Oxovanadium(IV) complexes of salicyl-L-aspartic acid and salicylglycyl-L-aspartic acid

Article abstract of DOI:10.1039/b506684k

The dipeptide and tripeptide analogues salicyl-L-aspartic acid (Sal-L-Asp) and salicylglycyl-L-aspartic acid (SalGly-L-Asp) were synthesized and their protonation and complex formation with V^IVO²⁺ were studied in aqueous solution through the use of pH-potentiometry and spectroscopic (UV-Vis, CD and EPR) techniques. The phenolate terminus proved to be a good anchoring site to promote (i) the metal ion-induced deprotonation and subsequent coordination of the peptide amide group(s) in the pH range 4-5 for the dipeptide analogue, (ii) and in the pH range 5-6 in a very cooperative way for the tripeptide analogue. The results suggest that the presence of good anchoring donors on both sides of the amide groups is responsible for the cooperative deprotonation of the two amide-NH groups. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b506684k

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F U L L P A P E R
Oxovanadium(IV) complexes of salicyl-L-aspartic acid and
salicylglycyl-L-aspartic acid†
a
b
c
b
b
Tam a´ s Jakusch, Susana Marc a˜ o, L ´ı gia Rodrigues, Isabel Correia, Jo a˜ o Costa Pessoa* and
a,d
Tam a´ s Kiss*
a
Bioinorganic Chemistry Research Group of the Hungarian Academy of Sciences, Department
of Inorganic and Analytical Chemistry, University of Szeged, POBox 440, H-6701 Szeged,
Hungary
Centro Qu ´ı mica Estrutural, Instituto Superior T e´ cnico, Av. Rovisco Pais, 1049-001 Lisboa,
b
Portugal
c
Departamento de Qu ´ı mica, Universidade do Minho, Largo do Pa c¸ o, 4709 Braga, Portugal
Department of Inorganic and Analytical Chemistry, University of Szeged, POBox 440,
d
H-6701 Szeged, Hungary
Received 12th May 2005, Accepted 15th July 2005
First published as an Advance Article on the web 11th August 2005
The dipeptide and tripeptide analogues salicyl-L-aspartic acid (Sal-L-Asp) and salicylglycyl-L-aspartic acid
IV 2+
(
SalGly-L-Asp) were synthesized and their protonation and complex formation with V O were studied in aqueous
solution through the use of pH-potentiometry and spectroscopic (UV-Vis, CD and EPR) techniques. The phenolate
terminus proved to be a good anchoring site to promote (i) the metal ion-induced deprotonation and subsequent
coordination of the peptide amide group(s) in the pH range 4–5 for the dipeptide analogue, (ii) and in the pH range 5–6
in a very cooperative way for the tripeptide analogue. The results suggest that the presence of good anchoring donors
on both sides of the amide groups is responsible for the cooperative deprotonation of the two amide-NH groups.
Introduction
The metal binding ability of a simple oligopeptide is strongly
determined by the presence in the ligand molecule of a suitable
anchoring donor, which can bind metal ions strongly enough to
promote deprotonation of the amide-NH. Various metal ions
have been found to be able to do this, e.g. Pt(II), Pd(II), Cu(II),
Ni(II), and in some special cases Zn(II), Co(II), and certain
1
−
−
others. This results in an (anchoring donor, CON , COO )eq
−
−
binding mode [below we represent phenolate-O , and amide-N
−
−
by O and CON , respectively, while (X,Y,. . .)eq and (X)ax, mean
that the donor atoms X, Y,. . . are coordinated in the equatorial
or the axial position, respectively], corresponding to a strong in-
teraction between the metal ion and the oligopeptide. These ba-
sic binding modes may be modified considerably by strongly co-
Scheme 1
ordinating side-chain donors, such as imidazole-N or thiolate-S.
Interestingly, deprotonation of the two adjacent amide-
NH groups in VO(SalGly-L-Ala) takes place in a strongly
overlapping way (pK(VOL) = 5.37 and pK(VOLH ) = 5.39);
IV 2+
With V O , neither the terminal NH
COO is a sufficiently strong binder to behave as an efficient
anchoring donor in the promotion of amide deprotonation.
2
nor the terminal
−
−
1
2
the intermediate species containing one deprotonated amide-
−
However, phenolate is known to have a much higher affinity
N
group being formed only in low concentration. Amide
IV 2+
for V O . Accordingly, replacement of the terminal NH
2
group
coordination is accompanied by a characteristic colour change
from blue to pinkish-red at pH ∼ 5.5, with hardly any subsequent
change up to pH ∼ 11. The changes in the EPR parameters
indicate more covalent bonding in the equatorial plane, which
is in accordance with the above-mentioned rearrangement
of the binding modes. A detailed analysis of the CD data
(including deconvolution of the data for the individual species)
−
by the phenolate-O enhances the metal-binding ability of the
molecule significantly. In fact, both the dipeptide analogue 2-
OH-hippuric acid (salicylglycine, SalGly) and the tripeptide
analogue SalGly-L-Ala (for their formulae, see Scheme 1) proved
to be strong V O binders: they keep V O in solution in
the pH range 2–12, even in equimolar ratio. No precipitation
or slow equilibration indicative of hydrolysis of the metal
ion or its complexes is observed in these systems. Detailed
IV 2+
IV 2+
IV
on the V O–SalGly-L-Ala system revealed that a strong CD
2
−
signal accompanies only the formation of [VOLH ] , with both
−
2
3,4
−
pH-metric and spectral studies have indicated that strong
coordination of the ligands to V O can induce deprotonation
peptide-N atoms equatorially coordinated. This indicates that,
although the deprotonation of the two amides take place in a
cooperative way, it starts on the one closer to the phenolate
terminal. This is in agreement with what has been observed
for most metal ions, but is in contrast with what was found
IV 2+
of the peptide-NH group(s), and the complexes containing
−
−
−
the binding mode (O , CON , COO , H O)eq for SalGly,
2
−
−
−
−
and (O ,CON ,CON ,COO )eq for SalGly-L-Ala, become the
−
predominant species by pH ∼ 6.
for R Sn(IV), where the C-terminal COO proved to be the
2
5
anchoring donor.
