Novel properties of cooperative dinuclear zinc(II) ions: The selective recognition of phosphomonoesters and their P-O ester bond cleavage by a new dinuclear zinc(II) cryptate

Article abstract of DOI:10.1021/ja953413m

A propanol-bridged octaazacryptand (26-hydroxy-1,4,7,10,13,16,19,22-octaazabicyclo[11.11.3]heptacosane, HL) has been synthesized from diethylenetriamine and [2-oxo-6-(aminomethyl)morpholyl]-N,N',N'-triacetic acid triethyl ester by refluxing in MeOH follow

Full text of DOI:10.1021/ja953413m

J. Am. Chem. Soc. 1996, 118, 3091-3099
3091
Novel Properties of Cooperative Dinuclear Zinc(II) Ions: The
Selective Recognition of Phosphomonoesters and Their P-O
Ester Bond Cleavage by a New Dinuclear Zinc(II) Cryptate
Tohru Koike,† Masaki Inoue,† Eiichi Kimura,*,† and Motoo Shiro‡
Contribution from the Department of Medicinal Chemistry, School of Medicine, Hiroshima
UniVersity, Kasumi 1-2-3, Minami-ku, Hiroshima, 734, Japan, and Rigaku Corporation X-ray
Research Laboratory, Matsubaracho 3-9-12, Akishima, Tokyo, 196, Japan
ReceiVed October 10, 1995X
Abstract: A propanol-bridged octaazacryptand (26-hydroxy-1,4,7,10,13,16,19,22-octaazabicyclo[11.11.3]heptacosane,
HL) has been synthesized from diethylenetriamine and [2-oxo-6-(aminomethyl)morpholyl]-N,N′,N′-triacetic acid triethyl
ester by refluxing in MeOH followed by BH3‚THF reduction. This octaazacryptand forms a novel dinuclear zinc(II)
cryptate (Zn2L) (L ) alkoxide form of HL) in aqueous solution. The X-ray crystal structure of the cryptate showed
each zinc(II) ion in a distorted trigonal-bipyramidal environment involving two NH’s and an alkoxide O- anion as
equatorial donors, with tertiary amine and an NH stand in apical positions. Crystals of the triperchlorate salt of
Zn2L (C19H43N8O13Cl3Zn2) are monoclinic, space group P21/n (No. 14) with a ) 15.037(5) Å, b ) 13.862(5) Å, c
) 15.780(4) Å, â ) 90.29(2)°, Z ) 4, and R ) 0.109. Although the two zinc(II) ions (separated with a distance of
3.42 Å) in the cryptate appeared to be coordinately saturated and hence were assumed unreactive, they work together
to selectively interact with a phosphomonoester, 4-nitrophenyl phosphate dianion (NPP2-), and promote the cleavage
of its P-O ester bond by nucleophilic attack of one of the apically coordinated NH’s at pH 4.9-9.5 in aqueous
solution. The reaction product was isolated as a phosphoramide derivative (Zn2L-PO3H) from aqueous solution at
pH 3 and characterized by X-ray crystal analysis. Crystals of Zn2L-PO3H‚2H2O‚(ClO4)3 (C19H48N8O18Cl3Zn2) are
monoclinic, space group Cc (No. 9) with a ) 19.573(3) Å, b ) 12.454(4) Å, c ) 15.066(3) Å, â ) 103.94(1)°, Z
) 4, and R ) 0.036. The structure of Zn2L-PO3H featured the two phosphoryl oxygens bound to each zinc(II) ion
in place of the original two apical NH’s. The kinetics were followed for liberation of 4-nitrophenol (the maximum
second-order rate constant kNPP ) (1.52 ( 0.05) × 10-3 M-1 s-1 at pH 5.9 and 35 °C). The rate vs pH profile with
5 mM Zn2L and 10 mM 4-nitrophenyl phosphate showed a bell-shaped curve with pK1 of 5.2 and pK2 of 6.3, which
were assigned to the protonation constants for NPP2- + H a HNPP- and NH (a freed apical donor in the associated
reaction intermediate) + H+ a HNH+, respectively. The most remarkable character of the present new zinc(II)
cryptate was that it reacted only with phosphoester polyanions such as NPP2- and ATP4-, but not with bis(4-
nitrophenyl) phosphodiester monoanion, tris(4-nitrophenyl) phosphotriester, or 4-nitrophenyl acetate. The present
results may well be relevant to the significance of dinuclear metal centers in metallophosphatases such as alkaline
monophosphatase.
Introduction
In the course of an attempt to synthesize a new ligand 4 for
a phosphomonoesterase model (a bis(macrocyclic tetraamine)
linked by a 2-hydroxypropyl group from a carboxyester (1) and
diethylenetriamine (2)), we incidentally isolated an isomeric (of
3) tetraoxocryptand 6 as a main product, which was an
intermediate for a new octaazacryptand 7 (see Scheme 1). The
purpose of the synthesis of 4 was to prepare a dinuclear zinc-
(II) complex 5, which may lead to a novel alkaline phosphatase
model. With all the previous phosphatase models composite
of mononuclear metal complexes studied by us4,5 and other
groups,2,3 hydrolyses of carboxyesters, phosphotriesters, phos-
phodiesters, and phosphomonoesters were successful. However,
Alkaline phosphatase (AP) is a ZnII-containing phosphomo-
noesterase that selectively hydrolyzes phosphomonoester dianion
by using the two cooperative zinc(II) ions at the active center.1
There have been numerous reports of phosphoesterase model
systems using metal complexes, but most of these models were
aimed at achieving faster hydrolysis of phosphate,2,3 and as yet,
few systematical models concentrate on the selective reaction
toward the substrate phosphomonoester dianion.
† Hiroshima University.
‡ Rigaku Corp.
* e-mail: ekimura@ue.ipc.hiroshima-u.ac.jp.
R. J. Am. Chem. Soc. 1986, 108, 2388-2394. (j) Chin, J.; Banaszczyk,
M.; Jubian, V.; Zou, X. J. Am. Chem. Soc. 1989, 111, 186-190. (k) Hendry,
P.; Sargeson, A. M. J. Am. Chem. Soc. 1989, 111, 2521-2527. (l) Morrow,
J. R.; Trogler, W. C. Inorg. Chem. 1988, 27, 3387-3394.
(3) Dinuclear system: (a) Vance, D. H.; Czarnik, A. W. J. Am. Chem.
Soc. 1993, 115, 12165-12166. (b) Hikichi, S.; Tanaka, M.; Morooka, Y.;
Kitajima, N. J. Chem. Soc., Chem. Commun. 1992, 814-815. (c) Chung,
Y.; Akkaya, E. U.; Venkatachalam, T. K.; Czarnik, A. W. Tetrahedron
Lett. 1990, 31, 5413-5416. (d) Takasaki, B. K.; Chin, J. J. Am. Chem.
Soc. 1995, 117, 8582-8585. (e) Connolly, J. A.; Banaszczyk, M.; Hynes,
R. C.; Chin, J. Inorg. Chem. 1994, 33, 665-669. (f) Chapman, W. H., Jr.;
Breslow, R. J. Am. Chem. Soc. 1995, 117, 5462-5469. (g) Clewley, R.
G.; Slebocka-Tilk, H.; Brown, R. S. Inorg. Chim. Acta 1989, 157, 233-
238.
X Abstract published in AdVance ACS Abstracts, March 15, 1996.
(1) (a) Kim, E. E.; Wyckoff, H. W. J. Mol. Biol. 1991, 218, 449-464.
(b) Coleman, J. E. Annu. ReV. Biophys. Biomol. Struct. 1992, 21, 441-
483.
(2) Mononuclear system: (a) Jones, D. R.; Lindoy, L. F.; Sargeson, A.
M. J. Am. Chem. Soc. 1983, 105, 7327-7336. (b) Tsubouchi, A.; Bruice,
T. C. J. Am. Chem. Soc. 1994, 116, 11614-11615. (c) Sigman, D. S.;
Wahl, G. M.; Creighton, D. J. Biochemistry 1972, 11, 2236-2242. (d)
Tafesse, F.; Massoud, S. S.; Milburn, R. M. Inorg. Chem. 1993, 32, 1864-
1865. (e) Norman, P. R.; Cornelius, R. D. J. Am. Chem. Soc. 1982, 104,
2356-2361. (f) De Rosch, M. A.; Trogler, W. C. Inorg. Chem. 1990, 29,
2409-2416. (g) Hay, R. W.; Govan, N. J. Chem. Soc., Chem. Commun.
