Versatility and trends in the interaction between Pd(ii) and peptide hydroxamic acids

Article abstract of DOI:10.1039/c9nj00296k

Primary and secondary di- and tripeptide hydroxamic acids, Ala-Ala-NHOH, Ala-Ala-N(Me)OH, Ala-Gly-Gly-NHOH and Ala-Gly-Gly-N(Me)OH were synthesized and their interaction with Pd(ii) (as a Pt(ii) model but with faster ligand exchange reactions) was studied in aqueous solution in the presence of the Cl^- competitor ion by pH-potentiometric and ¹H NMR methods. To the best of our knowledge, this is the first detailed solution study on Pd(ii)-peptide hydroxamate systems revealing that, except for Ala-Gly-Gly-NHOH, the other three ligands act not only as coordination compounds, but also the hydrolysis of the coordinated ligands and formation of the protonated hydroxylamine and Pd(ii) complexes of the corresponding peptides under acidic conditions occurred. The hydrolysis was rather slow with Ala-Gly-Gly-N(Me)OH (more than one week), and just a bit faster with Ala-Ala-NHOH, so speciation studies could also be performed successfully on the systems containing one of the latter two ligands. This was, however, hindered for the Pd(ii)-Ala-Ala-N(Me)OH system, where, in addition to the quite fast hydrolysis of the ligand, the reduction of Pd(ii) to elementary metal by the N(Me)-hydroxylamine formed was also observed. Speciation studies with Ala-Gly-Gly-NHOH revealed the predominance of a very stable 4N-donor complex, (NH₂, 2N_amide, N_hydr.) over a wide pH range. This ligand is also capable of binding the metal ion excess with the hydroxymate (O,O) set in dinuclear species. The formation of this latter type of complex is hindered with the secondary analogue, Ala-Gly-Gly-N(Me)OH, where, in addition to the 3N donor atoms, the hydroxamate-O is also involved in the coordination of the most stable complex. However, the formation of mixed hydroxo species at high pH and a bis-complex in a rather slow process with (NH₂, N_amide)₂ bonding mode in the presence of ligand excess was proven. Although the 3N coordination (NH₂, N_amide, N_hydx) results in a highly stable complex with the dipeptide derivative, Ala-Ala-NHOH, the fourth coordination site remains free for accepting an NH₂ moiety from the excess ligand, or a hydroxide ion at high pH. Likewise, the hydroxymate (O,O) set remains free to bind the metal ion excess in a trinuclear species. The results of this study may also contribute to the design and synthesis of novel Pt(ii) complexes with anticancer potential.

Full text of DOI:10.1039/c9nj00296k

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Versatility and trends in the interaction between Pd(II) and
peptide hydroxamic acids
Received 00th January 20xx,
Accepted 00th January 20xx
András Ozsváth, Etelka Farkas*, Róbert Diószegi, Péter Buglyó*
DOI: 10.1039/x0xx00000x
Primary and secondary di- and tripeptide hydroxamic acids, Ala-Ala-NHOH, Ala-Ala-N(Me)OH, Ala-Gly-Gly-NHOH and Ala-
Gly-Gly-N(Me)OH were synthesized and their interaction with Pd(II) (as a Pt(II) model but with faster ligand exchange
www.rsc.org/
–
1
reactions) was studied in aqueous solution in presence of Cl competitor ion by pH-potentiometric and H NMR methods.
To the best of our knowledge, this is the first detailed solution study on Pd(II)-peptide hydroxamate systems revealing
that, except Ala-Gly-Gly-NHOH, the other three ligands act not only as coordination compounds, but also the hydrolysis of
the coordinated ligands and formation of protonated hydroxylamine and Pd(II) complexes of the corresponding peptides
under acidic conditions occured. The hydrolysis was rather slow with Ala-Gly-Gly-N(Me)OH (more than one week), just a
bit faster with Ala-Ala-NHOH, so speciation studies could also be performed successully on the systems containing one of
the latter two ligands. This was, however, hindered for the Pd(II)-Ala-Ala-N(Me)OH system, where, in addition to the quite
fast hydrolysis of the ligand, the reduction of Pd(II) to elementary metal by the N(Me)-hyroxylamine formed was also
observed. Speciation studies with Ala-Gly-Gly-NHOH revealed the predominance of a very stable 4N-donor complex,
2
(NH ,2Namide, Nhydr.) over a wide pH-range. This is also capable of binding metal ion excess with the hydroxymate (O,O) set
in dinuclear species. The formation of this latter type complex is hindered with the secondary analogue, Ala-Gly-Gly-
N(Me)OH, where, in addition to the 3N donor atoms, the hydroxamate-O is also involved in the coordination of the most
stable complex. However, the formation of mixed hydroxo species at high pH and a bis-complex in a rather slow process
with (NH
hydx) results in a highly stable complex with the dipeptide derivative, Ala-Ala-NHOH, the fourth coordination site remains
free for accepting an NH moiety from excess ligand, or hydroxide ion at high pH. Likewise, the hydroxymate (O,O) set
2 2 2
,Namide) bonding mode in the presence of ligand excess was proven. Although the 3N coordination (NH ,Namide,
N
2
remains free to bind metal ion excess in a trinuclear species. The results of this study may also contribute to the design
and synthesis of novel Pt(II) complexes with anticancer potential.
matrix metalloproteinases and histone deacetylases.1,5,6 Some
may also behave as NO donors under certain conditions . The
7
Introduction
versatile biological activity is due in some part to the
Hydroxamic acids bear the characteristic weak acidic function -
C(O)N(R)OH (R = H in primary, alkyl or aryl moiety in secondary
derivatives).1 The most well-known natural representatives of
these very important bioligands belong to the siderophore’s
6
,8
significant H-bonding ability, but first of all to the metal-
binding ability of these biomolecules. This fact has initiated a
large number of investigations on metal complexes of
hydroxamic acids. Mostly, 3d transition metals have been
involved into the studies and much less literature results can
be found on complexes with other metals, like platinum group
metals. Although by far the most typical coordination mode of
a hydroxamate function has been found to involve chelation
via deprotonated hydroxyl and carbonyl oxygen atoms (O,O),
providing five-membered singly deprotonated hydroxamate
and doubly deprotonated hydroximato chelates. Other modes
,2
3
family and play a crucial role in the microbial iron uptake. A
wide range of potential applications of microbial siderophores
have been considered and some of these compounds, e.g. the
4
desferrioxamine B, are used in medicine for many years. Not
only the natural hydroxamic acid based molecules, but also
numerous synthesized ones are known to show biological
activities, such as hypotensive, anti-cancer, anti-malarial, or
anti-fungal properties, as well as effective and selective
inhibitory effect on numerous metalloenzymes like ureases,
6
,9
are also possible. According to the HSAB theory, the (O,O)
chelating set involves hard donor sites, therefore the most
stable hydroxamato chelates are formed with hard metal ions,
1
0-13
like Fe(III), Co(III), Al(III),
but high stability hydroxamato
Department of Inorganic and Analytical Chemistry, University of Debrecen, H-
032 Debrecen, Egyetem tér 1, Hungary. E-mail: efarkas@science.unideb.hu,
buglyo@science.unideb.hu; tel: + 36 52 512-900
4
(under certain conditions also hydroximato) complexes are
also known with borderline metal ions, such as Cu(II).
