CoIII complexes with square-planar N2S2- and N2(SO2)2-type ligands as an active site structural model for nitrile hydratase - Biological implications of an amidate coordination

Article abstract of DOI:10.1002/ejic.200600507

In an attempt to understand the unique active site structure of nitrile hydratase, four Co^III complexes with square-planar N ₂S₂- or N₂(SO₂)₂-type donor sets, Na[Co^III(L:N₂S₂)] (1-Na), PPh ₄[Co^III(L:N₂S₂)] (1-PPh ₄), PPh₄[Co^III(L:N₂S ₂)(tBuNC)₂] (2), and PPh₄[Co ^III{L:N₂(SO₂)₂}(tBuNC)₂] (3) were synthesized and characterized on the basis of electronic absorption spectroscopy, IR spectroscopy, cyclic voltammetry, and X-ray structural analysis. Both of the crystal structures of complexes 1-Na and 1-PPh₄ revealed a square planar structure with N₂S₂ donating atoms, and 2 exhibited an octahedral structure coordinated with two tert-butylisocyanide (tBuNC) molecules at the axial sites of complex 1-PPh ₄. Complex 3, which showed an octahedral structure with sulfinate sulfur atoms equatorially coordinated to the center, was synthesized by the treatment of 2 with a suitable oxidant. The reduction potential values from Co^III to Co^II for complex 3 in solution demonstrated a larger positive shift when compared with those of complexes 1-PPh₄ and 2, which indicates that the oxygenation of the sulfur atoms increased the Lewis acidity of the Co^III center. Interestingly, the coordination equilibrium, the C=O stretching frequency, and the redox potential for 1-PPh₄ were all closely related to the acceptor number (AN) of the solvents. Furthermore, the coordination of monodentate tBuNC to the axial position of 1-PPh₄ was dependent on the solvents used. These findings indicate that an electrophilic interaction between the carbonyl oxygen atoms and the solvent molecules control the Lewis acidity of the metal ion. On the other hand, such a solvent dependence was not detected in the S=O stretching frequency of sulfinates 3. We have concluded that the increase in the redox potential/ Lewis acidity of the metal center is a result of the oxygenation of sulfur, and that this increase is controlled by the interaction of the amidate carbonyl oxygen with the secondary coordination sphere. As demonstrated in previous mutation studies, this study suggests that the interaction of the nitrile hydratase active site with the functional groups from the peptide backbone is essential for the catalytic activity of the complex. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejic.200600507

FULL PAPER
DOI: 10.1002/ejic.200600507
CoIII Complexes with Square-Planar N2S2- and N2(SO2)2-Type Ligands as An
Active Site Structural Model for Nitrile Hydratase – Biological Implications of
an Amidate Coordination
Takuma Yano,[a] Hidekazu Arii,[a] Syuhei Yamaguchi,[a] Yasuhiro Funahashi,[a]
Koichiro Jitsukawa,[a] Tomohiro Ozawa,*[a] and Hideki Masuda*[a]
Keywords: Nitrile hydratase / Cobalt / Amidate ligand / Electrophilic interactions / Lewis acids
In an attempt to understand the unique active site structure
of nitrile hydratase, four CoIII complexes with square-planar
N2S2- or N2(SO2)2-type donor sets, Na[CoIII(L:N2S2)] (1-Na),
PPh4[CoIII(L:N2S2)] (1-PPh4), PPh4[CoIII(L:N2S2)(tBuNC)2] (2),
and PPh4[CoIII{L:N2(SO2)2}(tBuNC)2] (3) were synthesized
and characterized on the basis of electronic absorption spec-
troscopy, IR spectroscopy, cyclic voltammetry, and X-ray
rium, the C=O stretching frequency, and the redox potential
for 1-PPh4 were all closely related to the acceptor number
(AN) of the solvents. Furthermore, the coordination of mono-
dentate tBuNC to the axial position of 1-PPh4 was dependent
on the solvents used. These findings indicate that an electro-
philic interaction between the carbonyl oxygen atoms and
the solvent molecules control the Lewis acidity of the metal
structural analysis. Both of the crystal structures of com- ion. On the other hand, such a solvent dependence was not
plexes 1-Na and 1-PPh4 revealed a square planar structure
with N2S2 donating atoms, and 2 exhibited an octahedral
structure coordinated with two tert-butylisocyanide (tBuNC)
molecules at the axial sites of complex 1-PPh4. Complex 3,
which showed an octahedral structure with sulfinate sulfur
atoms equatorially coordinated to the center, was synthe-
detected in the S=O stretching frequency of sulfinates 3. We
have concluded that the increase in the redox potential/
Lewis acidity of the metal center is a result of the oxygena-
tion of sulfur, and that this increase is controlled by the inter-
action of the amidate carbonyl oxygen with the secondary
coordination sphere. As demonstrated in previous mutation
sized by the treatment of 2 with a suitable oxidant. The re- studies, this study suggests that the interaction of the nitrile
duction potential values from CoIII to CoII for complex 3 in
solution demonstrated a larger positive shift when compared
with those of complexes 1-PPh4 and 2, which indicates that
the oxygenation of the sulfur atoms increased the Lewis acid-
ity of the CoIII center. Interestingly, the coordination equilib-
hydratase active site with the functional groups from the
peptide backbone is essential for the catalytic activity of the
complex.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
and its pre-edge spectra[6,7] in addition to homology in sig-
nificant amino acid sequences.[8]
Introduction
Nitrile Hydratase (NHase) is an enzyme that hydrates a
nitrile compound to the corresponding amide. Because of
its high conversion ability, this enzyme has been employed
for the industrial production of acrylamide from acryloni-
trile under mild conditions.[1] The NHases are largely classi-
fied into FeIII-type and CoIII-type families.[2–4] Rhodococcus
Recent X-ray structure analysis of both NHase types re-
vealed that the metal centers are surrounded by two amide
nitrogen atoms and two cysteine sulfur atoms in the equato-
rial plane with a cysteine sulfur and an oxygen atom (H2O
or OH–), or a cysteine sulfur and a NO group, at the axial
sites for the CoIII- or FeIII-types, respectively.[9–12] Interest-
ingly, two equatorially coordinated cysteine sulfur atoms
are oxygenated to a sulfenate and a sulfinate, respectively,
and the two nitrogen atoms contribute as an amidate li-
gand. Such a unique structure is very interesting in relation
to its hydration mechanism in biological systems.