In the presence of an extra carboxylate in Asp-containing
IV 2+
†
Electronic supplementary information (ESI) available: Fig. SI-1–SI-5.
dipeptides, the V O -binding ability of the ligands is somewhat
See http://dx.doi.org/10.1039/b506684k
increased as compared with that of the Gly-type dipeptides,
3
0 7 2
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5

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−
−
because of the presence of new chelating sites: (COO , COO )
oroacetic acid (TFA) (25 ml) and left at room temperature,
protected from moisture, with occasional stirring for 1 h. The
solution was then concentrated and diethyl ether was added.
The mixture was cooled. The ether was decanted off and the oil
obtained was dried. A solid foam was obtained in quantitative
yield and used in the next step without further purification.
−
in GlyAsp and (NH
2
, COO ) in AspGly. Although these sites
are more efficient for V O binding, the extent of amide
deprotonation/coordination is hardly enhanced as compared
IV 2+
6
with the Gly-type dipeptides.
In this work the pseudo di- and tri-peptides analogues of
salicylic acid, containing aspartic acid in the C terminal position:
Sal-L-Asp 1 (L ) and SalGly-L-Asp 2 (L ) were synthesized and
NMR-DMSOd6 2.78–2.82 (2H, m, aCH Asp); 3.63 (3H, s,
2
1
2
OCH
3
); 3.61 (5H, s + overlapped, CH
2
Gly + OCH
3
); 4.71 (1H,
); 8.95 (1H, d J
IV 2+
+
their V O -binding abilities were studied by means of pH-
apq J 7.2 Hz, aCHAsp); 8.09 (3H, br s, NH
3
potentiometric and spectroscopic techniques.
8 Hz, NH-Asp).
SalGly-L-Asp(OMe)OMe. Salicylic acid (0.69 g, 5 mmol)
Experimental
was dissolved in ethyl acetate (20 ml) and the solution was
◦
Preparation of Sal-L-Asp 1
cooled to 0 C. DCC (1.08 g, 5.2 mmol), compound TFA·Gly-L-
Asp(OMe)OMe (1.66 g, 5 mmol) and triethylamine (0.9 ml,
5 mmol) were added successively. The reaction mixture was
stirred overnight at room temperature. The insolubles were
filtered off and the solvents were removed under reduced
pressure. The residue was taken up in a minimum amount of
Synthesis of L-Asp(OMe)OMe. Thionyl chloride (25 ml)
◦
was added dropwise to stirred and cooled (−10 C) methanol
(
100 ml), followed by the addition of L-Asp (6.66 g, 50 mmol).
◦
The temperature of the solution was then increased to 40 C for
2
h. The solvent was removed next and the oil obtained, after
◦
acetone and left at 0 C for 4 h. The precipitated solid was
crystallization from methanol–diethyl ether, afforded the pure
compound (7.8 g, 79%, mp 115–116 C).
NMR-DMSOd6 3.00–3.03 (2H, m, CH
◦
filtered off (DCHU) and the solution was concentrated. The oil
obtained resisted crystallization (1.091 g, 67%).
NMR-DMSOd6 2.70 and 2.80 (2H, dd J 1.5 and 6.0 Hz,
2
); 3.74 and 3.64 (6H,
+
2
s, 2 × OCH ); 4.32 (1H, t J 5.7 Hz, CH); 8.79 (3H, br s, NH
3
3
).
aCH
5.4 Hz, CH
2
Asp); 3.60 and 3.62 (6H, 2s, 2 × OCH
3
); 3.94 (2H, d J
Gly); 4.70 (1H, apq J 6.0 Hz, aCHAsp); 6.90–6.92
Sal-L-Asp(OMe)OMe. Salicylic acid (2.76 g, 20 mmol) was
2
dissolved in ethyl acetate (80 ml) and the solution was cooled
to 0 C. Dicyclohexylcarbodiimide (DCC) (4.3 g, 21 mmol), L-
Asp(OMe)OMe (3.95 g, 20 mmol) and triethylamine (3.6 ml,
(2H, m, 3-H and 5-H); 7.39 (1H, tap J 8.1 Hz, 4-H) 7.86 (1H,
dd J 0.9 and 8.4 Hz, 6-H); 8.54 (1H, d J 8.1 Hz, NHAsp); 9.08
(1H, t J 6.0 Hz, NHGly); 12.20 (1H, s, Ar-OH).
◦
2
0 mmol) were added successively. The reaction mixture was
stirred overnight at room temperature. The insolubles were then
filtered off and the solution was washed in turn with 5% citric
acid, water, 5% NaHCO
SalGly-L-Asp. SalGly-L-Asp(OMe)OMe
(1.090
g,
3.22 mmol) was dissolved in methanol (10 ml) and an
aqueous solution of 1 M NaOH was added (9.69 ml). The
mixture was stirred at room temperature for 4 h, and 1 M HCl
(3.3 ml) was then added. The methanol was removed under
reduced pressure and the solution obtained was cooled in an
ice-bath and acidified with 1 M HCl (6.4 ml) under vigorous
stirring. This aqueous solution was extracted three times with
ethyl acetate; the organic layers were collected, dried over
magnesium sulfate and filtered and the solvent was removed.
