ZnII complex with a phenanthroline-containing macrocycle as receptor for amino acids and dipeptides - Hydrolysis of an activated peptide bond

Article abstract of DOI:10.1002/ejic.200200648

The interaction between several L-amino acids and dipeptides and the Zn^II complex with ligand L, a macrocycle containing a triamine chain linking the 2,9 positions of a phenanthroline moiety, has been studied by means of potentiometric and

Full text of DOI:10.1002/ejic.200200648

FULL PAPER
ZnII Complex with a Phenanthroline-Containing Macrocycle as Receptor for
Amino Acids and Dipeptides ؊ Hydrolysis of an Activated Peptide Bond
Carla Bazzicalupi,[a] Andrea Bencini,*[a] Emanuela Berni,[a] Antonio Bianchi,*[a]
Patrizia Fornasari,[a] Claudia Giorgi,[a] and Barbara Valtancoli[a]
Keywords: Macrocyclic ligands / Zinc / Amino acids / Enzyme models / Stacking interactions
The interaction between several
L
-amino acids and dipept-
most stable complexes, due to hydrophobic and/or π-stack-
ing interactions between the aromatic subunits of the sub-
strates and the phenanthroline moiety of the metal receptor.
The hydrolytic properties of the ZnII complex with L, invol-
ving the cleavage of the peptide bond, have been tested by
ides and the ZnII complex with ligand L, a macrocycle con-
taining a triamine chain linking the 2,9 positions of a phen-
anthroline moiety, has been studied by means of potentio-
metric and 1H NMR spectroscopic measurements in aqueous
solutions. In the [ZnL]2+ complex, the metal ion displays an
unsaturated coordination environment and the metal can act
as a binding site for these substrates. Amino acids and di-
using
L-leucine-p-nitroanilide (LNA), which contains an
activated peptide bond. This substrate forms stable
[ZnL(LNA)]2+ and [ZnL(OH)(LNA)]+ complexes. The forma-
peptides in their neutral (zwitterionic) form bind to ZnII tion of the [ZnL(OH)(LNA)]+ complex is followed by LNA hy-
through the carboxylate function and, when in their anionic
drolysis, through a nucleophilic attack of the Zn−OH function
form, through the amine group. In this case the carbonyl of on the peptide bond.
the amide function is probably also involved in metal coor-
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
dination. Amino acids containing aromatic pendants form the Germany, 2003)
Introduction
used as simple model systems for hydrolytic enzymes.[16Ϫ50]
Examples of functional models for aminopeptidases are less
common, due to the marked hydrolytic inertness of the pep-
tide bond to hydrolysis. Examples of CuII,[17,18,51Ϫ53] PtII,[54]
PdII,[55Ϫ57] CoIII [18,58] and CeIV [59] complexes able to cleave
the peptide bond in aqueous solutions are known; peptide
bond cleavage induced by ZnII complexes has rarely been
observed,[40] and occurs only at high temperature in aque-
ous solution, due to the low tendency of ZnII to hydrolyze
the amide bonds. As a matter of fact, binding of free ZnII
to peptides may even inhibit the cleavage of the peptide
bond.[37]
Over the years a great deal of interest has focused on
the role of metal ions in the active centers of hydrolytic
metalloenzymes (e.g. phosphatase, amino- and carboxypep-
tidase, carbonic anhydrase, P1-nuclease).[1Ϫ15] Hydrolytic
metalloenzymes generally contain one or more ZnII ions in
their active site. The catalytic role of zinc is generally as-
cribed to two main functions: i) binding and activation of
the substrates, and ii) deprotonation of ZnII-coordinated
water molecules to give ZnϪOH functions, which can act
as nucleophiles in the hydrolytic mechanism. The catalytic
site is often lodged in a hydrophobic pocket, which may
contribute to substrate binding and can also assist in the
formation of the nucleophilic ZnIIϪOH functions.
We have recently reported on the synthesis of a series
of phenanthroline-containing polyamine macrocycles,[60,61]
such as L (Figure 1), able to form stable ZnII complexes in
aqueous solutions.[62]
Aminopeptidases are one of the ZnII enzymes and con-
tain either one or two zinc ions per active site and specifi-
cally hydrolyze the N-terminal residue of polypeptide
chains, through a catalytic mechanism which involves coor-
dination of the peptide function to zinc, followed by a nu-
cleophilic attack of a ZnϪOH group on the carbonyl car-
bon and cleavage of the peptide bond.[4,5,7,12Ϫ15]
As one of the approaches to the study of ZnII enzymes,
a number of synthetic ZnII complexes have recently been
[a]
`
Dipartimento di Chimica, Polo Scientifico, Universita di
Firenze,
Figure 1. L drawing (with atom labelling used in NMR spectro-
scopic experiments) and crystal structure of the [ZnL(H2O)]2ϩ cat-
ion.
Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
E-mail: andrea.bencini@unifi.it
1974
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejic.200200648
Eur. J. Inorg. Chem. 2003, 1974Ϫ1983

ZnII Complex with a Phenanthroline-Containing Macrocycle
FULL PAPER
The rather high stability of the complexes is mainly due [ZnL(HGly)]2ϩ cation contains the amino acid in its neutral
to the heteroaromatic nitrogens, which can offer an optimal form, herein indicated by HGly. The asymmetric unit con-
binding site for the metal ion. At the same time, the rigidity tains two [ZnL(HGly)]2ϩ independent cations. The OR-
of the phenanthroline unit does not allow for the involve- TEP[63] drawings of the two [ZnL(HGly)]2ϩ cations (herein
ment of all the aliphatic amine groups in the metal binding. indicated A and B) with atom labelling are shown in Fig-
Actually, the crystal structure of the [ZnL(H2O)]2ϩ cation ure 2 and Table 1 lists selected bond angles and distances
(Figure 1) shows a ZnII coordination environment not satu- for the coordination sphere of ZnII. The coordination en-
rated by the ligand donors, since the two benzylic nitrogens vironment for the ZnII ion is almost equal in both the A
are not involved in metal binding.[62] A water molecule is and B cations and can be described as a distorted tetra-
coordinated to ZnII, to give an almost tetrahedral coordi- hedron. In complex A, the Zn1 ion is coordinated by the
nation environment for the metal. The presence of ‘‘free’’ N1 and N2 heteroaromatic nitrogens, the central N4 amine
binding sites at the ZnII ion makes this complex a promising of the aliphatic chain and the O1g oxygen of the car-
metallo-receptor for substrate molecules.
boxylate group of the amino acid.