1990, 714-715. (h) Ruf, M.; Weis, K.; Vahrenkamp, H. J. Chem. Soc.,
Chem. Commun. 1994, 135-136. (i) Gellman, S. H.; Petter, R.; Breslow,
(4) Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935-8941.
0002-7863/96/1518-3091$12.00/0 © 1996 American Chemical Society

3092 J. Am. Chem. Soc., Vol. 118, No. 13, 1996
Koike et al.
Scheme 1
Figure 1. Typical titration curves for propanol-bridged octaazacryptand
7 at 35 °C with I ) 0.10 (NaNO3): (a) 1.0 mM 7‚8HCl; (b) a + 2.0
mM ZnIISO4. a(OH-) is the number of equivalents of base added.
from most of the mononuclear metal complexes.2,4 Thus, the
newly synthesized 8 was proven to be an extremely interesting
cryptate to selectively recognize the phosphate dianion and
cleave its P-O ester bond. Herein, we report the synthesis,
structure, and reactivity of this novel cryptate 8.
Results and Discussion
Synthesis of 26-Hydroxy-1,4,7,10,13,16,19,22-octaazabicyclo-
[11.11.3]heptacosane (Propanol-Bridged Octaazacryptand 7)
(Scheme 1). Contrary to our initial expectations, the reaction
of [2-oxo-6-(aminomethyl)morpholyl]-N,N′,N′-triacetic acid tri-
ethyl ester (1) with 2 equiv of diethylenetriamine (2) (refluxing
for 5 days) gave a new bicyclic tetraoxo intermediate 6 in 50%
yield with no sign of other macrocyclic products such as 3. Due
to the rigidity of the two amido bonds, formation of the 16-
membered ring in 6 is probably more facile than that of the
two 12-membered rings in 3. All the carbonyl groups were
reduced with BH3‚THF complex in THF to give the propanol-
bridged octaazacryptand 7, which was purified as its crystalline
8HCl salt in 33% yield.
Scheme 2
Protonation and Zinc(II) Complexation Constants of
Propanol-Bridged Octaazacryptand 7. The protonation con-
stants (Kn) of 7 (HL) were determined by potentiometric pH
titration of 7‚8HCl (1.0 mM) at 35 °C with I ) 0.10 (NaNO3).
A typical pH titration curve with 1.0 mM 7‚8HCl is shown in
Figure 1a. The titration data were analyzed for the acid-base
+
+
equilibrium (eq 1), where aH is the activity of H . The mixed
protonation constants log K1 - log K8 are 10.16 ( 0.03, 9.27
( 0.03, 8.47 ( 0.02, 7.30 ( 0.02, 2.5 ( 0.1, <2, <2, and <2.
they did not focus on the selective hydrolysis of the substrate
phosphomonoesters. In our recent work,5 we discovered that
the zinc(II) complexes with macrocyclic polyamines bearing
an alcohol pendant (N-hydroxyethyl group) promote efficiently
the hydrolysis of acetate and phosphodiester. Therefore, we
attempted to combine dinuclear zinc(II) and an alcohol pendant
in 5 to build a phosphomonoesterase model.
However, a new dinuclear zinc(II) cryptate 8 was obtained
from 7 and zinc(II) in aqueous solution and was anticipated to
be less reactive toward phosphates because the zinc(II) ions
appeared to be coordinately saturated in a distorted trigonal-
bipyramidal (five-coordinate) environment. To our surprise, 8
reacted exclusively with a phosphomonoester dianion, 4-nitro-
phenyl phosphate (NPP2-), to produce its phosphoramide
derivative 9 (see Scheme 2), but not with bis(4-nitrophenyl)
phosphate (BNP-, a phosphodiester monoanion), tris(4-nitro-
phenyl) phosphate (TNP, a neutral phosphotriester), or 4-nitro-
phenyl acetate (NA, a neutral carboxyester), while the latter ester
substrates are generally more reactive with nucleophiles derived
Hn-1‚HL + H+ a Hn‚HL
Kn ) [Hn‚HL]/[Hn-1‚HL]aH
+
(1)
The titration curve of 7‚8HCl (1 mM) in the presence of 2
equiv of ZnII (Figure 1b) reveals the formation of a stable
complex at pH > 4, with simultaneous deprotonation of the
alcohol OH, a conclusion being derived from the observation
of the sole neutralization break at a(OH-) ) 9. Further
deprotonation or precipitation of Zn(OH)2 was not observed at
a(OH-) > 9, indicating that the alkoxide-bridged zinc(II)
cryptate 8 (Zn2L) remains stable until pH ca. 12, where L is
the alkoxide form of 7. It should be noted that only the
obtention of dinuclear species, 8 and its monoprotonated
complex Zn2HL 10 (or Zn2L‚H+ 11) (see eqs 2 and 3), was
(5) (a) Kimura, E.; Nakamura, I.; Koike, T.; Shionoya, M.; Kodama,
Y.; Ikeda, T.; Shiro, M. J. Am. Chem. Soc. 1994, 116, 4764-4771. (b)
Koike, T.; Kajitani, S.; Nakamura, I.; Kimura, E.; Shiro, M. J. Am. Chem.
Soc. 1995, 117, 1210-1219. (c) Kimura, E.; Kodama, Y.; Koike, T.; Shiro,
M. J. Am. Chem. Soc. 1995, 117, 8304-8311.

NoVel Properties of CooperatiVe Dinuclear Zinc(II) Ions
J. Am. Chem. Soc., Vol. 118, No. 13, 1996 3093
Figure 2. Distribution diagram for the zinc(II) species in 5 mM
propanol-bridged octaazacryptand 7/10 mM zinc(II) system as a
function of pH at 35 °C.
confirmed under the experimental conditions employed. There-
fore, as one zinc(II) ion binds to 7, the second zinc(II) ion
simultaneously comes in the 1:1 ZnII-cryptand (e.g., ZnHL‚nH+).
The final structural assignment for the alkoxide O--bridged
dinuclear zinc(II) cryptate 8 comes from the X-ray crystal
structure analysis presented below. The dinuclear zinc(II)
complexation constants K(Zn2HL) and deprotonation constant
K(Zn2L) as defined as follows:
Figure 3. Crystal structure of 8 and its coordination bonding scheme
with three perchlorates omitted for clarity. Thermal ellipsoids for zinc,
nitrogen and oxygen atoms and circles for carbon atoms are at 30%
probablilty level. Bond angles (deg) around zinc(II) ions are as
follows: O28-Zn1-N1, 79.2(6); O28-Zn1-N4, 103.1(6); O28-Zn1-
N19, 100.7(5); O28-Zn1-N22, 130.8(9); N1-Zn1-N4, 84.9(7); N1-Zn1-
N19, 159.0(6); N1-Zn1-N22, 80.2(9); N4-Zn1-N19, 115.2(7); N4-Zn1-
N22, 118.8(9); N19-Zn1-N22, 84.4(9); O28-Zn2-N7, 102.9(6); O28-
Zn2-N10, 130.6(9); O28-Zn2-N13, 77.7(6); O28-Zn2-N16, 101.8(6);
N7-Zn2-N10, 82.5(10); N7-Zn2-N13, 159.3(6); N7-Zn2-N16, 115.6-
(7); N10-Zn2-N13, 81.8(10); N10-Zn2-N16, 120.2(9); N13-Zn2-N16,
84.0(7).