A solid foam was obtained (585 mg, 58%). An attempt of
crystallization from a mixture of methanol, diethyl ether and
3
and water. After drying over MgSO ,
4
the solution was concentrated. The residue was taken up in
◦
a minimum amount of acetone and left at ∼0 C for 4 h.
The precipitated solid was filtered off and the solution was
concentrated. The oil obtained proved resistant to crystallization
(
4.48 g, 79%).
NMR-DMSOd6 2.90–3.20 (2H, m, CH Asp); 3.69–3.78 (1H,
2
m, aCHAsp); 6.91 (2H, apparent t J 7.8 Hz); 7.40 (1H, apparent
t J 8.0 Hz) 7.86 (1H, dd J 1.2 and 8.0 Hz,); 8.90 (1H, d J 8.1 Hz,
NHAsp); 12.40 (1H, br s, OH).
◦
petroleum spirit afforded a white solid, melting at 105–107 C.
NMR-DMSOd6 2.60 and 2.70 (2H, dd J 1.5 and 6.0 Hz,
Sal-L-Asp 1. Sal-L-Asp(OMe)OMe (4.47 g, 15.9 mmol) was
dissolved in methanol (30 ml) and 1 M NaOH (50 ml, 50 mmol)
was added. The mixture was stirred at room temperature for
bCH
.0 Hz, aCHAsp); 6.86–6.92 (2H, m, 3-H and 5-H); 7.39 (1H,
tap J 8.1 Hz, 4-H) 7.86 (1H, dd J 0.9 and 8.4 Hz, 6-H); 8.37
2
Asp); 3.94 (2H, d J 5.7 Hz, CH Gly); 4.56 (1H, apq J
2
6
2
h, and 1 M HCl (16 ml, 16 mmol) was added. The methanol
was removed at reduced pressure and the solution thus obtained
was cooled in an ice bath and acidified with 1 M HCl (34 ml)
under vigorous stirring. The white solid that precipitated out
(
(
1H, d J 7.8 Hz, NHAsp); 9.06 (1H, t J 5.7 Hz, NHGly); 12.20
1H, s, Ar–OH): 12.60 (2H, br s, CO H).
Anal. Calcd. for C14
+ 1/2 C
0.85, H 5.08, N 7.91. Found: C 50.2, H 5.4, N 7.9.
2
H
18
N
2
O
5
4
H
8
O (ethyl acetate): C
2
was filtered off, washed with water and dried (1.2 g, 30%, mp
5
◦
171–172 C).
NMR-DMSOd6 2.84–2.81(2H, m, bCH2Asp); 4.75 (1H, ap-
parent q J 8.1 Hz, aCHAsp), 6.91 (2H, apparent t J 7.2 Hz);
pH-Potentiometric measurements
7
9
.39 (1H, td J 1.8 and 8.0 Hz), 7.88 (1H, dd J 1.8 and 8.2 Hz);
.13 (1H, d J 8.1 Hz, NH); 12.85 (1H, s, OH); 12.60 (2H, br s,
The protonation constants of Sal-L-Asp and SalGly-L-Asp
and the stability constants of their V O complexes were
IV 2+
OH).
Found: C, 52.4; H, 4.6; N, 5.5. Calcd. for C11
H, 4.65; N, 5.18%.
◦
3
determined at 25 C by pH-metric titration of 10 cm sam-
ples. The pHs were measured with an Orion 720A pH-meter
equipped with an Metrohm 6.0234.100 combined glass electrode
H
11
6
O N: C, 52.18;
7
calibrated for hydrogen ion concentration. The ligand concen-
−
3
Preparation of SalGly-L-Asp 2
trations were 0.0020 and 0.0040 mol dm and the ligand to
metal molar ratios were 4 : 4, 4 : 2, 4 : 1, 2 : 2 and 2 : 1. Titrations
were performed with KOH solution of known concentration
Boc·Gly-L-Asp(OMe)OMe. This was prepared by the DCC
method on a 10 mmol scale. The chromatographically pure oil
obtained (2.75 g, 86%) resisted crystallization.
−
3
(
ca. 0.2 mol dm ) under a purified argon atmosphere, to
avoid interference from oxygen and carbon dioxide in the
air. The pH range studied was normally from 2 to 10 unless
extensive hydrolysis or a very slow equilibrium was detected.
The reproducibility of titration points included in the evaluation
NMR-DMSOd6 1.36 (9H, s, Boc); 2.72–2.77 (2H, m,
CH
2
Asp); 3.50 (2H, d J 6 Hz, CH
2
Gly); 3.61 and 3.59 (3 +
3
H, s, 2 × OCH
3
); 4.66 (1H, apq J 7.8 Hz, aCHAsp); 6.97 (1H,
t J 6 Hz, NHGly); 8.23 (1H, d J 8 Hz, NH-Asp).
was within 0.005 pH units in the whole pH range. A pK
w
◦
value
TFA·Gly-L-Asp(OMe)OMe. The protected peptide Boc·Gly-
of 13.76 was determined and used for the titrations at 25 C and
I = 0.20 mol dm− KCl.
