [11].
Scheme 1. Structure of peptides: a) characteristic fragment of somatostatin and its an-
alogues, b) NH2-[His2,7]P1, c) Ac-[His2,7]P2.
analysis of the potentiometric together with spectroscopic results al-
lows us to characterize the binding abilities of investigated ligands.
We performed also molecular modeling investigations in order to de-
termine the possible structures of formed complexes.
2.3. Spectroscopic measurements
Visible spectra of complexes were recorded on Varian Carry 50 Bio
spectrophotometer. The EPR spectra were recorded on Bruker
ELEXSYS E500 CW-EPR, X-Band spectrometer, equipped with ER
036TM NMR Teslameter and E 41 FC frequency counter. Circular di-
chroism (CD) spectra were recorded on a JASCO J 600 spectro-
polarimeter in 300–800 nm range. The same concentrations were
used for both spectroscopic and potentiometric studies.
2. Materials and methods
2.1. Synthesis of the peptides
Peptides were synthesized by the standard manual Fmoc (9-
Fluorenylmethoxycarbonyl) solid-phase peptide synthesis method
on the Fmoc-Rink Amide MBHA resin (0.65 mM/g, Iris Biotech
GmbH). Synthesis was carried out in single-use plastic reactors (Intavis
GmBH). The following amino acid derivatives were used for the
synthesis: Fmoc-Thr (tBu)-OH, Fmoc-His (Trt)-OH, Fmoc-Lys (Boc)-
OH, Fmoc-Trp (Boc)-OH, Fmoc-Phe-OH, and Fmoc-Asn (Trt)-OH (Boc —
t-butoxycarbonyl, Trt — triphenylmethyl). Subsequent Fmoc-protected
amino acids (3 eq) were attached by using 3 eq TCTU (O-(6-
Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluorobo-
rate) as a coupling reagent in the presence of N-hydroxybenzotriazole
(3 eq) and diisopropylethylamine (6 eq) for 2 h at room temperature.
Fmoc protecting groups were removed by 25% piperidine in di-
methylformamide. Acetylation of the N-terminal amino group was
achieved on the resin by 1:1 mixture of acetic anhydride and 0.4 M
N-methylmorpholine in dimethylformamide. Final cleavage of the pep-
tides was achieved by “Reagent K” (81.5% trifluoroacetic acid, 5% phe-
nol, 5% thioanisole, 5% water, 2.5% ethanedithiol, 1% triisopropylsilane)
in 2 h at room temperature. Crude peptides were precipitated by cold
diethylether, washed with ether, dissolved in water and lyophilized.
Peptides were purified by semipreparative HPLC using Varian
ProStar apparatus equipped with TOSOH Bioscience C18 column
(300 Å, 21.5 mm, 10 μm beads) and 220 nm UV detector. Water–
acetonitrile gradients containing 0.1% TFA (trifluoroacetic acid) at a
flow rate of 7 mL/min were used for the purifications. Final purity
2.4. Fluorescence measurements
Fluorescence measurements were performed with Cary Eclipse
fluorescence spectrophotometers using 280 nm excitations for both
N-protected and unprotected derivative of octreotide containing tryp-
tophane. The fluorescence spectra were recorded using 8×10−5
M
peptide solutions at 25 °C. The titration was performed at fixed pH 7
and pH 11.5 as a function of metal concentration. The measurements
were performed for pure ligands and ligand–metal ratios in the
range of 1:0.1–1:8.7.
2.5. Molecular modeling
The geometry optimizations of all complexes were carried out tak-
ing into account solvent effects using the continuum PCM model at
the B3LYP/6-31G (d) level of theory [12,13]. Harmonic frequency
analysis was performed to verify that the structures correspond to a
stationary point on the potential energy surface. The previous study
on the coordination abilities of peptides have confirmed that this
level of theoretical approximation may provide reliable description
of structural as well as energetic properties for bioinorganic com-
plexes [14]. Molecular modeling was performed with the aid of the
Gaussian 09 program [15].
Table 1
Analytical data of the peptides.