HL (7) + 2ZnII a Zn2HL (10 or 11)
K(Zn2HL) ) [[Zn2HL]/[ZnII]2[HL] (M-2) (2)
Zn2HL a Zn2L (8) + H+
K(Zn2L) ) [Zn2L]aH+/[Zn2HL] (M) (3)
The obtained log K(Zn2HL) and -log K(Zn2L) values are
20.6 ( 0.1 and 4.0 ( 0.1, respectively. A typical diagram for
species distribution as a function of pH at [total zinc] ) 10
mM and [total ligand] ) 5 mM is displayed in Figure 2, identical
conditions being used for kinetic studies described below. The
extremely small -log K(Zn2L) value (i.e., deprotonation
constant of 10 or 11) indicates the facile deprotonation of the
alcohol with pKa e 4 involved in a double coordination with
two zinc(II) ions. It is of interest to recall that the pendant
alcoholic OH in 1:1 benzyl alcohol-pendant cyclen zinc(II)
complex 12a deprotonates to bind to a zinc(II) in 12b with a
larger pKa value of 7.3 under the same conditions.5c
complex. The zinc(II) complexation constant for 13, log
K(Zn2L′OH) (see eq 4), was reported to be 12.6, which is much
smaller than our overall complexation constant for Zn2L (8),
+
log K′(Zn2L) of 16.6 () log(K(Zn2HL)/(K(Zn2L)fH )), see eq
5). The comparison demonstrates the stronger stabilization by
the alkoxide bridge in 8 than the external OH- bridge in 13,
which is probably due to the well-preorganized ligand structure
of the cryptand.
K(Zn2L′OH) ) [Zn2L′OH][H+]/[ZnII]2[L′] (M-1) (4)
HL(7) + 2ZnII a Zn2L(8) + H+
K′(Zn2L) ) [Zn2L][H+]/[ZnII]2[HL] (M-1) (5)
X-ray Crystal Structure of the Dinuclear Zinc(II) Cryptate
8. The cryptand 7 (in its acid-free form HL, see the Experi-
mental Section) in EtOH was mixed with 2.3 equiv of Zn(ClO4)2
at 55 °C for 14 h. After the solvent was evaporated, the residue
was crystallized from water to obtain the dinuclear zinc(II)
cryptate 8‚(ClO4)3 as colorless crystals in 89% yield. The
elemental analysis (C, H, N) suggested the formula Zn2L‚3ClO4
containing no water. The crystal was subjected to X-ray crystal
analysis. The crystal structure provided unequivocal evidence
for the alkoxide-bridged dinuclear zinc(II) cryptate 8, which is
shown as an ORTEP drawing with 30% probability thermal
ellipsoids for N, O, and Zn atoms and circles for C atoms in
Figure 3. The methylene carbons and one of the perchlorate
ions are very disordered, which is reflected upon the relatively
large R value of 0.109. Selected crystal data and collection
parameters are displayed in Table 1. Selected bond distances
around zinc(II) ions are illustrated in Figure 3.
Previously, dinuclear zinc(II) complexation with a 24-
membered monocyclic octaamine ([24]aneN8, L′) was reported
by Bencini et al.6 In this case, a water molecule simultaneously
deprotonates with a pKa value below 7 to bridge the two zinc-
(II) to yield 13 (Zn2L′OH), which is somewhat similar to our
(6) Bencini, A.; Bianchi, A.; Dapporto, P.; Garcia-Espan˜a, E.; Micheloni,
M.; Paoletti, P. Inorg. Chem. 1989, 28, 1188-1191.

3094 J. Am. Chem. Soc., Vol. 118, No. 13, 1996
Koike et al.
complexes, ZnII-cyclen 14 (cyclen ) 1,4,7,10-tetraazacy-
clododecane)4 and alcohol-pendant derivatives 12b5c and 155b
Table 1. Selected Crystallographic Data for 8‚3ClO4 and
9‚3ClO4‚2H2O
8
9
emp form
form wt
crystal color, habit
crystal system
space group
lattice params
a (Å)
C19H43N8O13Cl3Zn2
828.7
colorless, prismatic
monoclinic
C19H48N8O18Cl3PZn2
944.7
colorless, prismatic
monoclinic
P21/n (no. 14)
Cc (no. 9)
15.037(5)
13.862(5)
15.780(4)
90.29(2)
3289(1)
4
1.710
47.05
20
19.573(3)
12.454(4)
15.066(3)
103.94(1)
3564(1)
4
1.760
49.15
23
b (Å)
c (Å)
promote hydrolysis of the neutral (TNP and NA) and monoan-
ionic diesters (BNP-), but none practically reacted with the
dianionic phosphomonoester NPP2-. This time, however, the
dinuclear complex 8 selectively reacts with a phosphomonoester
dianion NPP2- to release 4-nitrophenol (NP) in aqueous solution
(see Scheme 2). The other product was proven to be a novel
intramolecularly phosphoryl oxygen-coordinating cryptate 9 (i.e.,
a phosphoryl-transferred complex, Zn2L-PO3H) by X-ray
crystal analysis.10 The new complex 9 remained very stable,
and we did not see any degradation of 9 (such as hydrolysis of
the N-P bond) at higher pH (ca. 12) and higher temperature
(ca. 60 °C) even after 1 week. Hence, we could not convert 9
back to 8 as a hydrolysis catalyst.
The ligand 7 alone at pH 8.2 (which practically exists as HL‚
3H+ on the basis of the log Kn values described above) and pH
6.1 (as HL‚4H+) did not cleave the P-O ester bond at all, i.e.,
the same NPP2- hydrolysis rates were obtained as the back-
ground reaction rates shown in dotted line of Figure 5.11
Isolation of the Phosphoryl-Transferred Product (9‚
3ClO4‚2H2O) and Its X-ray Crystal Structure. The phos-
phoryl-transferred product 9 was isolated as its triperchlorate
salt in the reaction of 8 and 1.1 equiv of NPP2- in aqueous
solution at 60 °C for 2 days. The reaction was neat, and no
other product (such as diphosphoramide derivative) was detected
by 31P NMR. After the solution was washed with CHCl3 and
the solvent was evaporated, the residue became crystalline from
water at pH 3 in 69% yield. The elemental analysis (C, H, N)
and 31P NMR suggested the formula 9‚3ClO4‚2H2O. A crystal
of 9‚3ClO4‚2H2O was subjected to X-ray crystal analysis. The
crystal structure is shown as an ORTEP drawing with 30%
probability thermal ellipsoids in Figure 4. Selected crystal data
and collection parameters are displayed in Table 1. Selected
bond distances around zinc(II) ions are illustrated in Figure 4.
In the cryptate 9, both zinc(II) ions are almost equivalent in
a distorted trigonal-bipyramidal environment: Zn1 (or Zn2) is
coordinated by the two secondary amines N4 (N10) and N22 (or
N16) and an alkoxide O- anion (O31) as equatorial donors and
the tertiary amine N1 (or N13) and one of the phosphoramide
anionic oxygen O28 (or O29) as apical donors. The average
equatorial Zn-O- bond distance of 2.017 Å in 9 is elongated
â (deg)
V (Å3)
Z
D
calcd (g cm-3
)
µ(Cu KR) (cm-1
temp (°C)
)
scan type
ω-2θ
ω-2θ
scan rate
16.0 (9 scans)
16.0 (10 scans)
(deg min-1, in ω)
2θmax (deg)
110.7
120.1
no. of reflns measd
total 4556
unique 4357
(Rint ) 0.135)
direct method
full-matrix
least-squares
2591
total 2896
unique 2778
(Rint ) 0.022)
direct method
full-matrix
least-squares
2553
structure solution
refinement
no. of obsns
(I > 3.00σ(I))
residuals: R, Rw
0.109, 0.111
0.036, 0.051
In the cryptate 8, both zinc(II) atoms are almost equivalent:
Zn1 (or Zn2) is surrounded in a distorted trigonal-bipyramidal
environment by the two secondary amines N4 (or N10) and N22
(or N16) and an alkoxide O- anion (O28) as equatorial donors,
and the tertiary amine N1 (or N13) and another secondary amine
N19 (or N7) as apical donors (see Figure 3) (later, we found
that the apical NH donors N19 and N7 are labile and dissociable,
see below). The average Zn-O- bond distance of 1.93 Å is
much shorter than that of 2.07 Å for the Zn-N equatorial bond
distances and 2.23 Å for the Zn-N apical bond distances. Each
zinc(II) ion lies almost in each basal plane defined by N4, N22,
and alkoxide O28 and N10, N16, and O28, where the total angles
around zinc(II) atoms are 352.7° (N4-Zn1-N22, N22-Zn1-O28,
and O28-Zn1-N4) and 352.6° (N10-Zn2-N16, N16-Zn2-O28,
and O28-Zn2-N10). The apical angles are bent at 159.0(6)°
for N1-Zn1-N19 and 159.3(6)° for N13-Zn2-N7, suggesting
some strain at the apical coordination sites, which is caused by
the rigid ligand structure of the cryptand. The two zinc(II) ions
are separated with a distance of 3.42 Å,7 which we later realized
to be the appropriate distance to accept the two oxygen anions
of phosphate.
