A model for Zn(II)-containing-β-lactamase: Synthesis, X-ray crystal structure of a Zinc(II) complex bearing thiol group and hydrolysis of phosphate diester

Article abstract of DOI:10.1016/S0960-894X(00)00238-9

A novel N₃S₁ typed tripodal ligand bearing an SH group, N-(mercaptoethyl)-di(2-pyridylmethyl)amine, DPASH, was prepared and its zinc(II) complex, [Zn(II)(DPAS)Cl], was structurally characterized by X-ray crystallography. [Zn(II)(DPAS)Cl] promoted hydrolysis of sodium bis(p-nitrophenyl)hydrogenphosphate), BNP^-. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00238-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1333±1337
II
A Model for Zn -Containing-ꢀ-Lactamase: Synthesis, X-Ray
Crystal Structure of a Zinc(II) Complex Bearing Thiol Group
and Hydrolysis of Phosphate Diester
Hiromasa Kurosaki, Toshiaki Tawada, Satomi Kawasoe, Yoko Ohashi
and Masafumi Goto*
Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan
Received 19 November 1999; accepted 13 April 2000
3 1
AbstractÐA novel N S typed tripodal ligand bearing an SH group, N-(mercaptoethyl)-di(2-pyridylmethyl)amine, DPASH, was
II
II
prepared and its zinc(II) complex, [Zn (DPAS)Cl], was structurally characterized by X-ray crystallography. [Zn (DPAS)Cl] pro-
�
moted hydrolysis of sodium bis(p-nitrophenyl)hydrogenphosphate), BNP . # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
tamase II using a zinc(II) complex derived from macrocylic
polyamine, 1,4,7,10-tetraazacyclododecane (cyclen). In the
metallo-b-lactamase, the tetrahedral zinc center is recog-
nized as the catalytic center but the role of the second tri-
gonal bipyramidal zinc center remains to be answered.
Zinc ion is biologically important and has been found in
the active site of many enzymes such as carbonic anhy-
1
2
3
drase, carboxypeptidase, alkaline phosphatase, and
4
b-lactamases. b-Lactamases, for example, are a family
of enzymes that hydrolyze the antibiotic agents and
metallo-b-lactamases have been recently recognized as
potent threat in medicinal community because of their
wide spectrum of substrates including carbapenems.
More recently, X-ray crystallographic study of B. fra-
In order to determine the role of sulfur and the function
of zinc(II) ion as a Lewis acid, we synthesized an N3S1
typed tripodal ligand bearing a mercaptoethyl group, N-(2-
mercaptoethyl)-di(2-pyridylmethyl)amine, DPASH. To
our knowledge, only a few of such tripodal ligand with
4
c
7
gillis metallo-b-lactamase by Concha et al. has shown
that this enzyme has a dinuclear zinc active site in which
the ®rst zinc is coordinated by the nitrogen atoms of
His99, 101, and 162 and by a water/hydroxide with a
tetrahedral geometry while the second one is coordinated
by a nitrogen of His223, a carboxylate of Asp103, a
thiolate of Cys181, and a water molecule and the water/
hydroxide, which acts as a bridge between two zinc ions,
with trigonal bipyramidal geometry. In this structure,
the thiolate is thought to be important for enzymatic
activity. Therefore, the structure of active site has been
a topic of interest with respect to the discovery or design
of the inhibitors against this enzyme in the ®elds of
medicine and pharmacy.5
N S donor set have been reported. We described here the
1
3
®rst synthesis of DPASH, where H denotes a dissociable
proton, and of its zinc(II) complex, chloro{N-mercap-
II
toethyl-di(2-pyridylmethyl)amine}zinc(II), [Zn (DPAS)
Cl], and the ability of hydrolysis of diphosphoester, BNP-
along with that of dichloro{di(2-pyridylmethyl)amine=
II
8
DPA}zinc(II) complex, [Zn (DPA)Cl ] (Chart 1).