A solution study showed that the coordinated water mol-
ecule deprotonates at a slightly alkaline pH to give a
ZnIIϪOH function. A dihydroxo complex [ZnL(OH)2] is
also formed at an alkaline pH. Finally, phenanthroline can
generate a rather hydrophobic environment for the metal.
These characteristics make this complex a potential candi-
date for a model system of hydrolytic enzymes.
With this in mind, we decided to investigate the effective
binding properties of this complex for amino acids and di-
peptides as well as its hydrolysis of a peptide bond. The
substrates involved in this study, along with the atom labe-
ling used in the NMR spectroscopy experiments, are shown
in Scheme 1.
Figure 2. ORTEP drawing of the [ZnL(HGly)]2ϩ complex, rep-
resenting the A and B independent units coupled by π-stacking and
CϪH···O hydrogen bond interactions
˚
Table 1. Bond lengths [A] and angles [°] for the metal coordination
environment
in
the
A
and
B
cations
of
[ZnL(HGly)]2(ClO4)4·1.5H2O
A Unit
Zn1ϪO1g
Zn1ϪN2
O1gϪZn1ϪN1
O1gϪZn1ϪN2
O1gϪZn1ϪN4
1.989(4)
2.171(5)
103.79(19)
104.79(17)
135.5(2)
Zn1ϪN1
Zn1ϪN4
N1ϪZn1ϪN2
N4ϪZn1ϪN1
N4ϪZn1ϪN2
2.165(5)
2.094(6)
74.64(19)
110.5(2)
110.9(2)
B unit
Zn2ϪO3g
Zn2ϪN6
O3gϪZn2ϪN6
O3gϪZn2ϪN7
O3gϪZn2ϪN9
1.990(4)
2.150(5)
104.83(18)
104.42(19)
136.1(2)
Zn2ϪN7
Zn2ϪN9
N6ϪZn2ϪN7
N9ϪZn2ϪN6
N9ϪZn2ϪN7
2.167(5)
2.095(6)
75.0(2)
110.7(2)
109.0(2)
Scheme 1
˚
The ZnϪN bond lengths are in the range 1.99Ϫ2.17 A.
Their values are typical for ZnII complexes with polyamine
compounds. The benzylic nitrogens N3 and N5 are not co-
ordinated, lying far apart from the metal ion [Zn1···N3
Results and Discussion
Crystal Structure of [ZnL(HGly)]2(ClO4)4·1.5H2O
The crystal structure consists of [ZnL(HGly)]2ϩ cations,
˚
˚
2.440(6) A, Zn1···N5 2.448(7) A].
In complex B, the Zn2 ion is coordinated by three donor
perchlorate anions and water solvent molecules. The atoms of the ligand molecule (N6, N7 and N9) and the
Eur. J. Inorg. Chem. 2003, 1974Ϫ1983
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1975

A. Bencini, A. Bianchi et al.
FULL PAPER
O3g oxygen atom of glycine, while the N8 and N10 benzylic
nitrogen atoms are not coordinated (Table 1).
All of the amino acids under investigation, with the ex-
ception of leucine, interact with the [ZnL]2ϩ complex, both
It is worth noting that, in both [ZnL(HGly)]2ϩ indepen- in their neutral and deprotonated forms, herein indicated
dent cations, the amino acid is in its zwitterionic form. In as HA and AϪ, respectively, to give the [ZnL(HA)]2ϩ and
both A and B complexes, the CϪO bond lengths of the [ZnL(A)]ϩ species (Table 2). Among the six amino acids,
carboxylic group of HGly are equal within their e.s.d. leucine gives the weakest interaction, and only the complex
˚
˚
[C1gϪO1g 1.234(8) A, C1gϪO2g 1.238(8) A, C3gϪO3g with its deprotonated form is formed in aqueous solutions.
˚
˚
1.259(7) A, C3gϪO4g 1.226(8) A], indicating that this On the contrary, tryptophan gives the strongest interaction
group is in its anionic form. The unbound amine group is with [ZnL]2ϩ. Figure 3 shows the distribution diagram of
therefore protonated. Such a monodentate binding mode of complexes formed by tryptophan in the presence of our
Gly is rather common for amino acids in their zwitterionic ZnII complex. The formation of the complex with the zwit-
form.[24Ϫ26,35Ϫ37,64]
terionic form of the amino acid [ZnL(HTrp)]2ϩ occurs at
In both A and B, the coordination geometry of the metal an acidic pH and is followed by deprotonation of the amino
ion is very similar to that found in the crystal structure acid at neutral pH to give the [ZnL(Trp)] complex. In this
of [ZnL(H2O)](ClO4)2. Actually, in the present structure an case a third species, [ZnL(Trp)OH]ϩ, is found at an alka-
oxygen donor of the carboxylate unit simply replaces the line pH.
ZnII-coordinated water molecule of [ZnL(H2O)](ClO4)2.