The five-coordinate zinc(II) ions surrounded in such a rigid
cryptate appeared to be coordinately almost saturated (or
shielded) and, hence, initially were assumed to be totally
unreactive. HoweVer, we discoVered that 8 reacted with a
phosphomonoester, 4-nitrophenyl phosphate (NPP2-), at physi-
ological pH in aqueous solution to liberate 4-nitrophenolate.
(8) (a) Kimura, E.; Koike, T. Comments Inorg. Chem. 1991, 11, 285-
301. (b) Kimura, E. Progress in Inorganic Chemistry; Karlin, K. D., Ed.;
John Wiley & Sons: New York, 1994; Vol. 41, pp 443-491. (c) Kimura,
E.; Shionoya, M. Transition Metals in Supramolecular Chemistry; Fabbrizzi,
L., Poggi, A., Eds.; Kluwer Academic Publishers: London, 1994; pp 245-
259.
(9) (a) Kimura, E.; Koike, T.; Shiota, T.; Iitaka, Y. Inorg. Chem. 1990,
29, 4621-4629. (b) Kimura, E.; Koike, T.; Shionoya, M.; Shiro, M. Chem.
Lett. 1992, 787-790. (c) Koike, T.; Kimura, E.; Nakamura, I.; Hashimoto,
Y.; Shiro, M. J. Am. Chem. Soc. 1992, 114, 7338-7345. (d) Zhang, X.;
van Eldic, R.; Koike, T.; Kimura, E. Inorg. Chem. 1993, 32, 5749-5755.
(e) Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc. 1994, 116,
8443-8449.
P-O Ester Bond Cleavage of 4-Nitrophenyl Phosphate
(NPP2-) with 8. As an extension to our studies for intrinsic
properties of zinc(II) and metallohydrolase models with mac-
rocyclic polyamine complexes,4,5,8,9 we tested the reactivity of
8 with 4-nitrophenyl phosphate (NPP2-), bis(4-nitrophenyl)
phosphate (BNP-), tris(4-nitrophenyl) phosphate (TNP), and
4-nitrophenyl acetate (NA). All the earlier mononuclear
(10) A similar intramolecular phosphoryl-transfer reaction with mono-
nuclear cobalt(III) pentaammine complex and ethyl 4-nitrophenyl phosphate
was reported: Hendry, P.; Sargeson, A. M. Inorg. Chem. 1990, 29, 92-
97.
(11) We also synthesized a dinuclear copper(II) complex with 7
(analogous to 8) as its triperchlorate salt, which has no activity of the NPP2-
ester bond cleavage under the same condition.
(7) The earlier reported dinuclear zinc(II) complex 13 was not isolated,
and hence, no structural data to be compared with 8 are available.

NoVel Properties of CooperatiVe Dinuclear Zinc(II) Ions
J. Am. Chem. Soc., Vol. 118, No. 13, 1996 3095
Scheme 3
pH g 5.9. At lower pH (5.4 and 4.9), the concentration of
produced NP was determined by dilution of the test solution in
100 mM CAPS buffer (pH 10.4). The second-order dependence
of the rate constant, kNPP (M-1 s-1), on the concentration of 8
and on the total concentration of NPP2- and its monoprotonated
species HNPP- fits to the kinetic eq 6, where VNP is the rate of
NP production promoted by 8. The protonation constant of
Figure 4. Crystal structure of 9 and its coordination bonding scheme
with three perchlorates and two waters omitted for clarity. Thermal
ellipsoids are at the 30% probability level. Bond angles (deg) around
zinc(II) ions are as follows: O28-Zn1-O31, 98.3(2); O28-Zn1-N1,
178.6(2); O28-Zn1-N4, 95.7(2); O28-Zn1-N22, 97.5(2); O31-Zn1-
N1, 83.0(2); O31-Zn1-N4, 127.0(2); O31-Zn1-N22, 110.1(2); N1-Zn1-
N4, 83.1(2); N1-Zn1-N22, 82.4(3); N4-Zn1-N22, 118.3(3); O29-Zn2-
O31, 96.9(2); O29-Zn2-N10, 95.7(2); O29-Zn2-N13, 179.4(2); O29-
Zn2-N16, 97.6(2); O31-Zn2-N10, 124.8(2); O31-Zn2-N13, 83.5(2);
O31-Zn2-N16, 109.5(2); N10-Zn2-N13, 83.6(2); N10-Zn2-N16, 121.6-
(2); N13-Zn2-N16, 82.7(2).
NPP2- () log([HNPP-]/[NPP2-]aH )) was determined to be
+
5.22 ( 0.03 by potentiometric pH titration at 35 °C with I )
0.10 (NaNO3). The maximum kNPP value of (1.52 ( 0.05) ×
10-3 M-1 s-1 was obtained at pH 5.9.
VNP ) kNPP[8][NPP2- + HNPP-]
) kobs[NPP2- + HNPP-]
(6)
(7)
The observed pseudo-first-order rate constant, kobs (s-1) for
the NP production from 4-nitrophenyl phosphate (10 mM) in
the presence of 8 (5 mM) is plotted as a function of pH (see
Figure 5 and eq 7). The background reaction (i.e., hydrolysis
of 4-nitrophenyl phosphate)13 in the absence of 8 was also
determined using the same buffer solution (see dotted line in
Figure 5). The rate vs pH profile shows a bell-shaped
relationship with pK1 of 5.2 and pK2 of 6.3 in the acidic pH
region and a pH-independent reaction with a constant kobs value
of (1.1 ( 0.1) × 10-6 s-1 in the alkaline pH region (pH > 8).
The kinetic pK1 value (5.2) is almost the same as the protonation
constant (Ka ) 105.22) for NPP2- + H+ a HNPP-.
We propose the overall mechanism of the P-O ester bond
cleavage as shown in Scheme 4. First, the reactive form
(NPP2-) of the phosphomonoester approaches 8 and both O-
donors of NPP2- start to associate with suitably separated zinc-
(II) ions of 8 (from sixth coordination site). Simultaneously,
the weakly bound apical NH’s (N7 and N19) start to dissociate.
Then, one of them (N7) attacks the incoming P atom (see 19)
to release 4-nitrophenolate and form a new N-P bond. The
acidic conditions (below pK2) assist the dissociation of one of
the apical NH’s (N19) by protonation (see 20) so as to help the
stronger association of the phosphate substrate, which accounts
for the higher rate at lower pH (down to pH 5.9). We thus
assign the pK2 value of 6.3 to the protonation constant for the
apical NH of the associative reaction intermediate, 19 + H+ a
20. As proposed earlier (see Scheme 3), the protonated N19 in
the final product 9 deprotonates with a higher pK2 (8.50) to
form 17 and 18, indicating the stronger interaction between zinc-
(II) and N19 in the intermediate 19 than that in 9. Further
from 1.93 Å for 8, along with widened Zn1-Zn2 distance of
3.65 Å as of 3.42 Å in 8, which indicates the determining
influence of the phosphoramide bridging the zinc(II) ions. The
apical Zn-O bond distances (2.024 and 2.016 Å) are as short
as those for the equatorial Zn-O- bonds (reflecting the strong
Zn-O-(phosphoramide) interaction). Each zinc(II) ion lies
almost in each basal plane defined by N4, N22, and alkoxide
O31 and N10, N16, and O31, where the total angles around zinc-
(II) atoms are 355.4° (N4-Zn1-N22, N22-Zn1-O31, and O31-
Zn1-N4) and 355.9° (N10-Zn2-N16, N16-Zn2-O31, and O31-
Zn2-N10). The apical angles are almost linear at 178.6(2)° for
N1-Zn1-O28 and 179.4(2)° for N13-Zn2-O29, suggesting
smaller strain than that shown in 8, which is probably due to
the more flexible ligand structue of the phosphoryl cryptand.
