Interaction of urea with a hydroxide-bridged dinuclear nickel center: An alternative model for the mechanism of urease

Article abstract of DOI:10.1021/ja000202v

A hydroxide-bridged dinuclear nickel complex with a urea molecule linking the two metal ions through its carbonyl oxygen atom has been prepared as a model for the metalloenzyme urease. This complex, [Ni₂(μOH)(μ-urea)(bdptz)(urea)(CH₃CN)](ClO₄)₃, where bdptz is the dinucleating ligand 1,4-bis(2,2'-dipyridylmethyl)phthalazine, effects the hydrolysis of urea upon heating in a two-step reaction. In the first step, a molecule of ammonia is eliminated from urea with concomitant production of cyanate, the first-order rate constant in acetonitrile being (7.7 ± 0.5) x 10^-4 h^-1. This reaction is at least 500 times faster than the spontaneous decomposition of urea under the same conditions. When the cyanate-containing product is further heated in the presence of water, the cyanate is hydrolyzed with a second-order rate constant of (9.5 ± 1) x 10^-4 M^-1 h^-1. Reaction of [Ni₂(μ-OH)(μ-urea)(bdptz)(urea)(CH₃CN)](ClO₄)₃ in 50% aqueous acetonitrile afforded ammonia with no appreciable buildup of the cyanate-containing species. A possible analogue of the cyanate-containing product, [Ni₂(μ-OH)(μ-H₂O)(bdptz)(μ-OCN)]₂(OTs)₄, was independently synthesized and structurally characterized. These results establish the precedence for hydrolysis of urea via a cyanate intermediate as an alternative mechanism for the urease-catalyzed hydrolysis of urea.

Full text of DOI:10.1021/ja000202v

9172
J. Am. Chem. Soc. 2000, 122, 9172-9177
Interaction of Urea with a Hydroxide-Bridged Dinuclear Nickel
Center: An Alternative Model for the Mechanism of Urease
Amy M. Barrios and Stephen J. Lippard*
Contribution from the Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139
ReceiVed January 18, 2000
Abstract: A hydroxide-bridged dinuclear nickel complex with a urea molecule linking the two metal ions
through its carbonyl oxygen atom has been prepared as a model for the metalloenzyme urease. This complex,
[Ni2(µ-OH)(µ-urea)(bdptz)(urea)(CH3CN)](ClO4)3, where bdptz is the dinucleating ligand 1,4-bis(2,2′-
dipyridylmethyl)phthalazine, effects the hydrolysis of urea upon heating in a two-step reaction. In the first
step, a molecule of ammonia is eliminated from urea with concomitant production of cyanate, the first-order
rate constant in acetonitrile being (7.7 ( 0.5) × 10-4 h-1. This reaction is at least 500 times faster than the
spontaneous decomposition of urea under the same conditions. When the cyanate-containing product is further
heated in the presence of water, the cyanate is hydrolyzed with a second-order rate constant of (9.5 ( 1) ×
10-4 M-1 h-1. Reaction of [Ni2(µ-OH)(µ-urea)(bdptz)(urea)(CH3CN)](ClO4)3 in 50% aqueous acetonitrile
afforded ammonia with no appreciable buildup of the cyanate-containing species. A possible analogue of the
cyanate-containing product, [Ni2(µ-OH)(µ-H2O)(bdptz)(µ-OCN)]2(OTs)4, was independently synthesized and
structurally characterized. These results establish the precedence for hydrolysis of urea via a cyanate intermediate
as an alternative mechanism for the urease-catalyzed hydrolysis of urea.
Introduction
The metalloenzyme urease holds a distinguished place in the
history of enzymology.1 First crystallized in 1926,2 the nickel
dependence of urease was not recognized until nearly 50 years
later.3 Urease is the only metallohydrolase that utilizes nickel,
and a detailed picture of its active site has emerged over the
past several years through X-ray crystallographic studies of the
enzyme isolated from different sources.4-8 A schematic view
of the dinickel center in urease from Klebsiella aerogenes is
shown in Figure 1. Each nickel ion is coordinated to two
histidine residues from the protein, and a carbamylated lysine
residue bridges the two metal ions. The second nickel ion is
additionally ligated by an aspartate residue. Two terminally
coordinated water molecules and one bridging water molecule
or hydroxide ion complete the coordination spheres of the metal
ions, resulting in a distorted square pyramidal environment for
Ni(1) and a pseudooctahedral ligand environment for Ni(2).