1
0
Electronic Supplementary Information (ESI) available: [details of any In fact, there is a linear correlation between the first hydrolytic
supplementary information available should be included here]. See
constant of the metal ions and the stability of the
DOI: 10.1039/x0xx00000x
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hydroxamato chelate with them.11,13 Consequently, the
stability of a hydroxamato chelate is not too high with soft
metal ions. However, the hydroximato-N, in primary Materials and methods
derivatives and in specially designed coordination
environments, was found to become a very effective donor
towards soft metals. This is well-demonstrated by the few
results published on relevant complexes with Pd(II) or with
the half-sandwich type cation of Ru(II).
importance of the well-known H-bonding ability of the
hydroxamic acid moiety in the formation of a trinuclear cluster
ion, was also supported.
Experimental
View Article Online
DOI: 10.1039/C9NJ00296K
Methanol and THF were purified and dried according to
32
literature methods.
Ala-Ala-NHOH·TFA and Ala-Ala-
28-29
N(Me)OH·TFA were prepared as previously described.
Z-
1
4
Ala-Gly-Gly-OH was purchased from Bachem, hydroxylamine-
and N-methylhydroxylamine-hydrochloride from Fluka, Pd/C
(10 % (m/m)) from Merck. All chemicals have the highest
purity commercially available and were used without further
purification. The Pd(II) stock solution was prepared from
1
5,16
In the latter case,
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The metal ion binding behaviour of a hydroxamate group can
dramatically be altered by the incorporation of further groups,
2 4
K [PdCl ] (Merck) using deionised and ultra-filtered water
obtained from a Milli-Q RG (Millipore) water purification
system. The metal ion solution also contained two equivalents
of hydrochloric acid to prevent hydrolysis. The exact
concentration of the stock solution was determined by pH-
1
7
18
such as amino group, or pyridyl-based moiety in the
molecule. Aminohydroxamic acids, in addition to the 5-
membered hydroxamate-type (O,O)-chelate, are also capable
of forming an (N,N)-chelate via the amino-N and deprotonated
hydroxamate/hydroximate-N resulting in 5-membered (N,N)-
type chelate with α-aminohydroxamic acids, 6-membered with Synthesis of the ligands
β ones and 7-membered with γ derivatives. In the interaction
with borderline metal ions (e.g. Cu(II)), both chelation modes
are likely, leading to the formation of interesting clusters, the
most well-known of them belong to the metallacrowns family,
n
in which –[M–N–O] – units are repeated in a cyclic
arrangement.1
preferred by hard metal ions, while the N-donors are favoured
by soft ones. Taking advantage of this different preference of
different metal ions towards the (O,O) and (N,N) chelating
sites large number of supramolecular coordination complexes
have been developed during the past several years via the
33
potentiometric titration using Gran’s method.
Z-Ala-Gly-Gly-NHOH (1). Hydroxylamine hydrochloride (1.92 g,
27.62 mmol) was dissolved in dry methanol (20 mL) and chilled
in an ice-bath. KOH (1.55 g, 27.62 mmol) was added as pellets
and chilled to 0 °C under nitrogen. The reaction mixture was
stirred for 15 min, meanwhile a white precipitate (KCl) was
formed. In another flask Z-Ala-Gly-Gly-OH (3.11 g, 9.22 mmol)
was dissolved in dry THF (150 mL) under nitrogen, and chilled
in an ice-bath to 0 °C. Ethyl chloroformiate (2.11 mL, 22.17
mmol) followed by N-methylmorpholine (2.65 mL, 24.10
mmol) was added and stirred for 30 min under nitrogen,
meanwhile a fine white precipitate (N-methylmorpholinium
chloride) was formed. The free hydroxylamine solution was
filtered into a three-neck flask kept in an ice-bath under
nitrogen, while the mixed anhydride solution was filtered into
a dropping funnel under nitrogen. The latter solution was
added dropwise to the former one within 5 min; the obtained
opalescent reaction mixture was stirred under nitrogen
overnight and kept in an ice-bath. After removal the solvent,
yellowish oil was obtained, which was dissolved in water and
extracted at pH ~ 3–4 with EtOAc (5 × 100 mL). The collected
9-21
However, only the (O,O)-type chelation is
2
2-24
selective coordination to different metal centres.
Pd(II) as typical soft metal ion was chosen to coordinate to the
N,N) donor set and [Pd(en)Cl ] (en = ethane-1,2-diamine), in
Often
(
2
which the soft nitrogen donors of the en group occupy two
coordination sites of the metal centre, was selected as
palladium source.2
even simple dipeptides (without any side-chain donor) are very
effective ligands of Pd(II). Since this metal ion was reported to
be one of the most effective one to promote deprotonation
and coordination of peptide-amide groups,
arises, whether, alternatively, [PdCl
metal ion source to develop supramolecular entities with
hydroxamic acid derivatives of simple oligopeptides. Previous
results with various metal ions also supported that with these
hydroxamic based derivatives the hard metal ions are
coordinated exclusively to the (O,O)-chelate, while e.g. the
rather soft Ni(II) prefers the nitrogens of the peptide
backbone.2 It is also a question, whether only complexation
processes or also Pd(II)-assisted hydrolysis (as it was previously
observed in the case of Pt(II)-glycinehydroxamic acid
system)3 occur. The above open questions initiated the aim
of our study, the investigation of the interaction of Pd(II)-ions
with primary and secondary di- and tripeptide hydroxamic
acids in solution, namely with Ala-Gly-Gly-NHOH, Ala-Gly-Gly-
N(Me)OH, Ala-Ala-NHOH and Ala-Ala-N(Me)OH (their
structures are shown in Chart 1).