In such a hydration enzyme, a higher Lewis acidity of the
metal center should be required, although M–OH– (or M–
OH2) or M–NCR are proposed as possible active interme-
diates. On the basis of the HSAB rule, the N– and S– ions
employed in the enzymes may stabilize the higher oxidation
state of the metal center, and the attached electron-with-
drawing carbonyl group along with the sulfenyl and sulfinyl
rhodochrous J1, which is a bacterium with the typical CoIII
-
type NHase, has been used to produce several kilotons of
acrylamide per year.[3–5] The common amino acid sequence
(C–X–Y–C–S–C) of the peptide in the FeIII-type and the
CoIII-type NHase has been proposed as the metal binding
ligand on the basis of the similarity between the EXAFS
[a] Graduate School of Engineering, Nagoya Institute of Technol-
ogy,
Gokiso-cho, Showa-ku, Nagoya 466–8555, Japan
Fax: +81-52-735-5228
E-mail: masuda.hideki@nitech.ac.jp
Supporting information for this article is available on the
WWW under http://www.eurjic.org or from the author.
Eur. J. Inorg. Chem. 2006, 3753–3761
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. Yano, H. Arii, S. Yamaguchi, Y. Funahashi, K. Jitsukawa, T. Ozawa, H. Masuda
FULL PAPER
oxygen atoms may reduce the electron density on the metal that βR56K, whose Arg56 residue is replaced by lysine and
center to induce a higher acidity. It is quite apparent that coordinated to the sulfinate and sulfenate oxygen atoms
both functional groups are very important for the enzy- through a hydrogen bond, demonstrated only ca. 1% of the
matic reaction. However, the use of the mutant Fe-type reactivity of the native species.[21] For the βY68F mutant
NHases that did not contain oxygenated sulfur atoms did of the CoIII-type NHase, the enzymatic activity drastically
not show any hydration reaction, which indicates that the decreased, although it was positioned at a distance from the
oxygenated sulfur atoms are essential for the enzymatic ac- active site.[22] Moreover, the OH group of tyrosine in
tivity.[13]Most studies that use model complexes have fo- βTyr68 is linked to the carbonyl oxygen of the active site
cused on the effect of the oxidation of sulfur. For example, through water molecules and the hydrogen bond network
several CoIII and FeIII complexes with pentadentate ligands around βArg56. βArg56 is, itself, also connected to βTyr68.
that contain an imine,[14] an amide,[15,16] or an amine[17] These facts indicate that the hydrogen-bonding interactions
group and sulfur atoms have been synthesized and studied. of the coordinating functional groups with the outer sphere
Among them, Mascharak and coworkers[15a] have prepared strongly contribute to the appearance of the NHase activity.
the complex [Co(PyPS)(CN)] with the use of the amide-type Therefore, we focused on the biological role of the amidate
ligand, which was easily replaced by a H2O molecule. The carbonyl group and synthesized two CoIII complexes with
coordinated water molecule catalytically hydrated acetoni- N2S2-type ligands, N,NЈ-bis(2-mercapto-2-methylpropi-
trile to acetamide at pH = 9.5. The addition of an oxidant onyl)-1,3-diaminopropane (H4L) and the corresponding
to the solutions of the complexes afforded the correspond- sulfinic acid derivative [L:N2(SO2)2; the two sulfur atoms
ing sulfur-oxidized species.[15b] For the CoIII mononuclear of H4L are oxygenated to sulfinates]. In order to evaluate
complex, the CoIII species with a sulfinate was isolated. Hy- the influence of the outer sphere, the coordination behav-
dration of acrylonitrile with this sulfonate–CoIII complex iors of the axial sites of the CoIII complex with tert-butyl-
proceeded three times faster than that of the complex with isocyanide (tBuNC) were evaluated. Isocyanides such as
two unoxidized sulfur atoms even in acidic media (pH = isobutylisocyanide[23] and cyclohexylisocyanide[24] are often
8.0). They concluded that the oxygenation of sulfur would employed because of their well-known ability to act as coor-
make the acidity of the coordinated H2O molecule increase. dinative monodentate ligands for NHase.
Other groups have prepared and characterized the CoIII
and FeIII complexes with tetradentate planar ligands that
Results and Discussion
contain two amide nitrogen atoms and two thiolate sulfur
atoms in the same coordination environment as the NHase
active sites,[18,19] and they also succeeded in the generation
of sulfinate and sulfenate species by the oxidation of the
coordinated sulfur atoms.[18a,18b,19a,19b] However, all of
them showed octahedral structures with two monodentate
ligands at the axial positions.[18a,18b,19a,19b] Recently, Artaud
Crystal Structures of Na[Co(L:N2S2)]3H2O (1-Na),
PPh4[CoIII(L:N2S2)]H2O (1-PPh4), PPh4[CoIII(L:N2S2)2-
(tBuNC)2] (2), and PPh4[CoIII{L:N2(SO2)2}(tBuNC)2]-
EtOH (3)
The N2S2-type ligand, H4L, which provides electron-do-
and coworkers reported that in the synthesis of the N2(SO2)2- nating atoms similar to the equatorial coordination envi-
type CoIII complex with a CN– ion at the apical position, ronment of the NHase active center, was used for the con-
the octahedral complex with a hydroxo ion at another axial struction of the model complexes. The CoIII complex with
position was derived.[20] Thus, the oxygenation of sulfur in L, Na[Co(L:N2S2)] (1-Na), was prepared by the air-oxi-
the equatorial plane has been described to be one of the dation of the corresponding CoII complex derived from the
important factors that leads to an octahedral geometry.
reaction of CoCl2 and H4L in DMF. Recrystallization of
Few have paid attention to the amidate group. However, the compound from an acetonitrile/diethyl ether solution
a mutation study of Fe-containing NHase demonstrated afforded a single crystal suitable for X-ray analysis, whose
Figure 1. An ORTEP view of Na[Co(L:N2S2)] (1-Na) with solvated water molecules, which shows 50% probability ellipsoids. H atoms
are omitted for clarity except for those of the water molecules.