2
Results and Discussion
Syntheses
II
The synthetic routes of DPASH and [Zn (DPAS)Cl] are
9
shown in Scheme 1. The tripodal ligand, DPASH, was
10
synthesized by the reaction of di(2-pyridylmethyl)amine
II
In a model study on Zn -containing-b-lactamase, Kimura
et al. have investigated the role of zinc(II) and the hydro-
6
ꢀ
with 3 equiv of ethylene sul®de in dry toluene at 50 C for
lysis mechanism of b-lactam ring in Bacillus cereus b-lac-
4 days under argon. The solvent was evaporated under
reduced pressure and the resulting residue was puri®ed by
column chromatography with silica gel (eluent, AcOEt:
i-PrOH=1:1) to give DPASH as a yellow oil in 50% yield.
*
4
Corresponding author. Tel.: +81-96-371-4310; fax: +81-96-371-
314; e-mail: gotomphi@gpo.kumamoto-u.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00238-9

1
334
H. Kurosaki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1333±1337
II
Figure 1. ORTEP drawing of [Zn (DPAS)Cl], showing with 50%
Ê
probability thermal ellipsoids. Selected bond lengths (A) and angles
ꢀ
(
): Zn(1)-Cl(1) 2.337(1), Zn(1)-S(1) 2.303(1), Zn(1)-N(1) 2.313(3),
Zn(1)-N(2) 2.113(3), Zn(1)-N(3) 2.130(4), Cl(1)-Zn(1)-S(1) 106.57(5),
Cl(1)-Zn(1)-N(1) 167.59(9), Cl(1)-Zn(1)-N(2) 98.6(1), Cl(1)-Zn(1)-N(3)
9
7.0(1), S(1)-Zn(1)-N(1) 85.80(9), S(1)-Zn(1)-N(2) 118.7(1), S(1)-
Zn(1)-N(3) 116.0(1), N(1)-Zn(1)-N(2) 75.6(1), N(1)-Zn(1)-N(3)
6.1(1), N(2)-Zn(1)-N(3) 115.0(1).
7
de®ned by two pyridine nitrogen atoms, N(2) and N(3),
and the thiolate ion S(1). The apical coordination sites
are occupied by the tertiary amine N(1) and chloride ion
Cl(1). This structure is di€erent from that of the related
Chart 1.
II
11
II
8
The zinc(II) complex, [Zn (DPAS)Cl], was prepared
by mixing a solution of DPASH with equimolar amounts
of ZnCl and NaOH in methanol at room temperature.
2
zinc(II) complex, [Zn (DPA)Cl ], which takes a dis-
2
13
torted square pyramid with t value of 0.15. The average
Ê
Zn-Npyridine (N(2) and N(3)) bond lengths of 2.122 A are
II
8
Ê
shorter than those found in [Zn (DPA)Cl ] of 2.169 A
The insoluble materials formed were ®ltered o€ and the
®ltrate was concentrated in vacuo. The resulting residue
was recrystallized from 1:1 methanol:water to give
2
while the Zn-N(1) and Zn-Cl bond lengths of 2.313(3)
Ê
and 2.337(1) A respectively are longer than those of
II
II
8
Ê
[Zn (DPA)Cl ] (2.161(6) A for Zn-N and 2.275(1) and
2
[
Zn (DPAS)Cl] as colorless crystals in 45% yield.
Ê
Ê
2.265(A) for Zn-Cl). The Zn-S bond length of 2.303(1) A
is within normal range as compared to the other zinc
ꢀ
II
The molar conductance at 25 C of a 0.1 mM solution
of the zinc(II) complex, [Zn (DPAS)Cl], in water is
�
14
complexes. The angles of S(1)-Zn-N(2), S(1)-Zn-N(3),
and N(2)-Zn-N(3) are nearly 120 , while the angle of
2
2
ꢀ
Cl(1)-Zn-N(1) is 167.59(9) . The conformations of ®ve-
3
.1Â10 S m /mol, indicating that a chloride ion might
II
ꢀ
be displaced by a solvent water molecule to yield [Zn
(
+
DPAS)(H O)] complex as a 1:1 electrolyte.
2
membered chelate rings formed by coordination of thiol
group to zinc are the same with either the d,d or l,l form.