Properties of 9 and the Phosphoryl-Transfer Reaction
Mechanism from 8 to 9. The cryptate 9 has two dissociable
protons at the phosphoryl group and uncoordinated secondary
ammonium. The deprotonation constants, pK1 of 3.0 ( 0.1 (9
a 16 + H+) and pK2 of 8.50 ( 0.03 (16 a 17 (or 18) + H+),
were found by potentiometric pH titration with 9 (1 mM) at 35
°C with I ) 0.10 (NaNO3). We assigned the pK1 and pK2 values
of 9 as shown in Scheme 3. pK1 is assigned for 9 a 16, since
it is similar to the pKa value of 3.3 for hydrogen amidophosphate
(H2NPO3H2 a H2NPO3H- + H+).12 for pK2, see the following
discussion.
The reactivity of the cryptate 8 in NPP2- ester bond cleavage
was evaluated at various pH (9.5-4.9, 20 mM buffer) in
aqueous solution at 35 °C with I ) 0.10 (NaNO3). The reaction
(i.e., the NP production from 4-nitrophenyl phosphate) was
followed by the appearance of 4-nitrophenolate at 400 nm at
(13) The pH-dependent NPP2- hydrolysis at 39 °C with I ) 1.0 was
reported: Kirby, A. J.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 3209-
3216.
(12) Smith, R. M.; Martell, A. E. Critical Stability Constants; Plenum
Press: New York, 1976; Vol. 4, p 132.

3096 J. Am. Chem. Soc., Vol. 118, No. 13, 1996
Koike et al.
Scheme 6
Scheme 7
Figure 5. Rate vs pH profile for the pseudo-first-order rate constants
kobs (s-1) of the P-O ester bond cleavage of NPP2- (10 mM) in the
presence of 8 (5 mM) at 35 °C. The data in a dotted line are the first-
order rate constants of background reaction (NPP2- hydrolysis) in the
same buffer solution.
Scheme 4
As another reference experiment, the ATP4- hydrolysis with
the metal-free cryptand 7 (10 mM) was investigated at pH 7.4
(50 mM HEPES buffer) and 35 °C with I ) 0.5 (NaNO3) by
31P NMR, where 7 is considered to be in the mixture of tri-
and tetra-protonated forms on the basis of its pKa values. The
ATP4- hydrolysis was promoted by 7 with the kATP value of
(2.5 ( 0.3) × 10-5 M-1 s-1, which incidentally is almost the
same as that for the phosphoryl-transfer reaction 8 + ATP4-
f 16 + ADP3- 15
However, unlike the reaction of the zinc(II)
.
cryptate 8, the products detected were inorganic phosphate and
equimolar ADP3- (see Scheme 6).
An analogous phosphoryl-transfer reaction was earlier re-
ported to occur between ATP4- and macrocyclic polyamines
(e.g., 22, see Scheme 7).16 The tetraprotonated macrocyclic
hexaamine 22 at neutral pH is the reactive species and
hydrolyzes ATP4- (to ADP3-) with second-order kinetics. The
established mechanism involves a phosphoryl-transferred in-
termediate 23, which is only transiently detected and almost
immediately releases inorganic phosphate and goes back to 22.17
One can extrapolate the second-order rate constant (kATP) of 6
× 10-4 M-1 s-1 at 35 °C using the given thermodynamic
activation parameters at 60 °C and pH 7.6.16b
Scheme 5
By comparing the reactions of 7 and 8 with NPP2- and
ATP4-, it is evident that both zinc(II) ions play an additional
role in stabilizing the phosphoryl-transferred species, while in
the metal-free system 7, the phosphoryl-transferred intermediate
may be unstable like 23.17 Such a function of the cooperative
dinuclear zinc(II) ions may be important in phosphoryl-transfer
reactions in dinuclear metallophosphatases.1,18
Selective Phosphomonoester Dianion Recognition by 8 and
the Relevance to Dinuclear Metallophosphatase Mechanism.
Given the finding of the selective interaction of dianionic
phosphomonoester NPP2- (or more anionic ATP4-) with 8 in
the phosphoryl-transfer mechanism and the isolation of the stable
phosphoramide bridging between each zinc(II) in 9, we sus-
acidification, however, converts the reactive substrate NPP2-
into an unreactive, protonated species HNPP- (with pK1 value
of 5.2), which has weaker affinity for the cryptate 8,14 resulting
in the slower reaction.
Reaction of 8 with ATP4-. In order to see if a similar
phosphoryl-transfer reaction from adenosine triphosphate (ATP4-)
(10 mM) to 8 (10 mM) occurs, we followed the reaction by 31
P
NMR at pH 7.4 (100 mM HEPES buffer) and 35 °C with I )
0.5 (NaNO3). Indeed, the reaction took place. After 1 week,
the products were identified as the phosphoryl-transferred
cryptate 16 and an equimolar adenosine diphosphate (ADP3-
)
in ca. 20% yield, along with unreacting ATP4- and 8, but no
other products such as inorganic phosphate or adenosine
monophosphate (AMP2-) were detected. We thus propose a
reaction mechanism via an ATP4--bound intermediate 21 as
shown in Scheme 5, which is similar to the NPP2- ester bond
cleavage with 8. The second-order rate constant kATP for our
phosphoryl-transfer reaction (i.e., ATP4- + 8 f ADP3- + 16)
is (3.0 ( 0.3) × 10-5 M-1 s-1 at 35 °C.
(15) Note that 7, however, did not react with NPP2- as 8 did; see above
discussion.
(16) (a) Hosseini, M. W.; Lehn, J.-M.; Mertes, M. P. HelV. Chim. Acta
1983, 66, 2454-2466. (b) Hosseini, M. W.; Lehn, J.-M.; Maggiora, L.;
Mertes, K. B.; Mertes, M. P. J. Am. Chem. Soc. 1987, 109, 537-544. (c)
Hosseini, M. W.; Lehn, J.-M.; Jones, K. C.; Plute, K. E.; Mertes, K. B.;
Mertes, M. P. J. Am. Chem. Soc. 1989, 111, 6330-6335. (d) Mertes, M.
P.; Mertes, K. B. Acc. Chem. Res. 1990, 23, 413-418.
(17) Although we failed to see such an intermediate (a N-phosphorylated
cryptand, an analogue of 23) in our reaction of 7 with ATP4-, we postulate
a similar mechanism as shown in Scheme 6. The two zinc(II) ions in 9
could be removed with a 10 times excess amount of ethylenediaminetet-
raacetate at pH 7 and ca. 60 °C in H2O. Then, the P-N bond of the
N-phosphorylated cryptand was immediately hydrolyzed to yield the free
cryptand 7 and inorganic phosphate, as was determined by 1H and 31P NMR.
(14) Earlier, we reported that the 1,5,9-triazacyclododecane zinc(II)
complex has a larger affinity for dianionic NPP2- (K ) [ZnL-NPP2-]/
[ZnL][NPP2-] ) 103.1 M-1 at 25 °C) than for monoanionic BNP- (K <
100.5 M-1) (see ref 4). This fact is similarly related to the smaller affinity
of zinc(II) cryptate 8 for monoanionic HNPP-.

NoVel Properties of CooperatiVe Dinuclear Zinc(II) Ions
J. Am. Chem. Soc., Vol. 118, No. 13, 1996 3097
Conclusions
Scheme 8
In an attempt to synthesize a bis(macrocyclic tetraamine)
bridged with a 2-propyl alcohol 4, we incidentally isolated a
cryptand 7 in a good yield, which eventually gave a dinuclear
zinc(II) cryptate 8 having novel functions that may be relevant
to the properties of dinuclear metal-containing phosphatases.