3
L-Asp(OMe)OMe (5 mmol, 1.598 g) was treated with triflu-
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8
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The concentration stability constants bpqr = [M
p
L
q
H
r
]/
constants (log K values) of 2.93 and 4.50 for 1 and of 2.90 and
4.49 for 2 can be assigned to the COOH functions present in the
peptides, while the log KOH values of 8.38 for 1 and 7.94 for 2
in the basic pH range are assigned to the phenolic OH groups.
The concentration distribution curves of the complexes formed
p
q
r
[
M] [L] [H] were calculated using the PSEQUAD computer
8
IV 2+
program. The formation of the hydroxo complexes of V O
was taken into account. The following species were assumed:
IV
+
IV
2+
[
−
V O(OH)] (log b10−1 = −5.94), [(V O)
2
(OH)
2
]
(log b20−2
=
IV
IV
6.95), with stability constants calculated from the data of
Henry et al. and corrected for the different ionic strength by
using the Davies equation. [(V O)
and [V O(OH)
in V O-Sal-L-Asp and V O-SalGly-L-Asp systems are shown
in Fig. 1.
9
IV
−
2
(OH)
5
] (log b20 −5 = −22.5)
IV
−
10,11
3
] (log b10−3 = −18.0) were also included.
Instrumentation and procedures
Physical measurements on the ligands. Melting points were
determined on a Gallenkamp melting point apparatus and are
uncorrected. NMR data were recorded on a Varian Unity Plus
3
00 Spectrometer in the solvent indicated. Elemental analyses
were carried out on a Leco CHNS 932 instrument.
Spectroscopic measurements. The CD spectra were recorded
with a JASCO 720 spectropolarimeter with a red-sensitive
photomultiplier (EXEL-308), and visible spectra with a HI-
TACHI U2000 or a OOIChem S2000/PC2000 (the spectral
range covered was normally-Vis-400–900 or 370–880, and in
the case of CD 400–1000 nm). The EPR spectra were recorded
at 77 K with a Bruker ESR-ER 200D X-band spectrometer. The
EPR parameters were obtained by simulation of the spectra with
1
2
the computer program of Rockenbauer and Korecz.
IV 2+
CD, Vis and EPR spectra of V O –Sal-L-Asp system (I)
and V O –SalGly-L-Asp system (II) systems were recorded
IV 2+
by varying the pH at approximately fixed total vanadium and
1
ligand concentrations. For system I, the L : M ratio was 2.6
−
3
2
(
with CVO ∼ 0.003 mol dm ). For system II the L : M ratios
used were as follows: 1.4 for IIa, 2.7 for IIb, and 1.2 for IIc
−
3
−3
(
C
VO ∼ 3 mol dm in IIa and IIb, and CVO ∼ 5.5 mol dm
in IIc). Unless otherwise stated, by visible (Vis) spectra and
CD spectra we mean representations of either e or De
= (differential
= total
Fig. 1 Concentration distribution curves of complexes formed in solu-
IV 2+
−3
tions containing (a) V O and Sal-L-Asp, with CVO = 0.001 mol dm
m
m
IV 2+
and L : M = 4, and (b) V O and SalGly-L-Asp with CVO = 1 mM
and L : M = 4, calculated by using the stability constants listed in Table 1.
values versus k [e
absorption)/(bC
metal concentration].
m
= absorption/(bC
M
), and De
m
M
) where b = optical pathlength and C
M
It is noteworthy that there is some uncertainty in the
2
calculation of b(VOL H−1) of system II from pH-metric data.
Change of the value of log b(VOL H−1) from 0 to 1.5 has almost
Results and discussion
2
no effect on the fitting parameter and the other log b values.
Sal-L-Asp 1 and SalGly-L-Asp 2 were synthesized by the
procedures described in the Experimental. Their formulae are
depicted in Scheme 1. The protonation and V O complex
formation constants of the ligands are listed in Table 1. Both
ligands contain three dissociable protons: on the phenolic OH
group and the two terminal COOH groups. The protonation
2
2
Several calculations for log b(VOL H−1) and log b(VOL H−2
)
IV
based on the Vis, CD and EPR spectra (see ESI†) resulted
2
2
in log b(VOL H−1) ∼ 0.9 and log b(VOL H−2) = −4.53 ±
0
.10. As discussed below, the spectroscopic data indicate the
2
2−
formation of only a small amount of [VOL H−1
10%), therefore the formation constants obtained from the
spectroscopic data were accepted. pH-metry has a tendency to
overestimate the extent of formation of the species [VOL H−1] ;
this is possibly due to the uncertainty in the log b[(VO) (OH) ]
]
(maximum
∼
IV
Table 1 Protonation (log K) and V O complex formation constants
2
2−
(
2
log b with 3 S.D. values in parentheses) of the ligands studied at t =
◦
−3
−
5 C and I = 0.2 mol dm (KCl)
Species Sal-L-Asp
2
5
value, which appears in low concentration in the same pH range.
SalGly-L-Asp
Sal-L-Asp system
log K
log K
log K
VOL
1
2
3
2.93(2)
4.50(1)
8.38(1)
15.09(8)
11.88(3)
7.88(3)
3.09(2)
−5.00(5)
3.21
4.00
4.79
8.09
345
2.90(2)
4.49(1)
7.94(1)
14,1(2)
11.10(6)
7.46(3)
The Vis spectra recorded for this system at different pH values
are depicted in Fig. S1 of ESI†. At very low pH, the spectra
resemble that of [VO(H
1
,2
,2
,2
,2
,2
H
H
2
2
+
1
2
O) ] . In agreement with the speciation
5
VOL
VOL
VOL
VOL
1
1
1
curves, the visible spectra start to deviate from that of the aqua
b
H
H
−1
−2
∼0.9
ion at pH > 2, where the formation of the protonated species
c
1
+
1
−4.53(10)
[VOL H ] and [VOL H] are indicated by the speciation curves.