Peptide
[M+H]+
Calc.a
[M+H]2+
Calc.a
Rtc
Preparative gradientd
b
b
Found
Found
[min]
NH2-[His2,7]P1
Ac-[His2,7]P2
1069.533
1111.543
1069.531
1111.544
535.270
556.275
535.521
556.28
17.25
16.54
15–25% B in 45 min
15–23% B in 45 min
a
Monoisotopic mass calculated for the indicated ion formed by the peptide.
Monoisotopic mass found by ESI-MS.
Retention time of the crude peptide found by analytical HPLC.
HPLC gradient used for the semipreparative purification of the peptides.
b
c
d

12
A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
3. Results
peptide hormones [6]. The value of logβ*≈6 of the discussed complex
is comparable to the one calculated for the 3N complex with –NH2,
N−
NIm chromophores (logβCuH−1L ≈5.5–6) [21–23], where
logβ*=logβCuHL−(pKLys+pKHis). It supports the –NH2, NIm, N−
3.1. The coordination abilities of the NH2-[His2,7]P1
,
amide
amide
Stability constants for proton and complexes of copper are collect-
ed in Table 2. The studied peptide has four protonation constants
assigned to both His (pK=5.56 and 6.24), free N-terminal amino
group (pK=6.87) and Lys (pK=10.12) amino acid residues and
they are comparable to the protonation constants of these groups
found in the literature [16].
atoms as donors in the CuHL complex. This assumption is also
supported by comparison of the experimental value of λmax for d–d
transition with the theoretical value of λmax calculated for these
chromophores. The value of λmax for d–d transition in absorption
spectrum occurs at 596 nm and corresponds more to the –NH2, NIm
,
N−amide, H2O donor sets with the λmax(theoretical)=599 nm than to the
The NH2-[His2,7]P1 starts copper coordination around pH 3 which is
manifested by the formation of the CuHL species with the highest con-
centration around pH 4 (Fig. 1). The EPR parameters (AII=189 [G], gII=
2.229, Table 2) obtained at pH 4 strongly support binding of three nitro-
gen atoms to the metal ion [17]. There are two main possibilities of
formation of 3N complex by this peptide: with –NH2, NIm2, N−amide or
–NH2, NIm2, NIm7(macrochelate) binding modes. The first type of coordina-
tion is characteristic for the peptides with XaaHis motif [18–20]
while the second one was observed in the case of di-His‐analogues of
–NH2, NIm, NIm(macrochelate), H2O donor sets with λmax(theoretical)=
625 nm [24,25].
Above pH 4 the CuL complex appears in the system and dominates
up to pH 9 (Fig. 1). Blue shift of λmax for d–d transition in absorption
spectrum (≈30 nm) to λmax =567 nm strongly supports binding of
next nitrogen to metal ion. The EPR parameters obtained for the sys-
tem at pH 7.5 (AII =200 [G] and gII =2.211) confirm coordination of
four nitrogens to metal ion [26]. There are two options: i/ binding of
the amide nitrogen of Phe3 amino acid residue and formation of the
Table 2
Stability constants of H+ and Cu (II)-peptide complexes with NH2-[His2,7]P1 and Ac-[His2,7]P2 and the spectroscopic parameters at 25 °C, I=0.1 M (KCl).