The reaction catalyzed by the dinickel site in urease is the
hydrolysis of urea to form ammonia and carbon dioxide.9 The
hydrolysis of urea is not a trivial task; the uncatalyzed reaction
Figure 1. Schematic view of the active site of urease from K.
aerogenes.4
has never been observed.10 In aqueous solutions, urea spontane-
ously eliminates ammonia to form cyanic acid, in a reaction
that is pH independent from pH 2 to 12, with a half-life of 3.6
years at 38 °C.10 The stability of urea is attributed to its
resonance energy, which has been estimated at 30-40 kcal/
mol.11 Urease converts urea to products at a rate at least 1014
times faster than the spontaneous decomposition rate.10 The
proposed mechanism of urea hydrolysis at the active site of the
enzyme, shown in Scheme 1, involves (i) production of a
hydroxide ion at the dinickel(II) center, (ii) activation of the
substrate by coordination to one or both metal ions, and (iii)
nucleophilic attack of the hydroxide ion at the carbonyl carbon
atom of the substrate, producing ammonia and carbamic acid,
which spontaneously decompose to form a second equivalent
of ammonia and carbon dioxide. Carbamate is the first species
released from the enzyme,12 but there is no direct evidence that
it is the first intermediate formed in the reaction.
(1) Lippard, S. J. Science 1995, 268, 996-997.
(2) Sumner, J. B. J. Biol. Chem. 1926, 69, 435-441.
(3) Dixon, N. E.; Gazzola, C.; Blakeley, R. L.; Zerner, B. J. Am. Chem.
Soc. 1975, 97, 7, 4131-4132.
(4) Jabri, E.; Carr, M. B.; Hausinger, R. P.; Karplus, P. A. Science 1995,
268, 998-1004.
(5) Pearson, M. A.; Michel, L. O.; Hausinger, R. P.; Karplus, P. A.
Biochemistry 1997, 36, 8164-8172.
(6) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Ciurli, S.; Mangani,
S. JBIC 1998, 3, 268-273.
(7) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.;
Mangani, S. Structure 1999, 7, 205-216.
(10) Blakeley, R. L.; Treston, A.; Andrews, R. K.; Zerner, B. J. Am.
Chem. Soc. 1982, 104, 612-614.
(11) Wheland, G. W. Resonance in Organic Chemistry; Wiley: New
York, 1955.
(12) Blakeley, R. L.; Hinds, J. A.; Kunze, H. E.; Webb, E. C.; Zerner,
B. Biochemistry 1969, 5, 1991-2000.
(8) Benini, S.; Rypniewski, W. R.; Wilson, K. S.; Miletti, S.; Ciurli, S.;
Mangani, S. JBIC 2000, 5, 110-118.
(9) (a) Fearon, W. R. Biochem. J. 1923, 17, 84-93. (b) Mack, E.; Villars,
D. S. J. Am. Chem. Soc. 1923, 45, 505-510. (c) Karplus, P. A.; Pearson,
M. A.; Hausinger, R. P. Acc. Chem. Res. 1997, 30, 330-337.
10.1021/ja000202v CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/07/2000

Urea and Dinuclear Nickel Center Interaction
J. Am. Chem. Soc., Vol. 122, No. 38, 2000 9173
Scheme 1
seemed a likely candidate to effect the hydrolysis of urea. In
the present report we describe the synthesis of a related dinuclear
nickel complex containing a unique bridging urea molecule.
Upon heating, this complex effects the hydrolysis of urea by a
pathway involving cyanate ion that is distinct from that
previously proposed for the enzyme. These results provide the
first direct evidence for a mechanism long considered as an
alternative for the enzymatic hydrolysis of urea.9a,b
Experimental Section
General Considerations. All reagents were obtained from com-
mercially available sources and used without further purification, unless
otherwise noted. The ligand 1,4-bis(2,2′-dipyridylmethyl)phthalazine
was prepared as previously described.28 The complexes [Ni(terpy)-
(H2O)3](OTs)2 and [Ni2(µ-OH)(µ-H2O)(bdptz)(H2O)2](OTs)3 were pre-
pared according to published procedures.28 Fourier transform infrared
spectra were recorded on a Bio-Rad FTS-135 instrument, and UV-
vis spectra were obtained by using a Hewlett-Packard 8453-A diode
array spectrophotometer.
CAUTION: The syntheses and procedures described below involve
compounds that contain the perchlorate ion, which can detonate
explosively and without warning. Although we have not encountered
any problems with the reported compounds, all due precautions should
be taken.