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CoIII Complexes with Square-Planar N2S2- and N2(SO2)2-Type Ligands
FULL PAPER
crystal data and molecular structure are shown in Table 2 Table 1. Selected bond lengths [Å] and angles [°] for Na[Co-
and Figure 1, respectively. Two [CoIII(L:N2S2)]– and two
(L:N2S2)](1-Na) and PPh4[Co(L:N2S2)](1-PPh4) and PPh4-
[Co(L:N2S2)(tBuNC)2](2) and PPh4[Co{L:N2(SO2)2}(tBuNC)2]-
Na+ ions are included together with six water molecules in
a unit cell (Figure S1). Selected bond lengths and angles
are listed in Table 1. Complex 1-Na has a square-planar
geometry with N2S2 donor atoms as well as the equatorial
(3).[a]
1-Na
Bond lengths [Å]
coordination in the active site of NHase.[9–12] The bond
lengths around the metal center [Co1–N1 = 1.919(3), Co1–
N2 = 1.918(3), Co1–S1 = 2.141(1), Co1–S2 = 2.142(1) Å]
are comparable to those of the CoIII complexes with amide
(Co–N = 1.86–1.90 Å) and thiolate coordinations (Co–S =
2.13–2.14 Å) that were previously reported.[18c,19c] Each car-
bonyl oxygen of the ligand L was significantly coordinated
to three water molecules [Ocarbonyl···Owater = 2.84–2.91 Å]
in the crystal. Furthermore, one water molecule also weakly
approaches the CoIII ion with a bond length of 3.20 Å at
the axial position, which is very similar to the case of Co-
type NHase.[11]
Co1–N1
Co1–N2
Co1–S1
Co1–S2
O1–C4
O2–C8
N1–C4
N2–C8
1.919(3)
1.918(3)
2.141(1)
2.142(1)
1.263(4)
1.257(4)
1.330(4)
1.332(3)
O1···O3
O1···O4
O1···O5
O2···O3
O2···O4
O2···O5
2.86
2.91
2.84
2.86
2.91
2.84
Bond angles [°]
N1–Co1–N2
S1–Co1–S2
S1–Co1–N1
98.0(1)
86.12(4)
87.89(9)
S1–Co1–N2
S2–Co1–N1
S2–Co1–N2
173.99(9)
173.96(9)
87.92(9)
1-PPh4
Bond lengths [Å]
One more CoIII complex with L, PPh4[Co(L:N2S2)] (1-
PPh4), was also prepared by the ion-exchange of 1-Na in
an aqueous solution of PPh4Cl. Complex 1-PPh4 was ob-
tained as a single crystal suitable for X-ray analysis, whose
crystal data and molecular structure are shown in Table 2
and Figure 2, respectively. Four [CoIII(L:N2S2)]– anions and
Co1–N1
Co1–N2
Co1–S1
Co1–S2
O1–C4
1.902(3)
N1–C4
N2–C8
O1···O3
O2···O3
1.341(4)
1.348(4)
2.83
1.896(3)
2.138(1)
2.144(1)
1.250(4)
1.243(4)
2.82
O2–C8
+
four PPh4 cations are included together with four water
Bond angles [°]
molecules in a unit cell (Figure S2). Selected bond lengths
and angles are listed in Table 1. The molecular structure of
complex 1-PPh4 was essentially the same as that of 1-Na.
The bond lengths around the metal center [Co1–N1 =
1.902(3), Co1–N2 = 1.896(3), Co1–S1 = 2.138(1), Co1–S2
= 2.144(1) Å] are in the range of those of the CoIII com-
plexes with amide (Co–N = 1.86–1.90 Å) and thiolate coor-
dinations (Co–S = 2.13–2.14 Å) that were previously re-
ported.[18c,19c] However, a detailed comparison of those of
1-Na and 1-PPh4 indicated that the Co–N bonds of 1-PPh4
were significantly different from those of 1-Na, although
the Co–S bonds was almost the same as those of 1-Na.
This behavior is apparently influenced by the number of
hydrogen bonds between water molecules and the amidate
carbonyl oxygen atoms. The two carbonyl oxygen atoms of
1-PPh4 are hydrogen-bonded with one water molecule each.
However, each carbonyl oxygen of 1-Na is coordinated to
three water molecules of a water–sodium cluster. The C=O
[1.250(4), 1.243(4)], Camide–N [1.341(4), 1.348(4)], and Co–
N [1.902(3), 1.896(3)] bond lengths of 1-PPh4 with one
water molecule for one carbonyl oxygen demonstrated sig-
nificant alternations of shorter, longer, and shorter lengths
in comparison with those of 1-Na, C=O [1.263(4),
N1–Co1–N2
S1–Co1–S2
S1–Co1–N1
97.4(1)
86.51(4)
87.95(9)
S1–Co1–N2
S2–Co1–N1
S2–Co1–N2
174.07(9)
171.07(9)
88.50(9)
2
Bond lengths [Å]
Co1–N1
Co1–S1
Co1–C7
1.973(2)
2.2491(8)
1.844(3)
O1–C3
N1–C3
N2–C7
1.258(3)
1.318(3)
1.150(4)
Bond angles [°]
N1–Co1–N1*
S1–Co1–S1*
S1–Co1–N1
99.7(1)
89.12(4)
85.57(6)
S1–Co1–N1*
C7–Co1–C7*
Co1–C7–N2
174.69(6)
179.8(2)
176.8(3)
3
Bond lengths [Å]
Co1–N1
Co1–N2
Co1–S1
Co1–S2
Co1–C12
Co1–C17
1.996(2)
O1–C4
O4–C8
N1–C4
N2–C8
N2–C7
N4–C17
O1···O7
1.249(4)
1.252(3)
1.320(3)
1.322(3)
1.148(4)
1.148(3)
2.66
1.986(2)
2.1830(7)
2.1723(7)
1.874(3)
1.847(3)
Bond angles [°]
N1–Co1–N2
S1–Co1–S2
S1–Co1–N1
S1–Co1–N2
97.64(8)
93.07(3)
85.25(6)
176.70(6)
S2–Co1–N1
S2–Co1–N2
C12–Co1–C17
Co1–C12–N3
Co1–C17–N4
176.85(6)
83.97(6)
175.6(1)
174.8(2)
174.4(2)
1,257(4)],
Camide–N [1.330(4), 1.332(3)], and Co–N
[1.919(3), 1.918(3)], with three hydrogen bonds for each car-
bonyl oxygen. This alternation in bond lengths provides evi-
dence for the fact that the amidate carbonyl oxygen is affec-
ted by the interaction of the secondary sphere.
