Hydrolytic activity of A FeIIIZnII complex toward Di(p-nitrophenyl) phosphate: A functional model of heterobimetallic phosphodiestelase

Article abstract of DOI:10.1246/cl.2002.716

A Fe^IIIZn^III complex, [FeZn(L)(AcO)₃]BPh₄·H₂O, of 2-{N-[2-(dimethylamino)ethyl]iminomethyl}-6-[N, N-di(2-pyridyl-methyl)aminomethyl]-4-methylphenolate (L^-) hydrolyses tri(p-nitrophenyl) phosphate (TNP) into di(p-nitrophenyl) phosphate (DNP^-) and DNP^- into mono(p-nitrophenyl) phosphate (MNP^2-) in aqueous DMF.

Full text of DOI:10.1246/cl.2002.716

716
Chemistry Letters 2002
Hydrolytic Activity of A FeIIIZnII Complex toward Di( p-nitrophenyl) Phosphate:
A Functional Model of Heterobimetallic Phosphodiestelase
y
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¯
Hironobu Machinaga, Kanako Matsufuji, Masaaki Ohba, Masahito Kodera, and Hisashi Okawa
Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581
yDepartment of Molecular Science and Technology, Doshisya University, Kyotanabe, Kyoto 610-0321
(Received April 12, 2002; CL-020313)
A FeIIIZnIII complex, [FeZn(L)(AcO)3]BPh4ÁH2O, of 2-{N-
two oxygen atoms (O2and O4) from the bridging acetate groups
and the oxygen atom (O6) from a unidentate acetate group. The
Zn is bound to the tridentate arm and has a six-coordinate
geometry together with two oxygen atoms (O3 and O5) from the
bridging acetate groups. The average of the Fe-to-donor bond
[2-(dimethylamino)ethyl]iminomethylg-6-[N; N-di(2-pyridyl-
methyl)aminomethyl]-4-methylphenolate (LÀ) hydrolyses tri( p-
nitrophenyl) phosphate (TNP) into di( p-nitrophenyl) phosphate
(DNPÀ) and DNPÀ into mono( p-nitrophenyl) phosphate
(MNP2À) in aqueous DMF.
ꢀ
distances is 2.038 A and the average of the Zn-to-donor distances
ꢀ
is 2.123 A. The site specificity of metal ions in the FeZn complex
is in accord with our recent finding that a smaller metal ion is
bound to the bidentate arm and a larger metal ion to the tridentate
arm.14
Bimetallic cores exist at the active sites of many metallo-
enzymes and play an essential role in biological systems.1
Dinuclear Zn cores are found at the active sites of phospho-
esterases.1{4 It is known that phosphotriesterase has only two Zn
ions at the active site5 whereas phospholipase C6 and P1
nuclease7 have an additional Zn ion to hydrolyze phosphodiester.
It is supposed that these phosphodiesterases require a trinuclear
Zn core to bind a substrate at the dinuclear Zn unit and to provide
the nucleophile (OHÀ or water) on the third Zn center (Figure 1,
A). Moreover, heterodinuclear FeZn core was recognized at the
active sites of purple acid phosphatase8 and human calcineurin9
that facilitate the hydrolysis of phosphodiesters into phospho-
monoesters. It is considered that these enzymes employ a FeZn
core instead of trinuclear Zn core to accommodate a substrate in
chelating mode on the Fe center and provide the nucleophile on
the Zn center10 (Figure 1, B). Here we report a FeZn complex that
has a hydrolytic function relevant to phosphodiesterase.
Figure 2. Crystal structure of [FeZn(L)(AcO)3]BPh4ÁH2O.
The complex shows two absorption bands at 340 nm (":
5500 MÀ1cmÀ1) and 480 nm (": 1000 MÀ1cmÀ1) in DMF. The
spectrum is virtually invariant at a moderate complex concent-
ration (1:0 Â 10À3{2:0 Â 10À4 M). FAB mass spectrometry in m-
nitrobenzylalcohol matrix indicated the parent ion peaks centered
around m=z 654.2corresponding to {FeZn(L)(AcO) 2gþ.
Hydrolytic activity of the FeZn complex toward tri( p-nitro-
phenyl) phosphate (TNP) and hydrogen di( p-nitrophenyl) phos-
phate (HDNP) was examined in aqueous DMF (H2O : DMF ¼
2 : 98 in volume) at 25 ꢀC by means of UV-visible spectroscopy.
An aqueous DMF solution containing the FeZn complex
(2:0 Â 10À4 M) and the substrate (TNP or HDNP; 6:7Â
10À5 M) was prepared and subjected to spectroscopic measure-
ments, using a complex solution in aqueous DMF (2:0 Â 10À4 M)
as reference.
Spectral changes in the hydrolysis of TNP by the FeZn
complex are shown in Figure 3. The absorption band of TNP at
280 nm decreased with time with a concomitant increase at
304 nm due to the formation of DNPÀ. Another absorption band
observed at 422 nm is characteristic of p-nitrophenolate ion.
Based on the absorbance at 304 nm, the hydrolysis of TNP into
DNPÀ must be completed in 100 min, but the absorbance at
304 nm, and that at 422 nm as well, showed a tendency to increase
further. This fact suggests that the hydrolysis of BNPÀ into
MNP2À occurs after the completion of the hydrolysis of TNP into
Figure 1. Supposed interation of phosphodiester
with (A) triuclear Zn core and (B) dinuclear FeZn
core in biological phosphodiester.
The FeZn complex [FeZn(L)(AcO)3]BPh4ÁH2O of the end-
off compartmental ligand HL (Fig. 2) was prepared as reddish
brown crystals by the reaction of HL with Fe(AcO)3 and
Zn(AcO)2Á2H2O in methanol in the presence of sodium tetra-
phenylborate.11
The crystal structure of the FeZn complex was determined by
single-crystal X-ray analysis.12 An ORTEP view of the complex
is shown in Fig. 2.13 The two metal ions are bridged by the
phenolic oxygen atom of LÀ and two acetate groups in a ‘syn-syn’
ꢀ
mode in the Fe–Zn separation of 3.385(1) A. The Fe is bound to
the bidentate arm and has a six-coordinate geometry together with
Copyright Ó 2002 The Chemical Society of Japan