As a result, the dihedral angle between two pyridine rings
X-ray crystal structure
ꢀ
II
8
ꢀ
increased from 14.05 for [Zn (DPA)Cl ] to 47.57 .
2
II
The structure of [Zn (DPAS)Cl] was determined by an
1
2
II
X-ray analysis. The ORTEP drawing of [Zn (D-
PAS)Cl] is shown in Figure 1. The complex is neutral
molecule in the solid state. The geometry around zinc(II)
is a trigonal bipyramid with t value of 0.82 (t=(b-a)/60,
where b=Cl(1)-Zn-N(1); 167.59(9) and a=S(1)-Zn-N
Potentiometric pH-titrations
The protonation constants (K ) of DPASH were deter-
n
13
.
mined by potentiometric pH-titration of DPASH 3HCl
ꢀ
ꢀ
(1 mM) at 25 C and I=0.10 M (NaNO ). A typical
3
ꢀ
(
2); 118.68(10) : t is zero for an ideal square pyramid,
titration curve is shown in Figure 2a. The computed pKa
values (=� logK ꢁ� logK ) are 9.2 Æ 0.2 (SH), 5.0 Æ 0.1
whereas t=1 for an ideal trigonal bipyramid). The zinc
1
4
Ê
atom is located above 0.409 A from the trigonal plane
(tertiary amine), and <3 (pyridines) and the pK value
a
Scheme 1.

H. Kurosaki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1333±1337
1335
�
.
Figure 2. pH titration curves of triprotonated ligand DPASH 3HCl in
Figure 3. pH rate pro®le for the rate of hydrolysis of BNP (15 mM) at
ꢀ
II
ꢀ
the absence and presence of equimolar Zn at 25 C and I=0.10 M
35 C and I=0.10 M (NaNO ) in 10% (v/v) MeOH: (a) 1.25 mM of
3
2
[Zn (DPAS)Cl]; (b) 1.25 mM of [Zn (DPA)Cl ]; (c) background BNP .
II
II
II
�
.
). Key: (a) 1.0 mM DPASH 3HCl; (b) (a)+1.0 mM Zn
2.
3 2
NO ) 6H O.
(
(
NaNO
3
Ms 1. Therefore, [Zn (DPAS)Cl] and [Zn (DPA)Cl ]
�
II
II
of the thiol group is lower than that of ethanethiol (pK
II
a
2
1
5
.
0.61). The 1:1 DPASH 3HCl-Zn titration curve is
�
II
1
hydrolyzed BNP 8.4 and 55 times faster than Zn Cl ,
2
shown in Figure 2b where two in¯ections at a=4 and 5
were observed. The ®rst bu€er region until a=4 is
ascribed to the loss of four protons from two pyridine
nitrogens, a tertiary amine, and a thiol group. The sec-
ond bu€er region at 4<a<5 represents the removal of a
respectively.
II
The rate of hydrolysis of both Zn complexes increased
linearly with the concentration of the complexes at low
concentration range but the magnitude of slope was
gradually decreased at the higher concentration range as
shown in Fig. 4. These results suggest that an equili-
brium between monomers and a dimer exists in solution
as shown in Scheme 2 and the monomer is probably an
active species for hydrolysis. Such equilibrium in solu-
tion has been also observed in analogous Cu complex
in which the thiol group in DPASH is replaced with an
alcohol.18
II
proton from the Zn -bound water molecule at the ®fth
site with a pK value of 8.6 Æ 0.1, which is lower than
a
II
2+
6
16
that of Zn (H O) (pK =9.5). Under the same con-
2
a
ditions, the pK of the coordinated water molecule of
a
II
[
thiolate anion in DPASH slightly lowers the pK of the
Zn (DPA)Cl ] complex is 9.0 Æ 0.1, indicating that
2
II
a
II
Zn -bound water molecule and the zinc(II) is acting as
Lewis acid.