2-24
It is well-known from the literature that
2
5,26
the question
] ion can be used as soft
4
organic phases were dried on MgSO , after that the solution
2
-
4
(
B)
O
(C)
O
(D)
O
H
N
H
N
H
N
H3N CH C
(A) CH3
CH2C
CH2C
OH
I
(
B)
H3N CH C
A) CH3
O
(C)
O
O
(D)
H
H
N
CH2C
N
CH2C
N
OH
7-29
(
(E) CH3
II
0,31
(B)
H3N CH C
A) CH3
O
(C)
O
(B)
O
H
O
(C)
H
N
H
CH C
N
OH
H3N CH C
N
CH C
N
OH
(
(D) CH3
(A) CH3
(D) CH3 (E) CH3
III
Chart 1 Structures of the fully protonated ligands
IV
2
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was evaporated to one third and cooled to –20 °C, while fluffy argon. The titrations were performed in the pH-range 2.0-11.0
precipitate was obtained. The crude product was filtered off in equilibrium controlled mode (where equilibrium was
and recrystallized from MeCN. Yield: 0.72 g (22 %). H NMR assumed to be reached when dpH/dt was less than 0.015
View Article Online
DOI: 10.1039/C9NJ00296K
1
(400 MHz, DMSO-d6) δ (ppm): 10.46 (1H, s, CONHOH), 8.84 mV/11 s). The waiting time between two titration points was
(1H, s, CONHOH), 8.04 (1H, t, NH), 8.02 (1H, t, NH), 7.55 (1H, d, found to fall into the range of 150-1800 s because of the quite
NH), 7.36 (5H, m, C
CH), 3.73 (2H, d, CH ), 3.63 (2H, d, CH
Z-Ala-Gly-Gly-N(Me)OH (2). Z-Ala-Gly-Gly-N(Me)OH was stability constants (βpqr
prepared as described for 1 by using N-methylhydroxylamine determined via fitting the titration curves by the PSEQUAD and
hydrochloride (2.31 g, 27.66 mmol) and obtained after SUPERQUAD programs.3
6
H
5
), 5.02 (2H, q, C
6
H
5
-CH
2
), 4.06 (1H, q, slow equilibrium processes. For this reason, relatively high
2
2
), 1.21 (2H, d, CH
3
). fitting parameters were obtained during the calculations. The
p
q
r
=
p q r
[Pd H L ]/[Pd] [H] [L] ) were
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5-36
The stability constants for the
recrystallization of the crude product from EtOAc. Yield: 1.16 g complexes of Pd(II) with chloride ions (logβ
1
= 4.47, logβ
= 11.54 for the species [PdCl] ,
2
=
1
+
(
34 %). H NMR (400 MHz, DMSO-d6) δ (ppm): 9.95 (1H, s, 7.76, logβ
3
= 10.17, logβ
4
–
2–
OH), 8.15 (1H, t, NH), 7.81 (1H, t, NH), 7.50 (1H, d, NH), 7.35 [PdCl
5H, m, C ), 5.02 (2H, q, C -CH
s, CH ), 3.75 (2H, d, CH ), 3.10 (3H, s, N-CH
2
], [PdCl
3
] , [PdCl
4
] , respectively) were taken from the
), 4.06 (1H, q, CH), 3.98 (2H, literature. These fixed values were involved into the
), 1.22 (2H, d, CH ). equilibrium models during the calculations, but stability
3
7
(
H
6 5
H
6 5
2
2
2
3
3
Ala-Gly-Gly-NHOH·TFA (3). Z-Ala-Gly-Gly-NHOH (380 mg, 1.08 constants for various ternary complexes, involving chloride
mmol) was dissolved in dry methanol (20 ml) then TFA (100 µL, ions, were not involved. Consequently, the stability constants
1
.3 mmol) and Pd/C (10 % (w/w), 110 mg) were added. The determined for the various complexes formed between Pd(II)
mixture was stirred under H (2 bar) for 4 h. The catalyst was and the peptide hydroxamic acids are conditional values, valid
filtered off and the solvent was removed. Abs. diethyl ether under the experimental conditions used, and the free
was added to the residue, then the solvent was evaporated coordination site(s) of the metal ion is(are) occupied by
under vacuum. The product is a white hygroscopic solid. Yield: chloride ion(s) or water molecule(s). A negative stoichiometric
2
1
3
30 mg (92 %). H NMR (400 MHz, D
2
O) δ (ppm): 4.11 (1H, q, number for H in the formulae of the Pd(II) complexes indicates
), 1.51 (3H, d, CH ). a proton, which dissociates only from the complex but neither
CH), 3.99 (2H, q, CH
2
), 3.87 (2H, s, CH
2
3
Ala-Gly-Gly-N(Me)OH·TFA (4). Ala-Gly-Gly-N(Me)OH·TFA was from the ligand nor from the metal aqua ion separately. This
prepared as described for 3 using 500 mg Z-Ala-Gly-Gly- proton can be liberated from a water molecule in the
N(Me)OH. The product is a white hygroscopic solid. Yield: 444 coordination sphere of the metal ion bound by the ligand,
1
mg (94 %). Analysis: H NMR (400 MHz, D
2
O) δ (ppm): 4.23 from a hydroxamate group or from the very weakly acidic
), 3.25 (3H, s, N- amide group(s) of the bound ligands. For the Pd(II)-Ala-Ala-
NHOH 1:1 system, a repeated titration was also performed by
(
2H, s, CH
2
), 4.17 (1H, q, CH), 4.07 (2H q, CH
).
2
CH ), 1.57 (3H, d, CH
3
3
acidifying the sample to pH ~ 2.0 after titration then the
pH-potentiometric and spectroscopic studies
The pH-potentiometric titrations were carried out at t = 25.0
measurement was repeated after four days.
1
H NMR titrations were carried out on a Bruker Avance 400
°
C using 0.10 M KCl ionic strength, which was adjusted to 0.20 instrument at t = 25.0 °C in the presence of 0.10 M KCl and
M by addition of KNO . The application of that mixture of salts 0.10 M KNO (Itot = 0.20 M). The chemical shifts are reported in
to keep the ionic strength constant was taken from previous ppm (δ ) from TSP as internal standard. Titrations were
work on Pd(II)-peptides. In fact, in the absence of any performed using 5-10 individual samples in D O (99.8%) at clig
3
3
H
2
5
2
chloride ions the complexation was found to occur already = 5.0 mM, the metal ion to ligand ratios were 1:2, 1:1, 1.5:1
under very acidic conditions (below the measurable pH-range), and 2:1, the pH was varied within the range 1.7 – 11.5. The pH
but in the presence of the competitor chloride ions, the of the samples was set up with NaOD or DNO
complexation processes with the peptides were shifted into pH* values (direct pH-meter readings in a D O solution of a
the measurable pH range. On the other hand, more than 0.10 pH-meter calibrated in H O according to Irving et al. ) were
M chloride concentration was found to prevent the solution converted to pH values using the following equation: pH =
3
solutions. The
2
3
4
2
2
5
38
equilibrium studies due to slow complexation processes.
Carbonate free, ca. 0.2 KOH solution of known determine the fraction of each species allowing further
0.936·pH* + 0.412. NMR spectroscopy was also used to
M
concentration was used as titrant. The exact concentration of additional equilibrium information about those systems,
the HCl and KOH was determined by potentiometric titrations. where the signals of each complexes were well-separated.
A Mettler Toledo DL 50 titrator equipped with a Mettler Regarding the slow exchange in the NMR time scale, each
Toledo combined electrode (DGI114SC) was used for the pH- species has an individual resonance. At this condition the
metric measurements. The electrode system was calibrated integrated peak areas assigned to the different species were
3
4
according to Irving et al. The water ionization constant was converted to molar fraction and are also displayed on the
pK
w
= 13.76 ± 0.01 under the conditions used. The initial speciation curves. In case of Pd(II)-Ala-Ala-NHOH system, fast
+
volume of the samples was 15.0 mL, the ligand concentrations exchange in the NMR time scale was observed between [PdL]
were varied in the range 2.0-3.0 mM, the metal ion to ligand and [PdH–1L], where a single resonance can be observed at the
ratios were 1:2, 1:1, 1.5:1 and 2:1, where the equimolar molar fraction weighted average of the chemical shifts of the
solutions contained 5-10 % excess of ligand. The samples were two species.
stirred and completely deoxygenated by bubbling purified
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5
5
5
5
5
5
5
5
5
5
6
Time-dependent investigations were also carried out on all of processes, e.g. Pd(II) promoted irreversible hydrVoielwysAirsticolefOntlhinee
the systems. The first spectra were registered within 1 hr from coordinated ligands were observedDOinI: 10t.1i0m3e9/-Cd9eNpJe0n0d2e96nKt
preparation (freshly prepared samples), then the experiments under acidic conditions. As a consequence, the
measurements were repeated several times on the individual equilibrium results reported below are valid for freshly
samples over a period of 7-9 days. The pH of the samples was prepared samples. Ligand dependent differences were
checked before the NMR measurements, but because the observed in the rate of the irreversible process(es), which
samples were kept in NMR tube on air the pH was shifted were slower with tripeptide derivatives than with dipeptide
slightly in many cases which were taken into account during ones and slower with primary derivatives compared to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the evaluation
secondary ones (vide infra). The only system, in which
complete reversibility was found, is the Pd(II)-Ala-Gly-Gly-
NHOH one. Representative examples of pH-potentiometric
titration curves registered for this system are shown in Fig. 1.