2
1
,2
,2
pK(VOL
pK(VOL H)
pK(VOL
pK(VOL
No. of points
H
2
)
3.0
The spectral intensity in the near UV region (350–400 nm) starts
to increase at lower pH than for the formation of the [VOL ]
suggesting that the deprotonated phenolate oxygen takes part
of the coordination in both species [VOL H] and [VOL H ] . In
Scheme 2, some of the most probable isomeric binding modes
1
3.54
∼6.5
∼5.5
348
1
−
1
,2
,2
)
H
1
−1
)
1
1
+
2
a
3
−3
−3
Fitting (Dcm )
2.37 × 10
5.95 × 10
1
+
−
−
are presented for [VOL H
2
] : (O , Oamide, 2 × H
2
O)eq, (COO ,
a
Average difference between the experimental and calculated titration
curves expressed in the volume of the titrant. Estimation from the Vis
and the LN EPR spectra Average value, calculated from pH-metry,
Vis-, CD-, LN and RT EPR spectra.
1
−
−
O
amide, 2 × H
2
O)eq and for [VOL H]: (COO , Oamide, O , H
2
O)eq
,
b
−
c
(2 × COO , 2 × H
2
O)eq.
As the pH is increased (up to pH 7.5) there is a small shift of
the kmax to higher wavelengths. Between pH 5 and 8, a new band
3
0 7 4
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8

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Scheme 2
is observed at kmax ∼ 510 nm. At pH > 9, binary and ternary
−
OH containing species are formed.
The CD spectra for system I (Fig. 2) are in good agreement
with the species distribution (Fig. 1). Starting from pH ∼ 3.0, in
1
−
parallel with the formation of [VOL ] , the intensity of the CD
spectra (a negative band at around k = 790–800 nm) increases
1
2−.
1
−
up to the maximum amount of [VOL H−1
]
As [VOL ] starts
to deprotonate (pH ∼ 4), a new negative band appears at
490 nm. At pH ∼ 6.3, the CD shows maximum intensity,
with two negative bands at ∼790 nm and ∼490 nm (De = −0.6
and −0.35 mol dm cm , respectively), which can be assigned
∼
−
1
−3
−1
1
2−
to the formation of [VOL H−1] . The pattern of the spectrum
IV
6
is similar to that for V O–N-acetyl aspartic acid system,
suggesting its related to the coordination of two carboxylate
groups. At higher pH values the intensity decreases, and for
pH > ∼ 8.5 the pattern of the CD spectra changes, indicating
the formation of a different species, a ternary hydroxo complex
1
3−
with [VOL H−2
]
stoichiometry. As the shape of the spectra
IV
differs from what was measured in the system V O–N-acetyl
aspartic acid, we assume that the side-chain carboxylate group
6
of Asp was already displaced from the coordination sphere of
the metal ion, probably because of the increased charge of the
species.
The experimental visible and CD spectra obtained at dif-
ferent pH values were used to calculate the spectra of each
individual species for the V O-Sal-L-Asp system, using the
PSEQUAD computer program. Good CD and visible spectra
could be simulated for each species (see Fig. 3). This con-
firms the validity of the speciation model proposed and the
reliability of the stability constants calculated from pH-metric
IV 2+
−3
IV
Fig. 2 CD spectra of solutions containing V O (0.003 mol dm
)
−
3
and Sal-L-Asp (0.008 mol dm ) at different pH values in the range:
.52–6.34 (a) and 6.34–10.96 (b).
2
studies and the spectroscopic data obtained from the spectral
measurements.
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8
3 0 7 5

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2
3−
stoichiometry [VOL H−2] . In these spectra two main bands
−
1
−3
−1
can be observed: at ∼625 nm (De = 0.23 mol dm cm ) and
760 nm (De = −0.1 mol dm cm ). Two other bands are seen
at ∼485 nm (De ∼ 0.11 mol dm cm ) and ∼430 nm (De ∼
.15 mol dm cm ). These CD features of V O –SalGly-L-
−
1
−3
−1
−3
∼
−
1
−1
−
1
−3
−1
IV 2+
0
IV 2+
8
Asp are very similar with those of V O -SalGly-L-Ala, which
strongly suggests similar binding modes (vide supra) in the two
systems, i.e. the carboxylate group of the Asp side-chain does
IV 2+
not coordinate to V O .
Plausible binding modes
In order to elucidate the binding modes of the species, EPR
spectra were measured both at room temperature and in frozen
solution. The high-field region of the EPR spectra at 77 K, is
depicted in Fig. 5 and 6. Table 2 includes the spin-Hamiltonian
Fig. 3 Calculated Vis spectra of each individual species formed in
IV
the V O-Sal-L-Asp system, using the program PSEQUAD and the
formation constants listed in Table 1.
SalGly-L-Asp system
Up to pH 4.9 the spectra recorded for the two systems are similar
IV 2+
in type and resemble those for the V O –N-acetyl-L-aspartic
6
acid system. One main negative band is observed at k ∼ 750 nm
−
1
−3
−1
(
De = −0.25 mol dm cm ), with a shoulder at ∼650 nm
and a low-intensity positive band at ∼540 nm (see Fig. 4a). This
is in agreement with the coordination of the two carboxylate
groups. In contrast with system I, the |De| values of the band
at ∼750 nm start to decrease at lower pH values (pH > 4.9),
2
in parallel with the deprotonation of the [VOL ] species. For a
further increase of the pH in the range 7.5–12 the spectra remain
roughly the same (Fig. 4b), corresponding to the formation of
Fig. 5 High-field region of frozen solution EPR spectra (X-band)
IV
recorded at 77 K on the V O-Sal-L-Asp system at a 1 : 2.6 metal ion to
ligand ratio, at different pH values, cVO = 0.003 mol dm−
3
.
