General acid-promoted hydrolysis of phosphate ester in [ZnII(bnp)(tpa)](ClO4) (tpa = tris(2-pyridylmethyl)amine, bnp = bis(p-nitro-phenyl) phosphate); plausible functional model for P1 nuclease

Article abstract of DOI:10.1246/cl.2002.594

The Zn^II phosphate complex, [Zn^II(bnp)(tpa)](ClO₄) (2) was structurally characterized. The hydrolysis reaction of phosphate ester in 2 was promoted under acidic aqueous media efficiently. The relevancy of this system to P1

Full text of DOI:10.1246/cl.2002.594

594
Chemistry Letters 2002
General Acid-Promoted Hydrolysis of Phosphate Ester in [ZnII(bnp)(tpa)](ClO4)
(tpa ¼ trisð2-pyridylmethylÞamine, bnp ¼ bis( p-nitro-phenyl) phosphate);Plausible Functional
Model for P1 Nuclease
Masami Ito,ꢀ Koyu Fujita, Fumiaki Chitose, Takashi Takeuchi, Kazuaki Yoshida, and Yu-saku Takita
Research and Development Center, Oita University, 700 Dannoharu, Oita, Japan, 870-1192
(Received February 25, 2002; CL-020178)
The ZnII phosphate complex, [ZnII(bnp)(tpa)](ClO4) (2) was
structurally characterized. The hydrolysis reaction of phosphate
ester in 2 was promoted under acidic aqueous media efficiently. The
relevancy of this system to P1 nuclease, which has a low optimal pH
condition, is also discussed.
Hydrolytic enzymes such as phosphatases or nucleases are
known to catalyze the hydrolysis of phosphate ester.1;2 Many of
these enzymes contain metals such as zinc at the active sites. The
catalytic role of zinc is ascribed to the binding and activation of
substrates, while deprotonation of coordinated water to produce a
nucleophilic zinc hydroxide is also proposed as an essential
catalytic hydrolysis function. It is well known that model complex
system can mimic this enzymatic function under basic condition.
However, one of these enzymes, such as P1 nuclease2{7 have
relatively low optimal pH condition (pH 4–6) for their catalytic
reaction.1;8 From inorganic point of view, it seems unlikely that the
zinc(II)-bound water can deprotonate to form the hydroxo moiety in
such low pH condition. Thus, nucleophilic zinc hydroxide may not
be involved in their catalytic reaction. The possibility of other
Figure 1. An ortep drawing (40% probability drawing) of complex 2.
All hydrogen atoms and ClO4 anion is omitted for clarity. Selected bond
ꢀ
legths (A) and angles (deg): Zn-O1 1962(3); Zn-N1 2.075(3); Zn-N2
reaction mechanism cannot be neglected at the present stage.
In this report, we will describe hydrolysis reaction of phosphate
diester ligated to ZnII(tpa) (tpa ¼ trisð2-pyridylmethylÞamine)
complex. The pH-hydrolysis rate profile suggested that general
acid-catalyzed hydrolysis reaction for ligated phosphate ester was
efficient in our system.
2.066(3); Zn-N3 2.059(3); Zn-N4 2.201(4); O1-P 1.477(3); O2-P
1.454(3); O1-Zn-N4 175.1(1): Zn-O1-P 156.7(2).
The treatment of [ZnII(H2O)(tpa)](ClO4)2 (1)9 with bis( p-
nitro-phenyl) phosphate (bnpH) under the presence of 1 equiv. of
triethylamine affords the complex, [ZnII(bnp)(tpa)](ClO4) (2).10
The X-ray structure of 2 is shown in Figure 1.11 The angle of O1-Zn-
N4 (175.1 ꢁ) suggested that phosphate ester coordinates axially to
the slightly distorted trigonal-bypiramidal complex 2 where amine
ligand in tpa positions at the trans position to the phosphate oxygen
and three pyridine nitrogen atoms form trigonal plane.