If we assume that the monomeric ZnII complex with
hydroxide coordinated at the ®fth site (M) is exclusively
the catalytic active species, the concentration of M is
expressed by
Cleavage of phosphate diester
In order to determine whether hydrolysis of phospho-
II
�
diester, BNP , is promoted by [Zn (DPAS)Cl] or
[
II
�
Zn (DPA)Cl ], hydrolysis of BNP (15 mM) in 10%
2
II
MeOH (v/v) aqueous solution of Zn complex (1.25
mM) at pH 6.0 ±10.5 (20 mM HEPES or CHES bu€er)
ꢀ
at 35 C and I=0.10 M (NaNO ) was monitored by
3
following the increase in the visible absorption at 400
nm due to the release of 4-nitrophenolate.17 The initial
�
rate increases linearly with the concentration of BNP .
Therefore, v is expressed as eq (1).
ꢀinit ˆ kobs‰BNP� Š
ꢀ1†
II
The observed initial rates, n , for [Zn (DPAS)Cl] and
[
init
II
�
Zn (DPA)Cl ] at the concentration of [BNP ]=15
2
mM and the [Zn complex]=1.25 mM are plotted as a
function of pH (Fig. 3). Both reactions exhibited sig-
moidal pH-rate pro®les with in¯ection points at pH 8.9
II
II
for [Zn (DPAS)Cl] and at pH 9.2 for [Zn (DPA)Cl ].
2
These results are in good agreement with pK values
a
obtained from potentiometric pH-titrations. At pH
II
II
1
0.0, [Zn (DPAS)Cl] and [Zn (DPA)Cl ] gave rates of
2
�
Figure 4. Dependence of the rate for the hydrolysis of BNP (1.25
II
�
9
� 1
� 9
� 1
(
1.4 Æ 0.4)Â10 Ms and (9.2 Æ 0.3)Â10 Ms . Under
II
mM) on the concentration of [Zn (DPAS)Cl] (a) or [Zn (DPA)Cl
ꢀ
2
]
II
� 10
the same conditions, Zn Cl gave (1.7 Æ 0.3)Â10
(b) at 35 C and I=0.10 M (NaNO ) in 10% (v/v) MeOH.
3
2

1
336
H. Kurosaki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1333±1337
Scheme 2.
n
o
�
ꢀ1=2
‡
2
II
‡
II
� 9
� 1
� 1
Ms for
‰
MŠ ˆ ꢀ1 ‡ ‰H Š=Ka† ‡ 8K‰Zn Š
� ꢀ1 ‡ ‰H Š=Ka† =4K
by Zn complexes are (6.5 Æ 0.4)Â10
t
II
� 9
II
[
Zn (DPAS)Cl] and (25.2 Æ 0.2)Â10 Ms for [Zn (D-
� 9 � 1
2
2
‡
where K ˆ ‰Dimeric speciesŠ=‰MŠ , Ka ˆ ‰MŠ‰H Š=‰MHŠ,
PA)Cl ] compared with a rate of (3.4Æ 0.2)Â10 Ms
and ‰ZnŠ ˆ ‰MHŠ ‡ ‰MŠ ‡ 2‰Dimeric speciesŠ. The rate
for a control experiment. It seems that the intermolecular
II
t
expression can be written by
nucleophilic attack will not be enhanced since both Zn -
bound water/hydroxide ion and carbonyl group of b-
lactam ring of antibiotic agent can not simultaneously
�
�
ꢀ
ˆ kobs‰BNP Š ˆ k ‰MŠ‰BNP Š
II
II
coordinate to Zn ion in [Zn (DPAS)Cl].
and the pseudo-®rst order rate constant, kobs, is given in
+
II
In summary, a novel ZnII complex bearing a mercap-
toethyl group has been prepared and found to hydrolyze a
eq (2), where [Zn ] , [H ], K, and K represent the total
t
a
zinc complex concentration, the proton concentration,
apparent dimerization constant, and acid dissociation
constant, respectively.
�
II
diphosphoester, BNP . The rate of Zn -promoted
�
hydrolysis of BNP increases with the increase in pH,
indicating that zinc-bound hydroxide is an active species
n
o
�
ꢀ1=2
�
‡
2
II
‡
for the hydrolysis of BNP . The thiol group in DPASH
molecule contributes to stabilization of the dimer in
kobs ˆ k ꢀ1‡‰H Š=Ka† ‡8K‰Zn Š
� ꢀ1‡‰H Š=Ka† =4K
t
solution but decreases the hydrolytic activity, compared
II
ꢀ2†
with non-substituted [Zn (DPA)Cl ] complex.
