Structures and reactivity of synthetic zinc(II) complexes resembling the active sites and reaction intermediates of aminopeptidases

Article abstract of DOI:10.1039/b314089j

Herein we report the first crystallographic characterization and hydrolysis of a synthetic zinc(II) complex that resembles the active site and reaction intermediates proposed for aminopeptidases.

Full text of DOI:10.1039/b314089j

Structures and reactivity of synthetic zinc(II) complexes resembling the
active sites and reaction intermediates of aminopeptidases†
Juan C. Mareque Rivas,* Emiliano Salvagni and Simon Parsons
School of Chemistry, The University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh, UK EH9
3JJ. E-mail: juan.mareque@ed.ac.uk
Received (in Cambridge, UK) 5th November 2003, Accepted 4th December 2003
First published as an Advance Article on the web 22nd January 2004
Herein we report the first crystallographic characterization and
hydrolysis of a synthetic zinc(II) complex that resembles the
active site and reaction intermediates proposed for amino-
peptidases.
Furthermore, we demonstrate that these complexes have the ability
to hydrolyse the unactivated dipeptide glycylglycine (Gly-Gly) to
glycine (Gly).
Perchlorate salts of [(LH+)2Zn2(Gly-Gly)2]4+
1
and
[(LH+)Zn(Gly)]2+ 2 (L = 1-methyl-4-(6-amino-2-pyridinylme-
thyl)-piperazine; Gly = glycine anion, Gly-Gly = glycylglycine
anion) were assembled by stirring a mixture of equimolar amounts
of Zn(ClO4)2·6H2O and Gly-Gly for 1 and Gly for 2 in methanol.
Crystals of the two complexes were grown by slow evaporation of
a MeOH–water solution at room temperature and their structures
were determined by X-ray crystallography.‡
There is great interest in designing metal complexes that resemble
the active site of metallopeptidases and capable of hydrolysing
unactivated peptide bonds.1 Such complexes not only could be
useful in elucidating fundamental aspects of the enzyme chemistry
but also find important applications in protein sequencing.
Metallopeptidases typically require a zinc(II) ion with a co-
ordination environment consisting of a glutamate and two histidine
residues.2 In addition, active-site residues participate in substrate
binding and/or co-operate with the zinc(II) ion towards facilitating
the amide hydrolysis reaction, but the effects associated with the
second co-ordination sphere are not well understood.3 An example
of zinc metallopeptidase for which this co-ordination environment
has been proposed is aminopeptidase A (APA), which specifically
cleaves in vitro the N-terminal glutamyl or aspartyl residue from
peptide substrates (Scheme 1).4
In the crystal structures of 1 and 2 the zinc(II) centre is in a co-
ordination environment between square pyramidal and trigonal
bipyramidal (Figs. 1(i) and 2(i)).§ Whereas 1 adopts a dimeric
structure of formulation [(LH+)2Zn2(Gly-Gly)2](ClO4)4·2H2O (Fig.
1(ii)), 2 is a [(LH+)Zn(Gly)](ClO4)2 ‘polymer’(Fig. 2(ii)). The Zn–
N distances of Zn–N(1) 2.185(3) Å and Zn–N(2) 2.085(3) Å for 1
are slightly shorter than those of 2, (Zn–N(1) 2.1996(17) Å and Zn–