[Ni2(µ-OH)(µ-H2O)(bdptz)(urea)2](ClO4)3 (1). A 52.8 mg, 144
µmol portion of [Ni(H2O)6](ClO4)2 was dissolved in 1.5 mL of methanol
with stirring. A 33.4 mg, 71.5 µmol portion of bdptz was added as a
solid to the methanolic nickel solution. After the mixture was allowed
to stir for 2-3 min, 1 equiv of an aqueous 1 M NaOH solution was
added, followed by 10 equiv of urea. Diffusion of diethyl ether vapor
into the resulting brown methanolic solution resulted in X-ray quality
A number of researchers have reported the synthesis of
dinuclear nickel complexes of relevance to the active site of
urease.13-19 Several of these model complexes have a urea
molecule bound to the dinickel center.20-22 In all but one case,
the urea molecule binds through its carbonyl oxygen atom to
one of the nickel ions in the complex; this coordination mode
is likely to occur in the enzyme. In addition to these structural
models, a few of the complexes reported in the literature show
reactivity pertinent to that of urease. Ethanolysis of urea at a
dinickel center10,23,24 presumably takes place via nucleophilic
attack of ethanol solvent on coordinated urea. Complexes that
promote the elimination of ammonia from a coordinated urea
to form cyanates are also known,25-27 although the further
reactivity of the resulting dinickel cyanate compounds was not
investigated.
The fact that no dinuclear nickel complex reported thus far
has proved capable of hydrolyzing urea to ammonia and carbon
dioxide provides an intriguing challenge to the synthetic
bioinorganic chemist. The design of a complex that can produce
a sufficiently nucleophilic hydroxide ion and at the same time
activate a urea molecule by coordination to one or both metal
ions should facilitate the successful hydrolysis of urea under
the right conditions. To this end, we recently reported a dinuclear
nickel complex, [Ni2(µ-OH)(µ-H2O)(bdptz)(H2O)2](OTs)3 (where
bdptz is the dinucleating ligand 1,4-bis(2,2′-dipyridylmethyl)-
phthalazine), that is capable of hydrolyzing a bound amide
substrate by intramolecular attack of a coordinated hydroxide
ion.28 On the basis of the results of this study, this complex
brown-purple crystals of 1 (61 mg, 82% yield). UV-vis (CH3OH) (λmax
,
nm (ꢀ, M-1cm-1)): 562 (66), 770 (50), 940 (62). FTIR (KBr, cm-1):
3380 (s, br), 2982 (w), 1663 (s), 1609 (s), 1576 (s), 1477 (s), 1445 (s),
1373 (m), 1005 (s, br), 771 (m), 630 (m). Anal. Calcd for 1,
Ni2Cl3C32H33N10O16: C, 37.05; H, 3.21; N, 13.50. Found: C, 37.52;
H, 3.53; N, 13.26.
[Ni2(µ-OH)(µ-urea)(bdptz)(urea)(CH3CN)](ClO4)3 (2). A 55.9 mg,
153 µmol portion of [Ni(H2O)6](ClO4)2 was dissolved in 2 mL of
acetonitrile with stirring. To this solution, 35.3 mg, 75.6 µmol of bdptz
was added as a solid, portionwise. The mixture was allowed to stir
until all of the ligand had dissolved before 70 µL (70 µmol) of an
aqueous 1 M NaOH solution was added in a dropwise fashion. Finally,
10 equiv of urea was added, and the resulting brown solution was
allowed to stir for a few minutes more. A purple-brown crystalline
product (60 mg, 80% yield) suitable for X-ray structure determination
was isolated upon slow diffusion of diethyl ether vapor into the reaction
solution. UV-vis (CH3CN) (λmax, nm (ꢀ, M-1cm-1)): 549 (43), 771
(33), 940 (47). FTIR (KBr, cm-1): 3400 (s, br), 1661 (s), 1637 (s),
1611 (s), 1578 (m), 1477 (m), 1450 (m), 1373 (m), 1350 (w), 1090
(br). Anal. Calcd for 2, Ni2Cl3C34H34N11O15: C, 38.51; H, 3.23; N,
14.53. Found: C, 38.91; H, 3.65; N, 14.62.
[Ni2(µ-OH)(µ-H2O)(bdptz)(µ-OCN)]2(OTs)4 (3). A 43.7 mg, 37.4
µmol portion of [Ni2(µ-OH)(µ-H2O)(bdptz)(H2O)2](OTs)3 was dissolved
in 2 mL of ethanol with stirring. A 1.8 mL aliquot of 20 mM NaNCO
in 9:1 EtOH/H2O was added in a dropwise fashion, resulting in a green
solution. Upon slow evaporation of the resulting solution, green crystals
formed (70 mg, 93% crude yield). X-ray diffraction data obtained from
a single green crystal provided the structure of the compound. UV-
vis (CH3CN) (λmax, nm (ꢀ, M-1cm-1)): 537 (39), 785 (28), 931 (48).
FTIR (KBr, cm-1): 3450 (br), 3079 (w), 3044 (w), 3000 (w), 2915
(w), 2164 (vs), 1601 (s), 1572 (m), 1475 (m), 1443 (s), 1369 (w), 1184
(s), 1117 (s), 1027 (s), 1005 (s), 875 (w), 814 (w), 762 (m), 679 (m).