[a] The atoms with and without * are related by a crystallographic
mirror plane between each other.
An
octahedral
CoIII
complex,
PPh4[CoIII
-
(L:N2S2)(tBuNC)2] (2), where two of the axial positions of
1 are occupied by tert-butylisocyanide (tBuNC) molecules, able for X-ray analysis. Crystal data is listed in Table 2 and
was also isolated from an ethanol solution that contained a the ORTEP view of the anion moiety of 2 is shown in Fig-
large amount of tBuNC as a red-colored single-crystal suit- ure 3. Four [CoIII(L:N2S2)(tBuNC)2]– anions and four
Eur. J. Inorg. Chem. 2006, 3753–3761
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T. Yano, H. Arii, S. Yamaguchi, Y. Funahashi, K. Jitsukawa, T. Ozawa, H. Masuda
FULL PAPER
Figure 2. An ORTEP view of [Co(L:N2S2)]– anion part of complex 1 with solvated water molecules, which shows 50% probability
ellipsoids. H atoms are omitted for clarity except for those of the water molecules.
+
PPh4 cations are included in a unit cell. Selected bond thiolate groups were oxygenated to sulfinates. The crystal
lengths and angles are listed in Table 1. The tert-butyl group data of a single crystal obtained from an EtOH/ether solu-
of the axial ligands and the propylene group of the equato- tion is listed in Table 2, and the molecular structure re-
rial ligand are disordered. The octahedral coordination vealed the formation of complex 3, as depicted in Figure 4.
formed by the axial binding of tBuNC ligands to 1 induces Selected bond lengths and angles are listed in Table 1. The
longer Co–Namide and Co–S bonds in comparison with bond lengths around the metal center [Co1–N1 = 1.996(2),
those of 1, although the average bond lengths of Co–S Co1–N2 = 1.986(3), Co1–S1 = 2.1830(7), Co1–S2 =
[2.2491(8) Å] and Co–Namide [1.973(2) Å] are within the 2.1723(7) Å] lay within the range of those of the previously
range of those of the previously reported octahedral CoIII reported CoIII complexes with amide and sulfinate groups
complexes with thiols and/or amides (Co–S = 2.22–2.27 Å, (Co–N = 1.94–2.00 Å, Co–S = 2.21–2.23 Å).[18a,19b] The
Co–N = 1.91–2.00 Å).[18a,19a,19b,19d] The detailed examina- Co–N and Co–S bond lengths of 3 are longer than those
tion of the bond lengths around the Co center indicate that of 1-PPh4 and 1-Na, which is reasonably explained by the
the Co–N and Co–S bonds are clearly elongated in com- coordination of the tBuNC molecules. The oxygenation of
parison with those of complexes 1-PPh4 and 1-Na, which is sulfur in complexes 2 and 3 induced the lengthening of the
explained by the coordination of the tBuNC molecules.
Co–N bonds and the shortening of the Co–S bonds, al-
though it did not influence the Co–C(tBuNC) bond lengths.
The carbonyl oxygen atoms of the ligand L:N2(SO2)2 were
also significantly coordinated to an ethanol molecule. Such
a hydrogen bond was not found around the sulfinate oxygen
atoms.
Figure 3. An ORTEP view of [Co(L:N2S2)(tBuNC)2]– anion part
of complex 2, which shows 50% probability ellipsoids.
Furthermore, treatment of complex 2 with 30% H2O2
according to a previously-reported procedure[19b] with some
modifications successfully afforded the corresponding com-
Figure 4. An ORTEP view of [Co{L:N2(SO2)2}(tBuNC)2]– anion
part of complex 3, which shows 50% probability ellipsoids. H
plex, PPh4[Co{L:N2(SO2)2}(tBuNC)2] (3), of which the two atoms are omitted for clarity.
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CoIII Complexes with Square-Planar N2S2- and N2(SO2)2-Type Ligands
FULL PAPER
Coordination Equilibrium of tBuNC to 1 in Solution
been estimated. Equation (2) was derived on the basis of
Equation (1) with material balances taken into account.
The electronic absorption spectrum of complex 1 showed
a ligand-to-metal charge transfer (LMCT) band around
600 nm (ε = 3800 –1 cm–1), which is characteristic of a
square planar CoIII complex with sulfur atoms.[18c,19c] The
coordination behavior of the monodentate ligands at the
axial positions of 1 was studied with anionic ligands, such
as sodium phenolate, sodium thiophenolate, tetraethylam-
monium thiocyanate (Et4NSCN), and tetraphenylphospho-
nium cyanide (PPh4CN), and with neutral ligands, such as
water, 4-phenylpyridine, and tert-butylisocyanide (tBuNC).