The structure of 8 was characterized by potentiometric pH-
titration and X-ray crystal analysis. In the solid state, both zinc-
(II) ions are almost equivalent and have distorted trigonal-
bipyramidal structures; an alkoxide binds both zinc(II) ions as
a shared equatorial donor and two secondary amines (NH) as
apical donors to each zinc(II). In aqueous solution, as a
substrate phosphomonoester dianion (4-nitrophenyl phosphate)
approaches in order to bridge both zinc(II) ions, the apical NH’s
dissociate and one of them attacks the incoming P to perform
a phosphoryl-transfer reaction (i.e., a phosphoryl group migrates
from 4-nitrophenol to amine), yielding 4-nitrophenol and a new
phosphoramide-containing cryptate 16. The product structure
9 (monoprotonated species of 16) was fully characterized by
potentiometric pH-titration and X-ray crystal analysis. The
newly formed phosphoramide bridges both zinc(II) ions in the
new cryptate. The phosphoryl transfer by 8 stops at the stage
of 16, which is extremely stable, and hence 8 is not a catalyst
for phosphomonoester. The zinc(II) complex 8 cleaves ATP4-
to yield ADP3- and 16, while the metal-free cryptand 7 (as
multiprotonated species) promotes hydrolysis of ATP4- to
produce ADP3- and inorganic phosphate. The present new
findings may serve to rationalize the essence of dinuclear
metallophosphatases such as selective recognition of phospho-
monoesters, inhibition by inorganic phosphate, or stabilization
of the phosphoryl-transferred intermediates. Suitable modifica-
tion of the present serendipitous cryptand may lead to real and
practical artificial phosphomonoesterase molecules.
pected that inorganic phosphate dianion, HOPO32-, might
reversibly or irreversibly bind to 8 to inhibit the phorphoryl-
transfer reaction 8 f 9. Indeed, in the presence of 10 mM
2-
HOPO3 at pH 8.2 (20 mM TAPS) and 35 °C, the pseudo-
first-order rate constant (kobs ) 1.1 × 10-6 s-1) with 5 mM of
8 is reduced to (1.9 ( 0.1) × 10-7 s-1, supporting the notion
of competitive inhibition by inorganic phosphate. However,
30 mM SCN-, which has strong affinity to mononuclear zinc-
(II) (e.g., [ZnII-cyclen-SCN-]/[ZnII-cyclen][SCN-] ) 102.2
M-1 at 25 °C),9e did not interfere in the reaction 8 f 9 under
the same conditions. Accordingly, we may generalize that the
selective association of phosphate dianion is feasible with the
dinuclear zinc(II), provided that the distance (3-4 Å) is within
a range that allows the cooperative action for the phosphate
bridging and that some vacancy on the two zinc ions is available
for the access of the phosphate. In this regard, the role of the
strong alkoxide bridge for the simultaneous coordination to two
zinc(II) should be critical (see Scheme 8). This brings both
zinc(II) atoms close enough and at the same time behaves as
strong equatorial donors for both zinc(II) ions. The phosphate
would then access from the apical sites. In most of the dinuclear
phosphatases,18 the hydroxo (HO-), alkoxide (RO-), or car-
boxylate (COO-) ligands similarly serve as a bridging ligand
between the two metal ions.
In addition, if a potential nucleophile (X in Scheme 8) is
available in the vicinity, the dinuclear ZnII-phosphate complex
would easily be subjected to the nucleophile attack (due to the
neutralization of the anionic electrophile) that leads to phos-
phoryl-transferred species (e.g., 19 f 16). An analogous
phosphoryl-transfer reaction using a serine OH nucleophile and
a dinuclear zinc(II) complex is well-known in phosphomonoester
hydrolysis by alkaline phosphatases.1
Experimental Section
General Information. All reagents were of analytical reagent grade
(purity > 99%) and were used without further purification. All aqueous
solutions were prepared using distilled water. An aqueous solution of
0.10 M NaOH was made by dilution of 10 M NaOH (Merck 6495).
The 10 M NaOH solution was kept in a refrigerator below 5 °C, where
Na2CO3 is less soluble (< 1 %), and taken out before raising the solution
temperature. The Good’s buffers (Dojindo, pKa at 20 °C) were
commercially available and were used without further purification:
CAPS (3-(cyclohexylamino)propanesulfonic acid, pKa ) 10.4), CAPSO
(3-(cyclohexylamino)-2-hydroxypropanesulfonic acid, 10.0), CHES (2-
(cyclohexylamino)ethanesulfonic acid, 9.5), TAPS 3-[tris[(hydroxy-
methyl)methyl]amino]propanesulfonic acid, 8.4), EPPS 3-[4-(2-
hydroxyethyl)-1-piperazinyl]propanesulfonic acid, 8.0), HEPES 2-[4-
(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 7.6), MOPS (3-
morpholinopropanesulfonic acid, 7.2), and MES (2-morpholino-
ethanesulfonic acid, 6.2). The sodium salts of 5′-ATP‚Na2 and 5′-
ADP‚Na were obtained from Kohjin Co.
Recently, the X-ray crystal structure of the mammalian protein
phosphatase-1 (PP-1) was reported,19 which contains two metal
ions that are brought close to each other (ca. 3.3 Å) by a
carboxylate oxygen anion and water (or OH-). Each of the
metal ions in PP-1 is coordinated by five ligands (such as water,
imidazol, and carboxylate). The presence of two 5-coordinated
metals at the active site and a histidine (or ZnII-OH-) nucleophile
near the substrate phosphomonoester suggests structural as well
as mechanistic similarities between PP-1 and 8. In the proposed
mechanism for PP-1, the substrate phosphomonoester could
provide two O- donors to the metal ions and then the ester
bond cleavage reaction could proceed indirectly via a covalent
phosphorylhistidine intermediate or directly with the metal-
activated H2O.
IR spectra were recorded on a Shimadzu FTIR-4200 spectropho-
tometer. 1H (400 MHz), 13C (100 MHz), and 31P (162 MHz) NMR
spectra at 35.0 ( 0.5 °C were recorded on a JEOL R-400 spectrometer.
3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt in aqueous
solution and tetramethylsilane in organic solution were used as internal
1
references for H and 13C NMR measurements. An aqueous solution
of 80% phosphoric acid was used as an external reference for 31P NMR
measurement. Silica gel column chromatography was carried out on
Fuji Silysia Chemical FL-100D.
(18) (a) Karlin, K. D. Science 1993, 261, 701-708. (b) Gani, D.; Wilkie,
J. Chem. Soc. ReV. 1995, 55-63. (c) Purple acid phosphtase: Stra¨ter, N.;
Klabunde, T.; Tucker, P.; Witzel, H.; Krebs, B. Science 1995, 268, 1489-
1492. (d) Phospholipase, C: Hough, E.; Hansen, L. K.; Birknes, B.; Jynge,
K.; Hansen, S.; Hordvik, A.; Little, C.; Dodson, E.; Derewenda, Z. Nature
1989, 338, 357-360. (e) P1 nuclease: Volbeda, A.; Lahm, A.; Sakiyama,
F.; Suck, D. EMBO J. 1991, 10, 1607-1618. (f) Inositol monophos-
phatase: Bone, R.; Frank, L.; Springer, J. P.; Atack, J. R. Biochemistry
1994, 33, 9468-9476.
Synthesis of 26-Hydroxy-1,4,7,10,13,16,19,22-octaazabicyclo-
[11.11.3]heptacosane Octahydrochloric Acid Salt 7‚8HCl. Thionyl
chloride (9.7 mL) was added dropwise to an EtOH solution (60 mL)
of 2-hydroxypropylenediamine-N,N,N′,N′-tetraacetic acid (10.3 g, 32
mmol) over 1 h at 0 °C. After the solution was heated to reflux for 1
day, the solvent was evaporated. The oily residue was dissolved in
wate (70 mL) saturated with K2CO3 at 0 °C, and then the solution was
(19) Goldberg, J.; Huang, H.; Kwon, Y.; Greengard, P.; Nairn, A. C.;
Kuriyan, J. Nature 1995, 376, 745-753.

3098 J. Am. Chem. Soc., Vol. 118, No. 13, 1996
Koike et al.
extracted with CH2Cl2 (100 mL × 4). After the combined organic
layers were dried over anhydrous Na2SO4, the solvent was evaporated.
The oily residue was purified by silica gel column chromatography
(eluent: CH2Cl2/EtOH ) 20:1) to obtain [2-oxo-6-(aminomethyl)-
morpholyl]-N,N′,N′-triacetic acid triethyl ester (1) as a yellowish oil
(7.2 g, 58% yield). TLC (Merck Art. 5554, eluent:hexane/AcEt )
1:1): Rf ) 0.33. IR (neat): 3463, 2984, 2940, 2909, 2874, 1740, 1449,
1416, 1372, 1345, 1194, 1098, 1069, 1030, 988, 920, 864, 733, 586
4-nitrophenyl phosphate disodium salts (104 mg, 0.28 mmol) was stirred
at 60 °C for 2 days. The solution pH was adjusted to 3 with 0.1 M
HClO4(aq). After the solution was washed with CHCl3 (30 mL × 5),
the solvent was evaporated. The residue was crystallized from H2O to
obtain 9‚(ClO4)3‚2H2O as colorless prisms (163 mg, 69% yield). IR
(KBr pellet): 3472, 3142, 3025, 1557, 1464, 1385, 1356, 1308, 1287,
1267, 1225, 1190, 1171, 1144, 1120, 1090, 1051, 1017, 990, 949, 918,
864, 818, 802, 691, 627 cm-1 1H NMR (D2O, pD 10): δ 2.44 (2H,
.
cm-1
.
