Pentaglycine-NiII complex: From kinetics to structure

Article abstract of DOI:10.1002/ejic.201300039

Binding Ni^II to the terminal amine of a peptide depresses totally its nucleophilic character as measured by the rate constant of the reaction of the peptide with 4-nitrophenyl acetate. The structure of Ni ^II(GGGGG) in aqueous solutions was determined by this technique. The results indicate that the ratio of complexes in which the nickel is bound to four deprotonated peptide nitrogen atoms to those in which the nickel is bound to the terminal amine and three deprotonated peptide nitrogen atoms increases as the pH increases. At pH 9.0, this ratio is approximately 1. We measured the nucleophilic properties of the terminal amine of a Ni(peptide) complex as a tool to elucidate the ligation sites of the cation. Copyright

Full text of DOI:10.1002/ejic.201300039

FULL PAPER
DOI:10.1002/ejic.201300039
Pentaglycine–NiII Complex: From Kinetics to Structure
Zeev Gaisin,[a] Gary Gellerman,[a] and Dan Meyerstein*[a,b]
Keywords: Peptides / Nickel / Nucleophiles / Amines / Kinetics
Binding NiII to the terminal amine of a peptide depresses tot-
ally its nucleophilic character as measured by the rate con- tonated peptide nitrogen atoms to those in which the nickel
ratio of complexes in which the nickel is bound to four depro-
stant of the reaction of the peptide with 4-nitrophenyl acet-
ate. The structure of NiII(GGGGG) in aqueous solutions was
determined by this technique. The results indicate that the
is bound to the terminal amine and three deprotonated pept-
ide nitrogen atoms increases as the pH increases. At pH 9.0,
this ratio is approximately 1.
pected to coordinate to the central cation. The deproton-
ated amide nitrogen atoms are considerably stronger σ do-
nors than the terminal amine, therefore one expects that the
cation will bind to the four deprotonated amide nitrogen
atoms. However, crystallographic studies of copper com-
plexes with pentapeptides[6] and longer peptides[7] and sev-
eral nickel complexes[8] indicate that the metal ion is always
bound to the terminal amine and to three deprotonated
amide nitrogen atoms. In addition, NMR spectroscopic
studies of NiII and CuII complexes of polypeptides and pro-
teins[9] indicate that the terminal NH2 group is one of the
coordination sites. The plausible structures of metal–penta-
peptide complexes are shown in Figure 2, for M = Ni, and
represented by the formulas MII(H–4L)2– and MII(H–5L)3–
in which M is CuII or NiII and L denotes the peptide ligand.
Introduction
The study of the nature of transition-metal complexes
with peptides and proteins is of major biological impor-
tance.[1] It is commonly accepted that the metal cation binds
first to the terminal amine group.[2] The complexes of glyc-
ine peptides with transition metals have been known since
the end of the 1960s, and their properties are discussed in
the literature. The chemical properties of these complexes in
general,[3] and of the triglycine–NiII complex in particular,[4]
have been extensively studied.[2–4] The structure of this com-
plex (Figure 1) prepared under alkaline conditions was con-
firmed by X-ray crystallography.[5]
Figure 1. The structure of the triglycine–NiII complex.
It can be seen that the coordination sites of the peptide
to the central nickel ion are the terminal amine group, two
deprotonated amide nitrogen atoms, and the carboxylate
oxygen anion.
However, the structure of pentaglycine–MII complexes is
more complex. In principle, the metal cation might coordi-
nate to six sites of the ligand: the terminal amine, four de-
protonated amide nitrogen atoms, and the carboxylate. For
CuII and NiII a planar coordination sphere is expected, and
the question is: Which sites coordinate to the cation?
Clearly, carboxylate is the weakest σ donor and is not ex-
Figure 2. Two plausible forms of the pentaglycine–NiII complex.
To the best of our knowledge, the structure of the penta-
glycine–NiII complex was not determined. To shed light on
this dilemma, it was decided to study the following ques-
tions: What is the impact of the complexed nickel ion on
the nucleophilicity of the amine group? And which of the
forms mentioned above of the pentaglycine complex is pres-
ent in solution?
To address these questions, it was decided to carry out
the following experiments. The first is the reaction of tri-
glycine and a triglycine–NiII complex with 4-nitrophenyl
acetate (p-NPA). This compound is a classic electrophilic
substrate that reacts with all ambient nucleophiles: anionic
[a] Biological Chemistry Department, Ariel University,
Ariel, Israel
Fax: +972-2-5343205
E-mail: Danm@ariel.ac.il
[b] Chemistry Department,
Ben-Gurion University of the Negev,
Beer-Sheva, Israel
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201300039.
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and neutral species (e.g., amines).[10] The products of the
nucleophilic attack on p-NPA are 4-nitrophenol (p-NP) and
the acetylated nucleophile, as shown in Scheme 1.
Scheme 1. A general equation of nucleophilic attack on p-NPA.