Anal. Calcd for 3‚2EtOH‚6H2O, C94H102N14O26Ni4S4: C, 51.16; H, 4.66;
N, 8.89. Found: C, 51.13; H, 4.25; N, 8.53.
(13) Chadhuri, P.; Ku¨ppers, H.-J.; Wieghardt, K.; Gehring, S.; Haase,
W.; Nuber, B.; Weiss, J. J. Chem. Soc., Dalton Trans. 1988, 1367-1370.
(14) Stemmler, A. J.; Kampf, J. W.; Kirk, M. L.; Pecoraro, V. L. J. Am.
Chem. Soc. 1995, 117, 6368-6369.
(15) Ito, M.; Takita, Y. Chem. Lett. 1996, 929-930.
(16) Volkmer, D.; Ho¨rstmann, A.; Griesar, K.; Haase, W.; Krebs, B.
Inorg. Chem. 1996, 35, 1132-1135.
(17) Volkmer, D.; Hommerich, B.; Griesar, K.; Haase, W.; Krebs, B.
Inorg. Chem. 1996, 35, 3792-3803.
(18) Hosokawa, Y.; Yamane, H.; Nakao, Y.; Matsumoto, K.; Takam-
izawa, S.; Mori, W.; Suzuki, S. Chem. Lett. 1997, 891-892.
(19) Hommerich, B.; Schwo¨ppe, H.; Volkmer, D.; Krebs, B. Z. Anorg.
Allg. Chem. 1999, 625, 75-82.
(20) Wages, H. E.; Taft, K. L.; Lippard, S. J. Inorg. Chem. 1993, 32,
4985-4987.
(21) Koga, T.; Furutachi, H.; Nakamura, T.; Fukita, N.; Ohba, M.;
Takahashi, K.; Okawa, H. Inorg. Chem. 1998, 37, 989-996.
(22) Meyer, F.; Pritzkow, H. J. Chem. Soc., Chem. Commun. 1998,
1555-1556.
(23) Yamaguchi, K.; Koshino, S.; Akagi, F.; Suzuki, M.; Uehara, A.;
Suzuki, S. J. Am. Chem. Soc. 1997, 119, 5752-5753.
(24) Konrad, M.; Meyer, F.; Jacobi, A.; Kircher, P.; Rutsch, P.; Zsolnai,
L. Inorg. Chem. 1999, 38, 4559-4566.
(25) Uozumi, S.; Furutachi, H.; Ohba, M.; Okawa, H.; Fenton, D. E.;
Shindo, K.; Murata, S.; Kitko, D. J. Inorg. Chem. 1998, 37, 6281-6287.
(26) Roecker, L.; Akande, J.; Elam, L. N.; Gauga, I.; Helton, B. W.;
Prewitt, M. C.; Sargeson, A. M.; Swango, J. H.; Willis, A. C.; Xin, T.; Xu,
J. Inorg. Chem. 1999, 38, 1269-1275.
(27) Meyer, F.; Kaifer, E.; Kircher, P.; Heinze, K.; Pritzkow, H. Chem.-
Eur. J. 1999, 5, 1617-1630.
(28) Barrios, A. M.; Lippard, S. J. J. Am. Chem. Soc. 1999, 121, 11751-
11757.
Collection and Reduction of X-ray Data. Procedures for the
collection and reduction of X-ray data have been reported previously.29
(29) Feig, A. L.; Bautista, M. T.; Lippard, S. J. Inorg. Chem. 1996, 35,
6892-6898.

9174 J. Am. Chem. Soc., Vol. 122, No. 38, 2000
Barrios and Lippard
Table 1. Summary of X-ray Crystallographic Data
1‚2CH3OH‚2H2O
2‚CH3CN‚CH3OH‚CH3CH2OCH2CH3
3‚6CH3CH2OH
formula
fw
space group
a, Å
b, Å
c, Å
C33H43N10O20Ni2Cl3
1123.54
C42H57N12O19Ni2Cl3
1257.77
C98H102N14O22Ni4S4
1095.51
Pnma
P2(1)/m
P2(1)/n
17.6122(8)
21.1269(10)
14.3975(7)
10.429(2)
13.520(14)
12.434(9)
31.21(3)
21.129(4)
12.695(4)
R, deg
â, deg
γ, deg
V, Å3
Z
106.129(14)
100.938(11)
5357.2(4)
4
2687.2(12)
2
5151(8)
2
1.413
-85
0.877
3-57
31442
11910
5604
667
5.98
14.29
1.063, -1.200
F
calcd, g/cm3
1.393
-85
1.353
-85
T, °C
µ(Mo KR), mm-1
2θ limits, deg
total no. of data
no. of unique data
observed dataa
no. of parameters
R1,b %
0.928
3-57
32874
6609
0.912
3-57
17030
6412
4243
3831
314
423
6.68
6.57
wR2,c %
22.50
0.974, -0.508
16.58
1.094, -0.765
max, min peaks, e/Å3
a Observation criterion: I > 2σ(I). b R1 ) Σ||Fo| - |Fc||/Σ|Fo|. c wR2 ) {Σ[w(Fo - Fc2)2]/Σ[w(Fo )2]}1/2
.
