Z. Gaisin et al. / Inorganica Chimica Acta 450 (2016) 211–215
213
II
stock solution of 0.20 M, were added and the volume of the solu-
tion was adjusted to 25 ml, yielding solution containing
.1 mM Cu (peptide). The pH of the solutions was adjusted with
.010 M NaOH. (As GGGGG has a poor solubility it was dissolved
solution was added very
slowly and the pH was adjusted with HClO .)
ing out that also in this system the reaction of Cu (GGGGG)(OH) is
observed, the results clearly point out that in less than 10% of the
complex the terminal amine is not bound to the central copper
cation, as 10% of acetylation of the ligand would have been
detected by the ESI-MS analysis. The conclusion that hydroxide
a
II
5
0
in an alkaline solution to which the CuSO
4
II
II
4
ions are axially bound at pH 9.0 to Cu (GGG) and Cu (GGGGG) dif-
fers from the structures suggested in the literature for copper pep-
tides at similar pHs [13,18,19]. However, this result is not totally
surprising as it was proposed that ca. 11% of the triply deproto-
2.4. Product analysis
II
2À
nated Cu (GGGG) are present as [Cu(H-2
G
4
(OH)]
where the
The products of the reaction were analyzed by ESI-MS.
hydroxide is probably bound in an equatorial position [26], though
clearly the results presented herein indicate that for the triply-
3
. Results and discussion
II
deprotonated, or tetra-deprotonated, Cu (GGGGG) a considerably
2À
larger fraction is present as [Cu(H-2
G
5
(OH)]
or as [Cu(H
, where the
-
II
First the reaction of Cu (GGG) with 4-nitrophenyl-acetate, 4-
3À
2À
3À
G
3 5
(OH)] and not as [Cu(H-3
5
G ]
, or [Cu(H-4
G
5
]
NPA, was studied as in this complex clearly the central copper
cation cannot move and the terminal amine is always bound to
the copper cation. The results are presented in Fig. 1. The results
hydroxide is probably bound in an axial position. As potentiomet-
ric titrations [18] of Cu (GGGGG) indicate that even at pH 10.0 only
is nearly the only form of the complex present in
solution and at pH 9.0 a mixture of ca. 1:1 of [Cu(H-2
is present [18] our results indicate that the species
present in the solution, at least in part, is [Cu(H-2
II
2À
[
Cu(H-3
5
G ]
II
at pH 8.0 are analogous to those earlier reported for Ni (GGG),
À
5
G ]
and
II
[
22] i.e. the observed rate constant is independent on [Cu (GGG)].
2À
[
Cu(H-3
5
G ]
II
However, the linear dependence of the rate constant on [Cu (-
GGG)] at pH 9.0 is surprising, as clearly the rise of the pH cannot
affect the binding of the terminal amine to the copper central
cation. To verify this the products of the reaction were analyzed
by ESI-MS and indeed no acetylation of the peptide is observed.
Thus one has to conclude that the raise in the pH causes a binding
of an axial hydroxide to the copper cation, and this hydroxide acts
as the nucleophile in the reaction (Scheme 3).
2À
G
5
(OH)] and
2À
not [Cu(H-3
5
G ]
. Clearly potentiometric titrations cannot differen-
2À
2À
tiate between [Cu(H-2
Similar results are obtained, as expected, also for the reaction of
G (OH)] and not [Cu(H-3G ] .
5
5
II
Cu (GGGGS), i.e. no acetylation of the peptide is observed. Surpris-
ingly the reaction of Ni (GGGGS) with p-NPA at pH 9.0 yields the
II
À
acylated peptide, CH
3
C(O)NH(CH
2
C(O)NH)
3
(CH
2
N(CH
3
)-CH
2
COO .
This result proves that the terminal amine is not bound to the cen-
tral Ni cation though clearly the sarcosine amide cannot be
It is of interest to note that the observed rate constant for the
reaction at pH 9.0 is only somewhat lower than that reported for
II
deprotonated.