IV 2+
−3
Fig. 4 CD spectra solutions containing V O (0.003 mol dm ) and
Fig. 6 High-field region of frozen solution EPR spectra (X-band)
−
3
IV
SalGly-L-Ala (0.004 mol dm ) at different pH values in the range:
recorded at 77 K on the V O-SalGly-L-Asp system at a 1 : 1.4 metal ion
to ligand ratio at different pH values, cVO = 0.004 mol dm−
3
.
(
a) 2.43–4.92 and (b) 4.92–11.78.
3
0 7 6
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8

View Article Online
IV
Table 2 Spin-Hamiltonian parameters (g|| and A||) obtained for the species formed in V O–Sal-L-Asp and –SalGly-L-Asp systems. The EPR
12
parameters were obtained by simulation of the spectra with the computer program of Rockenbauer and Korecz
Species
Sal-L-Asp system
Proposed binding set
est
g
||
A
||
A
||
4
−1
×
10 /cm
IV
1
−
−
V OL H
2
1.935
1.935
1.938
1.947
1.953
1.935
1.937
1.939
1.947
1.957
177.9
176.0
174.0
165.2
161.8
178.0
176.2
173.3
164
177; 174
176; 170
170
(a-COO , Oamide, 2 × H
2
O)eq; (O , Oamide, 2 × H
2
O)eq
O)eq
IV
1
−
−
−
V OL H
(2 × COO , 2 × H
2
O)eq; (a-COO ,Oamide, O , H
O)eq(b-COO )ax
2
IV
1
1
−
−
−
V OL
(O , Oamide, COO , H
2
IV
a
−
−
−
−
V OL H−1
165
158
(O , CON , COO , H
2
O)eq(b-COO )ax
IV
1
a
−
−
−
−
V OL H−2
(O , CON , COO , HO )eq
IV
2
−
−
V OL H
2
177; 174
176; 170
166.8
(a-COO , 2 × Oamide, H
2
O)eq; (a-COO , Oamide, 2 × H
2 ax
O)eq(Oamide)
IV
2
−
−
−
V OL H
(2 × COO , 2 × H
2
O)eq; (O , Oamide, COO , H
2
O)eq(Oamide
)
ax
,
IV
2
2
−
−
V OL
(O , Oamide, 2 × COO )eq(Oamide
)
ax
IV
b
−
−
−
V OL H−1
163
(O , CON , Oamide, COO )eq
IV
2
b
−
−
−
V OL H−2
159.2
157
(O , 2 × CON , COO )eq
a
−
b
−
For CON contribution 38 was used. For CON , a contribution of 36 was used (see text).
1
2
parameters obtained by simulation of the experimental spectra.
coordination sphere and for steric reasons probably occupies
1 1
IV
13
−
2−
For the V O systems, Chasteen introduced an additivity rule
the axial position in both [VOL ] and [VOL H−1] . This is
1
est
est
to estimate the hyperfine coupling constant A|| (A|| = RA||,i),
also in agreement with the significantly higher pK(VOL H−1)
IV 3
based on the contributions A||,i of each of the four equatorial
value of 8.09 as compared with that for V O–SalGly (=7.57 )
est
−4
−1
donor groups. The estimated accuracy of A|| is ± 3 10 cm .
Most of the A||,i were presented by Chasteen and values for
in which the axial position is occupied by an H O molecule.
2
1
2−
The proposed structure for the resulting species [VOL H−2
]
is
−
14
the A||(CON ) contributions were proposed by Cornman and
Costa Pessoa. Following the suggestion of Tasiopoulos, in
this work we use different A||(CON ) contributions in the
range 36–40 × 10 cm depending on the total charge of the
presented in Scheme 2.
For system II, the parallel deprotonation/coordination of the
two amide groups are strongly cooperative processes, as was
15
16
−
−
4
−1
IV
4
found for V O–SalGly-L-Ala system. The stepwise deprotona-
2
equatorial ligands.
tion constants of the two amide-NH groups are pK(VOL ) ∼ 6.6
−
4
−1
17
2
We use 43.7 × 10 cm for the A||(Oamide
)
and 42.1 ×
and pK(VOL H−1) ∼ 5.4. Overall, the CD spectra differ from
−
4
−1
− 18
10
cm for the A||(COO ) contribution. These parameters
those of system I, indicating distinct coordination modes of the
b-carboxylate group for the two ligands. The average pK of the
processes determined by the different techniques (pH-metry:
6.12, Vis: 6.01, RT-EPR: 5.98, LN-EPR: 5.95, CD: 5.92) is
approximately half a unit higher than that for the same processes
in the system VO–SalGly-L-Ala. The presumed equatorial
coordination of the carboxylate group of the Asp side-chain in
can be used to predict the probable binding mode of each of the
complexes formed, but care must be taken as the contributions
of the donor groups to the hyperfine coupling constant may also
19–20
21
depend also on their orientation,
or type of the axial ligand.
The influence of the axial donor groups (if any) is not taken into
account.