log β
pΚ
UV–VIS
λ
EPR
A(
CD
Ε
g(
λ
Δε
(nm)
(M−1 cm−1
)
(G)
(nm)
(M−1 cm−1
)
NH2-[His2,7]P1
HL
10.12 0.03
16.99 0.05
23.23 0.04
28.79 0.05
H2L
H3L
H4L
pKLys
⁎
10.12
6.87
6.24
5.56
pKNH2
⁎
pKHis
pKHis
⁎
⁎
CuHL
21.68 0.01
16.47 0.01
596
567
50
67
189
200
2.229
2.211
605a
358c
309b
568a
482a
343c
300b
–
0.23
0.053
−0.19
0.096
−0.089
0.043
-0.18
–
CuL
CuH−1
CuH−2
CuH−3
L
L
L
6.88 0.02
−3.45 0.02
−14.55 0.02
–
–
–
–
–
–
–
–
–
–
525
107
202
2.208
510a
−0.32
0.12
324c
pKCuHL–CuL⁎⁎
pKCuL–CuH−1L⁎⁎
5.21
9.59
pKCuH−1L–CuH−2L⁎⁎
pKCuH−2L–CuH−3L⁎⁎
10.33
11.10
Ac-[His2,7]P2
HL
H2L
H3L
10.01 0.01
16.59 0.03
22.41 0.03
pKLys⁎
pKHis⁎
pKHis⁎
CuHL
CuL
10.01
6.58
5.82
15.32 0.01
8.89 0.02
2.49 0.01
689
-
587
20
-
54
164
-
183
2.279
-
2.222
-
-
-
-
CuH−1
L
L
583a
365c
327b
632a
490a
352c
318c
−0.17
−0.071
0.059
0.31
−0.88
−0.17
0.28
CuH−2
−5.72 0.02
514
590
65
Sh
198
2.182
pKCuHL–CuL⁎⁎
6.43
6.40
8.21
pKCuL–CuH−1L⁎⁎
pKCuH−1L–CuH−2L⁎⁎
pKCuH−2L–CuH−3L⁎⁎
10.64
a
d–d transition.
b
NIm→Cu(II) CT.
c
N−→Cu(II) CT.
⁎
pKn=logβHn+1−logβHn
.
⁎⁎
pKn=logβCuHn+1L−logβCuHn−1L
.

A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
13
CuL
constants for both His and Lys are comparable to the calculated con-
stant for the unprotected peptide (Table 2).
Cu(II)
100
80
60
40
20
0
CuHL
Protection of the N-terminal amino group significantly changes
the coordination abilities of the peptide. The studied Ac-[His2,7]P2
starts copper (II) binding by formation of the CuHL (Fig. 3) species
similarly to its unprotected analogue. However, the spectroscopic
parameters suggest 2N coordination (Table 2). The value of λmax at
689 nm (Table 2) strongly supports the binding of two imidazole ni-
trogens of both His residues [25]. Due to this process the created
complex has a cyclic structure. Above pH 6 the other two protons
dissociate from the discussed complex and the CuL together with
CuH−1L complexes appears in the system. The CuL complex has the
highest concentration at pH 6.5 where the CuHL and CuH−1L com-
plexes exist in comparable concentrations. Therefore determination
of the spectroscopic parameters for this species was too difficult to
carry out. Appearance of the CuH−1L form causes a blue shift of
λmax in absorption spectrum (to 587 nm, Table 2). The experimental
CuH
L
-1
CuH
L
-2
CuH
L
-3
2
4
6
8
10
pH
value of λmax is in good agreement with the theoretical λmax
=
584 nm calculated for the two amides, one imidazole nitrogen and
one oxygen from water molecule donor sets [25] and EPR parame-
ters (AII =183 [G], gII =2.222, Table 2) confirm coordination of
three nitrogens to Cu (II). Involvement of amide and imidazole nitro-
gens in metal ion coordination is supported by the presence of two CT
bands: negative at 365 nm and positive at 327 nm (Table 2). The
studied peptide has two His residues in its sequence: in the 2nd and
7th positions. It starts copper binding by both His (CuHL species).
Then involvement of amide nitrogens is observed. In the literature it
can be found that His amino acid residue promotes coordination of
Fig 1. Potentiometric profile of NH2-[His2,7]P1 copper (II) complexes; ligand concen-
tration 8×10−4 mol/l; metal to ligand ratio 1:1; I=0.1 M (KCl).
species with the {NH2, 2xN−amide, Nim2} binding mode or ii/ coordination
of the imidazole nitrogen from His7 and formation of the complex with
{NH2, N−amide, Nim2, NIm7} chromophores. The involvement of the sec-
ond amide nitrogen should influence significantly the CD spectrum
between 300 and 350 nm where CT (charge transfer) transitions
N− →Cu (II) are observed. In the analyzed system this effect is not
observed (Fig. 2, Table 1) and the second, {NH2, N−amide, Nim2, NIm7}, co-
ordination mode can be proposed.
its peptidic nitrogen [27]. Due to this fact the {NIm7, N−
,
amide of His7
N−amide of Thr6} binding mode can be proposed.