Significant spectral changes were observed only when
PPh4CN or tBuNC was added to a solution of 1 at ambient
temperature. For PPh4CN and tBuNC, the absorption spec-
tra had a change in the isosbestic point at 301 nm and
317 nm in EtOH, respectively (Figure S3). It is clear that
both the monodentate ligands coordinate at the axial sites
of 1. In the case of tBuNC, such a spectral change in the
isosbestic point of the LMCT band was also observed in
other solvents, which indicates that the CoIII complex re-
acted with tBuNC to form octahedral complex 2.[18a,19c]
Formation of the octahedral complex was also supported
by the fact that the finally obtained spectrum was in good
agreement with that of 2. The axial coordination of tBuNC
in complex 2 is also clear from the fact that complexes 1
and 2 show a paramagnetism and a diamagnetism, respec-
(2)
Here, A0 and A represent the absorption intensities of
the [CoIII(L:N2S2)]– solution and that of the mixed solution
of [CoIII(L:N2S2)]– and [CoIII(L:N2S2)(tBuNC)2]–, respec-
tively. CBu and CCoL are the total concentrations of tBuNC
and the [CoIII(L:N2S2)]– solutions, respectively. The rela-
tionship between the absorbance term of the complex ver-
sus the concentration term of added tBuNC was plotted
according to Equation (1) and showed a linear relationship
with a slope of 2, within experimental error. This indicates
that two tBuNC molecules are coordinated to the CoIII
complex in all of the solvents. This result was also con-
firmed from the similarity in the absorption spectrum of 2
as described above. The equilibrium constant, Ks, clearly
showed the solvent dependence (Table S1); they gradually
decreased as follows: H2O/MeOH (3:7) Ͼ H2O/MeOH (1:9)
Ͼ methanol Ͼ ethanol Ͼ 2-PrOH Ͼ dichloromethane Ͼ
acetone. The constant, Ks, demonstrated a linear relation-
ship only for the acceptor number (AN)[25–28] of solvents
which is an index of electrophilicity[28,29] [Figure 5 (᭿)]. Ks
did not show this relationship for any other physical param-
eters such as a donor number[29,30] or a dielectric constant.
This result indicates that an interaction of the electrophilic
solvent with 1 makes the axial coordination of tBuNC eas-
ier.
1
tively, in their H NMR spectra (Figure S4). It should be
noted that the octahedral coordination of 2 was maintained
without any release of the axial ligands in an aqueous solu-
tion, although the coordination of the ligands was not con-
served in organic solvents (Figure S5). Therefore, we can
conclude that the octahedral structure of complex 2 is
maintained in water, which is comparable to the effect of
the oxygenation of the sulfur atoms.
For the complete formation of octahedral species in ace-
tone, the addition of a 1000-fold excess of tBuNC in meth-
anol was required. The electrostatic effect on the metal cen-
ter that is caused by the interaction between the coordi-
nated amide groups and the solvent is best understood
through the examination of the equilibrium experiment
with complex 1 for the following reasons: (1) the sulfur
atoms of the tetradentate ligand are not easily air-oxidized
in the preparation of complex 1; therefore, direct interac-
tions of the oxygenated sulfinate oxygen atoms with the sol-
vent molecules of the secondary coordination sphere can
be eliminated, (2) the solvation of the neutral monodentate
ligand tBuNC that is coordinated to the complex can be
excluded, (3) the axial coordination of the ligands to the
square-planar complex induces a higher lability of the com-
plex. Therefore, we investigated the effect of solvents on the
equilibrium constant (Ks) [Equation (1)].
Figure 5. Plots of logKs values (᭿) and C=O stretching vibration
values (᭺) for complex 1 versus ANs of organic solvents. (a) THF;
(b) acetone; (c) DMF; (d) MeCN; (e) CH2Cl2; (f) CHCl3; (g) 2-
propanol; (h) EtOH; (i) MeOH; (j) H2O/MeOH [1:9 (v/v)]; (k)
H2O/MeOH [3:7 (v/v)].
Infrared Spectra of 1 and 3 in Organic Solvents
As described above, the coordination of tBuNC at the
axial sites of 1 depends on the electrophilic solvents, whose
solvent dependence should be considered to be caused by
the interaction of complex 1 with the solvent molecules. The
electron-rich carbonyl oxygen atoms of the ligand L can
also interact with the electrophilic solvent. Therefore, IR
(1)
With the use of the intensity change in the LMCT band spectra were obtained for 1 and 3 in order to identify the
(700 nm), the equilibrium constants for the reaction have interaction site of the complexes with the solvent molecules.
Eur. J. Inorg. Chem. 2006, 3753–3761
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. Yano, H. Arii, S. Yamaguchi, Y. Funahashi, K. Jitsukawa, T. Ozawa, H. Masuda
FULL PAPER
The IR spectra of sample solutions without the CoIII com- erties of the complexes was investigated with the use of cy-
plex were measured as a base line. A difference spectrum clic voltammetry with an Ag/Ag+ reference electrode. The
between the solution of 1 and the base line was then voltammogram of 1 exhibited only one quasi reversible re-
adopted. Interestingly, the frequencies assigned to the dox wave assignable to CoIII/CoII with ∆E Ϸ 100 mV and
stretching vibration of C=O are dependent on the solvents ip/ic Ϸ 1.0 within the experimental potential range (–1.7 V
within the range of 1520–1590 cm–1 as listed in Table S2. to –1.1 V). The electrochemical parameters are listed in
The C=O stretching vibrations are linearly dependent on Table S5, in which the redox potentials have been standard-
the ANs as shown in Figure 5 (᭺), which suggests that the ized to the Fc/Fc+ potential. The redox potential is high
axial coordination is predominantly controlled by the inter- enough to maintain the Co center in the 3+ oxidation state
action of the solvent molecules with the carbonyl oxygen and is consistent with those of previously reported N2S2-
atoms.