1H NMR (CDCl3): δ 1.27 (6H, t, J ) 7.1 Hz, CH3), 1.29 (3H,
dd, J ) 10.9 and 12.7 Hz, CHCC), 2.55-2.74 (8H, m, NCH), 2.76
(2H, dd, J ) 2.2 and 12.7 Hz, CHCC), 2.80-3.18 (20H, m, NCH),
3.23-3.32 (2H, m, NCH), 3.43-3.54 (2H, m, NCH), 3.87 (1H, tt, J )
2.2 and 10.9 Hz, CCHC). 13C NMR (D2O, pD 10): δ 45.1, 48.0, 48.1,
49.7, 51.4, 52.4, 57.1, 57.4, 60.0, 66.9. 31P NMR (D2O): 12.7 at pD
10.0, 14.2 at pD 6.3, 14.3 at pD 2.9. Anal. (C19H48N8Cl3O18PZn2) C,
H, N: calcd, 24.2, 5.1, 11.9; found, 24.0, 5.1, 11.8.
t, J ) 7.1 Hz, CH3), 2.93 (1H, dd, J ) 7.7 and 12.8 Hz, COOCCHN),
3.02 (1H, dd, J ) 6.7 and 14.2 Hz, COOCCHN), 3.07 (1H, dd, J )
5.9 and 12.8 Hz, COOCCHN), 3.10 (1H, dd, J ) 4.9 and 14.2 Hz,
COOCCHN), 3.31 and 3.36 (2H, ABq, J ) 17.0 Hz, NCHCO), 3.42
(1H, d, J ) 16.3 Hz, NCHCO), 3.55 and 3.62 (4H, ABq, J ) 17.6 Hz,
NCHCO), 3.61 (1H, d, J ) 16.3 Hz, NCHCO), 4.16 (4H, q, J ) 7.1
Hz, OCH2), 4.19 (2H, q, J ) 7.1 Hz, OCH2), 4.61 (1H, m, CCHC).
A solution of 1 (7.0 g, 18 mmol) and diethylenetriamine (2) (3.7 g,
36 mmol) in MeOH (400 mL) was heated to reflux for 5 days. After
evaporation of the solvent, the residue was purified by silica gel column
chromatography (eluent: CH2Cl2/MeOH/28% NH3(aq) ) 5:2:0.1)
followed by crystallization from CH3CN to obtain 26-hydroxy-3,11,-
15,23-tetraoxo-1,4,7,10,13,16,19,22-octaazabicyclo[11.11.3]-
heptacosane (6) as colorless crystals (4.1 g, 50% yield, mp 148-149
°C). TLC (Merck Art. 5554, eluent: CH2Cl2/MeOH/28% NH3(aq) )
5:2:0.5): Rf ) 0.55. IR (KBr pellet): 3463, 3296, 3088, 2940, 2830,
2368, 1649, 1551, 1485, 1472, 1465, 1443, 1343, 1269, 1167, 1130,
Potentiometric pH Titrations. The pH meter (Horiba F-16) and
electrode system (a pH-glass electrode and a double-junction reference
electrode) was calibrated daily as follows: An aqueous solution (50.0
mL) containing 4.00 mM of HCl and 96.0 mM of NaNO3 (I ) 0.10)
was prepared under an argon atmosphere (99.999%) at 35.0 ( 0.1 °C
and then the first pH value (pH1) was read. After 4.0 mL of 0.10 M
NaOH was added to the acidic solution, the second pH value (pH2)
was read. The theoretical pH values corresponding to pH1 and pH2
are calculated to be pH1′ ) 2.483 and pH2′ ) 11.128, respectively,
using Kw () aH aOH ) ) 10-13.68, Kw′ () [H+][OH-]) ) 10-13.48, and
+
-
+
+
+
+
fH () aH /[H ]) ) 0.823. The correct pH values (-log aH ) can be
obtained using the following equations: a ) (pH2′ - pH1′)/(pH2 -
pH1); b ) pH2′ - apH2; pH ) a(pH-meter reading) + b.
1100, 1055, 978, 872, 596 cm-1 1H NMR (D2O, pD ) 5): δ 2.43
.
(2H, dd, J ) 10.5 and 14.1 Hz, NCHCC), 2.66 (2H, dd, J ) 2.2 and
14.1 Hz, NCHCC), 3.29-3.56 (12H, m, CH), 3.45 and 3.50 (8H, ABq,
J ) 17.1 Hz, NCHCO) 3.79 (1H, tt, J ) 2.2 and 10.5 Hz, NCCHC),
3.80-3.89 (4H, m, CH). 13C NMR (D2O, pD ) 5): δ 38.6, 50.9,
63.8, 64.2, 72.6, 178.3.
The potentiometric pH titrations of 7‚8HCl (1 mM) in the presence
and the absence of 2 equiv of zinc(II) were carried out at 35.0 ( 0.1
°C with I ) 0.10 (NaNO3) under an argon atmosphere, and two
independent titrations were made. The ligand protonation constants,
Ka′ ) [HnL]/[Hn-1L][H+] (M-1), deprotonation constant K′(Zn2L) )
[Zn2L][H+]/[Zn2HL] (M), and zinc(II) complexation constant, K(Zn2-
HL) ) [Zn2HL]/[ZnII]2[HL] (M-2), were originally determined using
a pH-titration program BEST.20 All σ pH fit values defined in the
program are smaller than 0.005. The mixed constants Ka ) [HnL]/
To a suspended solution of the tetraoxomacrocycle 6 (1.66 g, 3.6
mmol) in dry THF (33 mL) was added slowly a THF solution (75 mL)
of 1 M BH3‚THF at 0 °C. The mixture was stirred at room temperature
for 3 h and heated at 65 °C for 7 days. After decomposition of the
various borane complexes with water at 0 °C, the organic solvent was
evaporated. The residue was dissolved in 6 M HCl(aq) (80 mL), and
the solution was heated at 70 °C for 3 h. The mixture was cooled to
room temperature and washed with CH2Cl2 (30 mL × 2). After the
aqueous solution was concentrated to 10 mL, it was purified on an
anion exchange column of Amberlite IRA-400 with water. The
obtained acid-free residue was purified by silica gel column chroma-
tography (eluent: CH2Cl2/MeOH/28% NH3(aq) ) 5:2:0.3) followed
by crystallization from 6 M HCl(aq) to obtain 26-hydroxy-1,4,7,10,-
13,16,19,22-octaazabicyclo[11.11.3]heptacosane, 7‚8HCl‚2.5H2O, as
colorless prisms in 33% yield (269 °C dec). TLC (Merck Art. 5567,
eluent: CH2Cl2/MeOH/28% NH3(aq) ) 2:2:1): Rf ) 0.30. IR (KBr
pellet): 3440, 2988, 2780, 2689, 2484, 1617, 1574, 1472, 1375, 1329,
[Hn-1L]aH (M-1) and K(Zn2L) ) [Zn2L]aH /[Zn2HL] (M) are derived
+
+
from Ka′ and K′(Zn2L) using [H+] ) aH /fH
.
+
+
Crystallographic Study. Colorless prismatic crystals, 0.25 × 0.20
× 0.20 mm of 8‚(ClO4)3 and 0.30 × 0.20 × 0.10 mm of 9‚(ClO4)3‚
2H2O, were used for data collection. The lattice parameters and
intensity data were measured on a Rigaku AFC7R diffractometer with
graphite monochromated Cu KR radiation and a 12-kW rotating anode
generator at 20.0 and 23.0 °C, respectively. The structures were solved
by direct methods (SHELXS86) and expanded using Fourier techniques
(DIRDIF92). All calculations were performed using the teXsan crystal
structure analysis package developed by Molecular Structure Corp.