II
the reaction of Ni (GGGGG) with p-NPA at this pH [22]. This result
In an effort to determine the nature of the complexes in alkaline
pHs, 10 < pH < 11 the spectra of the solutions were measured. The
results are summed up in Table 1.
Clearly UV–Vis. spectra do not prove a structure but they might
support the kinetic results. The source of the discrepancy in the
II
suggests that the hydroxide bound to the Cu (GGG) is a nucle-
II
ophile with similar properties to the free terminal amine of Ni (-
GGGGG) at this pH. It should be noted that there is no solid
evidence for the site of binding of the hydroxide and it might
replace the carboxylate that might then be the axial ligand, see dis-
cussion below.
II
experimental results for Cu (GGG) is not clear. However, it was
suggested that axial binding to copper peptides shifts the absorp-
tion band to the red [27], this is in accord with our result. Clearly
we cannot determine whether the carboxylate or the hydroxide
II
Next the reaction of Cu (GGGGG) with p-NPA at pH 9.0 was
studied, the results are presented in Fig. 2. It should be noted that
II
at pH 8.0 a precipitate is formed in the presence of Cu (GGGGG)
II
is the axial ligand. The absorption band of Cu (GGGGG) is between
even in the absence of p-NPA. (The precipitate was shown to con-
tain both Cu and GGGGG.)
II
II
those calculated for Cu (H-3
5 5
G ) and Cu (H-4G ), however the
II
2À
kinetic results above suggest [Cu (H-2
5
G (OH)] . The absorption
The observed rate constant of the reaction is similar to that
II
II
bands of the alkaline solutions of Cu (GGGGG) and Cu (GGGGS)
are very similar clearly suggesting that Cu(H-4GGGGG) is not
II
II
observed for Cu (GGG) at this pH but also to that reported for Ni (-
GGGGG) at this pH and to that of GGGGG [22]. In order of deter-
mining the mechanism of reaction the products of reaction were
determined by ESI-MS: no acetylated peptide was observed point-
II
II
formed as Cu (H-4GGGGS) is impossible. The spectrum of Cu ((-
CH GGGGG) is considerably shifted to the red in comparison with
that of Cu (GGGGG) this might be due to the fact that the terminal
di-methylated amine is a considerably weaker -donor than the
3 2
)
II
r
terminal amine [24] or that it was replaced by a hydroxide or water
molecule as an equatorial ligand.
Reaction with p-NPA
pH 9.0
pH 8.0
1
1
1
1
8
6
4
2
.60E-03
.40E-03
.20E-03
.00E-03
.00E-04
.00E-04
.00E-04
.00E-04
y = 6.55E-02x + 3.25E-04
R² = 9.78E-01
4
. Concluding remarks
The kinetic results presented clearly point out that already at
II
II
pH 9.0 the complexes Cu (GGGGG) and Cu (GGGGS) are coordi-
nated, probably axially, at least partially by a hydroxide anion.
The degree of hydroxide coordination cannot be determined kinet-
ically as the rate constant of the bound hydroxide with p-NPA is
y = 4.74E-03x + 2.44E-04
R² = 8.04E-01
II
unknown. Also Cu (GGG) at this pH is coordinated at least partially
0
.00E+00
by a hydroxide anion, in this case the coordination site could not be
determined. The observation that hydroxo complexes are formed
0
.000
0.005
0.010
0.015
0.020
[
GGG-Cu(II)], M
II
in slightly alkaline solutions is not surprising as the Cu com-
2
+
2+
II
II
plexes: Cu(H
amino-acid) (H
Cu (Proteins) [37], are all penta-coordinated. Therefore the
2
O)
5
[29–33], Cu(NH
3
)
5
[34], cis- or trans-Cu (-
Fig. 1. Dependence of the rate of hydrolysis of p-NPA on [Cu (GGG)]. [GGG]/
II
À4
O)
[35],
Cu (Glycylglycine)(H O) [36],
[
CuSO
4
] = 1.19, [p-NPA] = 5 Â 10 M. (The rate constants are the average values of
2
2
2
2
II
all the measured kinetics ± 10%.)