The overall conformation of the ligand is also similar in
the two complexes. As already found in [ZnL(H2O)]2ϩ, the
ligand is folded along the line connecting the benzylic nitro-
gen atoms, giving rise to dihedral angles of 82.4(3)° (A com-
plex) and 84.5(3)° (B complex) between the mean planes
defined respectively by the benzylic nitrogen atoms and the
˚
aromatic unit (max deviations 0.15(1) A for C8 in the A
˚
complex and 0.154(8) A for C25 in the B complex) and by
the three secondary nitrogen atoms of the aliphatic chains.
Inspection of the crystal packing reveals the presence of
π-stacking interactions between the phenanthroline moiety
of the two independent cations. As shown in Figure 2, the
Figure 3. Species distribution diagram for the system [ZnL]2ϩ/Trp
two heteroaromatic groups are almost parallel [the dihedral
angle between the planes containing the aromatic groups is
in 1:1 molar ratio (NMe4NO3 0.1 , 298 K).
˚
7.1(2)°] and lie 3.55 A apart from each other. In addition,
The data in Table 2 clearly outline that the stability of the
two H-bonding interactions between a CϪH aromatic car- complexes of our metallo-receptor with the neutral form of
bon atom of each complex and a carboxylate oxygen atom amino acids (HA) is lower than that found with their
belonging to the other [C5···O3g 3.34(1) A and C28···O1g anionic form (AϪ). A similar behavior was found for the
˚
[65]
˚
3.412(9) A] contribute to the molecular pairing.
addition of amino acids to the ‘‘free’’ ZnII ion; it is gener-
ally accepted that coordination to ZnII involves the anionic
carboxylate function in the [Zn(HA)]2ϩ complex and both
the carboxylate and the amine groups, with the formation
of a six-membered chelate ring, in the deprotonated
[Zn(A)]ϩ complex.[35Ϫ37,47,64] To shed further light on the
binding mode of amino acids to our ZnII complex we re-
Binding of
Complex Containing L
L-Amino Acids and L
-Dipeptides to the ZnII
The binding of the amino acids glycine (HGly), -phenyl-
glycine (Hph-Gly), -alanine (HAla), -phenylalanine
(HPhe), -leucine (HLeu) and -tryptophan (HTrp) to the
[ZnL]2ϩ complex was studied by means of potentiometric
and 1H NMR spectroscopic measurements in aqueous solu-
tions; the results of the potentiometric investigation are re-
ported in Table 2.
1
corded H NMR spectra at different pH values. Figure 4
(a) shows the pH dependence of the Trp signals in the pres-
ence and in the absence of our ZnII complex, while Figure 4
1
(b) shows the H NMR spectrum of a D2O solution con-
taining [ZnL](ClO4)2·H2O and tryptophan in a 1:1 molar
ratio at pH 9.3, where the [ZnL(Trp)]ϩ is the prevalent spec-
ies in solution, compared with those of deprotonated tryp-
Table 2. Equilibrium constants for the addition of -amino acids
to the ZnII complex with the ligand L (NMe4NO3 0.1 , 298 K).
tophan (TrpϪ) and [ZnL]2ϩ
.
Reaction
log K
Phe
As shown in Figure 4(a), the formation of the
Gly
ph-Gly Ala
Leu
Trp
[ZnL(HTrp)]2ϩ complex in the pH range 3Ϫ7 does not lead
1
[ZnL]2ϩϩ AϪ
ϭ
2.96(4) 3.61(3) 2.95(4) 3.83(4) 2.11(4) 5.12(6)
to a significant variation in the chemical shifts of the H
[ZnL(A)]ϩ
NMR signals of the protons in the α-position to the car-
boxylate and the amine function (a in Scheme 1) with re-
spect to the same signals of the amino acid HA (prevalent
in solution in the pH range 3Ϫ8), where the acidic proton
is located on the amine group. This would indicate that in
[ZnL]2ϩϩAH ϭ
[ZnL(AH)]2ϩ
2.58(4) 2.71(5) 2.43(2) 2.79(3) Ϫ
3.71(3)
5.02(6)
[ZnL(OH)]ϩϩAϪ
[ZnL(OH)(A)]
ϭ
1976
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ZnII Complex with a Phenanthroline-Containing Macrocycle
FULL PAPER
pH 7 leads to a minor, but significant, downfield shift of
the a resonance [Figure 4 (a)]. Similar downfield shifts for
the signal of the a protons are also found for all the amino
acids under investigation [up to 0.4 ppm in the case of Trp,
see Figure 4 (b)]. This indicates the involvement of the NH2
group in ZnII binding. However, a bidentate coordination
mode of the deprotonated amino acids, involving both the
amine and the carboxylate functions, has generally been
found in binary and ternary ZnII complexes with these
substrates,[24Ϫ26,35Ϫ37,47,64] and can be presumed to a
reasonable extent in the present case.
The most interesting finding in Table 2 is the higher sta-
bility of the adducts with amino acids containing an aro-
matic side arm. In particular, tryptophan, which contains
the largest aromatic subunit, indole, shows addition con-
stants to the [ZnL]2ϩ complex that are two orders of magni-
tude higher than Gly, Ala or Leu. This is rather surprising,
since the addition constants of Gly or Ala to the ‘‘free’’
ZnII ion are somewhat higher than those reported for ph-
Gly, Phe and Trp.[66] Figure 4 (a) also shows that the forma-
tion of the [ZnL(Trp)]ϩ complex remarkably affects the
spectrum of the indole units, with a marked upfield shift
of the signals of the aromatic protons with respect to the
corresponding signals of ‘‘free’’ TrpϪ. At the same time,
the phenanthroline subspectrum displays a dramatic loss of
symmetry. Besides the downfield shift of the a signal of the
[ZnL(Trp)]ϩ complex, discussed above, Figure 4 (b) displays
a splitting into two doublets of each signal of the protons 2
and 3. The singlet attributed to 1 is also split into a doublet.