The potentiometric titration of 1 under the presence of bnpH in
water gave stability constants for the formation of complex 2 at
30 ꢁC (I ¼ 0:2 with NaClO4).12;13 Theoretical species distribution
diagram which is based on these measured stability constants
(Figure 2) suggests that ligation of bnp anion to (tpa)ZnII complex is
dominant in the whole range of pH condition in this system. The
existence of free bnp(H) in this solution is negligible in every pH
condition. The electron spray ionization mass (ESI-MS) spectrum
of aqueous solution at pH 5.6 of complex 1 (10.0 mM (1 mM ¼
1 mmol dmÀ3)) under the presence of bnpH (1.0 mM) presented
major peak at m/z 693 corresponding to the [ZnII(bnp)(tpa)]þ in
positive mode.14 This result is consistent with the data provided by
potentiometric titration. On the other hand, the existence of ion peak
corresponding to the ZnII(OH)(bnp)(tpa) which was proposed by
Figure 2. Distribution diagram for 1.2 mM ZNII, 1.2 mM tpa(HClO4)3 and
0.5mM bnp system as a function of pH at 30 ꢁC and I ¼ 0:20 (NaClO4).
potentiometiric titration was not observed from aqueous solution of
complex 1 (10.0 mM) with bnpH (1.0 mM) at pH 9.1 by ESI-MS,
possibly due to the difficulty for the detection of neutral complex by
ESI-MS, while the ESI-MS spectrum of this aqueous solution at pH
9.1 showed the peak at m/z 693 which was corresponded to the
[ZnII(bnp)(tpa)]þ in positive mode. These observations suggest the
stable ligation of bnp anion to (tpa)Zn(II) complex in the whole
Copyright Ó 2002 The Chemical Society of Japan

Chemistry Letters 2002
595
range of pH conditions measured in present study.
zinc ion in the phosphate ester complex relates to the hydrolysis
reaction in such low pH condition, the effort to prepare other zinc
phosphate complex by using other ligand systems is now in
progress.
The hydrolysis reactions of bnpH (0.5mM) under the presence
of complex 1 (5.0 mM) were studied in the pH range of 5.6–10 at
30 ꢁC.15 The reactions were followed by the increase in an
absorption of the released p-nitro-phenolate ion. The pseudo-first
order rate constant kobs(bnp) was obtained by a log plot method at
each pH condition. The pH versus rate constant (kobs(bnp)) profile is
shown in Figure 3. Interestingly the rate of hydrolysis reaction is
dramatically increased in proportion to the decrease of pH in acidic
condition. Indeed, the hydrolysis rate at the lowest pH condition
measured in this system, (pH 5.6) is 10 fold faster than that of free
bnp(H). In thispH condition, the slope of thelinear plots of kobs(bnp)
against [complex 1] gave the second order rate constant, k(bnp)
(2:13 Â 10À3 MÀ1 sÀ1) (See eq. (1)).
References
1
~
N. W. Lipscomb and N. Strater, Chem.Rev. , 96, 2375(1996) and references
therein.
D. E. Wilcox, Chem.Rev. , 96, 2435(1996).
M. J. Fraser and R. L. Low, ‘‘In nucleases,’’ 2nd ed., ed. by S. M. Linn, R. S. Lloyd,
and R. J. Roberts Cold Spring Harber Laboratory Press, Plainview, NY (1993),
pp 171–207.
2
3
4 M. Fujimoto, A. Kuninaka, and H. Yoshino, Agric.Biol.Chem. , 39, 1991 (1975).
5B. V. L. Potter, B. A. Connolly, and F. Eckstein, Biochemistry, 22, 1369 (1983).
6
7
A. Volbeda, A. Lahm, F. Sakiyama, and D. Suck, EMBO J., 10, 1607 (1991).
D. Suck, R. Dominguez, A. Lahm, and A. Volbeda, J.Cell.Biochem. Suppl. 17C,
154 (1993).
