A novel dizinc bridged hydroxamate model for hydroxamate inhibited zinc hydrolases

Article abstract of DOI:10.1039/b202612k

Reaction of Zn(OAc)₂·2H₂O with tmen leads to the formation of [Zn(tmen)(OAc)₂] (I) which reacts with benzohydroxamic acid to form Zn(BA)₂·H₂O (II) and the novel dizinc hydroxamate bridged complex [Zn₂(μ-OAc)₂-(OAc)(μ-BA)(tmen)] (III), which may also be prepared by self-assembly and whose structure closely mimics that of the native hydroxamate inhibited Aeromonas proteolytica aminopeptidase.

Full text of DOI:10.1039/b202612k

A novel dizinc bridged hydroxamate model for hydroxamate inhibited
zinc hydrolases†
David A. Brown,*a William Errington,b Noel J. Fitzpatrick,a William K. Glass,a Terence J. Kemp,b
Hassan Nimira and Áine T. Ryana
a Department of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.
E-mail: noel.fitzpatrick@ucd.ie
b Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: w.errington@warwick.ac.uk
Received (in Cambridge, UK) 15th March 2002, Accepted 18th April 2002
First published as an Advance Article on the web 2nd May 2002
Reaction of Zn(OAc)2·2H2O with tmen leads to the forma-
tion of [Zn(tmen)(OAc)2] (I) which reacts with benzohy-
droxamic acid to form Zn(BA)2·H2O (II) and the novel
dizinc hydroxamate bridged complex [Zn2(m-OAc)2-
(OAc)(m-BA)(tmen)] (III), which may also be prepared by
self-assembly and whose structure closely mimics that of the
native hydroxamate inhibited Aeromonas proteolytica ami-
nopeptidase.
Dinuclear metal hydrolases are an important group of metal-
loenzymes which catalyse the hydrolysis of a range of peptide
and phosphate ester bonds and include the amidohydrolases,
amidinohydrolases and peptide hydrolases.1 A common struc-
tural feature of these metallohydrolases is a dinuclear metal
active site featuring Zn(II), Ni(II), Co(II) and Mn(II) and
carboxylate bridges which occur respectively in leucine amino-
peptidase,2 urease,3 methionineaminopeptidase4 and arginase.5
Fig. 1 Molecular structure of complex I. Selected bond distances (Å) and
Hydroxamic acids are ubiquitous bioligands which inhibit many
enzymes including one-zinc endopeptidases such as thermoly-
sin6 and colleganases7 in which the catalytic zinc centre is
pentacoordinated by three protein ligands and chelated by the
hydroxy and carbonyl oxygen atoms of the inhibiting hydrox-
amate group, –CONHOH. In contrast, the acetohydroxamate-
inhibited C319A variant of Klebsiella aerogenes urease shows
the deprotonated hydroxy oxygen of the hydroxamic acid
bridging the two nickel centres with the carbonyl oxygen
bonding one nickel centre only.8 A similar structure occurs in
the dizinc enzyme, Aeromonas proteolytica aminopeptidase