The speciation curves (Fig. 1) indicate that complex forma-
tion starts at relatively low pH with the protonated species
] and [VOL H]. In both systems, the EPR parame-
ters and Vis spectra at low pH (ca. 2–3.3) indicate the presence
2
−
[VOL ] is the cause of the difference: a structural rearrangement
is necessary, in parallel with the deprotonation of the amide
nitrogens. Species containing one deprotonated amide–N seem
to be formed in negligible amounts, probably because the good
anchoring donors present, a negatively charged phenolate–O
on one side, and a negatively charged carboxylate–O on the
1
,2
+
1,2
[
VOL H
2
2
+
1,2
+
1,2
−
of a mixture of [VO(H
2
O)
5
] , [VOL H
2
]
and [VOL H]
] possibly involves both
1
,2
+
−
species. The binding mode of [VOL H
2
−
the carboxylate (or the phenolate-O ) and Oamide donors (see
other side, making the deprotonation/coordination strongly
cooperative. Low temperature EPR measurements were made
with nearly equimolar solutions in an effort to detect the
−
Scheme 2). The unambiguous appearance of the phenolate-O
IV
1,2
→
V O CT band, in the Vis spectra of [VOL H] indicates
−
2
2−
phenolate coordination, but probably Asp-type (2 × COO and
monodeprotonated species [VOL H−1] . Samples with the same
composition were measured both by CD and by EPR; some of
the spectra are presented in Fig. 6. The EPR spectra show that
0
, 1 or 2 × Oamide) coordination also occurs, and thus isomeric
structures coexist in the solution. It is difficult to determine
1
,2
2
−
the exact EPR parameters for each of the species VOL H
2
-
the concentration of [VOL ] is approximately equal to that of
1
,2
1,2
2
3−
VOL H-VO L, as the A|| values change almost continuously
in both systems in the pH range 3–5.5, and the individual spectra
strongly overlap each other.
For both systems, the formation of [VOL ] gives rise to
considerable changes in the CD spectra, due to the coordination
of the Asp side-chain, and the maximum concentrations of the
complexes corresponding to this stoichiometry are achieved at
pH ∼ 4.5 for system I, and ∼5 for system II. Plausible binding
modes are indicated in Scheme 2.
The next deprotonation step in both systems is ascribed
to the amide deprotonation/coordination. For system I the
pK(VOL ) value of 4.79 agrees with that for the correspond-
ing complex V O-SalGly (=4.76 ), indicating close structural
similarity between the complexes involved. The pattern of the
CD spectra up to pH 6.3 remains approximately the same and is
similar to that for V O –N-acetyl-L-aspartic acid system. This
suggests that the b-carboxylate of the Asp side-chain is in the
[VOL H−2
]
at around pH 5.8, and a low-intensity signal which
2
2−
can presumably be ascribed to [VOL H−1
]
is also observed,
its maximum relative concentration being ∼10%. Accordingly,
we can say that in system II there is high cooperativity in the
deprotonation of the two NHamide protons and the coordination
1
,2
−
−
of both CON donors (see also Table 2).
The room temperature EPR measurements (Fig. SI-2 in
ESI†) also confirmed these findings: the spectra in the pH
range 5–8 could be simulated within experimental error as a
2
−
linear combination of the individual spectra of [VOL ] and
2
3−
[VOL H−2] . It is not necessary to presume formation of
1
2
2−
[VOL H−1
]
to explain all the features of the room temperature
IV
3
EPR spectra. The CD measurements led to similar findings:
(Fig. SI-3 in ESI†) it was possible to describe the system in
the same pH range with two linearly independent CD spectra,
IV 2+
6
2
−
2
3−
those of [VOL ] and [VOL H−2] . This means again that
2
2−
complex [VOL H−1
]
has almost no contribution to the CD. The
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8
3 0 7 7

View Article Online
2
2−
log b value of species [VOL H−1
(
]
was estimated from the Vis
References
Fig. SI-4 in ESI†), and the LN EPR spectra (Fig. SI-5 ESI†).
The spectral uncertainty of [VOL H−1
1
I. S o´ v a´ g o´ , in Biocoordination Chemistry, ed. K. Burger, Ellis Hor-
wood, New York, 1990, ch. 4, p. 134.
2
2−
]
species does not allow
us to determine clearly which amide-NH deprotonates first,
though, the missing CD intensity suggests the same sequence
as in the case of the SalGly-L-Ala system, i.e. the amide-NH
near the phenolate terminus deprotonates first, and the amide-
NH near the carboxylate terminus second.
2
T. Kiss, T. Jakusch, J. Costa Pessoa and A. I. Tomaz, Coord. Chem.
Rev., 2003, 237, 123.
3 T. Kiss, K. Petrohan, P. Buglyo, D. Sanna, G. Micera, J. Costa Pessoa
and C. Madeira, Inorg. Chem., 1998, 37, 6389.
4
5
6
T. Jakusch, A. D o¨ rnyei, I. Correia, L. Rodrigues, G. T o´ th, J. Costa
Pessoa, T. Kiss and S. Marc a˜ o, Eur. J. Inorg. Chem., 2003, 11, 2113.
A. Jancs o´ , T. Gajda, A. Szorcsik, T. Kiss, B. Henry, Gy. Vank o´ and
P. Rubini, J. Inorg. Biochem., 2001, 83, 187.
Conclusions
J. Costa Pessoa, T. Gajda, R. D. Gillard, T. Kiss, S. M. Luz, J. J. G.
Moura, I. Tomaz, J. P. Telo and I. T o¨ r o¨ k, J. Chem. Soc., Dalton
Trans., 1998, 3587.