2
2
The crystals were mounted on glass fibers with Paratone-N (Exxon)
and cooled rapidly in the -85 °C cold stream of a Bruker CCD X-ray
diffraction system controlled by a Pentium-based PC running the
SMART software package.30 The structures were solved by using the
direct methods program XS, and refinements were carried out with
XL. Both programs are part of the SHELXTL program package.31 All
non-hydrogen atoms were refined by a series of least squares cycles.
Hydrogen atoms were assigned idealized positions and given a thermal
parameter 1.2 times that of the atom to which they are attached.
Empirical absorption corrections were calculated and applied for each
structure with SADABS,32 and PLATON33 was used to search for higher
symmetry.
The structure of 2 has disorder in the two terminal ligands of the
dinuclear nickel complex. There is a crystallographically imposed mirror
plane running through the molecule, bisecting the NisNi vector
perpendicular to the plane of the phthalazine rings. The terminal
Figure 2. ORTEP diagram of the cation in 1 showing 50% probability
ellipsoids for all non-hydrogen atoms. Selected interatomic distances
(Å) and angles (degrees): Ni1-O1, 2.204(3); Ni1-O2, 2.060(3); Ni1-
O20, 2.027(3); Ni1-N1, 2.044(4); Ni1-N2, 2.079(4); Ni1-N3, 2.072-
(4); N21-O2, 2.931(4); Ni1-Ni1A, 3.085(4); N1-Ni1-O20, 171.34-
(14); O1-Ni1-O2, 78.37(15).
coordination site of the nickel ion is occupied by an acetonitrile and
an oxygen-bound urea molecule. The two ligands were assigned
arbitrary positions and occupancies and then refined to the reported
positions with 50% occupancy each. All pertinent crystallographic
information for each complex, including bond distances and angles,
atomic coordinates, and equivalent isotropic displacement parameters,
is provided in Tables S1-S15 in the Supporting Information. Selected
crystallographic information for the complexes is provided in Table 1.
Kinetics. The decomposition of urea promoted by the dinickel center
in complexes 1 and 2 was studied by the method of initial rates. In a
typical experiment, an 18 mM solution of 1 in CH3CN was prepared
and serially diluted. The solutions of 1 were incubated at 60 °C for 20
h with swirling at 200 rpm, after which time the ammonia content of
the solutions was quantified by using a colorimetric assay reported
previously.28,34 In a control reaction, a solution 20 mM in [Ni(terpy)-
(H2O)3](OTs)2 and 20 mM in urea was heated and then assayed under
the same conditions described above to determine the rate of urea
decomposition promoted by a mononuclear nickel species. Another
control reaction involved heating 20 mM [Ni2(µ-OH)(µ-H2O)(bdptz)-
(H2O)2](OTs)3 in acetonitrile under the reaction conditions to ensure
that none of the ammonia formed resulted from the reaction of the
dinickel complex with acetonitrile. No ammonia was detected in either
control reaction.
The rate of cyanate hydrolysis in the presence of 1 was determined
for a series of substrate and complex concentrations. When the sodium
cyanate concentration was varied from 5 to 24 mM and the concentra-
tion of nickel complex was held constant at 11.5 mM, saturation kinetics
behavior was observed, indicating coordination of the cyanate to the
dinickel complex with a binding constant of 110 ( 30 M-1. The
concentration of dinickel complex could not be made sufficiently high
to observe saturation when the nickel concentration was varied. When
the concentration of the nickel complex was varied from 6 to 14 mM
and the cyanate concentration was held constant at 12 mM, the reaction
rate showed a first-order dependence on the concentration of dinickel
complex. The initial rate of cyanate hydrolysis also had a first-order
dependence on the concentration of water in the reaction. The second-
order rate constant for this reaction is (9.5 ( 1) × 10-4 M-1 h-1. When
the pH of the reaction was varied from 6.5 to 8.5 by using a buffered
solution (0.020 M in HEPES), the rate remained unchanged.
Results
(30) SMART; V5.05 ed.; Bruker AXS, Inc.: Madison, WI, 1998.
(31) Sheldrick, G. M. SHELXTL: Program for the Refinement of Crystal
Structures; 2nd ed.; University of Gottingen: Gottingen, Germany, 1997.