As pH increases other three complexes, namely CuH−1L, CuH−2
L
Formation of the next CuH−2L complex causes significant changes
in the spectroscopic parameters. The blue shift of λmax in the absorp-
tion spectrum together with the increase of EPR parameters (Table 2)
suggests binding of four nitrogens to the metal ion. The pK value of
proton dissociation and formation of this complex is equal to 8.21
and supports binding of the next amide nitrogen and formation of
the square planar complex with the {Nim, 3×N−amide} binding mode
[28].
Finally, the CuH−3L complex is created in the systems and domi-
nates above pH 11. The value of pK for proton dissociation and forma-
tion of this complex (pK=10.64) and any significant changes in the
spectroscopic parameters suggest proton dissociation from the Lys
residue. This has no impact on binding of the metal ion.
and CuH−3L, appear in the system (Fig. 1, Table 1) but spectroscopic
parameters could be obtained only for the last, CuH−3L, complex. The
appearance of this species causes significant changes in the spectro-
scopic abilities of the system, especially in the CD spectrum (Fig. 2,
Table 1). The blue shift of λmax for d–d transition to 525 nm and the ap-
pearance of positive CT transition at 324 nm show major involvement
of amide nitrogens in metal ion binding. Due to these facts the amino
and three amide nitrogen chromophores in square planar complexes
can be proposed for the CuH−3L species.
3.2. The coordination abilities of the Ac-[His2,7]P2
The protection of the N-terminal amino group affects the Ac-
[His2,7]P2 that has one protonation constant less. The other three
100
CuH
L
-2
Cu(II)
CuH
-1
L
CuH
L
-3
80
60
40
20
0
0,2
0,1
0,0
CuHL
CuL
300
400
500
600
700
800
Δε
λ [nm]
-0,1
-0,2
-0,3
pH 3,00
CuHL
4
6
8
10
CuL
pH
CuH
L
-3
Fig. 3. Potentiometric profile of Ac-[His2,7]P2 copper (II) complexes; ligand concentra-
tion 8×10−4 M; metal to ligand ratio 1:1; I=0.1 M (KCl).
Fig. 2. CD spectra of Cu (II)/NH2-[His2,7]P1 solutions at different pH levels.

14
A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
Fig 4. Fluorescence quenching of investigated complex in the presence of increasing concentrations.
3.3. The structural aspects of Cu (II) binding
Moreover, in the case of NH2-[His2,7]P1 at the same value of pH the
CuL species is formed. At pH=11.5 N-protected derivative of octreotide
forms CuH–L stable complex characterized by {NIm, 3 N−amide} binding
mode. As far as Ac-[His2,7]P2 pure ligand is concerned the maximum of
fluorescence intensities are equal to 10.71 and 11.72 for pH 7 and pH 11,
respectively. The intensity of fluorescence for NH2-[His2,7]P1 at pH 7 is
almost the same, on the other hand at pH 11.5 the intensity is smaller
by about 50%. Quenching of N-protected and N-terminal free deriva-
tives of ligand fluorescence by increasing concentrations of copper
presented in Fig. 4 follows hyperbolic curve. In the case Ac-[His2,7]P2
at pH 7 and pH11.5 and NH2-[His2,7]P1 at neutral pH after adding the
first portion of copper (peptide/metal ratio 1:0.1) lower than 20% fluo-
rescence quenching is observed. In alkaline 25% fluorescence quenching
for NH2-[His2,7]P1 is noticed after adding the same amount of Cu (II).