(1985 and 1992). 8‚(ClO4)3: Zn, N, Cl, and O atoms were refined
anisotropically, while C and H atoms were refined isotropically. The
final cycle of full-matrix least-squares refinement was based on 2591
observed reflections (I > 3.00σ(I)) and 337 variable parameters and
converged (largest parameter was 0.09 times its esd) with R () ∑||Fo|
1074, 1005, 987, 965, 754 cm-1 1H NMR (D2O, pD 2): δ 2.67 (2H,
.
dd, J ) 10.5 and 15.6 Hz, NCHCC), 2.83 (2H, dd, J ) 1.8 and 15.6
Hz, NCHCC), 2.98-3.12 (8H, m, NCH), 3.26-3.43 (8H, m, NCH),
3.48-3.64 (16H, m, NCH), 3.80 (1H, tt, 1.8 and 10.5 Hz, CCHC). 13
C
NMR (D2O, pD 2): δ 46.4, 46.8, 48.3, 55.9, 60.9, 78.1. Anal.
(C19H57N8O3.5Cl8) C, H, N: calcd, 31.0, 7.8, 15.2; found, 31.0, 7.7,
14.9.
2
- |Fc||/∑|Fo|) ) 0.109 and Rw () (∑w(|Fo| - |Fc|)2/∑wFo )0.5) ) 0.111.
-
The carbon atoms and the oxygen atom of one of ClO4 ions are
disordered at two locations. All the hydrogen atoms were not located.
We attempted to determine the X-ray crystal structure of 8 at a lower
temperature using liquid nitrogen, but it was unsuccessful due to the
formation of cracks in the crystal. 9‚(ClO4)3‚2H2O: The nonhydrogen
atoms were refined anisotropically, and the hydrogen atoms were
included but not refined. The final cycle was based on 2553 observed
reflections (I > 3.00σ(I)) and 459 variable parameters and converged
Synthesis of Dinuclear Zinc(II) Cryptate 8‚(ClO4)3. The octahy-
drochloric acid salt 7‚8HCl‚2.5H2O (1.2 g, 1.6 mmol) was passed
through an anion exchange column (Amberlite IRA-400) with water
to obtain acid-free ligand 7 as a colorless oil. After dissolution of 7 in
EtOH (60 mL), Zn(ClO4)2‚6H2O (1.4 g, 3.7 mmol) was added. The
solution was stirred at 55 °C for 14 h. After the solvent was evaporated,
the residue was crystallized from water to obtain colorless prisms at
triperchlorate salts 8‚(ClO4)3 in 89% yield. IR (KBr pellet): 3430,
3256, 2922, 2876, 1468, 1381, 1281, 1109, 1092, 1019, 980, 890, 845,
(largest parameter was 0.04 times its esd) with R ) 0.036 and Rw
0.051.
)
Kinetic Measurements. The P-O ester bond cleavage (i.e.,
4-nitrophenol and 4-nitrophenolate release reaction) rates of 4-nitro-
phenyl phosphate(2-) (NPP2-) were measured by an initial slope
method (following the increase in 400-nm absorption of released
4-nitrophenolate in aqueous solution at 35.0 ( 0.5 °C. All absorbance
833, 693, 627 cm-1 1H NMR (D2O, pD 7): δ 2.33-3.68 (36H, m,
.
NCH), 3.85-3.96 (1H, br, CCHC). 13C NMR (D2O, pD 7): δ 43.5,
43.9, 44.7, 45.5, 45.7, 46.0, 46.2, 46.6, 47.0, 47.4, 47.8, 47.9, 50.5,
51.0, 53.3, 53.8, 54.1, 58.9, 64.4. Anal. (C19H43N8Cl3O13Zn2) C, H,
N: calcd, 27.5, 5.2, 13.5; found, 27.1, 5.2, 13.3.
Synthesis of Dinuclear Zinc(II) Phosphocryptate 9‚(ClO4)3‚2H2O.
An aqueous solution (25 mL) of 8‚(ClO4)3 (207 mg, 0.25 mmol) and
(20) Martell, A. E.; Motekaitis, R. J. Determination and Use of Stability
Constants, 2nd ed.; VCH: New York, 1992.

NoVel Properties of CooperatiVe Dinuclear Zinc(II) Ions
J. Am. Chem. Soc., Vol. 118, No. 13, 1996 3099
measurements were made on a Hitachi U-3500 spectrophotometer
equipped with an electric temperature controller SPR-10. Buffer (20
mM) solutions (CAPSO, pH 9.5; CHES, pH 9.1; TAPS, pH 8.2, EPPS,
pH 7.8; HEPES, pH 7.4; MOPS, pH 7.0; MES, pH 6.7, 6.3, and 5.9;
acetate, pH 5.4 and 4.9) were used, and the ionic strength was adjusted
to 0.10 with NaNO3. For the initial rate determination, the following
typical procedure was employed: NPP2- (10, 5.0, and 2.5 mM) and 8
(5.0, 2.5, and 1.0 mM) were mixed in the buffer solution, and the visible
absorption increase was recorded immediately and then followed
generally until ca. 0.2% formation of 4-nitrophenolate (and 4-nitro-
phenol), were log ꢀ values for 4-nitrophenolate were 4.27 (pH 9.5),
4.26 (pH 9.1), 4.24 (pH 8.2), 4.21 (pH 7.8), 4.13 (pH 7.4), 4.00 (pH
7.0), 3.83 (pH 6.7), 3.53 (pH 6.3), and 3.08 (pH 5.9) at 400 nm. The
4-nitrophenol concentration at pH 5.4 and 4.9 was determined by
dilution of the test solution in 100 mM CAPS buffer (pH 10.4), where
log ꢀ value for 4-nitrophenolate is 4.27. The observed first-order rate
constant kobs (s-1) for the P-O ester bond cleavage reaction was
calculated from the decay slop (4-nitrophenol and 4-nitrophenolate
amide derivative 9. The NMR measurement was carried out until ca.
20% P-O-P bond cleavage by the non-decoupling method at 35.0 (
0.5 °C. The calculated standard deviation for the observed second-
order rates was within 10%. The control reactions for ATP hydrolysis
in the absence of 8 were performed with the same buffer solution. The
ATP (10 mM) hydrolysis reaction with ligand 7 (10 mM) was
determined under the same conditions apart from buffer (50 mM
HEPES, pH 7.4).
Kinetics procedures for reaction of 8 (5.0 and 10 mM) with
4-nitrophenyl acetate,5b bis(4-nitrophenyl) phosphate,5c and tris(4-
nitrophenyl) phosphate4 are the same as previously reported with
macrocyclic polyamine zinc(II) complexes.
Acknowledgment. We are thankful to the Ministry of
Education, Science and Culture in Japan for financial support
by a Grant-in-Aid for Scientific Research (B) (No. 07458144)
for E.K. and by a Grant-in-Aid for Scientific Research (C) (No.
07807206) for T.K. T.K. thanks the Ciba-Geigy Foundation
(Japan) for the Promotion of Science for financial support. An
NMR instrument in the Research Center for Molecular Medicine
(RCMM) of Hiroshima University was used.
release rate/[NPP2-]). The value of kobs(1 + KaaH )/[Zn2L] gave the
+
second-orde rate constant kNPP (M-1 s-1) for the P-O ester bond
cleavage reaction. Ka is the protonation constant of NPP2- (log Ka )
log([HNPP-]/[[NPP2-]aH ) ) 5.22 ( 0.03 at 35 °C with I ) 0.10
+
(NaNO3), where HNPP- is the monoprotonated species of NPP2-. The
products (i.e., phosphoramide derivative 9 and 4-nitrophenol) were
identified by 31P and 1H NMR. The control reaction for NPP2-
hydrolysis in the absence of 8 was performed with the same buffer
solutions. Na2HPO4 and NaSCN were used for anion inhibition study.
The terminal P-O-P bond cleavage (i.e., the phosphoryl-transfer
reaction 8 f 9) rates of ATP4- (10 mM) with 8 (10 mM) were
determined in pH 7.4 HEPES buffer (100 mM) with I ) 0.5 (NaNO3)
by the time-dependent change in the integrals from the resolved 31P
NMR signals of ATP4-, ADP3-, inorganic phosphate, and phorphor-
Supporting Information Available: Tables of crystal-
lographic parameters, atomic coordinates, equivalent isotropic
temperature factors, anisotropic temperature factors, bond
lengths, and bond angles in CIF format for 8‚(ClO4)3 and
9‚(ClO4)3‚2H2O. Ordering information is given on any current
masthead page.
JA953413M