angles (°): Zn1–O1 2.052(4), Zn1–O2 2.353(4), Zn1–N1 2.140(4), O1–C4
1.250(6), O2–C4 1.243(6), O1–Zn1–O2 58.76(14), N1–Zn1–N1#1 85.3(2),
O2–Zn1–O2#1 97.78(19).
Although there was no evidence for formation of a dimeric
species in the above reaction, further reaction of I with BHA in
a 1+1 molar ratio in methanol for 3 h at room temperature gave
a mixture of bisbenzohydroxamatozinc(II), Zn(BA)2·H2O (II)
and the dizinc hydroxamate III, which were separated by
filtration and work up.‡ The same products were obtained by
self-assembly from the reaction of Zn(OAc)2·2H2O, tmen and
BHA in molar ratio 1+1+1 in methanol for 3 h at room
temperature and identical work up (Scheme 1). The formation
of II was confirmed from previously reported data.13
The structure of III (Fig. 2) shows the presence of a bridging
benzohydroxamate with the same type of bonding previously
found in all model dinuclear bridged hydroxamates10–12 and in
the hydroxamate-inhibited urease8 and Zn2AAP,9 namely
bridging of the two metal centres by the deprotonated
(Zn2AAP) inhibited by p-iodo- -phenylalanine hydroxamic
D
acid.9 We have modelled the hydroxamate inhibition of urease
by the reaction of aceto- and benzo-hydroxamic acids (AHA,
BHA) with the model dinickel complexes, [Ni2(m-OAc)2-
(OAc)2(m-H2O)(tmen)2] and [Ni2(m-OAc)3(urea)(tmen)2]-
[OTf] which gave respectively the hydroxamate bridged
complexes, [Ni2(m-OAc)(m-AA)2(tmen)2][OAc] and [Ni2(m-
OAc)2(m-AA)(urea)(tmen)2][OTf] with structures very close to
the above inhibited C319A variant.10 Similar complexes are
11
formed by Co(II
)
and Mn(II).12 In this communication, the
isolation and structural characterisation of the novel dizinc
bridged hydroxamate complex, [Zn2(m-OAc)2(OAc)(m-BA)-
(tmen)] (III), which closely mimics a number of features of the
above hydroxamate inhibited Zn2AAP, are described.
In view of the relative ease of formation of [M2(m-
OAc)2(OAc)2(m-H2O)(tmen)2], M = Ni, Co, Mn, and their
reactions with hydroxamic acids,10–12attempts were made to
synthesise the analogous dinuclear zinc complex by the reaction
of a 1+1 molar ratio of Zn(OAc)2·2H2O and tmen in methanol
at room temperature, but resulted in the formation of the
mononuclear complex, [Zn(tmen)(OAc)2] (I) only. I is octahe-
dral with some distortion of the metal ligand bond angles from
90° (Fig. 1) and some slight asymmetry in the chelating acetates
with Zn1–O1 = 2.052(4) and Zn1–O2 = 2.353(4) Å.
† Abbreviations: OAc
BA = deprotonated hydroxamic acid, tmen
mine.
=
CH3CO22, BHA
=
=
benzohydroxamic acid,
tetramethylethylenedia-
Scheme 1
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CHEM. COMMUN., 2002, 1210–1211
This journal is © The Royal Society of Chemistry 2002

