Copper complexes of a new tridentate imidazole-containing ligand: Spectroscopy, structures and nitrite reductase reactivity: The molecular structures of [Cu(biap)(NO2)2] and [Cu(biap)Br2]

Article abstract of DOI:10.1016/S0020-1693(00)00271-1

The ligand N,N-bis(2-ethyl-5-methyl-imidazol-4-ylmethyl)-aminopropane (abbreviated as biap) forms several copper(II) coordination compounds with general formula [Cu(biap)X₂], with X^- = Cl^-, Br^-, NO₃^-, NO₂^- and NCS^-. In all these compounds the ligand biap acts as a tridentate ligand. Single crystals of [Cu(biap)(NO₂)₂] and [Cu(biap)Br₂] were used for X-ray structure determinations. All compounds were characterized by IR, ligand field and EPR spectroscopy, and cyclic voltammetry. The geometry of copper(II) in the (pseudo-) halide complexes is five-coordinate, whereas copper(II) is distorted octahedrally coordinated in the nitrate and nitrite complexes, whereby one of the anions binds didentate chelating and the other one monodentate. The affinity of nitrite for the [Cu(biap)]²⁺ cation was studied by determining the binding constant of the anion to the perchlorate salt of the complex in methanol solution. The data show that nitrite exhibits high affinity for the complex and its behavior is comparable with that of azide. The reduction of nitrite, to produce nitric oxide, by the reduced complex [Cu(biap)]⁺ has been studied anaerobically. Kinetic experiments showed that the reaction exhibits saturation behavior with respect to nitrite concentration. The kinetic data indicate that [Cu(biap)]⁺ has a moderate efficiency in the nitrite reductase reaction. (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S0020-1693(00)00271-1

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Inorganica Chimica Acta 310 (2000) 41–50
Copper complexes of a new tridentate imidazole-containing ligand:
spectroscopy, structures and nitrite reductase reactivity
The molecular structures of [Cu(biap)(NO2)2] and [Cu(biap)Br2]
Michela Beretta a, Elisabeth Bouwman a,*, Luigi Casella b, Be´ne´dicte Douziech a,
Willem L. Driessen a, Lucia Gutierrez-Soto a, Enrico Monzani b, Jan Reedijk a
a Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden Uni6ersity, PO Box 9502, 2300 RA Leiden, The Netherlands
b Dipartimento di Chimica Generale, Uni6ersita di Pa6ia, Via Taramelli 12, 27100 Pa6ia, Italy
Received 28 April 2000; accepted 8 August 2000
Abstract
The ligand N,N-bis(2-ethyl-5-methyl-imidazol-4-ylmethyl)-aminopropane (abbreviated as biap) forms several copper(II) coordi-
nation compounds with general formula [Cu(biap)X2], with X−=Cl−, Br−, NO3−, NO2− and NCS−. In all these compounds
the ligand biap acts as a tridentate ligand. Single crystals of [Cu(biap)(NO2)2] and [Cu(biap)Br2] were used for X-ray structure
determinations. All compounds were characterized by IR, ligand field and EPR spectroscopy, and cyclic voltammetry. The
geometry of copper(II) in the (pseudo-) halide complexes is five-coordinate, whereas copper(II) is distorted octahedrally
coordinated in the nitrate and nitrite complexes, whereby one of the anions binds didentate chelating and the other one
monodentate. The affinity of nitrite for the [Cu(biap)]2+ cation was studied by determining the binding constant of the anion to
the perchlorate salt of the complex in methanol solution. The data show that nitrite exhibits high affinity for the complex and its
behavior is comparable with that of azide. The reduction of nitrite, to produce nitric oxide, by the reduced complex [Cu(biap)]+
has been studied anaerobically. Kinetic experiments showed that the reaction exhibits saturation behavior with respect to nitrite
concentration. The kinetic data indicate that [Cu(biap)]+ has a moderate efficiency in the nitrite reductase reaction. © 2000
Elsevier Science B.V. All rights reserved.
Keywords: X-ray crystal structures; Copper(II) complexes; Tridentate imidazole-containing ligand; Cyclic voltammetry; EPR spectroscopy; Ligand
field
1. Introduction
one amine and two azole nitrogens, has been designed
and successfully synthesized. A synthesis scheme is
given in Fig. 1.
Among the biological copper sites, typical mononu-
clear centers with histidine coordination (type 2 centers)
are those contained in amine oxidases [2] and copper
nitrite reductases [3]. In the first case, a protein-derived
redox cofactor, topaquinone, produced by oxidative
The imidazole group of histidine is involved in the
coordination of most copper(II) sites which are present
in naturally occurring protein systems [1]. Low-molecu-
lar weight compounds, especially those containing imi-
dazole-based ligands, may mimic to a certain extent
structure or function of the active site of the copper
enzyme, while reaction studies with biomimetics may
shed light on the catalytic aspects. In this respect, the
ligand N,N-bis(2-ethyl-5-methyl-imidazol-4-ylmethyl)-
aminopropane (biap), a bulky tridentate ligand with
* Corresponding author. Tel.: +31-71-527 4550; fax; +31-71-527
4451.
E-mail address: bouwman@chem.leidenuniv.nl (E. Bouwman).
Fig. 1. Synthesis of the ligand biap.
0020-1693/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0020-1693(00)00271-1