type Co complexes that contain two amidate nitrogen
The solvent-dependent shift of the C=O stretching fre- atoms and two sulfur atoms as coordinating atoms.[31–33]
quency was also observed for 3, and the C=O stretching The redox potential in methanol is clearly much higher than
vibration for 3 (Table S3) showed a similar shift to that of that in acetone, which is in agreement with the tendencies
1, as shown in Figure 6 (᭿). However, in the same solvents for the equilibrium constant Ks and the C=O stretching vi-
the absorption bands in 3 appeared in a higher energy re- brations. The solvent dependence of the results led us to
gion compared with those of 1. An increase in the C=O expect that the Lewis acidity of the CoIII center could effec-
bond strength of 3 compared with that of 1 induced a de- tively be controlled by the interaction of 1 with the solvent
crease in the Co–N bond strength, which may be explained molecules. The redox potential value (E1/2) assignable to
in terms of the coordination saturation and strong trans CoIII/CoII of 1, as expected, exhibited a solvent dependency
influence of SO2.[15c] On the other hand, remarkably, the with a linear relation to the AN values of the solvents; that
stretching frequency of the S=O group, which is considered is, –1.12 V versus Fc/Fc+ in MeOH (AN = 41.3) and
as another possible interaction site for the solvents, did not –1.73 V in THF (AN = 8.0), respectively (Figure 7, Table
show any significant shift [Figure 6 (᭺), Table S4]. The vi- S6). The redox potential values showed a significantly
bration responsible for NϵC in 3 is also linearly dependent larger positive shift with an increase in AN, which suggests
on the ANs of the solvents (Figure S6, Table S5). The linear that the attractive interaction of the carbonyl oxygen atoms
relationship indicates that the interaction of the carbonyl with the solvent molecules causes an increase in the Lewis
oxygen atoms with the solvent molecules causes a change acidity of the CoIII center and promotes the axial coordina-
in the NϵC vibration, while the sulfinate oxygen did not tion of a monodentate ligand. On the basis of such a linear
show any interaction with the solvents as described above. relation, the ideal redox potential values of 1 in an aqueous
The NϵC vibration showed a significantly higher energy solution (AN = 54.8) and in a noninteracting solvent (AN
shift with an increase in AN, which is similar to the case of = 0) were estimated to be –0.93 and –1.89 V, respectively,
the oxidized sulfur atoms.[18a] This result suggests that the which implies that the interaction of the amidate carbonyl
attractive interaction of the carbonyl oxygen atoms with the group with the water molecules causes a shift in the redox
solvent molecules causes an increase in the Lewis acidity of potential for the CoIII complex by ca. 1 V in comparison
the CoIII center.
with that of a noninteracting solvent.
Figure 6. Plots of C=O (᭿) and S=O stretching vibration values
(᭺) for complex 3 versus ANs of organic solvents. (a) Acetone; (b)
MeCN; (c) CHCl3; (d) 2-PrOH; (e) MeOH.
Figure 7. Plots of E1/2 for complex 1 versus ANs of organic sol-
vents. (a) THF; (b) acetone; (c) DMF; (d) MeCN; (e) 2-PrOH; (f)
EtOH; (g) MeOH.
Furthermore, the reduction potential of complex 3 in
MeOH was observed at –1.27 V and that of 2 in the pres-
Redox Behaviors of 1, 2 and 3 in Organic Solvents
The solvent dependence of the C=O stretching vibrations ence of a large amount of tBuNC in a MeOH solution was
that appeared for complexes 1 and 3 must also influence detected at –2.18 V, which indicates that the oxygenation of
the acidity of the metal center, so the electrochemical prop- the sulfur atoms raises the reduction potential of the CoIII
3758
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Eur. J. Inorg. Chem. 2006, 3753–3761

CoIII Complexes with Square-Planar N2S2- and N2(SO2)2-Type Ligands
FULL PAPER
complex by ca. 0.9 V. The octahedral structure of complex
2 that is maintained in water is comparable to the effect of
the oxygenation of the sulfur atoms. These findings indicate
that the attractive interaction between the carbonyl oxygen
atoms and the water molecules can control the Lewis acid-
ity of the metal ion as well as the effect of the oxygenation
of the sulfur atoms. Consequently, complex 2, as described
above, can maintain the octahedral structure in an aqueous
solution (AN = 54.8) without the release of the axial li-
gands.
Biological Implications
In the crystal structures of both the Co- and Fe-contain-
ing NHases,[9–12] the sulfenate and the sulfinate oxygen
atoms interact with the arginine residue through hydrogen
bonds as described above, and the amidate carbonyl oxygen
atoms are coordinated to water molecules. Moreover, the
water molecules form a hydrogen bonded network with
other water molecules that starts from the tyrosyl oxygen
(βTyr68 in CoIII-type NHases, Figure 8). It has recently
Figure 8. Hydrogen bonding networks near the active site of
NHase: hydroxy oxygen of βTyr68 hydrogen-bonded to amide car-
bonyl oxygen atoms by water molecules.
Experimental Section
been reported by Miyanaga et al. that the mutant of CoIII
-
Reagents and Technique: All manipulations were performed with
Schlenk technique under an Ar atmosphere or in a glove box. Rea-
gents in this study were purchased in reagent grade from Wako
Pure Chemical Industries Ltd. or Tokyo Kasei Kogyo Co. Ltd. and
used without further purification. Solvents in reagent grade were
purchased from Wako Pure Chemical Industries Ltd. and Kanto
Kagaku Co. Inc. and distilled twice prior to use.