For ligand/metal ratio 1:0.4 fluorescence quenching increases by
about 50% in the case of both Ac-[His2,7]P2 and NH2-[His2,7]P1 in neutral
3.3.1. Fluorescence measurements
Both investigated ligands contain aromatic amino acids, phenylal-
anine and tryptophan which are responsible for the fluorescence
emission in proteins. Interpretation of peptide fluorescence in the
presence of multiple fluorescent amino acids is a very complicated
task. However, in the considered cases the absorption and emission
spectra of tryptophan residue overlap the spectra of phenylalanine.
In both considered pH, namely 7 and 11.5 the emission maxima of
360 nm are typical for tryptophan fluorophore. Due to the tendency
of excited-state indole to donate electrons Trp appears to be uniquely
sensitive to collisional quenching. Many reports on this subject have
been recorded in the literature [29–31]. As far as the coordination
abilities of Ac-[His2,7]P2 are concerned at pH=7 CuH−1L species
dominates in the solution with {H2O, NIm, 2 N−amide} binding mode.
Fig 5. Stern–Volmer plot for copper (II) quenching of 8∗10−5 M solution of investigated complex.

A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
15
and alkaline solutions. As far as Ac-[His2,7]P2 and NH2-[His2,7]P1
100
Cu(II)
peptides are concerned, fluorescence quenching is almost 100% for
peptide/metal ratio 1:1.2. The fluorescence quenching of investigated
peptides allows determination of the extent of fluorophore (trypto-
phan) exposure to the aqueous solution. To better understand the
quenching mechanism during tritration of peptides with Cu (II) ions,
the data were analyzed using Stern–Volmer equation [32]:
80
60
40
20
0
NH2-[His2,7]OCT
F0=F ¼ 1 þ KSV½Qꢀ;
GlyHisGly
where F0 and F are fluorescence intensities in the absence and presence
of copper, respectively. The KSV is the Stern–Volmer constant, and [Q] is
the concentration of the metal ion [32]. Constant appearing in the above
equation indicates the sensitivity of ligand to the quencher. The plotted
curves (Fig. 5) for quenching by Cu (II) at both considered pH show up-
ward curvature which is indicative for complex (static vs dynamic)
quenching mechanism. The first type of quenching is induced by forma-
tion of nonfluorescent complex while the latter is connected with colli-
sions arising from diffusive encounters between fluorophore and
quenchers. The obtained data indicate that aromatic tryptophan residue
is not buried inside the peptide but on the surface of the created com-
plexes in both considered pH. However, in the case of CuL complex of
NH2-[His2,7]P1 and CuH−1L complex of Ac-[His2,7]P2, aromatic ring of
tryptophan is located closer to the peptide chain. It is in agreement to
our molecular modeling findings.
2
4
6
8
10
pH
Fig. 7. Comparison of the efficacy of copper (II) binding between GlyHisGly and
NH2‐[His2,7]P1.
4. Discussion
Figs. 7, 8 and 9 show the competition diagrams of the ligand1/Cu
(II)/ligand2 systems. These types of diagrams allow comparison the ef-
ficiency in metal ion binding between two ligands. It allows obtainment
of the quantity of bounded metal ions by ligand1 and by ligand2 in each
pH.
3.3.2. Molecular modeling study
In order to analyze the structural aspects of the binding properties
of short-chain analogues of somatostatin towards copper ions we
performed quantum chemical calculations. The initial set of plausible
conformations of the complexes of ligands with Cu (II) at different pH
levels (in total around 30 structures) were optimized at PM6 level of
theory leading to many unique conformations. The most energetically
favored conformations (i.e. those of the lowest total energy including
zero-point energy) were reoptimized using the B3LYP functional. In
this paragraph, we report only on the most stable conformers of com-
plexes with cyclic structures. Fig. 6 contains the lowest-energy struc-
tures of Cu (II)–peptide complexes obtained based on quantum-
chemical calculations. The structural parameters, namely Cu (II)–N
distances vary from 1.9 to 2.2 Å which is in agreement with data
obtained from X-ray diffraction measurements collected in CSD
(Cambridge Structural Database) database [33].