Results and discussion
+
H complexes of the ligands
Pd(II)-Ala-Gly-Gly-NHOH system
The fully protonated ligands can release two protons (from the
ammonium and hydroxamic functions) in the pH range ca. 7-
1
0. Although the dissociation constants of the studied peptide
hydroxamic acids were determined in previous works in our
laboratory,2
8-29,39
the slightly different conditions used in the
present work and to obtain the exact concentration of the
stock solutions made the determination of the constants
necessary. The dissociation constants (pK values) calculated via
fitting the pH-potentiometric titration curves are shown in
Table 1. The values relating to the two functions of a ligand are
quite close to each other, indicating that the deprotonation
processes of -NH
considerably. Since the present macroconstants are in good
agreement with the earlier ones,
+
3
and -CON(R)OH groups overlap
2
8-29
and in these cited works
the microconstants (showing the basicity of the individual
Table 1 Protonation constants (log K
M KCl + 0.10 M KNO
i
) of the ligands at 25.0 °C, Itot = 0.20 M (0.10
a
3
)
Ligand
log K
1
log K
2
Ala-Gly-Gly-NHOH
Ala-Gly-Gly-N(Me)OH
Ala-Ala-NHOH
8.80(1)
8
7.70(1)
7.701
.821
8.55(1)
8.631
7.73(1)
7.661
8.80(1)
8
7.70(1)
7.662
.882
Ala-Ala-N(Me)OH
8.74(1)
.743
7.78(1)
7.743
8
a
3
σ standard deviations are in parentheses
Ref. 39 (t = 25.0 °C, I = 0.20 M KCl)
Ref. 28 (t = 25.0 °C, I = 0.20 M KCl)
Ref. 29 (t = 25.0 °C, I = 0.20 M KCl)
1
2
3
+
Fig. 1 Representative titration curves of H –Ala-Gly-Gly-NHOH (a) and Pd(II)–Ala-
Gly-Gly-NHOH systems at 1:2 ratio (b), 1:1 ratio (c), 1.5:1 ratio (d) and 2:1 ratio (e);
negative base equivalent refers to an excess of acid in the sample
groups) were also determined, the same basicity-trend under
our conditions is assumed, namely, the -NH
increased acidity compared to the -CON(R)OH moiety.
comparison of the corresponding values does not show any
measurable difference between the values relating to (i) the
primary versus secondary, (ii) dipeptides versus tripeptides.
+
3
group shows
2
8
A
Pd(II) complexes of peptide hydroxamic acids
Equilibrium studies on the Pd(II)-peptide hydroxamate systems
were performed by pH-potentiometric titrations and H NMR
method to obtain the speciation. It was clearly indicated by the
experimental findings that, except the Ala-Gly-Gly-NHOH
containing system, not only complexation, but also other
1
4
| J. Name., 2012, 00, 1-3
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Ple Na es we dJ oo u nr no at l ao df jCu hs et mm i as tr rgy ins
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ARTICLE
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
View Article Online
DOI: 10.1039/C9NJ00296K
Table 2 Stability constants (logβ) of Pd(II) complexes of peptide hydroxamic
acids at Itot = 0.20 M (0.10 M KCl + 0.10 M KNO ) at 25.0 °C
3
a
Ala-Gly-Gly-
NHOH
22.70(1)
18.98(3)
13.98(6)
22.96(5)
13.9(2)
–
Ala-Gly-Gly-
N(Me)OH
22.60(2)
18.69(2)
Species
PdL]+
Ala-Ala-NHOH
[
22.84(3)
20.25(2)
11.5(1)
–
[
PdH–1L]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
[
[
PdH–2L]
Pd –3L]
12.2(1)
2
H
–
–
–
–
–
–
[
[
Pd
Pd
2
H–4L]
–
3
H
PdL
–4
L
]
2
]
44.4(3)
33.4(3)
24.8(3)
[
2
–
–
[PdH–1
L
2
]–
Fitting
0
.0165
97
0.0250
214
0.0284
283
parameter
Number of
fitted data
pK[PdL]
5
3.72
5.00
3.92
6.50
2.59
8.8
pK[PdH–1L]
σ standard deviations in parentheses
a
3
Fig. 2 Concentration distribution curves of the Pd(II)–Ala-Gly-Gly-NHOH system at
(
a) 1:1 and (b) 2:1 metal ion to ligand ratio, at clig = 3.0 mM
As it can be seen in Fig. 1, the titration curves indicate the
beginning of the complexation at pH ~ 2, and already under
acidic conditions metal ion induced proton release of the
ligand takes place in overlapping steps. Fitting all of the
titration curves together led to the speciation model and
st
overall stability constants summarized in Table 2, 1 column.
These pH-metric results were used to calculate the
concentration distribution curves seen in Fig. 2. These curves
were also calculated under biologically more relevant
concentrations (cPd(II) = 20 μM, in presence of cCl- = 100 mM
and cCl- = 4 mM), which can be seen in Figures S1-S4. At lower
metal ion concentration and high chloride concentration the
competition with chloride ions is more significant than it was
assumed under the conditions of pH-metric studies. On the
other hand, at low chloride concentration the complexation
processes are shifted into the lower pH-range. These
observations are generally true for all of the investigated
systems. As it is seen in Fig. 1c, in approximately equimolar
solution, in addition to the release of the two dissociable
protons of the ligand, two extra deprotonation processes take
place. Fig. 2a clearly shows that the complexation starts with
+
the formation of the [PdL] complex, then an increase in pH
–
results in the formation of [PdH–1L] and [PdH–2L] in
overlapping processes; the latter complex predominates above
pH ~ 6 at this ratio. The four equivalents base consumption by
the end of the titration indicates the metal ion induced
deprotonation of the terminal ammonium, the two amides
and the hydroxamic groups and, consequently, a 4N-
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ARTICLE
Journal Name
mixed hydroxido species (Chart 2/IV). Similarly to the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3 Chemical shifts (δ) of non-dissociable protons of the ligand in ppm
measured in the Pd(II)-Ala-Gly-Gly-NHOH system at different metal ion to ligand
ratios and pH values
View Article Online
DOI: 10.1039/C9NJ00296K
3
9
appropriate Cu(II) system there was no indication for the
formation of trinuclear species with similar binding mode as XI
in Chart 2. At 1:2 metal ion to ligand ratio the titration curve
(Fig. 1/b) does not show any extra metal ion induced
deprotonation processes, therefore formation of bis-
complexes was not indicated by the pH-metric experimental
results.