In the specific case of OH– as the nucleophile, the prod-
uct is the acetate anion (Scheme 2).
Figure 4. The kinetic curve of the reaction of p-NPA and GGG.
The solid line is the first-order fit to the data and the dot is the
experimental data. [GGG] = 1.00ϫ10–2 m, pH 8.0, [p-NPA] =
0.50 mm, kobs = 6.17ϫ10–4 s–1, standard deviation: 1.10ϫ10–5,
400 nm.
Scheme 2. A nucleophilic attack of OH– on p-NPA.
Due to the strong absorbance of p-NP at 400 nm in alka-
line solutions, it is easy to follow the reaction kinetics spec-
trophotometrically. The effect of adding NiII to solutions of
triglycine is a measure of the effect of the nickel ion on the
nucleophilicity of the amine group.
And, secondly, from the analogous reaction of p-NPA with
pentaglycine and its NiII complex, one can determine the
coordination sites of the NiII in this complex.
175.8 (C=O), 177.8 (C=O), 177.8 (C=O) ppm. MS (ESI-
MS): m/z (%): 230 [M – H]– (100.00), 461 [2M – H]– (9.15)}
indicate that the product is 2-[2-(2-acetamidoacetamido)-
acetamido]acetic acid (1). The reaction observed is pre-
sented in Scheme 3.
Results and Discussion
Triglycine and Its Nickel(II) Complex
The reaction between 4-nitrophenyl acetate and the tri-
glycine peptide, GGG, was studied first. Mixing p-NPA
with GGG resulted in a color change due to the formation
of p-NP (Figures 3 and 4).
Scheme 3. The reaction of (Gly)3OH with p-NPA.
The kinetics of disappearance of p-NPA in the presence
of NiII(GGG) were studied. The results (Figure 5) indicate
that kobs is independent of [NiII(GGG)], which suggests
that the reaction observed is the reaction of OH– with p-
NPA. This was confirmed by performing the experiment in
1
an NMR spectroscopy tube with D2O as a solvent. The H
NMR spectrum was recorded at 15, 30, 40 min, and 18 h
after preparing the solution. During the reaction, the in-
Figure 3. Spectral changes during the reaction of p-NPA with
GGG. UV/Vis selected spectra of the reaction mixture at (a) 25,
(b) 675, and (c) 3525 s after mixing the solutions. The peak at
274 nm is due to p-NPA, and the peak at 400 nm is due to p-NP.
[GGG] = 1.00ϫ10–2 m, [p-NPA] = 5.0ϫ10–4 m, pH 8.0.
The product of the reaction of GGG with p-NPA was
1
analyzed by H and 13C NMR spectroscopy and ESI-MS.
The results {1H NMR (300 MHz, D2O): δ = 1.83 (s, 3 H,
CH3), 3.72 (s, 2 H, CH2), 3.75 (s, 2 H, CH2), 3.78 (s, 2 H,
CH2) ppm. 13C NMR (75 MHz, D2O): δ = 24.4 (CH3), 43.6
Figure 5. Dependence of kobs for the reaction of p-NPA with GGG
(CH2), 44.9 (CH2), 45.3 (CH2),174.6 (C=O), 175.0 (C=O), and with NiII(GGG) on the substrate concentration at pH 8.0.
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crease of a singlet peak at δ = 1.88 ppm, which is character-
istic for the acetate anion, was observed. After 18 h only
the signals due to p-NP and acetate were observed. These
results indicate that binding the nickel ion to the terminal
amine group leads to deactivation of its nucleophilic char-
acter.
Pentaglycine and Its NiII Complex
The analogous kinetics of the reaction of GGGGG and
NiII(GGGGG) with p-NPA were measured. The results
(Figure 6) indicate that in this system the complexation by
nickel slows down the reaction by a factor of 7.8 (i.e., some
nucleophilicity is retained). It should be noted that prepar-
ing the complex two weeks prior to the kinetic measure-
ments does not affect the observed rate constant of the re-
action.
Scheme 4. The reaction of (Gly)5OH with p-NPA.
As the binding of the central cation to the peptide is pH-
dependent, it was decided to measure the pH effect on the
rates of reaction. The pH effect on the rate constants of the
reaction of GGGGG with p-NPA (Figure 7) was measured;
the rate constants are (8.6, 9.1, and 14.4)ϫ10–2 –1 s–1 at
m
pH 8.0, 9.0, and 10.0, respectively. The small effect of the
pH on the rate constants is not surprising, because the pKa
of the terminal amine is approximately 8.0.[11] On the other
hand, the rate constant of the reaction of NiII(GGGGG)
with the p-NPA is considerably faster at pH 9.0 than at pH
8.0 (Figure 8). [The latter experiment cannot be performed
at pH 10.0 due to precipitation of Ni(OH)2.] Thus at pH
9.0 the results indicate that approximately 46% of the cen-
tral nickel ions are bound to four deprotonated amide nitro-
Figure 6. The dependence of kobs for the reaction of p-NPA with
GGGGG or NiII(GGGGG) on the substrate concentration; pH
8.0, [p-NPA] = 5.0ϫ10–4 m.