8
9
M. Fujimoto, A. Kuninaka, and H. Yoshino, Agric.Biol.Chem. , 38, 1555 (1974).
G. Anderegg, E. Hubmann, N. G. Podder, and F. Wenk, Helv.Chem.Acta , 60, 123
(1977). The mononuclear structure of [ZnII(H2O)(tpa)](ClO4)2 was confirmed by
Crystallography. M. Ito, unpublished results.
v ¼ kðbnpÞ½complex1ꢂ½bnpꢂ
ðv is the hydrolysis rate of bnpðHÞÞ
ð1Þ
10 A solution of [ZnII(H2O)(tpa)](ClO4)2 (1) (0.20 mmol) and bis( p-nitro-phenyl)
phosphate (bnpH)(0.20 mmol) in acetonitrile (10 ml) was treated with triethyla-
mine (0.20 mmol) and stirred for 5hours. The recrystallization by Et 2O vapour
diffusiton of this mixture gave colorless crystals of [ZnII(bnp)(tpa)](ClO4) (2)
(Yield 80%) which was suitable for X-ray diffraction. Caution! Perchlorate salts
of transition metal complexes are potentially explosive.These complexes should be
prepared only in small quantities and handled with care. Anal. Found C,45.59; H,
3.43; N, 10.90; Cl, 4.29%. Calcd for 2 Zn1C30N6H26O8P1Cl1: C. 45.47; H. 3.31; N.
10.61; Cl 4.47%. IR(KBr, cmÀ1), v(C ¼ C), 1608, v(P-O) 1215, v(ClO4), 1110.
1H-NMR (CD3CN, 300 MHz, 25 ꢁC); 4.21 (s, 6H, CH2), 7.56–7.60 (m, 10H, 3-
Hpy, 5 H- py and p-NO2-C6H4), 8.07 (t, 3H, J ¼ 7:8 Hz, 4-Hpy), 8.24 (d, 4H,
J ¼ 8:0 Hz, p-NO2-C6H4), 8.89 (d, 3H, J ¼ 5:4 Hz. 6Hpy).
Since the result of potentiometric titration suggested that ligation of
phosphate ester was dominant in this pH condition, monomeric
Zn(II) complex, [ZnII(bnp)(tpa)]þ can be regarded as ‘‘reactive
intermediate’’ for this hydrolysis reaction. Taken together, it is
concluded that Lewis acidity of Zn(II) ion facilitates the general
acid-promoted hydrolysis of ligated phosphate ester under acidic
condition. On the other hand, the pseudo-first order rate constants of
this system are comparable to those of free phosphate ion under
basic conditions respectively. While potentiometric titration
suggests that the ligation of phosphate to metal ion is dominant, it
does not appear that ZnII complex affects the hydrolysis reaction
under basic pH condition in our system. While we don’t have a clear
reason in this point, nucleophilic attack of hydroxide anion may not
be effectual for the hydrolysis of bnp anion in our system, even
though the phosphate diester seems to be ligated to zinc(II)
complex.
11 Crystal data for 2, Mr ¼ 697:7; 2 was crystallized in the triclinic space system with
ꢁ
ꢁ
ꢀ
ꢀ
ꢀ
P1, a ¼ 10:589ð1Þ A, b ¼ 17:708ð3Þ A, c ¼ 9:1498ð8Þ A, ꢀ ¼ 100:81ð1Þ , ꢁ ¼
101:68ð7Þ ꢁ, ꢂ ¼ 91:17ð1Þ ꢁ, V ¼ 1647:4ð4Þ A , Z ¼ 2, Dc ¼ 1:60 g cmÀ3
,
ꢀ 3
ꢃðMo-KÞ ¼ 9:49 cmÀ1. The R(Rw) value is 7.0 (10.4)% for 5547 reflections
(3 ꢁ < 2ꢄ < 55 ꢁ, Fo > 3ꢅðFoÞ). The X-ray data collection was carried outatroom
temperature. The structure was solved direct methods (SIR92) and the non-
hydrogen atoms were refined anisotropically. Hydrogen atoms were included but
not refined. Crystallographic data (excluding structure factors) for the structure
reported in this paper have been deposited with The Cambridge Crystallographic
Data Centre as supplementary publication no. CCDC-155611. Copies of the data
can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge
CB21EZ, UK (fax:(þ44)1223-336-033l; e-mail:deposit@ccdc. cam.ac.uk).