Similarly to the indole resonances, the signals of the phen-
anthroline protons show a marked upfield shift with respect
to the corresponding signals in [ZnL]2ϩ
.
These spectral characteristics account for the presence of
π-stacking interactions between phenanthroline and the
aromatic pendant arm of Trp (Scheme 2), which can con-
tribute to the stability of the complex.
Figure 4. (a): pH dependence of the 1H NMR chemical shifts of
selected protons of Trp in the absence (ᮀ: a proton; ᭞: g; ᭝: f; ᭛:
h; ᭺: e). and in the presence of the ZnII complex with L (᭿: a
proton; ᭢: g; ᭡: f; ᭜: h; ᭹: e). The signals of the protons b, c and d
1
are omitted for clarity. (b): H NMR spectra of [ZnL]2ϩ (a), TrpϪ
(b) and [ZnL(Trp)]ϩ (pH, 9.3) (c). See Scheme 1 and Figure 1 for
the labelling used.
Scheme 2
[ZnL(HA)]2ϩ the acidic proton localization is not changed
In the case of Phe and ph-Gly, substrate complexation by
by HA binding to zinc and, therefore, HA coordination [ZnL]2ϩ is also accompanied by upfield shifts of the reson-
takes place through the carboxylate group, while the amine ances of the aromatic protons of the substrates and phen-
remains protonated. This hypothesis is corroborated by the anthroline. The shifts, however, are generally smaller (ca.
crystal structure of the [ZnL(HGly)]2ϩ cation, which shows 0.1Ϫ0.2 ppm) than those observed for Trp and no loss of
the metal coordinated by one oxygen atom of the car- symmetry was found for the phenanthroline unit. This ac-
boxylate function, while the amine group is protonated.
counts for a reduced π-stacking pairing between the phenyl
At an alkaline pH, the a signal of tryptophan alone shifts units and phenanthroline. Accordingly, the stability of the
upfield, due to deprotonation of the amine group. On the Phe and ph-Gly complexes is lower than that of the Trp
contrary, the formation of the [ZnL(Trp)]ϩ complex above complex.
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A. Bencini, A. Bianchi et al.
FULL PAPER
Interestingly, no significant shift of the signals of the aro- to the [ZnL]2ϩ complex are about one order of magnitude
matic protons are observed at acidic pH, where the com- higher than those of the dipeptides in their neutral (zwit-
plexes with the protonated forms (HA) of the amino acids terionic) form. To clarify the binding mode of these dipep-
1
are present in solution [Figure 4(a)]. This experimental ob- tides to ZnII, we decided to record H NMR spectra on
servation points out that the pairing between the two aro- solutions containing the dipeptides in the presence of
matic moieties is disrupted by coordination to ZnII through [ZnL]2ϩ at different pH values. The formation of the
the carboxylate function, due to protonation of the amine [ZnL(HLeu-Gly)]2ϩ complex at pH 5 does not significantly
group in the [ZnL(HA)]2ϩ complex.
change the spectrum of the dipeptide in its neutral form,
Similar π-stacking interactions have already been ob- which contains a protonated amine and a carboxylate group
served by Sigel et al. in the case of ternary metal com- in its anionic form. Only a minor shift (ca. 0.05 ppm) of the
plexes[67,68] involving Trp and dipyridine or ATP. In the lat- CH2 protons in the α-position to the carboxylate group is
ter case the indole unit of Trp and the adenine moiety of observed. As in the case of amino acids, this may suggest
the nucleotide are paired via π-stacking.[67] Yamauchi et al. that in [ZnL(HLeu-Gly)]2ϩ the dipeptide is coordinated to
also found intramolecular stacking interactions in CuII ter- ZnII through the carboxylate group, while the amine group
nary complexes with Trp and dipyridine or phenanthroline, still remains protonated. On the contrary, the comparison
both in solution and in the solid state.[47][69Ϫ72]
of the 1H NMR spectra of the anionic form of the Leu-Gly
In conclusion, our ZnII complex may behave as a multi- dipeptide in the absence and in the presence of our [ZnL]2ϩ
functional receptor for amino acids, presenting two distinct complex at pH 8.5 shows that the formation of the
binding sites, i.e., the metal center, which forms coordi- [ZnL(Leu-Gly)]ϩ species leads to a marked upfield shift of
nation bonds with amino acids, and the heteroaromatic the signal of the proton in the α-position (a in Scheme 1)
unit, which can give stabilizing hydrophobic and/or π-stack- to the carbonyl carbon atom and the deprotonated NH2
ing interactions with the aromatic portion of the substrates. group (Figure 5).
These interactions lead to an unusual selectivity pattern in
amino acid binding by our ZnII complex, since the amino
acids with aromatic pendant arms are strongly bound with
respect to the aliphatic amino acids Gly or Ala.
In order to evaluate the effective binding properties of
our complex towards the peptide amide group, we decided
to extend our analysis to dipeptides. Unfortunately, most of
the dipeptides tested, such as tryptophan-containing dipep-
tides, form low soluble complexes with [ZnL]2ϩ. Our study
was therefore limited to -glycylglycine (HGly-Gly) and -
leucylglycine (HLeu-Gly). Table 3 reports the equilibrium
constants for the addition of these dipeptides to the
[ZnL]2ϩ complex in aqueous solution.
Table 3. Equilibrium constants for the addition of Gly-Gly and
Leu-Gly to the ZnII complex with the ligand L (NMe4NO3 0.1
, 298 K).