type NHase, βY68F, where the hydrogen bonded network
is destroyed by the replacement of tyrosine by phenylala-
nine, does not show significant hydratase function.[22] Mi-
yanaga et al. have explained it on the basis of the crystal
structures as follows: βTyr68 contributes to the capture of a
substrate or an imidate intermediate through the hydrogen
bonds.[22] With the consideration of this report[22] and our
results, we can propose that a higher Lewis acidity of the
central metal ion is required for the axial coordination of
the ligands, which is important for the enzymatic reaction,
and may finely be regulated by the hydrogen bonded net-
work from tyrosine to the amidate oxygen atoms through
water molecules. From the fact that the S=O group did not
show an electrophilic interaction,[28,29] we can conclude that
the sulfinate oxygen atoms do not contribute to the control
of the Lewis acidity of the central metal. If the Lewis acid-
ity of the central metal can be sensitively controlled by these
hydrogen bonding interactions, the reaction will rapidly
proceed and the reaction product will also be rapidly re-
leased from the central metal ion. In the crystal structures
of 1-PPh4, 1-Na, and 3 the carbonyl oxygen atoms of the
ligands L and L:N2(SO2)2 are coordinated to water and eth-
anol molecules, respectively. Furthermore, as shown in the
crystal structure of complex 1-Na, three hydrogen bond in-
teractions with three water molecules for each carbonyl
oxygen influenced the Co–N–C–O bonding system. How-
ever, such an interaction was not found in the sulfinyl oxy-
gen atoms of 3, which may suggest that the sulfinyl oxygen
groups do not significantly interact with the solvent mole-
cules.
Synthesis of H4L: 2-benzylthio-2-methylpropionic acid chloride
was synthesized as described previously.[34] The acid chloride
(12.77 g, 50.00 mmol) was added dropwise to a THF solution con-
taining 1,3-diaminopropane (1.85 g, 25.00 mmol) and triethylamine
(5.06 g, 50.00 mmol). After filtration, a 1  HCl aqueous solution
was added to the filtrate and evaporated. Addition of diethyl ether
to the concentrated solution afforded a white powder which was
1
identified as an S-benzylated L by H NMR spectroscopy, whose
powder (2.00 g, 4.36 mmol) was dissolved in THF (20 mL) and
treated with liquid ammonia at –78 °C. To this solution was added
sodium (1.91 g, 82.95 mmol), which was stirred vigorously for
30 min. After the excess ammonia was removed by evaporation at
room temperature, the solution was adjusted to pH 3 with 1 
KHSO4. The product was extracted with ethyl acetate and the sol-
1
vent evaporated. H NMR (300 MHz, CDCl3): δ = 1.63 (s, 12 H),
1.70 (m, 2 H), 2.29 (s, 2 H), 3.28 (q, 4 H), 7.83 (s, 2 H) ppm.
Preparation of Na[Co(L:N2S2)] (1-Na) and PPh4[Co(L:N2S2)] (1-
PPh4): Na[Co(L:N2S2)] (1-Na) was prepared according to the lit-
erature with some modifications.[18,19] Recrystallization of the com-
pound from acetonitrile/diethyl ether afforded a single crystal in
atmosphere. Addition of PPh4Cl (269.4 mg, 0.72 mmol) to an aque-
ous solution (20 mL) of the sodium salt (256.3 mg, 0.72 mmol) af-
forded a single crystal of PPh4[Co(L:N2S2)] (1-PPh4). C35H40CoN2-
O3PS2 (690.74): calcd. C 60.86, H 5.84, N 4.06; found C 60.93, H
5.77,
N 4.03. Electronic absorption spectra (CHCl3): λmax
[ε (–1 cm–1)] = 312 (7300), 443 (sh), 640 (2600) nm.
In conclusion, this paper describes that the hydration re-
action in NHase is significantly controlled by the amidate
groups as well as the oxygenated sulfur groups.
Preparation of PPh4[Co(L:N2S2)(tBuNC)2] (2): Monodentate li-
gand, tBuNC, (200 µL, 1.76 mmol) was added to an acetonitrile
solution
(150 µL)
containing
PPh4[Co(L:N2S2)]
(3 mg,
Eur. J. Inorg. Chem. 2006, 3753–3761
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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T. Yano, H. Arii, S. Yamaguchi, Y. Funahashi, K. Jitsukawa, T. Ozawa, H. Masuda
FULL PAPER
0.43·10–2 mmol). A red crystal was isolated by the slow diffusion of
diethyl ether into the solution at 0 °C. C45H56CoN4O2PS2 (838.99):
tallographic software package.[36] CCDC-606977, CCDC-252730,
CCDC-606976, and CCDC-281586 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
calcd. C 64.42, H 6.73, N 6.68; found C 64.14, H 6.60, N 6.47.
1
Selected IR bands (KBr): ν = 2176 (s, CN), 1535 (s, CO) cm–1. H
˜
NMR (300 MHz,D2O): δ = 1.36 (s, 12 H, CH3), 1.49 (s, 18 H, tBu),
1.75 (t, 2 H, CH2), 3.79 (t, 4 H, CH2), 7.72 (m, 20 H, Ph) ppm.
Electronic absorption spectra (H2O): λmax [ε (–1 cm–1)] = 294
(12800), 360 (sh) nm.
Other Physical Measurements
Elemental analysis was performed with a Perkin–Elmer 2400II
Preparation of PPh4[Co{L:N2(SO2)2}(tBuNC)2] (3): To an ethanol
CHNS/O full-automatic analyzer. All mass spectra were acquired
solution (1 mL) containing PPh4[Co(L:N2S2)] (83 mg, 0.12 mmol) with a LCT mass spectrometer equipped with an ionspray interface
was added tBuNC (191 µL, 1.44 mmol). After the solution was (Micromass Limited, Manchester, UK). Instrument settings, data
stirred for 10 min, 30% H2O2 (0.62 mL, 5.45 mmol) was added acquisition, and data processing were controlled by a computer
dropwise to the ethanol solution at –10 °C. The solution was left
to stand for 3 h at –10 °C. A crude compound was obtained by the
addition of diethyl ether into the solution. Recrystallization of the
compound from ethanol/diethyl ether afforded a single crystal suit-
able for X-ray analysis. C47H64CoN4O8PS2 (967.07): calcd. C 58.37,
H 6.67, N 5.79; found C 58.14, H 6.50, N 6.00. Selected IR bands
with Windows NT operating system. Samples were introduced with
a single syringe pump (KD scientific Inc., USA) fitted with Hamil-
ton syringes (Hamilton Co, Reno, NE). The samples for all spectral
1
measurements were prepared in MeCN or MeOH. H NMR spec-
tra were recorded with a Varian Gemini-300 FT-NMR instrument.