12 The formation constants of each product were determined by means of pH-titraton
II
program BEST. (aH is the activity of Hþ); pK1 ¼ 8:29 (K1 ¼ ½Zn ðH2OÞðtpaÞꢂ=
þ
½ZnIIOHðtpaÞꢂaH ),
pK2 ¼ 8:82
(K2 ¼ ½ZnIIðbnpÞðH2OÞðtpaÞꢂ=
þ
½ZnIIðbnpÞðOHÞðtpaÞꢂaH ),
pK3 ¼ 10:10
(K3 ¼ ½ZnIIðH2OÞðtpaÞꢂ2=
þ
½ðZnIIðtpaÞÞ2ðOHÞꢂaH ),
pK4 ¼ 10:05
(K4 ¼ ½ðZnIIðtpaÞÞ2ðOHÞ2ꢂaH
=
þ
þ
½ZnIIðtpaÞÞ ðOHÞꢂ), pK5 ¼ 13:59 (K5 ¼ ½ðtpaÞHꢂ=½tpaꢂaH ), pK6 ¼ 5:37, (K6 ¼
þ
½ðtpaÞH2ꢂ=½2ðtpaÞHꢂa ),
pK7 ¼ 10:61
(K7 ¼ ½ðtpaÞH2ꢂaH =½ðtpaÞH3ꢂ),
Hþ
þ
pK8 ¼ 2:94 (K8 ¼ ½bnpHꢂ=½bnpꢂ½Hꢂ), pKL1 ¼ 13:55, ([tpa][ZnII]/[(tpa)ZnII]),
log KL2 ¼ 6:27 (KL2 ¼ ½ZnIIðtpaÞꢂ½bnpꢂ=½ZnIIðbnpÞðtpaÞꢂ). While the dinuclear
structure of ZnII ꢃ-OH complex, [(tpa)ZnII(ꢃ-OH)2ZnII(tpa)](ClO4)2 complex
has been already reported, Distribution diagram based on potentiometiric titration
shows that mononuclear ZnII(tpa) species is dominant in every pH condition. See;
N. N. Murthy, and K. D. Karlin, J.C.S.Chem.Commun. , 1993, 1236.
13 E. Martell and R. J. Montekaitis, ‘‘Determination and Use of Stability Constants,’’
2nd ed., VCH, New York (1992).
14 In this pH condition, we also could observe the ion peaks at m/z 196 which is
corresponding to the [ZnII(H2O)2(tpa)] as a most intense peak. The ratio of
intensity of ion peaks at m/z 693 ([ZnII(bnp)(tpa)]þ
([ZnII(H2O)2(tpa)]2þ) was 0.92.
15The cleavage reaction rates of bnpH (0.m5 M) under the presence of complex
) toward 196
1
(5mM) (i.e. the rate of release of p-nitro-phenolate anion) were measured by
following 392 nm absorption at 30 ꢁC. In each experiment, buffer solution
containing50 mM Good buffer was used and the ionic strengthwas adjusted to 0.20
with NaClO4. The complex 1 was dissolved in the buffer solution, the UV
absorption increase recorded immediately and then followed the formation of p-
nitro-phenolate ion generally until ca. 5% formation of p-nitro phenolate ion (and
p-nitro-phenol), where log " values for p-nitro phenolate ion were 3.00 (pH 5.6),
3.32 (pH 6.2), 4.02 (pH 7.0), 4.10 (pH 7.5), 4.25 (pH 8.8) and 4.28 (pH 10)
respectively. Every experiment was run for three times to check the reproduci-
bility. Since the final conversionof hydrolysisfor bnp(H)is ca. 40% (in comparison
to the amount of added bnp(H) in each experiment, the initial slope method until
3% formation of p-nitro phenolate ion was applied to estimate the pseudo-first
order rate constants, kobs(bnp) which were obtained by a log plot method at each
pH. From the slopes of linear plots of kobs(bnp) against [complex 1], second order
rate constant, k(bnp) (2:13 Â 10À3 MÀ1 sÀ1) was obtained at pH 5.6.
Figure 3. The pH-rate profile for the first-order rate contants of bnp
(½bnpꢂ ¼ 0:5 mM) under the presence of complex 1 (½complex 1ꢂ ¼ 5 mM)
at 30 ꢁC and I ¼ 0:20 (NaClO4) (solid circle) and the experimenatal results
without complex 1 (solid square).
Since the present system shows that hydrolysis rate is
accelerated dramatically in acidic pH condition and no acceleration
of hydrolysis rate is observed in basic condition, the reaction
mechanism of present system may have a relevancy to that of
hydrolytic enzyme such as P1 nuclease, which has relatively low
optimal pH condition. To elucidate how the geometry around the