Reaction
log K
Gly-Gly
Leu-Gly
[ZnL]2ϩ ϩ AϪ ϭ [ZnL(A)]ϩ
[ZnL]2ϩϩ HA ϭ [ZnL(AH)]2ϩ
2.84(9)
1.9(1)
3.91(4)
3.06(2)
pKa
1
Figure 5. H NMR spectra of Leu-GlyϪ (a) and [ZnL(Leu-Gly)]ϩ
[ZnL(A)]ϩϩ H2O ϭ [ZnL(A)(OH)] ϩ Hϩ
8.77(6)
8.95(3)
(pH, 8.5) (b). (See Scheme 1 and Figure 1 for the labelling used).
The formation of the complex with the neutral form of
A similar behavior is also found in the case of Gly-Gly.
the dipeptide ([ZnL(HLeu-Gly)]2ϩ) at acidic pH is followed As already proposed in the case of amino acids, the
by deprotonation of the substrate to give [ZnL(Leu-Gly)]ϩ potentiometric and 1H NMR spectroscopic data suggest
at a slightly alkaline pH. A further deprotonation step is that Leu-Gly, in its anionic form, binds to ZnII by using
monitored above pH 9. This process could be attributed, in the amine group. However, the coordination of the amide
principle, to deprotonation of the amide function to give carbonyl function in metal binding can also be supposed,
the [ZnL(Leu-GlyHϪ1)]ϩ complex or to deprotonation of a in agreement with the binding mode generally found for
coordinated water to give a hydroxylated ternary complex. dipeptides to ZnII alone as well as in ternary ZnII
Gly-Gly shows similar binding features towards [ZnL]2ϩ complexes.[35Ϫ37] The deprotonation process observed in the
1
(Table 3). As already found in the case of binding amino pH range 9Ϫ12 does not significantly alter the H NMR
acids, the addition constants of the deprotonated dipeptides spectrum of coordinated Leu-GlyϪ, suggesting that the de-
1978
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Eur. J. Inorg. Chem. 2003, 1974Ϫ1983

ZnII Complex with a Phenanthroline-Containing Macrocycle
FULL PAPER
protonation process involves a coordinated water molecule [ZnL(LNA)]2ϩ complex, compared with those of LNA
to give a [ZnL(Leu-Gly)(OH)] complex.
and [ZnL]2ϩ
.
The latter complex contains both a ZnII-coordinated di-
peptide and a ZnϪOH function, as a potential nucleophile
in a hydrolytic process. On the other hand, dipeptide hy-
drolysis in the presence of our model complex is too slow
to be confidently followed at a physiological temperature.
This observation prompted us to test the effective hydrolytic
ability of our ZnII complex by using -leucine-p-nitroanilide
(LNA), a substrate containing an activated peptide bond.
L-Leucine-p-nitroanilide (LNA) Binding and Hydrolysis by
the ZnII Complex Containing L
LNA, a substrate generally used to test the hydrolytic
ability of leucine-aminopeptidases involving the N-terminal
peptide bond,[73] behaves as a weak base (log K ϭ 7.37(3)
for the equilibrium LNA ϩ Hϩ ϭ LNAHϩ at 308.1 K) and
can also cause deprotonation at a strongly alkaline pH to
give a [LNA(HϪ1)]Ϫ species (pKa ϭ 10.6).
LNA binding by [ZnL]2ϩ was studied by means of
1
potentiometric and H NMR spectroscopic experiments at
308.1 K.[74] The [ZnL]2ϩ complex forms rather stable ad-
ducts with this substrate, with a log K value of 5.55 for
the equilibrium [ZnL]2ϩ ϩ LNA ϭ [ZnL(LNA)]2ϩ. A small
interaction is also detected with the protonated form of the
substrate HLNAϩ {log K ϭ 2.1 for the equilibrium [ZnL]2ϩ
ϩ HLNAϩ ϭ [ZnL(HLNA)]3ϩ}. Finally, the potentio-
metric measurements show that the [ZnL(LNA)]2ϩ complex
releases a single proton at pH Ͼ 8 to give a deprotonated
species.[74] Once again, this process could be ascribed to de-
protonation of the amide group of LNA, giving a complex,
or a metal-bound water molecule, yielding, respectively, a
[ZnL(LNAHϪ1)]ϩ or a [ZnL(LNA)(OH)]ϩ species. The dis-
tribution diagram, derived from the potentiometric meas-
urements (Figure 6) shows that the formation of the
[ZnL(LNA)]2ϩ complex, in the pH range 4Ϫ7, is followed
by complex deprotonation in the pH range 8Ϫ10.
Figure 7. (a) pH dependence of the 1H NMR chemical shifts of
selected protons of LNA in the absence (ᮀ: a proton; ᭝: f; ᭺: e).
and in the presence of the [ZnL]2ϩ complex (᭿: a proton; ᭡: f; ᭹:
e). The signals of protons b, c and d are omitted for clarity. Their
chemical shift does not significantly change in the pH range investi-
gated. (b) 1H NMR spectra of [ZnL]2ϩ (a), LNA (b) and
[ZnL(LNA)]2ϩ (pH, 7.5) (c). (see Scheme 1 and Figure 1 for the
labelling used).
The signal of the a proton of ‘‘free’’ LNA displays an
upfield shift in the pH range 6Ϫ8.5, resulting from the de-
protonation of the amine group of HLNAϩ to give the neu-
tral LNA form. The 1H signals of the aromatic protons, on
the other hand, do not significantly shift, up to pH 10.