The speciation and solution spectral studies discussed above
−
indicate that both the terminal COO and the phenolate-
−
7 H. M. Irving, M. G. Miles and L. D. Pettit, Anal. Chim. Acta, 1967,
8, 475.
8
O
can behave as anchoring donors to chelate the dipeptide
3
and tripeptide-analogue ligands Sal-L-Asp and SalGly-L-Asp:
L. Z e´ k a´ ny and I. Nagyp a´ l, in Computational Methods for the
Determination of Stability Constants, ed. D. Leggett, Plenum, New
York, 1985, p. 291.
IV 2+
both ligands are strong V O binders and deprotonation of
the amide-NH group(s) is induced by the metal ion in both
systems.
9 R. P. Henry, P. C. H. Mitchell and J. E. Prue, J. Chem. Soc., Dalton.
Trans., 1973, 1156.
A comparison of the two ligands indicates that presence of
the b-carboxylate group has a different effect on the metal
ion induced deprotonation and subsequent coordination of the
1
1
0 A. Komura, M. Hayashi and H. Imagana, Bull. Chem. Soc. Jpn.,
1
977, 50, 2927.
1 L. F. Vilas Boas, J. Costa Pessoa, in Comprehensive Coordination
Chemistry, ed. G. Wilkinson, R. D. Gillard and J. A. McCleverty,
Pergamon Press, Oxford, 1987, vol. 3, p. 453.
12 A. Rockenbauer and L. Korecz, Appl. Magn. Reson., 1996, 10, 29.
13 N. D. Chasteen, in Biological Magnetic Resonance, ed. J. Lawrence,
L. J. Berliner and J. Reuben, Plenum, New York, 1981, vol. 3, p. 53.
IV
peptide amide group. For the V O–Sal-L-Asp system this takes
place with a pK of 4.79, which is practically the same as that for
a
3
SalGly (4.76). However, further deprotonation (substitution of
−
an H
2
O ligand by OH ) occurs with a significantly higher pK
a
IV
value (8.09), as compared with that of the corresponding V O–
1
4 C. R. Cornman, E. P. Zovinka, Y. D. Boyajian, K. M. Geiser-Bush,
P. D. Boyle and P. Singh, Inorg. Chem., 1995, 34, 4213.
5 J. Costa Pessoa, J. L. Antunes, L. F. Vilas Boas and R. D. Gillard,
Polyhedron, 1992, 6, 1449.
3
SalGly complex (= 7.57 ), in which the axial position is
−
occupied only by a water molecule instead of a b-COO
1
function.
As for SalGly-L-Ala, deprotonation of the two amide-NH
groups of SalGly-L-Asp occurs in a cooperative way, but at
16 A. J. Tasiopoulos, A. N. Troganis, A. Evangelou, C. P. Raptopoulou,
A. Terzis, Y. G. Deligiannakis and T. A. Kabanos, Chem.-Eur. J.,
1
999, 5, 910.
a significantly higher pH; the pK values are 6.00 (5.37 for
a
4
17 B. J. Hamstra, A. L. P. Houseman, G. J. Colpas, J. W. Kampf, R.
SalGly-L-Ala ), the difference being due to the effect of the
LoBrutto, W. D. Frasch and V. L. Pecoraro, Inorg, Chem., 1997, 36,
−
equatorial coordination of the b-COO function of the Asp
4
866.
moiety. Coordination of the b-carboxylate of the Asp has also
1
8 T. Jakusch, P. Bugly o´ , A. I. Tomaz, J. Costa Pessoa and T. Kiss, Inorg.
been observed to hinder amide-NH deprotonation in Cu(II)
Chim. Acta, 2002, 339, 119.
22–25
complexes.
19 T. S. Smith, C. H. Root, J. W. Kampf, P. G. Rasmussen and V. L.
Pecoraro, J. Am. Chem. Soc., 2000, 122, 767–775.
2
2
0 I. Cavaco, J. Costa Pessoa, M. T. Duarte, R. T. Henriques, P. M.
Matias and R. D. Gillard, J. Chem. Soc., Dalton. Trans., 1996, 1989.
1 J. T. Evangelos, K. D. Soulti, T. A. Kabanos, C. P. Themistoklis, C. P.
Raptopoulou, A. Terzis and Y. Deligiannakis, Chem. Commun., 2000,
601.
2 I. S o´ v a´ g o´ , T. Kiss and A. Gergely, Inorg. Chim. Acta, 1984, 93, L53.
3 B. Decock-Le Reverend, A. Lebriki, D. Livera and L. D. Pettit, Inorg.
Chim. Acta, 1986, 124, L19.
Acknowledgements
This work was carried out in the frame of a COST D21
project. The authors are grateful to the National Research
Fund (OTKA T49417/2004), the Hungarian Academy of
Sciences, the Fundo Europeu para o Desenvolvimento Re-
gional, Funda c¸ a˜ o para a Ci eˆ ncia e Tecnologia, the POCTI
Programme (project POCTI/QUI/56949/2000), the PhD grant
SFRH/BD/6371/2001 and the Hungarian-Portuguese Inter-
governmental S & T Co-operation Programme for 2004–2005.
2
2
2
4 I. S o´ v a´ g o´ , B. Radomska, I. Schn and O. Ny e´ ki, Polyhedron, 1990, 9,
8
25.
25 B. Decock-Le Reverend, L. Andrianariajona, C. Livera, L. D. Pettit,
I. Steel and H. Kozlowski, J. Chem. Soc., Dalton Trans., 1986, 2221.
3
0 7 8
D a l t o n T r a n s . , 2 0 0 5 , 3 0 7 2 – 3 0 7 8