As far as CuHL species for N-protected peptide is concerned, it is
found that the metal ion is coordinated by two nitrogen atoms of his-
tidine moieties. We analyzed also the structure of tetra coordinated
complex of N-terminal free derivative of octerotide. Contrary to
Ac-[His2,7]P2 ligand, the N-terminal part of NH2-[His2,7]P1 is involved
in copper coordination. This peptide is found to bind Cu (II) in the CuL
protonation state. In this case metal is coordinated by two imidazoles,
first amide and amino group nitrogens.
Fig. 7 shows the competition diagram for system NH2-[His2,7]P1/Cu
(II)/Gly-His-Gly [18].
The investigated NH2−[His2,7]P1 starts copper coordination in the
same way as Gly-His-Gly [18] peptide by formation of the first com-
plex with the {NH2, N−amide, NIm2} binding mode. Nevertheless, this
ligand is much more effective in Cu (II) binding than the simple
tripeptide. The significant higher efficiency in Cu (II) coordination of
the NH2−[His2,7]P1 can be explained by the fact that the investigated
peptide forms stable four nitrogen complexes with the {NH2, N−
Im2, NIm7(macrochelate)} binding mode already above pH 5.5 while in
,
amide
N
the same range of pH the Gly-His-Gly forms still 3N complex with
the NH2, N−amide, and NIm2 chromophores.
The comparisons of the efficiency in Cu (II) binding between the
protected ligand, Ac-[His2,7]P2, and similar protected peptides are
presented in Figs. 8 and 9. Fig. 8 shows the comparison of analyzed
peptide with AcGlyHisGlyGly [28] because this peptide has His resi-
due in the position 2 of the peptide chain. In Fig. 9 are compared
Ac-[His2,7]P2 and AcGlyGlyGlyHis [28] because both of them form
the same type of complexes. As it can be seen in both considered
cases the protected analogue of somatostatin is much more effective
in copper coordination. The reason for the higher efficiency in metal
binding by the investigated peptide is probably the same as in the
a) { NH ,N ,N ,N
}
b) {N ,N
}
2
Im1 Im6 amide1
Im1 Im6
Fig. 6. Structures of investigated cyclic complexes of NH2-[His2,7]P1 (a) and Ac-[His2,7]P2 (b). The data were obtained at the 6–31 G (d) level of theory.

16
A. Marciniak et al. / Journal of Inorganic Biochemistry 117 (2012) 10–17
100
80
60
40
20
0
Cu(II)
Ac-[His2,7]OCT
AcGlyHisGlyGly
Fig. 10. The comparison of the calculated structures of the Cys-analogues of analyzed
peptides (blue) and a) CuL complex for NH2-[His2,7]P1, b) CuHL complex for
Ac-[His2,7]P2 with the cyclic structures (red).
2
4
6
8
10
pH
of structures of the mentioned complexes and structure of their
Cys-analogues has shown that the location of the aromatic rings of
Phe and Trp is very similar in the case of protected peptide however
the whole structure of the part with the –Phe-Trp-Lys-Thr-sequence
is not strictly the same.
Fig. 8. Comparison of the efficacy of copper (II) binding between AcGlyHisGlyGly and
Ac-[His2,7]P2.
previously discussed system (unprotected peptide). The presence of
two histidines and formation of the complex with cyclic structure sig-
nificantly facilitate the process of Cu (II) binding.
As it was shown above, studied peptides are able to form com-
plexes with cyclic structure: CuL (in a case of NH2-[His2,7]P1) and
CuHL (in a case of Ac-[His2,7]P2). In order to determine the effects
of somatostatin modifications on the structural properties of aromatic
fragments of NH2-[His2,7]P1 and Ac-[His2,7]P2 the optimal geometries
of NH2-[Cys2,7]P1 and Ac-[Cys2,7]P2 were found.
Acknowledgements
Authors gratefully acknowledge the allotment of the CPU time in
Wroclaw Center of Networking and Supercomputing (WCSS).
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The comparison of aromatic part of complexes and Cys-analogues
of analyzed peptides structures shows that the modification of
N-protected peptide does not causes significant changes in the ar-
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