LNA deprotonation above pH 10 yields the [LNA(HϪ1)]Ϫ
anion, which is accompanied by a marked upfield shift of
the aromatic protons e and f. [Figure 7 (a)]. In the presence
of ZnII, the formation of the [ZnL(LNA)]2ϩ complex in the
pH range 4Ϫ7 (see Figure 6) gives rise to a slight, but sig-
nificant, downfield shift of the a resonance. Actually, the a
Figure 6. Species distribution diagram for the system [ZnL]2ϩ/LNA
in 1:1 molar ratio (NMe4NO3 0.1 , 308.1 K).
1
Figure 7 (a) displays the H chemical shifts of selected signal in [ZnL(LNA)]2ϩ is ca 0.5 ppm downfield shifted
protons of LNA in the presence and in the absence of the with respect to that of unbound LNA [Figure 7 (b)].
ZnII complex with L at different pHs, while Figure 7 (b)
These observations are indicative of the involvement of
shows the 1H NMR spectroscopic chemical shift of the the amine group in metal coordination. Although these
Eur. J. Inorg. Chem. 2003, 1974Ϫ1983
www.eurjic.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1979

A. Bencini, A. Bianchi et al.
FULL PAPER
NMR spectroscopic data do not allow conclusions to be LNA is bound to the ZnII complex, probably in a bidentate
inferred as to the role of the peptide bond in metal binding, binding mode, which involves the peptide bond. Hydro-
the rather high value of the equilibrium constant for the phobic interactions also contribute to complex stabilization.
addition of LNA to the ZnII complex and the analogies of Coordination of the peptide bond to the electrophilic ZnII
the LNA structure with amino acids and dipeptides, leads ion would activate the amide carbon atom leading to the
to the supposition that the carbonyl oxygen of the amide subsequent intramolecular nucleophilic attack of the Zn-
function is also involved, or partially involved, in ZnII bind- bound hydroxide or to intermolecular attack of hydroxide
ing, with the formation a stabilizing six-membered chelate from the medium. On the other hand, the nucleophilic
ring. Figure 7 (a and b) also shows that both the phen- character of a ZnϪOH function is only slightly lower
anthroline and phenyl protons are upfield shifted above pH than that of hydroxide.[75] At the same time, in
5, where the [ZnL(LNA)]2ϩcomplex occurs, due to π-stack- [ZnL(LNA)(OH)]ϩ the metal-bound hydroxide is close to
ing and hydrophobic interactions between the aromatic the substrate. This suggests that the peptide bond cleavage
units of metallo-receptor and substrate. Therefore, as al- occurs via an intramolecular pathway, as sketched in
ready observed in the binding of the amino acids, our ZnII Scheme 3.
complex behaves as a multifunctional binding site for LNA,
since both coordination bonds and supramolecular interac-
tions contribute to the complex stability.
According to Figure 6, the [ZnL(LNA)]2ϩ complex de-
1
protonates in the pH range 8Ϫ10; the H NMR signals of
coordinated LNA do not shift appreciably in this pH region
[Figure 7(a)], indicating that deprotonation occurs on a co-
ordinated water molecule, with the formation of the
[ZnL(LNA)(OH)]ϩ hydroxo complex.
Although the potentiometric measurements do not reveal
the formation of other complexes between LNA and
[ZnL]2ϩin the pH range investigated (2.5Ϫ10.5), the analy-
1
sis of the H NMR spectra of LNA recorded at a strongly
alkaline pH (pH Ͼ10.5) in the presence of [ZnL]2ϩ reveals a
marked upfield shift of the signals of the a, e and f protons
Figure 8. Plot of the distribution curve of [ZnL(LNA)(OH)]ϩ (solid
line, right y axis) and kLNA values (᭹, left y axis) as a function of
pH (0.1  NMe4NO3, 308.1 K).
[Figure 7 (a)]. A similar upfield shift is also observed at a
strongly alkaline pH for the corresponding signals of ‘‘free’’
LNA, due to deprotonation of the amide function to give
the N-deprotonated [LNA(HϪ1)]Ϫ species. This comparison
suggests that the [ZnL(LNA)(OH)]ϩ complex undergoes
deprotonation in strongly alkaline solutions to give a neu-
tral [ZnL(LNAHϪ1)(OH)] complex. A similar complex,
containing a N-deprotonated LNA unit coordinated to a
ZnII complex has recently been characterized by Vahrenk-
amp et al.[26] Our attention, however, was focused on the
[ZnL(LNA)(OH)]ϩ complex, which contains a ZnII-coordi-
nated neutral LNA molecule and a ZnϪOH function, in
close proximity, as a potential nucleophile for cleavage of
the peptide bond.
Indeed, the [ZnL(LNA)(OH)]ϩ complex promotes LNA
hydrolysis in aqueous solution at 308.1 K to give -leucine
and p-nitroanilide. The hydrolysis products were identified
by means of 1H NMR spectroscopic measurements.
Pseudo-first order rate constants kLNA have been deter-
mined at different pH values for the complex. In Figure 8
kLNA values for the Zn-L complexes are reported as a func-
tion of pH, together with the distribution curve of the
[ZnL(LNA)(OH)]ϩ species at 308.1 K. A good fit between
the kLNA values and the distribution curve of the
[ZnL(LNA)(OH)]ϩ was found. No promoted hydrolysis
was observed below pH 8, where the [ZnL(LNA)(OH)]ϩ is
absent from the solution. Accordingly, it can be concluded
that the [ZnL(LNA)(OH)]ϩ is the kinetically active species.