(32) Sheldrick, G. M. SADABS: Area-Detector Absorption Correction;
University of Gottingen: Gottingen, Germany, 1996.
(33) Spek, A. L. PLATON, A Multipurpose Crystallographic Tool;
Utrecht University: Utrecht, The Netherlands, 1998.
When nickel perchlorate and bdptz are allowed to react with
an excess of urea in methanol, complex 1 is obtained. The X-ray
crystal structure of 1, shown in Figure 2, reveals a dinuclear
nickel complex bridged by the phthalazine moiety of the bdptz
ligand, a water molecule, and a hydroxide ion. Each nickel ion
(34) Weatherburn, M. W. Anal. Chem. 1967, 39, 971-974.

Urea and Dinuclear Nickel Center Interaction
J. Am. Chem. Soc., Vol. 122, No. 38, 2000 9175
Figure 4. Plot of the initial rate of ammonia formation versus the
concentration of 1.
Figure 3. ORTEP diagram of the cation of 2 showing 50% probability
ellipsoids for all non-hydrogen atoms. Selected interatomic distances
(Å) and angles (degrees): Ni1-O1, 2.039(3); Ni1-O10, 2.158(3);
Ni1-O20, 2.14(2); Ni1-N20A, 1.88(3); N20-O1, 2.774(5); Ni1-
Ni1A, 3.079(4); Ni-Ni1-O20, 174.2(8); N1-Ni1-N20A, 173.2(10).
is ligated by two pyridine donor arms from the ligand, and the
pseudooctahedral coordination sphere of each is filled by a urea
molecule bound to nickel through its carbonyl oxygen atom.
The electronic spectrum of 1 shows intense π-π* transitions
below 400 nm, attributable to the ligand and three weak d-d
transitions in the visible region, indicating that the nickel ions
are in an octahedral environment in solution as well as in the
solid state. The solid-state FTIR spectrum of the complex
exhibits a shift in the carbonyl stretching frequency of urea from
1690 to 1663 cm-1 upon coordination to the dinickel center.
Complex 2 can be obtained through the same reaction as 1
by using acetonitrile as the solvent instead of methanol, or by
recrystallizing complex 1 from acetonitrile instead of methanol.
An ORTEP diagram of 2 is shown in Figure 3. In this complex,
the bdptz ligand coordinates to the dinickel center in the same
manner as in complex 1, with the phthalazine moiety bridging
the two metal ions and two pyridine donor arms coordinating
to each metal ion. The two remaining terminal coordination
positions are each 50% occupied by an acetonitrile molecule
and a urea molecule coordinated to the metal through its
carbonyl oxygen atom. The dinickel center is additionally
bridged by a hydroxide ion and by a urea molecule that forms
a single atom bridge between the two metal ions with its
carbonyl oxygen atom. The electronic spectrum of 2 confirms
the pseudooctahedral coordination environment of the nickel
ions in solution. The solid-state FTIR spectrum of 2 is very
similar to that of 1, with the carbonyl stretching bands of the
urea molecules producing a somewhat broad peak centered at
1661 cm-1. Comparison of the solution IR spectra of urea and
2 in acetonitrile reveals a shift in the urea stretching frequencies
from two sharp peaks at 1712 and 1695 cm-1 (CdO stretch
and NH2 bend, respectively) to two much broader peaks at 1660
and 1636 cm-1. Because of the breadth of the peaks and the
presence of ligand-derived bands in the same region, it was
difficult to distinguish and assign the terminal and bridging urea
molecules.
Figure 5. ORTEP diagram of the cation of 3 showing 50% probability
ellipsoids for all non-hydrogen atoms. Selected bond lengths (Å) and
angles (degrees): Ni1-O1, 2.123(3); Ni1-O2, 2.091(3); Ni1-O3B,
2.050(4); Ni2-O1, 2.109(3); Ni2-O2, 2.088(3); Ni2-O3, 2.050(3);
Ni1-Ni2, 3.048(4); N1-Ni1-O3B, 179.52(15); N2-Ni2-O3, 178.85-
(13); O3-C3A-N3A, 179.3(9).
an observed rate constant of (7.7 ( 0.5) × 10-4 h-1. When
urea, alone or with added [Ni(terpy)(H2O)3](OTs)2, was heated
under the same conditions in acetonitrile, no ammonia was
produced, indicating that two metal ions act in concert to effect
the decomposition of urea at least 500 times faster than the
spontaneous decomposition of urea under these conditions. The
dinickel complex promotes the decomposition of urea to
ammonia and cyanate rather than the hydrolysis of urea to
ammonia and carbamate, as evidenced by the presence of a
strong cyanate band in the FTIR spectrum of the reaction
product.