Ratio
(cPd(II):c )
L
pH
(A)
(B)
(C)
(D)
Species
0
:1
3.11
1.559 4.168 4.036 3.914
1.548 4.142 4.033 3.914
H
2
2
L+
6
.63
8.19
.44
0.22 1.278 3.557 3.956 3.800 L–
2.36 1.559 4.168 4.036 3.914
1.398 3.582 3.977 3.914 [PdL]+
H
L+/HL
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H NMR study was also performed for the determination of the
1.405 3.835 3.999 3.897 HL
1.290 3.585 3.963 3.828 HL/L–
1
binding mode of each complex. The reported H NMR spectra
9
were registered within one-hour equilibration; therefore the
obtained data are comparable with the pH-potentiometric
results. The chemical shifts of the species are reported in Table
3. In the NMR spectra at 1:1 Pd(II) to ligand ratio there are
three new sets of signals beside the signals of the
uncomplexed ligand. The obtained spectra demonstrate that
each complex has an individual signal in the NMR spectra,
indicating that the species show slow exchange in the NMR
time scale. This is an additional support to the fact that the
Pd(II) is coordinated by N-atoms in the 1:1 complex. A
comparison between the speciation curves in Fig. 2a and the
chemical shifts as well as integrated peak areas of the NMR
signals recorded at various pH-s clearly show that the above-
mentioned three signal-sets correlate well with the formation
1
1
:1
H
2
L+
L+
3
.18
1.559 4.168 4.036 3.914
H
2
1
1
.398 3.582 3.977 3.914 [PdL]+
.360 3.600 3.832 3.814 [PdH–1L]
4.98
1.360 3.600 3.832 3.814 [PdH–1L]
.374 3.651 3.876 3.779 [PdH–2L]–
1
7
.14
1.374 3.651 3.876 3.779 [PdH–2L]–
1.559 4.168 4.036 3.914
L+
1
.5:1
2.47
H
2
1.398 3.582 3.977 3.914 [PdL]+
1.398 3.582 3.977 3.914 [PdL]+
+
–
of the [PdL] , [PdH–1L] and [PdH–2L] complexes. At pH = 2.36
the quartet of B in Table 3 showed a significant upfield shift
4
.00
1
1
.360 3.600 3.832 3.814 [PdH–1L]
.365 3.602 3.841 3.723 [Pd
compared to the one of the free ligand supporting the (NH
2
,
+
2
H–3L]
N
amide) binding mode for the [PdL] complex. The exact
+
L+
stoichiometry of this complex is [Pd(H–1L)H] , where one of the
peptide moiety is already deprotonated, while the dissociable
proton is still on the hydroxamic function. This finding is also in
good agreement with the earlier results on palladium(II)
complexes of simple di- and tripeptides, where the interaction
2:1
2.47
1.559 4.168 4.036 3.914
H
2
.398 3.582 3.977 3.914 [PdL]+
1
6
.12
1.360 3.600 3.832 3.814 [PdH–1L]
1
.365 3.602 3.841 3.723 [Pd
1.365 3.602 3.841 3.723 [Pd
.363 3.651 3.886 3.752 [Pd
0.84 1.363 3.651 3.886 3.752 [Pd
2
H
2
H
2
H
2
H
–3L]
2
5
2
also starts with a (NH , Namide) coordination at pH ~ 2. As the
8.53
–3L]
pH increases new signals appear in two sets, which are
assigned to the formation of [PdH–1L] (with the exact
–4L]–
1
1
–4L]–
–
stoichiometry of [Pd(H–2L)H]) and [PdH–2L] complexes. Their
–
parallel existence is seen until pH = 7.14, where the [PdH–2L]
–
becomes the dominant one. At 2:1 metal ion to ligand ratio
above pH ~ 4 a new set of signals can also be detected
coordination (NH
2
, Namide, Namide, Nhydr.) in the [PdH–2L] . (More
–
precisely this complex has a [Pd(H–3L)H] composition, where
the dissociable hydroxamate-OH is still protonated.) The
assumed binding mode is shown in Chart 2/II. Fig. 1 also shows
that this ligand is capable of binding metal ion excess. This
observation is in agreement with the results obtained
previously for the Cu(II)-complexes of this tripeptide
hydroxamic acid. One-equivalent of Pd(II) excess is necessary
to substitute the dissociable proton of the hydroxamate
moiety from the total amount of the ligand up to pH ~ 6 (see
curves d and e in Fig. 1). However, at 2:1 metal ion to ligand
ratio all together two equivalents of base were titrated, in
addition to the one up to pH ~ 6, another one up to pH ~ 10,
which indicates additional metal ion-induced deprotonation
indicating the formation of a dinuclear [Pd
2
H–3L] complex
(
Chart 2/III), while above pH 8.5 another signal can be assigned
–
2
to the formation of the [Pd H–4L] species (Chart 2/IV). The
NMR measurements were repeated in 7 days. During this
period of time the samples were kept in the tubes.
Consequently, the pH values of the samples changed due to
dissolution of carbon-dioxide from the air, but the signals
detected in each sample were found to be changed according
to the slight change in the pH only. As a consequence, it can be
stated, that no other processes apart from the reversible
complexation take place in measurable extent in this system.
3
9
Pd(II)-Ala-Gly-Gly-N(Me)OH system
process. The best fit of the titration curves was obtained with
the involvement of [Pd
model. Out of the processes the first one corresponds to the
formation of [Pd
–
Substitution of the hydrogen at the hydroxamic-N by a methyl
moiety results in the elimination of this nitrogen as a potential
coordinating donor atom. Consequently, one ligand can
provide only three N-donors as a maximum, which are, unlike
2
H
–3L] and [Pd
2
H
–4L] species in the
2
H
–3L] dinuclear complex (Fig. 2/b and Chart
–
2
2/III), while the second one to the formation of [Pd H–4L]
6
| J. Name., 2012, 00, 1-3
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Ple Na es we dJ oo u nr no at l ao df jCu hs et mm i as tr rgy ins
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ARTICLE
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
N
O
O
N
O
View Article Online
DOI: 10.1039/C9NJ00296K
O
H N
2
H N
H N
2
N
N
H N
2
H2N
N
2
N
Pd
Pd
Pd
Pd
Pd
CONHOH
HO
N
O
N
O
O
N
O
O
O
N
O
N
O
O
HN
O
Pd
Pd
HO
I
II
III
IV
V
HO
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N
O
N
O
O
H N
2
N
N
H N
2
H2N
Pd
H N
2
H N
2
N
Pd
Pd
O
N
Pd
Pd
N
N
H
O
O
O
N
O
VII
N
O
N
O
NH2
2
O
N
HO
HO
O
HO
N
VI
VIII
IX
X
O
N
O
O
Pd NH2
H N Pd
N
Pd
N
2
O
O
N
XI
O
Chart 2 Suggested binding modes of the complexes
with the above discussed primary derivative Ala-Gly-Gly-
NHOH, not able to take all the four equatorial coordination
sites of one Pd(II). The experimental findings obtained for the
Pd(II)-Ala-Gly-Gly-N(Me)OH system by pH-potentiometric
method are as follows: (i) This ligand is not able to bind metal
ion excess at all suggesting some involvement of the
hydroxamic function in the coordination already at equimolar
conditions. (ii) As it is illustrated in Fig. S5, the titration curve
registered at 1:1 metal ion to ligand ratio shows three
equivalents base-consumption in the pH range ~ 2.5-4.5, which
indicates three overlapping processes in this region. In a
separated step, in the range of ca. pH 5-9 an additional
equivalent base is consumed. This latter process is shifted to
higher pH compared to that with the analogous primary ligand
Table 4 Chemical shifts (δ) of non-dissociable protons of the ligand in ppm
measured in the Pd(II)-Ala-Gly-Gly-N(Me)OH system at different metal ion to
ligand ratios and pH values
Ratio
(cPd(II):c )
L
pH
(A)
(B)
(C)
(D)
(E)
Species
0
:1
3.02
1.557 4.164 4.050 4.215 3.238
1.536 4.111 4.047 4.211 3.234
H
2
L+
7
8
9
.11
.36
.48
H
2
L+/HL
1.379 3.772 4.012 4.178 3.215 HL
1.293 3.580 3.991 4.134 3.188 HL/L–
1
1.60 1.281 3.550 3.989 4.123 3.178 L–
2.53 1.557 4.164 4.050 4.215 3.238
.428 3.564 4.232 4.029 3.260 [PdL]+
1.428 3.564 4.232 4.029 3.260 [PdL]+
.359 3.592 4.049 3.839 3.208 [PdH–1L]
1.359 3.592 4.049 3.839 3.208 [PdH–1L]
.348 3.576 3.884 3.863 3.178 [PdH–2L]–
1.348 3.576 3.884 3.863 3.178 [PdH–2L]–
1
:1
H
2
L+
1
(
cf. Figs. 1 and S5). (iii) The titration curve, registered at 1:2
4.05
metal ion to ligand ratio, does not show any “extra” metal-
induced process compared to those observed at 1:1 ratio.