We can propose a two-step hydrolytic mechanism where Scheme 3
1980  2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjic.org
Eur. J. Inorg. Chem. 2003, 1974Ϫ1983

ZnII Complex with a Phenanthroline-Containing Macrocycle
FULL PAPER
The [ZnL(LNA)(OH)]ϩ complex is formed in a yield of with phenanthroline-containing ligands, with the purpose
70% percentage, at most, in the pH range used in the kinetic to generate more strongly nucleophilic MϪOH functions in
measurements (Figure 8). As a consequence, the first order aqueous solutions.
rate constants, kЈLNA, have been determined from the maxi-
mum kLNA value by using the following equation:
Experimental Section
v ϭ kLNA[total ZnII complex] ϭ kЈLNA [ZnL(LNA)(OH)ϩ]
General Remarks: Ligand L and its ZnII complex [ZnL(H2O)]-
By using this expression, a kЈLNA value of 3.0·10Ϫ6 sϪ1
can be calculated. The hydrolysis rate is much lower than
those reported for aminopeptidases,[73] but, at the same
time, almost two orders of magnitude higher than the rate
constant for spontaneous hydrolysis of LNA.
1
(ClO4)2 were prepared as already described.[61][62] 300.07 MHz H
spectra in D2O solutions at different pH values were recorded at
298.1 K in a Varian Unity 300 MHz spectrometer. Peak positions
1
1
are reported relative to HOD at δ ϭ 4.79 ppm. H-1H and H-13C
2D correlation experiments were performed to assign the signals.
Small amounts of 0.01 mol dmϪ3 NaOD or DCl solutions were
added to solutions containing the substrates and [ZnL(H2O)]-
(ClO4)2 to adjust the pD. The pH was calculated from the measured
pD values using the following relationship:[76] pH ϭ pD Ϫ 0.40
The addition constant of LNA to the [ZnL]2ϩ is ca 3100
times higher than the corresponding constant of Leu; this
consideration implies that LNA hydrolysis promoted by our
ZnII is free, for the major part, of inhibiting agents gener-
ated by its own hydrolysis, due to the replacement of Leu
by the strongly bound LNA substrate in the coordination
UV/Vis spectra were recorded on a Shimadzu UV-2101PC spectro-
photometer.
sphere of [ZnL]2ϩ
.
Potentiometric Measurements: All the pH metric measurements
(pH ϭ Ϫlog [Hϩ]) were carried out in degassed 0.1 mol·dmϪ3
NMe4NO3 solutions, at 298.1 or 308.1 K, by using the equipment
and procedure that have already been described.[60] The combined
The hydrolytic process is too slow at 308.1 K to deter-
mine the reaction turnover. However, an experiment carried
out at 343.1 K and pH 9.7, with an excess of substrate
showed that 1 equiv. of the complex can completely cleave Ingold 405 S7/120 electrode was calibrated as a hydrogen concen-
tration probe by titrating known amounts of HCl with CO2-free
NMe4OH solutions and determining the equivalent point by the
Gran’s method[77][78] which allows for the determination of the
as many as 11 equiv. of LNA.
standard potential Eo, and the ionic product of water (pKw
ϭ
13.83(1) at 298.1 K and 13.40(1) at 308.1 K in 0.1 mol·dmϪ3
NMe4NO3). All equilibria involved in the studied systems were de-
termined, or redetermined,[79] under the present experimental con-
ditions in order to obtain a consistent set of data. The concen-
tration of [ZnL(H2O)](ClO4)2 was 1 ϫ 10Ϫ3 mol·dmϪ3 in all experi-
ments, while the concentrations of the substrates were varied in the
range 1 ϫ 10Ϫ3Ϫ4 ϫ 10Ϫ3 mol·dmϪ3. At least three measurements
(about 150 data points for each) were performed for each system
in the pH range 2.5Ϫ10.5 and the relevant e.m.f. data were treated
by means of the computer program HYPERQUAD[79] that fur-
nished the relevant equilibrium constants reported in Table 2 and 3.
Conclusion
The particular molecular topology of the phenanthro-
line-containing macrocycle L leads to an unsaturated coor-
dination sphere of the metal in the [ZnL]2ϩ complex. There-
fore, the metal ion can behave as a binding site for substrate
molecules and anions. Actually, the [ZnL]2ϩ forms stable
complexes with amino acids and dipeptides in their zwit-
terionic or anionic forms. The phenanthroline moiety can
act as a further binding site for substrates containing aro-
matic pendants, through stacking and/or hydrophobic inter-
actions, giving a peculiar selectivity pattern in amino acid
binding. In some cases, a [ZnL(A)OH] species is also
formed in aqueous solution. This species contains, in close
proximity, both a coordinated substrate and a ZnIIϪOH
unit, a potential nucleophile towards cleavage of the peptide
bond. Our complex is indeed able to hydrolyze the peptide
bond of the activated substrate -leucine-p-nitroanilide
(LNA). The cleavage process occurs in two successive steps
i) substrate binding, through coordination bonds to ZnII
and stacking interactions and ii) nucleophilic attack of a
ZnϪOH function, formed at alkaline pH values. Although
hydrolysis only occurs at alkaline pH values, this mecha-
nism resembles that proposed for peptide hydrolysis by ami-
nopeptidases.
Kinetics of Leucine-p-Nitroanilide (LNA) Hydrolysis: The hydrolysis
rate of LNA in the presence of the ZnII complexes with L to give
Leu and p-nitroaniline was measured by an initial slope method
following the increase in the 405 nm absorption of the released p-
nitroaniline at 308.1 K. The molar absorbance of p-nitroaniline was
determined at the pH of each measurement. The ionic strength was
adjusted to 0.1  with NMe4NO3. TAPS (pH, 7.8Ϫ8.9), CHES
(pH, 8.9Ϫ9.7) and CAPS (9.7Ϫ10.5) buffers were used (50 m). In
a typical experiment, with LNA (2Ϫ5 m) and [ZnL(H2O)](ClO4)2
(0.5 m) in aqueous solutions at the appropriate pH (the reference
experiment does not contain the ZnII complex) were mixed, the UV
absorption decay was recorded immediately and was followed until
5% of p-nitroaniline had been formed.