Although attempts to obtain X-ray quality crystals of the
cyanate-containing product were unsuccessful, an analogue was
synthesized independently by allowing [Ni2(µ-OH)(µ-H2O)-
(bdptz)(H2O)2](OTs)3 to react with 1 equiv of NaNCO in
aqueous ethanol. Green X-ray quality crystals of this cyanate
complex, [Ni2(µ-OH)(µ-H2O)(bdptz)(µ-OCN)]2(OTs)4, 3, were
harvested following slow evaporation of the solvent from the
reaction mixture. An ORTEP diagram of 3 is displayed in Figure
5. Complex 3 consists of two dinuclear nickel units, each bridged
by the phthalazine moiety of bdptz and ligated by its pyridine
donor arms, with a water molecule and a hydroxide ion filling
the two remaining bridging positions. Two cyanate ions link
the two dinickel units, each bridging through its oxygen atom.
The bridging atom can clearly be assigned as oxygen on the
basis of crystallographic data. When the structure was refined
with nitrogen assigned as the bridging atom, the Ueq values for
the oxygen and the nitrogen atom increased and decreased by
over 50%, respectively. Further evidence for the assignment of
the bridging atom as oxygen is provided by the cyanate bond
When either complex 1 or 2 is heated to 60 °C in acetonitrile
solution, ammonia is produced. The rates of reaction were
identical, suggesting that the complexes are the same in solution.
Because the initial rates of ammonia production from the two
compounds were the same, 1 was used for detailed kinetic
studies because it was easier to prepare in satisfactorily pure
form. The rate of ammonia production has a first-order
dependence on complex concentration, as seen in Figure 4, with

9176 J. Am. Chem. Soc., Vol. 122, No. 38, 2000
Barrios and Lippard
Discussion
In both complexes 1 and 2, there are two nickel ions in
pseudooctahedral ligand environments, a geometry imposed in
part by the hexadentate, dinucleating bdptz ligand. Both
complexes contain two coordinated urea molecules and a
bridging hydroxide ion that could potentially serve as a
nucleophile for substrate hydrolysis. The coordination of urea
to the dinuclear nickel centers in 1 and 2 reinforces the proposal
that urea most likely binds preferentially through its carbonyl
oxygen atom to an active site nickel center in urease. The
bridging coordination mode of urea in complex 2 has not
previously been encountered and may be of relevance to the
enzyme. Presumably, the urea molecule would be better
activated by coordination to two metal ions rather than just one.
In the urea decomposition chemistry reported here, however, it
is unknown which binding mode facilitates the elimination
reaction.
It is interesting to speculate why in the present work the urea
molecule undergoes an elimination reaction rather than being
hydrolyzed. We recently reported the hydrolysis of picolinamide
by an analogous hydroxide-bridged dinickel complex;28 in this
case, intramolecular nucleophilic attack of the bridging hydrox-
ide ion resulted in hydrolysis of a coordinated amide. Since 1
and 2 both contain such a bridging hydroxide ion, why does
intramolecular nucleophilic attack not occur? Perhaps the
hydroxide ion, although able to effect the hydrolysis of an amide
substrate, is not sufficiently nucleophilic to hydrolyze the
coordinated urea molecule. Another possibility is that the
coordinated hydroxide ion serves as a general base, aiding in
the deprotonation of urea to favor elimination. The terminally
coordinated urea molecule in both 1 and 2 is hydrogen bonded
through one of its amino groups to the bridging hydroxide ion
(Figures 2 and 3), providing a facile pathway by which the
hydroxide ion could participate in such a reaction.
Figure 6. Plots showing (a) the dependence of the initial rate of cyanate
hydrolysis on the initial concentration of [Ni2(OH)(H2O)3(bdptz)](OTs)3
and (b) the dependence of the observed rate constant of cyanate
hydrolysis (h-1) on the concentration of water in the reaction mixture.
lengths. The O-C distance of 1.290(14) Å is slightly longer
than that in other cyanate complexes, and the C-N distance is
1.149(14) Å, just shorter than the average distance in known
cyanate complexes. A slight lengthening of the C-O bond
accompanied by a similar shortening of the C-N bond would
be expected in an oxygen-bound cyanate complex, correspond-
ing to increased negative charge on the bridging oxygen atom
and a strengthening of the C-N bond. Both the solution and
solid-state FTIR spectra of 3 are virtually identical to that of
the reaction product. The νas of cyanate occurs at 2164 cm-1 in
both cases.