We thank the EU COST D21 programme, Project D21/
0001/00, for support.
Notes and references
‡
Satisfactory microanalyses were obtained for compounds I–III.
Preparation of I: to a solution of Zn(OAc)2·2H2O (20 mmol) in methanol
was added dropwise 20 mmol of tmen in methanol followed by stirring for
1 h. After work-up, recrystallisation from acetone gave colourless crystals
of I suitable for X-ray crystallography.
Preparation of II and III by self-assembly: to a solution of Zn(OA-
c)2·2H2O (20 mmol) and tmen (20 mmol) in methanol was added dropwise
BHA (20 mmol) in methanol followed by stirring for 3 h. II was filtered off
leaving on concentration a pale yellow oil which on stirring in diethyl ether
for 1 h gave a white solid which on recrystallisation from acetone gave
colourless crystals of III suitable for X-ray crystallography.
Preparation of II and III from I: to a solution of I (20 mmol) in methanol
was added dropwise a solution of BHA (20 mmol) in methanol followed by
stirring for 3 h. Identical work-up as in the preceding method gave II and III
as above.
Crystallography: crystal data: for I: C10H22N2O4Zn, M = 299.67,
monoclinic, space group C2/c, a = 14.9905(19), b = 7.3672(14), c =
12.5105(17) Å, b = 97.015(3)°, U = 1371.3(4) Å3, Z = 4, l = 0.71073 Å,
m = 1.796 mm21, 1327 independent reflections were measured. Final R1 =
0.0661 and wR2 = 0.1666.
For III: C19H31N3O8Zn2, M = 560.21, orthorhombic, space group Pbca,
a = 8.9976(11), b = 15.9235(19), c = 33.533(2) Å, U = 4804.4(9) Å3, Z
= 8, l = 0.71073 Å, m = 2.044 mm21, 6118 independent reflections were
measured. Final R1 = 0.0355 and wR2 = 0.0663.
Data were collected using a Siemens SMART CCD area-detector
diffractometer. Refinement was by full-matrix least squares on F2 for all
data using SHELXL-97.14 Hydrogen atoms were added at calculated
positions and refined using a riding model.
Fig. 2 Molecular structure of complex III. Selected bond distances (Å) and
angles (°): Zn1–O6 2.0760(16), Zn2–O6 1.9729(16), Zn1–O5 2.1160(16),
Zn1–O1 2.1140(16), Zn2–O2 1.9614(16), Zn1–N2 2.1469(16), Zn2–O7
1.9320(17), Zn2–O8 2.9922(19), N1–H…O8 2.7138(28), Zn1–Zn2
3.2368(5); O6–Zn1–O1 92.74(6), O6–Zn1–O5 78.13(6), O1–Zn1–N2
94.36(7), O7–Zn1–O2 106.63(7), O4–Zn2–O2 105.66(8), O4–Zn2–O6
109.00(7).
CCDC reference numbers 182248 and 182249. See http://www.rsc.org/
suppdata/cc/b2/b202612k/ for crystallographic data in CIF or other
electronic format.
hydroxamate hydroxy group and bonding of only one metal
centre by the hydroxamate carbonyl oxygen. In addition, III
shows a number of structural features very close to that of the
native Zn2AAP inhibited by p-iodo- -phenylalanine hydroxa-
D
mic acid.9 Firstly, in both cases the zinc atoms in III and in
inhibited Zn2AAP have different coordination numbers (Zn1, 6
and Zn2, 4 in III and Zn1, 5 and Zn2, 4 in the inhibited enzyme).
Secondly, the bridge bonds in III are unequal, Zn1–O6 =
2.0760(16) and Zn2–O6 = 1.9729(16) Å, as are those in the
inhibited enzyme with values of 2.4 and 1.8 Å, respectively,
consistent with the different coordination numbers of the two
1 D. E. Wilcox, Chem. Rev., 1996, 96, 2435.
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zinc centres in both cases. Thirdly, the capping acetate in III is
1
clearly h with Zn2–O7
= 1.9320(17) and Zn2–O8 =
2.9922(19) Å due to a hydrogen bond between the N1H of the
hydroxamate and the free acetate oxygen, O8 with an N1–
H…O8 distance of 2.7138(28) Å. This behaviour in III mimics
the hydrogen bonding occurring between an oxygen of the
carbonyl group of Glu151 and the hydroxylamino nitrogen (3.3
Å) in inhibited Zn2AAP.9
9 B. Chevrier, H. DAorchymount, C. Schaik, C. Tamus and D. Moras, Eur.
J. Biochem., 1996, 237, 6388.
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T. J. Kemp, H. Nimir and R. Werner, Inorg. Chem., 1998, 37, 2920.
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5962.
12 A. H. Mahdi, personal communication.
13 D. A. Brown, W. K. Glass and S. J. C. McGardle, Inorg. Chim. Acta,
1983, 80, 13.
In conclusion, the very similar structures of III and
hydroxamate inhibited Zn2AAP illustrate the ability of hy-
droxamic acids to induce dimerisation of mononuclear species
and to then mimic closely the inhibition of related dinuclear
metalloenzymes. Extension to other hydroxamic acids, includ-
ing the secondary series, is in progress to establish criteria for
their possible pharmaceutical efficacy in enzyme inhibition.
14 G. M. Sheldrick, SHELXL 97, Program for the Refinement of Crystal
Structures, University of Göttingen, Germany, 1997.
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