2
A non linear least-squares ®tting19 of the data using eq
�
1
(
2) gave K values of 762 Æ 4 and 316 Æ 6 M and k values
Finally, the major role of the thiolate coordination will
be structural stabilization rather than enzymatic activity.
�
4
� 4
� 1 � 1
M s , and
of (1.5 Æ 0.2)Â10 and (5.8 Æ 0.3)Â10
II
pK values of 9.1 Æ 0.1 and 9.4 Æ 0.2 for [Zn (DPAS)Cl]
a
II
and [Zn (DPA)Cl ], respectively. The curves based on
2
these values are included as solid curves in Figure 4.
References and Notes
II
The dimerization constant of [Zn (DPAS)Cl] with the
mercaptoethyl group is about two times larger than that
1
2
2
. (a) Eriksson, A. E.; Jones, T. A.; Lilijas, A. Proteins 1988, 4,
74. (b) Vallee, B. L.; Auld, D. S. Biochemistry 1990, 29, 5647.
. Lipscomb, W. N. Acc. Chem. Res. 1970, 3, 81.
II
of [Zn (DPA)Cl ], indicating that the dimerization
2
II
II
occurs readily as compared to [Zn (DPA)Cl ]. [Zn (D-
2
3. Kim, E. E.; Wycko€, H. W. J. Mol. Biol. 1991, 218, 449.
. (a) Car®, A.; Pares, S.; Duee, E.; Galleni, M.; Duez, C.; Frere,
Á
II
PAS)Cl] is about four times less reactive than [Zn (D-
PA)Cl ] due to the decreased overall charge or steric
4
Â
2
J. M.; Dideberg, O. EMBO, J. 1995, 14, 4914. (b) Baldwin, G. S.;
Galdes, A.; Hill, H. A. O.; Smith, B. E.; Waley, S. G.; Abraham,
E. P. Biochem. J. 1978, 175, 441. (c) Concha, N. O.; Rasmussen,
B. A.; Bush, K.; Herzberg, O. Structure, 1996, 4, 823.
e€ect of mercaptoethyl substituent. Furthermore, these
pK values obtained by ®tting the concentration-rate
a
pro®les are in agreement with those obtained from pH-
rate pro®les.
5
. (a) Payne, D. J.; Bateson, J. H.; Gasson, B. C.; Proctor, D.;
Khushi, T.; Farmer, T. H.; Tolson, D. A.; Bell, D.; Skett, P.
W.; Marshall, A. C.; Reid, R.; Ghosez, L.; Combret, Y.;
Marchand-Brynaert, J. Antimicrob. Agents Chemother. 1997,
II
Finally, in order to test whether Zn complex can hydro-
lyze a b-lactam antibiotic agent, nitroce®n, preliminary
kinetics studies were carried out under the conditions; the
concentrations of the complex and nitroce®n are 4.18
4
1, 135. (b) Goto, M.; Takahashi, T.; Yamashita, F.; Koreeda,
A.; Mori, H. T.; Ohta, M.; Arakawa, Y. Biol. Pharm. Bull.
997, 20, 1136.
1
ꢀ
mM, 20 mM CAPS bu€er (pH 9.0) at 35 C and I=0.10 M
6. Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc.
1994, 116, 8443.
(
NaNO ). The obtained rates of hydrolysis of nitroce®n
3

H. Kurosaki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1333±1337
1337
ꢀ
7
. (a) Burth, R.; Stange, A.; Scha
È
fer, M.; Vahrenkamp, H.
T=20 Æ 1 C. The structure was solved by direct methods.
Non-hydrogen atoms were re®ned anisotropically and all the
hydrogen atoms were included in the models at their calcu-
lated positions but were not re®ned. Atomic coordinates, bond
angles, bond lengths and thermal parameters have been
deposited at the Cambridge Crystallographic Data Center in
CIF ®le. CCDC number: 144305.