All of the pH-potentiometric experimental data were fitted
together and the results obtained are shown in the second
column of Table 2. The speciation model indicates that only
mononuclear complexes can be detected in this system and
1
6
8
.51
.10
1
1
:2
2.74
1.557 4.164 4.050 4.215 3.238
H
2
L+
+
–
the formulae of them are [PdL] , [PdH–1L] and [PdH–2L] . Due to
the very similar basicity of the functions in the two
investigated tripeptide derivatives (see Table 1), simple
comparison of the corresponding stability constants is
possible. The similarity of the stability constants suggests
1
.428 3.564 4.232 4.029 3.260 [PdL]+
L+
6
7
.00
.91
1.557 4.164 4.050 4.215 3.238
H
2
1.359 3.592 4.049 3.839 3.208 [PdH–1L]
.407 3.600 3.742 4.239 3.258 [PdL
1.550 4.143 4.038 4.212 3.238
L+/HL
.407 3.600 3.742 4.239 3.258 [PdL
.348 3.576 3.884 3.863 3.178 [PdH–2L]–
1
2
]
+
analogy in the coordination modes in the complexes [PdL] ,
H
2
[PdH–1L] formed with Ala-Gly-Gly-NHOH and Ala-Gly-Gly-
N(Me)OH. Namely, in both systems, a 5-membered chelate via
2
, Namide) donor set can be assumed as main coordinating
, Namide, Namide) joined
chelates in the latter one. As it could be expected, the stability
1
1
2
]
(NH
mode in the former species, while (NH
2
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Journal Name
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
–
constants of [PdH–2L] are significantly different in the two function is still protonated. Therefore the exact composition of
View Article Online
DOI: 10.1039/C9NJ00296K
+
systems. In fact, the value for the complex formed with the this complex is [Pd(H–1L)H] (structure V in Chart 2), although
secondary derivative is ca. two-orders of magnitude lower the coordination of the carbonyl oxygen of the neighbouring
than the one with the primary counterpart. 4N coordination amide function cannot be completely ruled out either.
was suggested in the complex of the primary derivative (see (ii) The new signals appeared in the pH range 6.46-7.20 were
above), but, because the hydroxamate nitrogen of the assigned to [PdH–1L] with (NH , Namide, Namide, Ocarb.) binding
2
secondary ligand, Ala-Gly-Gly-N(Me)OH, is unavailable for mode, as a consequence the precisely assigned formula of this
coordination, most likely, the deprotonated hydroxamate-O species is [Pd(H–2L)H] (Chart 2/VI). A further increase of pH
occupies the fourth coordination site in [PdH–2L] (see above ca. 8 resulted in the deprotonation and coordination of
structure VII in Chart 2). This binding mode is the same as it hydroxamic OH, what is confirmed by the upfield shift of the E
was determined in the corresponding Cu(II) complex of Ala- peak (Δδ = –0.029 ppm) in the mentioned pH-range.
Gly-Gly-N(Me)OH.39 This also explains why binding of any Pd(II) Consequently, both the NMR and pH-metric results show that
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
–
excess is not possible by this ligand.
under equimolar conditions, above pH ca. 8, [PdH–2L] is the
major complex in this system. (iii) At twofold excess of ligand,
in the pH-range ca. 6.0 - 9.5, in addition to the signals
belonging to the free ligand and 1:1 complexes, one additional
low intensity “extra” set could be observed. Although, pH-
potentiometry did not indicate the formation of bis-complex at
ligand excess, still its appearance after 1 hour under the
mentioned conditions seems a reasonable interpretation of
these NMR results. For further information in this question
time dependence of the spectra was monitored (as detailed in
Fig. S6). In fact, the intensity of the “extra” signals was
increased for five days, when the maximum intensity was
reached. This result shows that the formation of a bis-complex
is rather slow, five days being necessary for its complete
formation. The bis-complex has a single set of signals, which
indicating its symmetrical structure, therefore, this complex
should contain the two ligands in the same binding mode.
Most probably, each of the two ligands is coordinating via a
4
.0
3.5
3.0
2.5
δ (ppm)
2.0
1.5
1.0
1
Fig. 3 H NMR spectra of Pd(II)–Ala-Gly-Gly-N(Me)OH = 1:1 in 9 days (a), freshly
prepared samples of Pd(II)-HN(Me)-OH = 1:1 (b) and Pd(II)–Ala-Gly-Gly-OH = 1:1
(c) at pH = 2.4
2
five-membered (NH , Namide)-chelate (see structure VIII in
Chart 2). (iv) Below pH ~ 5, no additional signals were observed
in the spectra registered after 1 hour at 1:2 metal ion to ligand
ratio, compared to 1:1. However, in one day, at both ratios
new signals were detected in the acidic region and their
intensity increased continuously during the period of the
investigation, 7-9 days (see Fig. S6). Taking into account the
previous result on the hydrolysis of the ligand in the Pt(II)-
1H NMR measurements were carried out at various metal ion
to ligand ratios, pH values and time-dependence of the
interaction was also monitored on samples summarized in Fig.
S6. Although the registered NMR spectra supported the
formation of all the three species suggested by pH-metry,
some different and unexpected results were found, too. If the
spectra were registered in 1 hour after the preparation of the
samples, the following main results were obtained: (i)
Compared to those of the free ligand, NMR spectra registered
at equimolar conditions, showed a new set of signals in the
3
1
glycinehydroxamate complex to glycine and hydroxylamine,
the possibility of a similar process in our system was assumed.
Spectra registered on “nine-days samples” were compared
with those of Pd(II)-HN(Me)OH and Pd(II)-Ala-Gly-Gly-OH and
for illustration are shown in Fig. 3.
As Fig. 3 shows, the new signals observed under the above
conditions in the Pd(II)-Ala-Gly-Gly-N(Me)OH system
correspond to HN(Me)OH and that of Pd(II)-Ala-Gly-Gly-OH.
Although detailed investigation on the mechanism was not
performed, the facts that the hydrolysis was observed only
below pH 5 and the pH-dependence of the relative intensity of
E signals (see Table 4) fits the speciation curve of [PdL],
support the kinetic activity of this complex in the hydrolysis.
+
range pH = 1.93-2.53, which was assigned to [PdL] (the values
of the chemical shifts are summarized in Table 4). In this pH
range the B quartet of the ligand showed a significant upfield
shift, the E singlet a downfield shift. Based on this observation,
it was assumed, that the carbonyl oxygen of the –CON(Me)OH
moiety is also coordinated to the metal ion beside the main
2
coordinating donors, NH - and Namide, while the hydroxamic
(
2
see Fig. 4). Because in [PdL], in addition to the (NH , Namide)
chelate also the coordination of the hydroxamic Ocarb. was
supported by the NMR results, a similar hydrolysis of our
coordinated ligand might be possible that it was published in
the above already mentioned literature for glycinehydroxamic
3
1‡
acid in its Pt(II) complex.
8
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Fig. 4 The extent of ligand hydrolysis (expressed in the ratio of the N-CH
3
signals of
the ligand and the free HN(Me)OH) after 24 hours (dots) on concentration
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distribution curve of Pd(II)-Ala-Gly-Gly-N(Me)OH = 1:1 system

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6
Pd(II)-dipeptide hydroxamic acid systems
to bind Pd(II) excess, but significant differences were observed
View Article Online
DOI: 10.1039/C9NJ00296K
between the two systems in this question. Namely, while one
equivalent Pd(II) excess was bound by the former molecule, it
was only half equivalent for the dipeptide hydroxamic acid (cf.