N(2) 2.0957(16) Å) presumably because Gly is a slightly stronger
ligand than Gly-Gly. As expected, Zn–N distances in 1 and 2 are
longer than those of the tetrahedral complex (L)Zn(Cl)2.10 Binding
provided by L mimics the bis-histidine zinc-ligation in aminopepti-
dases. Binding of the Gly and Gly-Gly through the COO2 group
mimics glutamate binding to the zinc(II) centres of aminopepti-
dases. In 1, the peptide (Gly-Gly) is co-ordinated through the
terminal NH2 group and the carbonyl amide oxygen atom. This
mode of co-ordination was recently suggested to account for the
ease of hydrolysis of Gly-Gly with a cis,cis-1,3,5-triaminocyclo-
hexane–copper(II) complex9 (tach-Cu(II)), and also resembles the
peptide binding mode proposed for APA.11,12 Remarkably, the
positioning of a water molecule O(1W) in the crystal structure of 1
would be suitable for attack on the carbonyl carbon atom C(3G)
(Fig. 1, O(1W)…C(3G) 3.050 Å), which should be activated for
Several metal complexes have been used as synthetic models for
metallopeptidases. Substitutionally inert metal complexes of Co(III
)
have demonstrated that amide hydrolysis can be promoted through
activation of the carbonyl and/or intramolecular attack of a metal
bound hydroxide.5 Complexes of Cu(II) and Zn(II) were also used to
study the mechanism and reaction intermediates of metal-promoted
amide-bond hydrolysis.6 Because of the great stability of peptide
bonds, however, these studies generally involved activated amides
or amide tethered to the ligand. Recently, it has been shown that
Pd(II) complexes spontaneously bind to the side chains of
methionine and histidine residues and effect hydrolytic cleavage of
short peptides.7 To our knowledge the only metal complexes of
biologically relevant Cu(II) and Zn(II) ions that are able to cleave
unactivated peptides and/or proteins have been two Cu(II) com-
plexes of two N3 ligands.8,9 Thus to date, no synthetic zinc(II
)
complex with double nitrogen and single carboxylate ligation has
been reported. In this Communication, we report the structure of
two zinc(II) complexes that resemble quite faithfully the zinc(II
)
ligation and several of the peripheral active site features of APA.
Fig. 1 (i) Thermal ellipsoid plot (50% probability) showing the zinc(II) co-
ordination environment of 1; (ii) structure of the [(LH+)2Zn2(Gly-Gly)2]4+
cation with water molecules close to the amide bond. Selected bond lengths
(Å) and angles (°): Zn(1)–N(1) 2.185(3), Zn(1)–N(2) 2.085(3), Zn–N(4G)
2.099(3), Zn–O(1GA) 1.952(2), Zn–O(3G) 2.094(2), O(1GA)–Zn–N(2)
112.98(11), O(1GA)–Zn–N(4G) 115.76(13), O(1GA)–Zn–O(3) 94.14(10),
O(1GA)–Zn–N(1) 98.61(10), N(2)–Zn–O(3G) 151.52(11), N(2)–Zn–
N(4G) 95.38(11), N(2)–Zn–N(1) 78.78(10), O(3G)–Zn–N(4G) 79.54(11),
O(3G)–Zn–N(1) 89.08(10), N(4G)–Zn–N(1) 144.29(13).
Scheme 1
† Electronic supplementary information (ESI) available: Experimental
details (synthesis, characterization and hydrolysis studies). See http://
www.rsc.org/suppdata/cc/b3/b314089j/
460
C h e m . C o m m u n . , 2 0 0 4 , 4 6 0 – 4 6 1
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 4