Electronic absorption spectra were measured with a JASCO V-550
(KBr) : ν = 2199 (s, CN), 1552 (s, CO), 1214 and 1071 (s, SO) cm–1. or V-570 spectrophotometer in the wavelength range of 900 –
˜
1H NMR (300 MHz, [D6]DMSO): 1.15 (s, 12 H, CH3), 1.34 (s, 18 250 nm. A match paired quartz cell was used with a 10 mm length.
H, tBu), 3.16 (t, 6 H, CH2), 7.83 (m, 20 H, Ph) ppm. Electronic Infrared spectral measurements were carried out with a JASCO
absorption spectrum (EtOH): λmax [ε (–1 cm–1)] = 273 (9700), 313 FT/IR 410 spectrophotometer. Solid samples were prepared with a
(21400), 400 (sh) nm. ESI-MS ([CoIII(L:N2(SO2)2)(tBuNC)2]–):
m/z (%) = 563.
pressure greater than 5 tons in KBr powder. Spectra in solution
were measured with the use of a double-piled CaF2 or KBr cell
with 0.1 mm thick walls for the C=O and S=O stretching fre-
quencies of the sulfinates. Differential spectra between the sample
and the corresponding solvent were used to verify the spectral fea-
tures. Electrochemical measurements were performed with a BAS
BIOANALYTICAL Systems model CV-50W or with a ALS/CH
Instruments Electrochemical Analyzer Model 600A. Pt electrodes
were adopted as the working- and counter-electrode and an Ag/
Ag+ electrode was used as a reference. Cyclic voltammograms were
collected in a glove box under an Ar atmosphere. The potential
values were revised on the Fc/Fc+ standard.
X-ray Structural Analysis: Crystals suitable for X-ray diffraction
measurements were mounted on glass fibers. The diffraction data
were collected with a Rigaku Mercury diffractometer using graph-
ite-monochromated Mo-Kα radiation at –100 °C with the oscilla-
tion technique. Crystal data and experimental details are listed in
Table 2. All structures were solved by a combination of direct
methods and Fourier techniques. Non-hydrogen atoms were aniso-
tropically refined by full-matrix least-squares calculations. Hydro-
gen atoms were included but not refined. Refinements were contin-
ued until all shifts were smaller than one-tenth of the standard
deviations of the parameters involved. Atomic scattering factors
and anomalous dispersion terms were taken from the International
Tables for X-ray Crystallography.[35] All calculations were carried
out with a Japan SGI workstation computer with the teXsan crys-
Determination of Equilibrium Constants
Equilibrium constants were spectrophotometrically determined by
means of spectral changes of 1 upon the addition of a suitable
Table 2. Crystallographic data and experimental details for Na[Co(L:N2S2)] (1-Na), PPh4[Co(L:N2S2)] (1-PPh4),
PPh4[Co(L:N2S2)(tBuNC)2] (2) and PPh4[Co{L:N2(SO2)2}(tBuNC)2] (3).
1-Na
1-PPh4
2
3
Empirical formula
Formula mass
C11H24CoN2O5S2Na
410.36
C35H40CoO3N2PS2
690.74
C22.5H28Co0.5ON2P0.5
419.49
S
C47H62CoO7N4PS2
949.06
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
triclinic
P1 (No. 2)
monoclinic
P21/n (No. 14)
11.658(1)
14.085(1)
20.977(2)
tetragonal
P4 (No. 81)
16.7400(7)
monoclinic
P21/a (No. 14)
10.4277(4)
33.484(1)
14.1350(5)
¯
¯
9.949(3)
9.958(3)
10.156(3)
69.48(2)
69.54(2)
74.05(2)
869.5(5)
2
7.8718(5)
100.159(4)
95.288
γ [°]
V [A3]
3390.3(5)
4
1.353
1448.00
7.14
2205.9(2)
4
1.263
888.00
5.60
4914.4(3)
4
1.283
2008.00
5.18
Z
Dcalcd. [gcm–3]
1.567
428.00
12.73
F(000)
µ [cm–1]
λ [Å]
T [K]
0.71070
173
0.71070
173
0.71070
173
0.17070
173
No. of refls. measured
No. of refls. used [I Ͼ 2σ(I0)]
6948
2892
0.041/0.117
1.05
27401
5649
0.049/0.102
1.08
17735
4584
0.040/0.110
1.12
37445
8175
0.051/0.13
1.11
[b]
R1[a]/Rw
GOF
3760
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CoIII Complexes with Square-Planar N2S2- and N2(SO2)2-Type Ligands
FULL PAPER
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stant at 5.00·10–5 moldm–3.
Supporting Information (see footnote on the first page of this arti-
cle):Crystal packing of 1-Na with hydrogen bonding interactions
(Figure S1), crystal packing of 1-PPh4 (Figure S2), representative
UV/Vis spectral change of 1-PPh4 due to the addition of mono-
dentate ligand (Figure S3), 1H NMR spectra of 1-PPh4 and 2 (Fig-
ure S4), UV/Vis spectra of 2 (Figure S5), plots of NϵC stretching
vibration values for 3 versus ANs (Figure S6), tables of equilibrium
constants, C=O stretching vibration values for 1 and 3, S=O
stretching vibration values for 3, NϵC stretching vibration values
for 3, electrochemical parameters for 1 (Tables S1–S6) as PDF files.
Acknowledgments
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This work was supported partly by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports, and Cul-
ture of Japan (H.M. and Y. F.) and supported in part by a grant
from the NITECH 21st Century COE Program, to which our
thanks are due. We also thank to Prof. Masafumi Odaka, Tokyo
University of Agriculture and Technology, who kindly provided
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Received: May 15, 2006
Published Online: August 3, 2006
Eur. J. Inorg. Chem. 2006, 3753–3761
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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