Fig. 1 and S7). (iv) Unlike with the two tripeptide hydroxamic
acids, additional pH-effect compared to the equimolar ratio
was observed in the titration curve registered at 1:2 metal ion
to ligand ratio with Ala-Ala-NHOH, what is a direct indication
for the measurable formation of bis-complex(es) being
detectable by pH-metry in this system.
Despite the clear indication for the hydrolysis of the
coordinated ligand under acidic conditions after a period of
few days (see point (ii) above), yet the pH-metric curves
registered in freshly prepared samples were adequate to carry
out equilibrium calculations. The best fit of all titration curves
provided the speciation model and overall stability constants
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 5 Concentration distribution curves of Pd(II)–Ala-Ala-NHOH system at cPd(II)
=
c
lig = 3.0 mM
rd
presented in Table 2, 3 column. The obtained results showed
+
–
the measurable existence of [PdL] , [PdH–1L], [PdH–2L] , [PdL
2
]
Although Ala-Ala-NHOH has three N-donor atoms (NH
2
, Namide,
and [PdH- ] mononuclear, as well as [Pd ] trinuclear
complexes. By using these results, concentration distribution
curves were calculated and, for illustration, those obtained at
:1 and 1.5:1 metal ion to ligand ratio are shown in Fig. 5 and
Fig. 6b., while the concentration distribution curves at 1:2
metal to ligand ratio can be seen in fig. S8. The stability
constant of [PdL] is very similar to those determined for the
corresponding complexes formed with the two tripeptide
hydroxamic acids (see Table 2), suggesting the most likely
L
1 2
3 –4 2
H L
Nhydr.) and two hydroxamate O-donors to interact with Pd(II) all
the four coordination sites of Pd(II) cannot be occupied by
them in a mononuclear species. The pH-metric measurements
were performed as it is detailed in the Experimental part and
provided the following experimental findings: (i) The pH-
potentiometric titration curve registered in an equimolar
sample showed the consumption of three equivalents of base
until pH ~ 5, and an additional one above pH 7 (Fig. S7, curve
d).
1
+
existence of a 5-membered (NH
stoichiometry of [Pd(H–1L)H] ) (Chart 2/I).
2
, Namide) chelate (and the exact
(ii) Interestingly, this latter process was not observed in the
+
titration curve registered on an equimolar sample, which was
once already titrated than acidified for a 4 days period prior to
this “repeated titration” (see curve c in Fig. S7). This finding
was a clear indication for the hydrolysis of the hydroxamic
function from the coordinated ligand during the mentioned
period of time. (iii) Like the primary tripeptide hydroxamic
acid, Ala-Gly-Gly-NHOH, this ligand was also found to be able
a)
b)
pH
7.06
4
3
.90
.38
2 2
H H
L+ L+
2
.40
2.05
1
Fig. 6 pH-dependence of the H NMR spectra of the Pd(II)-Ala-Ala-NHOH = 1.5:1 system (a); concentration distribution curves calculated using the stability constants in
Table 2 (solid lines, c
1
+
]2–
L
= 5 mM.) and from H NMR spectra at 1.5:1 metal ion to ligand ratio (b); [PdL] ( ), [PdH–1L] ( ), [Pd
3
H
–4
L
2
( )
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2
2
2
3
3
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3
3
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3
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4
4
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4
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6
indicates fast exchange between these two species in the NMR
View Article Online
DOI: 10.1039/C9NJ00296K
time scale, which is a significant difference compared to the
tripeptide hydroxamic acid containing systems, where each of
the complexes showed individual signals. (see Fig 7.) Based on
this difference, it is assumed that the third coordination site of
the metal ion in [PdH–1L] might be occupied (at least in a
transient species) by a rather labile bond, e.g. by Ohydr. Getting
additional information in this question (e.g. from time-
dependent measurements), were hindered by the above
hydrolytic process, which were also found to occur in this
acidic pH-range. (ii) At 1.5:1 Pd(II) to ligand ratio above pH ca.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
.4, a new pair of quartets appeared at δ = 4.015 ppm and δ =
.556 ppm, which were assigned to [Pd ] complex. To
3 –4 2
H L
demonstrate the good fit between the pH-metric and NMR
speciation profiles Fig. 6 is shown. (iii) In the case of ligand
excess above pH = 5.8 two pairs of new signals appeared
beside the signals of the free ligand, which were assigned to
the formation of bis-complex(es) (see Fig. S10). Existence of
two sets of signals suggested that the coordinated ligands have
different chemical environments in the bis-complexes. This
finding supports the coordination mode discussed above
based on the pH-metric results. Similarly, different binding
modes of the two coordinated ligands in the Pd(II)-tripeptide
bis-complexes were previously published, where, one of the
ligands was assumed to bind via tridentate, while the other via
1
Fig. 7 pH-dependence of the H NMR spectra of Pd(II)-Ala-Ala-NHOH system at 1:1
metal ion to ligand ratio
Deprotonation of [PdL] (most probably at the hydroxamic-NH
function) occurs already in the pH-range, pH < 5, resulting in
the formation of [PdH–1L] (Chart 2/IX). In the last
deprotonation process [PdH–2L] is formed and predominates
in equimolar sample above pH 8. Simply the pH-
potentiometric effect does not provide information for the
–
–
question, whether, [PdH–2L] is either a mixed hydroxo species,
or a (NH , Namide, Namide) coordinated one with doubly
2
2
5
a monodentate manner.
deprotonated hydroxymate function. However, the latter
binding mode is supported by the lack of this fourth
deprotonation process in the “repeated titration” on the “4-
day sample”, where the hydrolysis of the hydroxamic function
from the coordinated ligand and formation of the simple
dipeptide was assumed (see above in point (ii)). In the
presence of Pd(II) excess the deprotonation and coordination
of the hydroxamate group is assumed above pH ~ 3-4 and a Pd(II)-Ala-Ala-N(Me)OH system
trinuclear complex, [Pd ] is formed. The coordination
mode is suggested to be analogous with the one found
The hydrolysis of the coordinating ligand) detected after 4
days with Ala-Gly-Gly-N(Me)OH) was already measurable after
days with Ala-Ala-NHOH. Significant changes in the NMR
spectra were monitored in the pH-range 2-7 although some
minor changes in the spectra could also be seen at neutral pH.
2
3 –4 2
H L
During the titration of the Pd(II)-Ala-Ala-N(Me)OH system,
above pH 4.5 elementary palladium was formed as fine black
powder. This also occurred during the NMR measurements, as
a Pd-mirror was formed on the inner surface of the NMR tube.
For this reason, equilibrium study on this system was not
2
8
previously in the corresponding Cu(II) complex and shown in
Chart 2/ XI). In the case of twofold ligand excess, formation of
bis complexes was shown by the pH-metric results. The
2 2
stoichiometry of these are [PdL ] and [PdH–1L ], and each of
them most probably contains a [PdH–1L] unit, to which the
second ligand coordinates at the fourth equatorial site via its
terminal amino-N donor, and contains the hydroxamic
function still in protonated or already in the deprotonated
form. Consequently, the exact stoichiometries are [Pd(H
L)(HL)] and [Pd(H–1L)(L)], respectively (see Chart 2/X). The
–
1
formation of all the complexes discussed above was supported
1
by H NMR measurements, monitoring the dependence of the
chemical shifts on the metal ion to ligand ratio and pH. The
obtained chemical shifts are shown in Fig. S9. Several new
information obtained from the NMR results are as follows: (i)
At 1:1 metal ion to ligand ratio one pair of quartets was
detected beside the quartets of the free ligand at pH ca. 1.7
+
which was assigned to [PdL] . On increasing the pH up to ca.