nucleophilic attack by the zinc(II) centre. Moreover, like in the case
of Gly-Gly hydrolysis with the tach-Cu(II) complex, the peptide
chelate ring would not be required to be broken for hydrolysis to
occur, as shown by the retention of this chelation motif in crystal
structure of 2. Thus Gly is chelated to one zinc(II) ion via the
nitrogen atom and oxygen atom of the carboxylate, and bridges to
the next zinc via the other oxygen atom. To our knowledge the only
other example with this polymeric chain structure is [Cu(Gly)-
(NO3)(H2O)].13 The positioning of the NH2 group of the ligand in
2 is such that forms H-bonding with both of the oxygens of the
carboxylate group of Gly (N(7)…O(1G) 3.059 Å, N(7)…O(2G)
3.225 Å). Similarly, the amino group of the ligand in 1 is H-bonded
to one of the oxygens of the zinc(II) bound carboxylate group of
Gly-Gly (N(7)…O(2G) 2.957 Å). In principle, these H-bonding
interactions may assist peptide binding and orient the bound
peptide.
We have investigated the hydrolysis of 1 by NMR and found that
Gly-Gly is hydrolysed to Gly at 70 °C and pH 7 ± 0.1 (50 mM
HEPES) (Fig. 3).† Gly-Gly was cleaved at 1 and pH 7 with similar
efficiency to copper(II) complexes of 1,4,7-triazacyclononane
([9]aneN3) and cis,cis-1,3,5-triaminocyclohexane (tach) at pH 8.1.
Unlike previously reported synthetic model complexes of metallo-
peptidases or peptide cleaving agents, however, 1 utilizes a
biologically-relevant metal ion, zinc(II), in a biologically-relevant
co-ordination environment and achieves cleavage of an otherwise
unactivated peptide bond under mild physiological conditions.14
In summary, the structures of two zinc(II) complexes with Gly-
Gly and Gly co-ordinated to the zinc(II) centre are reported. These
two complexes represent the first crystallographically characterized
zinc(II) complexes in which the zinc(II) centre is in co-ordination
environment that closely resembles the active site and reaction
intermediates proposed for aminopeptidases. Furthermore, hydrol-
ysis of Gly-Gly at the zinc(II)–Gly-Gly complex is achieved at
physiological pH. Thus, to our knowledge, this is the first example
of an unactivated peptide being hydrolysed by a small biomimetic
zinc(II) complex.
We gratefully acknowledge the EPSRC (GR/R25743/01), the
Royal Society (RSRG:22702), the Nuffield Foundation (NAL/
00286/G) and The University of Edinburgh for funding.
Notes and references
‡
Crystal data for 1·(ClO4)4·4H2O: C30H60Cl4N12O26Zn2, M = 1277.44,
¯
triclinic, space group P1, a
= 9.0776(10), b = 10.2002(11), c =
14.5845(16) Å, a = 104.467(2), b = 101.407(2), g = 98.007(2)°, U =
1256.2(2) Å3, T = 150(2) K, l(Mo–Ka) = 0.71073 Å, Z = 1, Dc = 1.689
g cm23, m = 1.265 mm21, 11411 reflections measured, 5900 unique, Rint
=
0.0274 (all data), R1 = 0.0657 (all data), wR2 = 0.1438 (all data), S =
1.069 (all data), largest difference peak, hole 1.317, 20.550 e Å23
.
For 2·(ClO4)2: C13H23Cl2N5O10Zn, M = 545.63, Monoclinic, space
group P21/n, a = 16.0691(10), b = 7.9677(5), c = 16.3441(10) Å, a = 90,
b = 96.9230(10), g = 90°, U = 2077.3(2) Å3, T = 150(2) K, l(Mo–Ka)
= 0.71073 Å, Z = 4, Dc = 1.745 g cm23, m = 1.502 mm21, 18298
reflections measured, 5145 unique, Rint = 0.0260 (all data), R1 = 0.0389,
wR2 = 0.0909 (all data), S = 1.047 (all data), largest difference peak, hole
0.885, 20.504 e Å23
.
§ CCDC 223725–223726. See http://www.rsc.org/suppdata/cc/b3/
b314089j/ for crystallographic data in .cif or other electronic format.
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Fig. 2 (i) Thermal ellipsoid plot (50% probability) showing the zinc(II) co-
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[(LH+)Zn(gly)]2+ cation. Selected bond lengths (Å) and angles (°): Zn–N(1)
2.1996(17), Zn–N(2) 2.0957(16), Zn–N(3G) 2.0799(17), Zn–O(1G)
2.0645(14), Zn–O(2GA) 2.0020(14), O(2GA)–Zn–O(1) 111.13(6),
O(2GA)–Zn–N(3G) 102.08(6), O(1G)–Zn–N(3G) 81.41(6), O(2GA)–Zn–
N(2) 94.52(6), O(1G)–Zn–N(2) 90.61(6), N(3G)–Zn–N(2) 163.24(7),
O(2GA)–Zn–N(1) 103.88(6), O(1G)–Zn–N(1) 143.95(6), N(3G)–Zn–N(1)
99.79(7), N(2)–Zn–N(1) 78.09(6).
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11 It is interesting that the proposed H-bonding between Glu-386 and Glu-
352 of APA and the nucleophilic water/hydroxide and co-ordinated NH2
from the peptide substrates (Scheme 1), which are believed to be
functionally critical,12 are mimicked by similar H-bonds to the
perchlorate anions in the crystal structure of 1.
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14 The half-life of hydrolysis of Gly-Gly at pH 7 and 70 °C is ca. 2 years:
see A. Radzicka and R. Wolfenden, J. Am. Chem. Soc., 1996, 118,
6105.
Fig. 3 Extent of hydrolysis of Gly-Gly of 1 and Gly-Gly to Gly at 70 °C and
pH 7 ± 0.1 (50 mM HEPES) at different times.
C h e m . C o m m u n . , 2 0 0 4 , 4 6 0 – 4 6 1
461