13. Addison, A. W.; Rao, T. N.; Reedijk, J.; Rijn, J.; Ver-
schoor, G. C. J. Chem. Soc., Dalton Trans. 1984, 1349.
14. (a) Brand, U.; Vahrenkamp, H. Inorg. Chem. 1995, 34,
3285. (b) Corwin, D. T.; Koch, S. A. Inorg. Chem. 1988, 27,
493. (c) Diahum, G. P.; Fairall, L.; Klug, A. Nature 1986, 324,
698.
Eur. J. Inorg. Chem. 1998, 1759. (b) Nishida, Y.; Takahashi,
K. Inorg. Chem. 1988, 27, 1406. (c) Ambundo, E. A.; Deydier,
M.-H.; Grall, A. J.; Aguera-Vega, N.; Dressel, L. T.; Cooper,
T. H.; Heeg, M. J.; Ochrymowycz, L. A.; Rorabacher, D. B.
Inorg. Chem. 1999, 38, 4233.
8
9
3
7
. Wirbser, J.; Vahrenkamp, H. Z. Naturforsch 1992, 47b, 962.
1
. H NMR(CDCl
3
): d 1.62 (br, 1H), 2.68 (t, J=5.5 Hz, 2H),
.82 (t, J=5.5 Hz, 2H), 3.86 (s, 4H), 7.16 (t, J=5.5 Hz, 2H),
.54 (d, J=7.9 Hz, 2H), 7.66 (t, J=6.1 Hz, 2H), 8.52 (d,
1
3
J=5.3 Hz, 2H). C NMR (CDCl
1
3
) d: 22.5, 57.1, 60.2, 122.1,
+
23.1, 136.5, 149.1, 1593.2. MS(M ): m/z 258.
1
J. Chem. Soc. C. 1968, 2884.
0. Romary, J. K.; Zachariasen, R. D.; Barger, J. D.; Sciesser.
15. Kagaku Binnrann; Nippon Kagaku Kai; 4th Ed. 1993; Vol.
2, pp 318.
1
1
3
1. H NMR (CD OD): d 2.75 (t, J=5.5 Hz, 2H), 2.84 (t,
J=5.5 Hz, 2H), 4.00 (AB, J=5.5 Hz, 2H), 4.25 (AB, J=16.5
Hz, 2H), 7.63 (d, J=7.9 Hz, 2H), 7.66 (t, J=5.5 Hz, 2H), 8.12
16. Rabenstein, D. L.; Blakney, G. Inorg. Chem. 1973, 12, 128.
17. The concentration of released p-nitrophenolate was calcu-
�
1
(
C
4
1
P2
t, J=6.1 Hz, 2H), 8.86 (d, J=5.3 Hz, 2H). Anal. calcd for
Cl Zn : C, 46.81; H, 4.49; N, 11.70. Found: C,
6.85; H, 4.44; N, 11.47
lated from the extinction coecient at 400 nm (E=18700 M
�
cm ).
1
14
H
16
N
3
S
1
1
1
18. (a) Wahon, D.; Hynes, R. C.; Chin, J. J. Chem. Soc.,
Chem. Commun. 1994, 1441. (b) Young, M. J.; Wahnon, D.;
Hynes, R. C.; Chin, J. J. Am. Chem. Soc. 1995, 117, 9441.
19. The second-order rate constant k, apparent dimerization
constant, and dissociation constant were obtained by ®tting
the experimental data according to eq (2) using non-linear
least-square curve ®tting program (Kaleida Graph Inc.).
II
2. Crystal data for [Zn (DPAS)Cl]: monoclinic, space group
Ê
ꢀ
1
/c, a=13.077(3), b=7.338(3), c=15.815(1) A, b=96.25(1) ,
Ê
3
V=1508.5(6) A , and Z=4; 3900 measured re¯ections, 1906
with I>3 . 0 s(I), 182 re®ned parameters, R=0.038, R =0.029.
w
Intensity measurement: Rigaku AFC-7R di€ractometer, Mo K
radiation, graphite monochromator, o-2y scan, 2ymax=55 ,
a
ꢀ