Chemistry Letters 2002
717
BNPÀ. It is worth notingthat the FeCu complex has a high activity
in the hydrolysis of TNP relative to analogous ZnZn complex
[Zn2(L)(AcO)2]ClO4 when compared under the same conditions
(see Insert).
is released more or less in a dilute solution, providing two vacant
sites on the Fe center and one vacant site on the Zn center. The
resulting [FeZn(L)(AcO)]3þ can accommodate DNPÀ in the
chelating mode on the Fe center and OHÀ (or H2O) on the Zn
center (Fig. 1, B), allowing the nucleophilic attack of the OHÀ (or
H2O) to the phosphorus nucleus of DNPÀ.
This work was supported by a Grant-in-Aid for COE
Research ‘Design and Control of Advanced Molecular Assembly
System’ (No. 08CE2005) and a Grant-in-Aid for Scientific
Research Program (No. 13640561) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology.
References and Notes
1
¯
D. E. Fenton and H. Okawa, ‘‘Perspectives on Bioinorganic
Chemistry,’’ JAI Press, London (1993), Vol. 2, p 81.
2K. D. Karlin, Science, 261, 701 (1993).
3
4
5
D. E. Wilcox, Chem. Rev., 96, 243 (1996).
Figure 3. Spectral changes in the hydrolysis of TNP by the
FeZn complex (measured every 10 minutes). The insert is the
spectral changes in the hdrolysis of TNP by [Zn2(L)(AcO)2]-
ClO4 (measured every 10 minutes).
G. C. Dismukes, Chem. Rev., 96, 2357 (1996).
J. L. Vanhooke, M. M. Benning, F. M. Raushel, and H. M.
Holden, Biochemistry, 35, 6020 (1996).
E. Hough, L. K. Hansen, B. Birkness, K. Jynge, S. Hansen, A.
Hardvik, C. Little, E. J. Dodson, and Z. Derewenda, Nature,
338, 357 (1989).
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215, 207 (1990).
N. Strater, T. Klabunde, P. Tucker, H. Witzel, and B. Krebs,
Science, 268, 1489 (1995).
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Pelletier, A. Tempczyk, V. J. Kalish, K. D. Tucker, R. E.
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F. Maldonado, J. E. Barker, R. Bacquet, and E. Villafranca,
Nature, 378, 641 (1995).
6
The hydrolysis of HDNP by the FeZn complex was studied by
a separate run (Figure 4). In this case spectral changes in the near
UV region are small because the absorption band of HDNP and
that of MNP2À are located at close wavelength (304 and 308 nm,
respectively). However, the hydrolysis of HDNP by the FeZn
complex is evident from the absorption band of p-nitrophenolate
ion appearing at 425 nm. The solution soon after dissolution gave
spectrum a which changed to spectrum b after 100 min and then
gradually to c after 700 min. The spectral feature of b showing