4
.2, where the [PdH–1L] was found to become the major
Fig. 8 Time dependence of the 1H NMR spectra of Pd(II)-Ala-Ala-N(Me)OH 1:1
+
species, not only the intensity of these signals was increased,
but surprisingly, they showed a continuous up-field shift. This
system at pH = 2.0 showing the formation of H
Pd(II)-dipeptide complex
2
N(Me)OH and the corresponding
1
0 | J. Name., 2012, 00, 1-3
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possible. H NMR spectra showed that beside the signals of the monodentate coordination of the second ligand via its
View Article Online
DOI: 10.1039/C9NJ00296K
free ligand, the registered new signals correspond to the terminal amino-N to [PdH-1L] is assumed (Chat 2, X).
protonated HN(Me)OH and the Pd(II)-Ala-Ala-OH complex (Fig. Unlike in the Pd(II)-Ala-Gly-Gly-NHOH system, parallel to
8
). This finding indicates the very fast hydrolysis of the complexation, Pd(II)-assisted hydrolysis of the coordinated
coordinated Ala-Ala-N(Me)OH. The protonated hydroxylamine ligands was also found under acidic conditions in the other
formed in the hydrolytic process can reduce Pd(II) to metallic three systems. The results supported the formation of
palladium.
protonated hydroxylamine and the corresponding peptides in
these processes. Hydrolysis hindered the equilibrium
measurements for the Pd(II)-Ala-Ala-N(Me)OH system, where
the ligand can donate maximum two N-donors during the
complexation and reduction of Pd(II) to Pd(0) by the
hydroxylamine formed was also observed. Fortunately, the
significantly slower hydrolysis with the two 3N-donor ligands,
Ala-Ala-NHOH and Ala-Gly-Gly-N(Me)OH, allowed us to carry
out equilibrium measurements (speciation studies) on these
systems (as it is discussed above). Although detailed study of
the hydrolysis was beyond the scope of this work, an order of
the rate of hydrolysis could be determined. No indication for
the hydrolysis was found with Ala-Gly-Gly-NHOH while some
days were necessary to observe the measurable extent of it in
the Pd(II)-Ala-Gly-Gly-N(Me)OH system. The process was faster
in the Pd(II)-Ala-Ala-NHOH, while very fast for Pd(II)-Ala-Ala-
N(Me)OH system. Plausible suggestion was also made for the
kinetically active species in the case of Pd(II)-Ala-Gly-Gly-
N(Me)OH. Since Pd(II) and Pt(II) share very similar coordination
behaviour (square planar complexes and soft donor atom
preference) but different ligand exchange kinetics the results
obtained in this study may provide useful information for the
design of hydroxamate based Pt(II) complexes with antitumor
potential.
0
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Conclusions
The present study provides important new information
relating to the Pd(II)-peptide hydroxamic acids interactions.
This is the first case, when [PdCl
used. The previous results refer to systems, in which the metal
ion source was [Pd(en)Cl ], so the formation of ternary
2
–
4
]
as metal ion source was
2
1
8
complexes with a peptide hydroxamic acid was studied. This
study indicates that peptide hydroxamic acids are very
effective chelators of Pd(II) where the nitrogen atoms are the
most preferred donors by the metal ion. For primary ligands, in
addition to the amino and amide-nitrogen(s), the
deprotonated hydroxamate-N was also found to be an
effective donor. Out of the investigated molecules only the
primary tripeptide derivative, Ala-Gly-Gly-NHOH, is capable of
saturating the coordination sphere of the Pd(II) by N-donors in
a mononuclear complex. In fact, this 4N-coordinated species
with very high stability predominates in equimolar solution, pH
> 6 (Chart 2, II). Because in this complex the hydroxamic
oxygens are not involved in the coordination, in the presence
of metal ion excess a second Pd(II) will also be bound via the 5-
membered hydroximate-O,O chelate resulting in the formation
of a dinuclear species (Chart 2, III and IV).
Both Ala-Gly-Gly-N(Me)OH and Ala-Ala-NHOH are capable of
providing up to three N-donors, (NH
former and (NH
Conflicts of interest
2
,Namide,Namide) by the
,Namide,Nhydr.) by the latter ligand. In these There are no conflicts to declare.
2
systems, in mononuclear complexes, saturation of the fourth
coordination site by a strongly coordinating donor can be
achieved by the Ohydr. (Chart 2, VII) in the former case, but, due
to steric reasons this could happen with the latter ligand only
in intermolecular interaction. As a result, unlike with Ala-Ala-
NHOH (Chart 2, XI), binding of metal ion excess is not possible
by Ala-Gly-Gly-N(Me)OH. Steric reason might be responsible
for the difference between the stoichiometry of the complexes
formed in presence of metal ion excess with Ala-Gly-Gly-NHOH
and Ala-Ala-NHOH. Namely, dinuclear species was found to
form with the tripeptide derivate but a trinuclear one with the
dipeptide derivate.
With the two 3N-donor ligands, formation of bis-complexes
was also found in presence of ligand excess. Interestingly, the
formation of the bis-complex was very slow with the
secondary tripeptide derivative, Ala-Gly-Gly-N(Me)OH, and
monitoring the necessary structural changes identical binding
mode of the two coordinating ligands (Chart 2, VIII) was
supported by the NMR results. For the primary dipeptide
derivative, however, the bis complex formation was relatively
Abbreviations
Ala – alanyl
DMSO – dimethyl-sulfoxide
EtOAc – ethyl-acetate
Gly – glycyl
L – completely deprotonated forms of the ligands studied
MeCN –acetonitrile
N
N
amide – deprotonated amide group
hydr. – deprotonated hydroxamic-N
2
NH – terminal amino moiety of the ligand
Ocarb. – carbonyl-O
Ohydr. – deprotonated hydroxamic-O
Pd – Pd(II)aq./Cl
TFA – trifluoroacetic acid
THF – tetrahydrofuran
Z – benzyloxycarbonyl
fast and the results supported the different binding modes of Acknowledgements
the two coordinating ligands. In this latter complex, simple
This journal is © The Royal Society of Chemistry 20xx
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The research was supported by the EU and co-financed by the
European Regional Development Fund under the project GINOP-
28 P. Buglyó, E. M. Nagy, E. Farkas, I. Sóvágó, D. VSiaewnnAraticalenOdnliGne.
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9 P. Buglyó, E. M. Nagy, I. Sóvágó, A. Ozsváth, D. Sanna and E.
Micera, Polyedron, 2007, 26, 1625.
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Notes and references
‡
In the cited work protonation of the (NH
2
, Nhydr.)-bound
0
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9
0
1
2
3
4
5
6
7
8
9
0
+
2+
[
PtL] to [PtHL] with (NH , Ocarb.) binding mode was found to be
2
33 G. Gran, Acta Chem. Scand., 1950, 4, 559.
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of an uncoordinated water on the coordinated and activated
3
4 H. M. Irving, M. G. Miles and L. D. Pettit, Anal. Chim. Acta,
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5 I. Zékány, I. Nagypál, in: D. Leggett (Ed.), Computational
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Plenum, New York, 1985
1
3
31
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S. P. Gupta (ed), Hydroxamic acids: A unique Family of 36 P. Gans, A. Sabatini and A. Vacca, J. Chem. Soc., Dalton
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37 L. I. Elding, Inorg. Chim. Acta, 1972, 6, 647.
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