The products of the reaction between urea and the dinuclear
nickel complex are ammonia and cyanate. Attempts to crystallize
the cyanate-containing product afforded crystals too small to
be characterized by X-ray crystallography, but the IR spectrum
of these crystals closely resembled that of the independently
synthesized cyanate complex 3. It is difficult to distinguish
between bridging and terminally coordinated cyanate on the
35
basis of νCN
,
and the other cyanate bands are obscured by
ligand vibrations. Although there is insufficient evidence to
demonstrate that 3 is the same as the product formed upon
decomposition of bound urea in 1 or 2, the similarities in the
spectra and properties indicate 3 to be a reasonable candidate
for the urea decomposition product.
When either 1 or 2 was heated in a 50% aqueous acetonitrile
solution, ammonia was produced at a rate comparable to that
in dry acetonitrile but with no appreciable buildup of the
cyanate-containing product. This result indicates that the cy-
anate-containing product is hydrolyzed in the presence of water.
Further evidence to support this hypothesis was obtained by
first heating 1 in dry acetonitrile for several days to allow the
cyanate product to accumulate, then adding several equivalents
of water, and observing the characteristic cyanate peak in the
IR spectrum decrease over time. Cyanate hydrolysis was
followed quantitatively by allowing sodium cyanate to react with
the hydroxide-bridged dinickel complex [Ni2(µ-OH)(µ-H2O)-
(bdptz)(H2O)2](OTs)3. The reaction exhibited saturation kinetics
behavior as the concentration of cyanate ion was varied. A
binding constant of 110 ( 30 Μ-1 for coordination of cyanate
to the dinickel complex was obtained from the data, as shown
in Figure 6a. The reaction has a first-order dependence on the
concentration of water, as indicated in Figure 6b, with a second-
order rate constant of (9.5 ( 1) × 10-4 M-1 h-1. When the pH
of the reaction was varied from 6.5 to 8.5, the rate was
unaffected.
The cyanate product formed upon heating complexes 1 or 2
can be further decomposed hydrolytically in the presence of
water. This hydrolysis reaction was quantitated by following
the hydrolysis of NaNCO by [Ni2(µ-OH)(µ-H2O)(bdptz)(H2O)2]-
(OTs)3. As the concentration of cyanate was varied, saturation
kinetics were observed. The calculated nickel-cyanate binding
constant was 110 ( 30 M-1. The concentration of the nickel
complex could not be made sufficiently high to observe
saturation behavior when the nickel concentration was varied.
Under these reaction conditions, the rate showed a first-order
dependence on the concentration of dinickel complex. The
reaction rate was also first-order in water, indicating that the
hydrolysis proceeds by attack of an external water molecule on
the coordinated cyanate ion. The pH independence of the
reaction in the range 6.5 e pH e 8.5 indicates that the bridging
(35) Bailey, R. A.; Kozak, S. L.; Michelsen, T. W.; Mills, W. N. Coord.
Chem. ReV. 1971, 6, 407-445.

Urea and Dinuclear Nickel Center Interaction
J. Am. Chem. Soc., Vol. 122, No. 38, 2000 9177
Scheme 2
500 times as fast as the reaction between urea and a mononuclear
nickel complex or urea alone. The final hydrolysis step is
significant. Other researchers have reported the formation of
cyanate from urea at a dinickel site but have not investigated
the reactivity of the resulting cyanate complex. To the best of
our knowledge, there is no definitive evidence ruling out the
possibility of a transient nickel-bound cyanate intermediate in
urease. Such an intermediate would be consistent with the results
of the present and other model studies.25-27
Acknowledgment. This work was supported by grants from
the National Science Foundation and the National Institutes of
Health. A.M.B. thanks the NIH for a predoctoral fellowship.
We thank Dr. Natalia Kaminskaia for helpful discussions.
hydroxide ion could be acting as a general base in this reaction,
helping to deprotonate the external nucleophile. The pKa of this
bridging hydroxide ion is 4.4,28 well below 6.5.
The net reaction effected at the dinickel center is hydrolysis
of urea to ammonia and carbon dioxide through formation of a
cyanate intermediate. This reaction proceeds via a pathway
distinct from that previously proposed for the enzymatic
hydrolysis of urea (Scheme 1).1,7,9c As shown in Scheme 2, the
important steps in the hydrolysis of urea at the dinickel center
in 1 and 2 are (i) elimination of ammonia from the coordinated
urea molecule, and (ii) hydrolysis of the resulting cyanate by
an external water molecule. Coordination of urea to the dinuclear
nickel center is crucial; complexes 1 and 2 promote the
elimination of ammonia from coordinated urea at a rate at least
Supporting Information Available: Figures S1-S3, show-
ing fully labeled ORTEP diagrams for complexes 1-3, respec-
tively. Figure S4 shows the nickel complex concentration
dependence of the initial rate of cyanate hydrolysis, and Figure
S5 shows the pH independence of the rate over the range 6.5-
8.5 (PDF). X-ray crystallographic files, in CIF format, are
available on the Internet only. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA000202V