‘negative absorption’ around 370 nm implies that a FeZn-DNP
adduct is formed at the initial stage and the bound DNPÀ is slowly
hydrolyzed into MNP2À. The hydrolysis of DNPÀ into BNP2À is
almost completed in 700 min judged from the time-course of the
absorbance at 425 nm.
7
8
9
¯
10 K. Arimura, M. Ohba, T. Yokoyama, and H. Okawa, Chem.
Lett., 2001, 1134.
11 Found: C, 63.14; H, 5.95; N, 6.69; Fe, 5.48; Zn, 6.17%. Clcd for
BC55FeH61N5O8Zn: C, 62.78; H, 5.84; N, 6.66; Fe, 5.31; Zn,
6.21%.
12Crystal data: [FeZn(L)(AcO) 3]BPh4ÁH2O, F.W. 1052.15;
monoclinic space group P21/n(#14), a ¼ 18:8620ð4Þ, b ¼
15:3948ð4Þ, c ¼ 20:0449ð4Þ A, ꢀ ¼ 117:222ð1Þ ꢀ, V ¼
ꢀ
3
ꢀ 3
5157:9ð2Þ A , Z ¼ 4, Dc ¼ 1:350 g/cm . Intensity data were
collected at À90 ꢀC on a Rigaku RAXIS-RAPID Imaging Plate
using graphite-monochromated Mo Kꢁ radiation (ꢂðMo
KꢁÞ ¼ 8:02 cmÀ1). No. of measured ¼ 44600 and No. of
unique reflections ¼ 11633. R ¼ 0:110 (all data), RW
0:159, R1 ¼ 0:065 (I > 2:00ꢃðIÞ).
¼
13 Fe–O1 1.970(3), Fe–O2.2036(3), Fe–O4 1.949(3), Fe–O6
1.955(3), Fe–N1 2.086(4), Fe–N2 2.230(4), Zn–O1 2.092(3),
Zn–O3 2.009(3), Zn–O5 2.144(3), Zn–N3 2.191(3), Zn–N4
ꢀ
2.131(3), Zn–N5 2.171(3) A; Fe–O1–Zn 112.9(1), O1–Fe–O2
92.7(1), O1–Fe–O4 99.9(1), O1–Fe–O6 91.6(1), O1–Fe–N1
86.1(1), O1–Fe–N2 166.6(1), O2–Fe–O4 90.8(1), O2–Fe–O6
172.4(1), O2–Fe–N1 84.9(1), O2–Fe–N2 87.3(1), O2–Fe–O6
94.7(2), O4–Fe–N1 172.8(1), O4–Fe–N2 93.5(1), O6–Fe–N1
89.1(2), O6–Fe–N2 87.1(1), N1–Fe–N2 80.5(1), O1–Zn–O3
101.0(1), O1–Zn–O5 86.1(1), O1–Zn–N3 90.2(1), O1–Zn–N4
163.1(1), O1–Zn–N5 88.4(1), O3–Zn–O5 98.8(1),O3–Zn–N3,
164.8(1), O3–Zn–N4 93.6(1), O3–Zn–N5 91.5(1), O5–Zn–N3
92.3(1), O5–Zn–N4 83.3(1), O5–Zn–N5 169.1(1), N3–Zn–N4
77.2(1), N3–Zn–N5 78.4(1), N4–Zn–N5 99.7(1) ꢀ.
Figure 4. Spectral changes in the hydrolysis of DNPÀ by the
FeZn complex (measured every 100 minutes).
It must be emphasized that analogous ZnZn complex has no
activity to hydrolyze DNPÀ.15 We have confirmed that the
absorption bands at 340 and 480 nm of the FeZn complex in aq.
DMF (H2O : DMF ¼ 2 : 98) diminish their intensities upon high
dilution (<2 Â 10À4 M). Furthermore, the molar conductance of
the complex in aq. DMF increased upon dilution from
50 S cm2molÀ1 at 2 Â 10À4 M to 90 S cm2molÀ1 at 4 Â 10À5 M.
These facts imply that one acetate bridge of [FeZn(L)(AcO)2]2þ
¯
14 K. Abe, K. Matsufuji, M. Ohba, and H. Okawa, Inorg. Chem., in
press.
¯
15 K. Abe, J. Izumi, M. Ohba, T. Yokoyama, and H. Okawa, Bull.
Chem. Soc. Jpn., 74, 85 (2001).