At
a given pH value, a plot of the hydrolysis rates vs.
[ZnL(LNA)(OH)ϩ] concentration, calculated at the different LNA
concentrations, gave a straight line, allowing for the determination
of the pseudo-first order rate constants kLNA from the slope of the
line. Errors on kLNA values were about 5%.
In particular, selective recognition of amino acids, due to
stabilizing π-stacking interactions, is a striking result; it
could be used, in principle, for anchoring the complex onto
a specific peptide bond of polypeptide chains. This effect,
coupled with the hydrolytic properties of the ZnϪOH func-
X-ray Crystallographic Study: Crystals of [ZnL(HGly)]2-
(ClO4)4·1.5H2O were obtained by slow evaporation of an aqueous
tion, encourage the development of other metal complexes solution containing [ZnL(H2O)](ClO4)2 and HGly in a 1:1 molar
Eur. J. Inorg. Chem. 2003, 1974Ϫ1983
www.eurjic.org  2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1981

A. Bencini, A. Bianchi et al.
FULL PAPER
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1320.50; a ϭ 18.520(1) A, b ϭ 15.171(1) A, c ϭ 19.884(2) A, β ϭ
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P21/a. Data Collection: P4 SIEMENS X-ray diffractometer, λ ϭ
˚
1.54178 A (Cu-Kα), graphite monochromated, 0.3 ϫ 0.2 ϫ
0.15 mm, θϪ2θ up to θ ϭ 60° at room temperature, 7885 reflections
collected, 6637 symmetry-independent reflections, I Ͼ 2σ(I), µ ϭ
3.710 mmϪ1, Psi-Scan method, transmission factors 0.44Ϫ0.57.
Structural analysis and refinement: direct method of the SIR97
program;[81] full-matrix least-squares method of SHELXL-97;[82]
All the non-hydrogen atoms were anisotropically refined. The hy-
drogen atoms were introduced in calculated positions and their co-
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the linked atoms. Parameter/Fo ratio ϭ 0.11, final agreement fac-
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0.2013(all data).
CCDC-198421 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Inorg. Chem. 2003, 1974Ϫ1983

ZnII Complex with a Phenanthroline-Containing Macrocycle
FULL PAPER
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determined in NMeNO3 0.1  at 308.1 K): Zn2ϩ ϩ L ϭ
[ZnL]2ϩ, log K ϭ 15.98; [ZnL]2ϩ ϩ OHϪ ϭ [ZnL(OH)]ϩ,
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HLNAϩ ϭ [ZnL(HLNA)]3ϩ, logK ϭ 2.1; [ZnL(LNA)]2ϩ
H2O ϭ [ZnL(LNA)(OH)]ϩ ϩ Hϩ, logK ϭ 10.0.
ϩ
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F. J. Rossotti, H. Rossotti, J. Chem. Educ. 1965, 42, 375Ϫ378.
Protonation and ZnII addition constant for amino acids and
dipeptides (A) determined in the present work (potentio-
metrically determined in NMeNO3 0.1  at 298.1 K): Gly, log
˚
only if the C···O distance is lower than 3.25 A (F. A. Cotton,
L. M. Daniels, G. T. Jordan, C. A. Murrillo, Chem. Commun.
1997, 1673Ϫ1674; J. Donohue, in Structural Chemistry and
Molecular Biology, A. Rich, N. Davidson (Eds.), Freeman: San
Francisco, 1968, 443Ϫ465.)
KHA ϭ 9.52(1), log KAH2
ϭ 2.37(1), log KZnA ϭ 4.94(1),
A
A
HA
log KZnA2
ϭ 4.38(1); Ala, log KHA ϭ 9.66(1), log
ZnA
A
KAH2
ϭ 2.38(1), log KZnA ϭ 4.54(3), log KZnA2
ϭ
ZnA
HA
A
[66]
R. M. Smith, A. E. Martell, NIST Stability Constants Datab-
4.45(4); PhGly, log KHAA ϭ 8.87(1), log KAH2HA ϭ 1.97(1), log
KZnA2 ϭ 8.20(2); Phe, log KHA ϭ 9.03(1), log KAH2
ase, Version 4.0, National Institute of Standard and Technol-
ogy: Washington, DC, 1997.
ϭ
HA
2A
A
2.31(1), log KZnA2 ϭ 8.94(2); Try: log KHA ϭ 9.50(2), log
2A
A
[67]
H. Sigel, C. F. Naumann, J. Am. Chem. Soc. 1976, 98,
KAH2
ϭ 2.23(3), log KZnA2
ϭ 9.70(3); Gly-Gly, log
HA
2A
730Ϫ739.
KHA ϭ 8.00(1), log KAH2
ϭ 3.12(1), log KZnA ϭ 3.48(9),
HA A
A
[68]
H. Sigel, Angew. Chem. Int. Ed. Engl. 1982, 2, 389Ϫ391. H.
log KZnA2
ϭ 3.18(8); Leu-Gly log KHA ϭ 7.99(3), log
ZnA A
Sigel, Pure Appl. Chem. 1989, 61, 923Ϫ931.
KH2A
ϭ 3.28(2), log KZnA ϭ 2.84(3), log KZnA2
ϭ
ZnA
HA
A
[69]
O. Yamauchi, A. Odani, J. Am. Chem. Soc. 1985, 107,
2.77(2), where KAB ϭ [AB]/[A][B] and KAB2
ϭ [AB2]/
A
AB
5938Ϫ5945.
[AB][B].
[70]
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T. Sugimori, H. Masuda, N. Ohata, K. Koiwai, A. Odani, O.
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G. M. Sheldrick, SHELXL-97, Göttingen, 1997.
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www.eurjic.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1983