The results presented in Figure 6 are similar to those pre-
sented in Figure 5. Indeed GGGGG also reacts with p-NPA
as a nucleophile. The nature of the final product was deter-
mined by 1H and 13C NMR spectroscopy and ESI-MS. The
results indicate that the product is indeed N–Ac–(Gly)5OH
(2) {1H NMR (500 MHz, D2O + NaOD): δ = 1.96 (s, 3 H,
CH3), 3.70 (s, 2 H, CH2), 3.87 (s, 2 H, CH2), 3.89 (s, 2 H,
CH2), 3.90 (s, 2 H, CH2), 3.92 (s, 2 H, CH2) ppm. 13C NMR
(125 MHz, D2O + NaOD): δ = 21.7 (CH3), 41.8–42.6
(4CH2), 43.2 (CH2), 168.2 (C=O), 171.1 (C=O), 172.1
(C=O), 172.3 (C=O), 172.6 (C=O),175.1 (C=O), 176.5
(C=O) ppm. MS (ESI-MS): m/z (%): 344 [M – H]–
(100.00)}, which is to say, the reaction occurs as outlined
in Scheme 4.
Figure 7. pH effect on the kinetics of the reaction of
NiII(GGGGG) with p-NPA.
However, the binding of NiII to GGGGG does not elim-
inate its nucleophilic properties, although it does decrease
them by a factor of approximately 8, and the same product
is obtained. Formation of acetylated pentaglycine and p-NP
was confirmed by ESI-MS analysis of the reaction mixture.
The results thus suggest that at pH 8.0 in approximately
(1.1/8.6)ϫ100 = 13% of the NiII(GGGGG) complex, the
central nickel cation is bound to four deprotonated amide
nitrogen atoms and not to the terminal amine.
Figure 8. UV/Vis spectra of (a) NiSO4(aq) at pH 5.5, and
NiII(GGGGG) at pH (b) 7.0, (c) 8.0, and (d) 9.0, respectively.
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(peptide or complex in large excess amount relative to 4-ni-
trophenyl acetate). All reaction rate constants are the average of
results obtained from at least two independently prepared samples.
The error limit on the rate constants is ϽϮ10%. In all cases an
excellent correlation to a first-order calculated kinetic curve (Ϯ2%)
was observed.
gen atoms and not to the terminal amine as is commonly
accepted.
Conclusion
The results indicate that the nucleophilic character of the
Complex Preparation: In the general procedure, a peptide and
terminal amine of peptides, as measured by its rate of reac- NiSO4·6H2O [2 equiv. (to avoid uncomplexed peptide)] were dis-
solved in a minimal amount of deionized water. A buffer solution
was added to reach the required volume of the solution. The forma-
tion of the complex was followed by the change of the color of the
solution from blue-green to yellow [note that the spectrum of the
complex at pH 8.0 and 9.0 differs significantly (Figure 8)]. It should
be noted that at pH 8 all the peptide is bound to nickel as the
kinetic data (Figure 6) indicates. After the precipitation of the ex-
cess amount of nickel as a white solid Ni(OH)2, the solution was
centrifuged (3000 rpm, 4 min, room temp.) and its pH was mea-
sured.
tion with p-NPA, can be used to determine the binding site
of a cation to the peptide. Specifically for the complex
NiII(GGGGG) the rate results indicate that the mechanism
of this reaction is as outlined in Scheme 5. The results sug-
gest that the binding site is shifted to the deprotonated
amide nitrogen atoms as the pH increases.
Supporting Information (see footnote on the first page of this arti-
cle): Analytical data of the reactions products.
Acknowledgments
Z. G. is indebted to Mr. M. Gross for his fellowship towards Ph. D.
studies at Ariel University that enabled this study.
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Scheme 5. The reaction mechanism between NiII(GGGGG) and p-
NPA.
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Experimental Section
Materials: Tri- and pentaglycine were purchased from Chem-Impex
International. 4-Nitrophenyl acetate was purchased from Sigma–
Aldrich. The structure of these materials was confirmed by 1H
NMR spectroscopy in D2O as a solvent in the case of peptides,
and CDCl3 for 4-nitrophenyl acetate (Varian 300 spectrometer).
[7] R. A. Bradshaw, W. T. Shearer, F. R. N. Gurd, J. Biol. Chem.
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Kinetics: All kinetic measurements were carried out with an Agilent
8453 Diode Array UV/Vis spectrophotometer. The reactions were
monitored by following the increase in absorbance at 400 nm,
which corresponded to the appearance of the product p-NP. The
reactions were carried out under pseudo-first-order conditions
Received: January 13, 2013
Published Online: May 8, 2013
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