42
M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
modification of a tyrosine residue, is also contained [4].
This organic cofactor has an active role in the enzy-
matic reaction and, therefore, the activity of amine
oxidases is difficult to reproduce in a model system. In
the case of copper nitrite reductases, the enzyme con-
tains additionally a type 1 (‘blue’) center, which delivers
an electron to the adduct formed by the type 2 center
and the nitrite substrate [5]. The mechanism of the
reaction is complicated by the participation of protons,
according to the equation:
formed at room temperature in the solid state, or at 77
K as frozen dmso solutions with a JEOL Esprit RE-2X
spectrometer and a JEOL Esprit 330 ESRE data sys-
tem. The electrochemistry measurements were per-
formed with an Autolab PGstat10 potentiostat
controlled by GPES4 software. A three-electrode sys-
tem was used, containing a platinum working electrode,
a platinum auxiliary electrode and an Ag/AgCl refer-
ence electrode. The experiments were carried out in
acetonitrile at room temperature (r.t.) under an argon
atmosphere with tetrabutylammonium hexafluorophos-
phate as supporting electrolyte. Under these conditions,
the ferricenium–ferrocene couple was located at +0.44
V with a peak separation of 100 mV.
NO2− +e− +2H+“NO+H2O
(1)
and no details of how the reaction proceeds are avail-
able [5]. This enzymatic reaction is easier to handle in a
model system, since only a reduced copper species and
protons are necessary to reduce bound nitrite.
2.2. Synthesis of the coordination compounds
In the past decade, the research of our group was
focused on the design and development of chelating
azole containing ligands for first row transition metals
[6]. The present paper describes the synthesis and char-
acterization of coordination compounds of the ligand
biap with various copper salts, and includes two single-
crystal X-ray structures.
The complex containing nitrite has biomimetic rele-
vance, prompting us to give a more complete character-
ization of the behavior of the Cu(biap)-nitrite adducts
in solution, in relation to the activity of copper nitrite
reductases. To this end, we have investigated the bind-
ing affinity of nitrite towards the [Cu(biap)]2+ complex,
and its reductive activation to NO by the reduced
Cu–biap complex in the presence of protons, reproduc-
ing the biological reaction described by Eq. (1). Coordi-
nation compounds of biap with other transition metals
are described elsewhere [6a].
2.2.1. [Cu(biap)Cl2], [Cu(biap)Br2] and [Cu(biap)(NO3)2]
Triethylorthoformate (3 ml) was added to the hy-
drated copper salt (1 mmol) for dehydration, followed
by 10 ml of absolute ethanol. The ligand biap (1 mmol)
was dissolved in 15 ml of absolute ethanol. The two
solutions were heated to boiling before mixing. The hot
reaction mixture was filtered through a paper filter in
order to remove any solid impurities, and the complex
crystallized on cooling or after standing for several days
(slow evaporation of the solvent). Yields varied from 40
to 60%.
2.2.1.1. [Cu(biap)Cl2]. Anal. Calc. for C17H29CuCl2N5:
C, 46.6; H, 6.67; Cl, 16.2; Cu, 14.5; N, 16.0. Found: C,
46.26; H, 6.56; Cl, 15.8; Cu, 14.7; N, 16.15%. IR (KBr,
cm−1) 3200–2700 (vs, multiple bands), 1645 (m), 1540
(m), 1465 (vs), 1389 (w), 1369 (w), 1352 (w), 1326 (w),
1289 (m), 1237 (w), 1137 (w), 1100 (m), 1085 (s), 1047
(s), 996 (m), 958 (m), 918 (m), 854 (w), 826 (w), 801 (s),
786 (s), 732 (w), 564 (m), 344 (w), 304 (m).
2. Experimental
2.1. General
2.2.1.2. [Cu(biap)Br2]. Anal. Calc. for C17H29Br2CuN5:
C, 38.8; H, 5.55; Br, 30.3;Cu, 12.1; N, 13.3. Found: C,
36.39; H, 5.32; Br, 29.9; Cu, 11.8; N, 12.83%. IR (KBr,
cm−1) 3200–2700 (vs, multiple bands), 1637 (m), 1541
(m), 1464 (vs), 1431 (s), 1408 (w), 1373 (w), 1336 (m),
1325 (m), 1287 (m), 1255 (w), 1089 (s), 1066 (s), 991
(w), 957 (w), 932 (w), 862 (m), 826 (w), 758 (s), 726 (w),
487 (w), 349 (w).
All starting materials were commercially available
and used as purchased. The synthesis of the ligand biap
has been described in detail elsewhere [6a].
Metal analyses were carried out complexometrically
with Na2H2edta as the complexing agent. Halogen
analyses were performed by potentiometric titrations
with silver nitrate. C, H, N, S-analyses were performed
on a Perkin–Elmer 2400 series. IR spectra in the 4000–
300 cm−1 range of the samples, pelleted in KBr or as
nujol mulls, were recorded on a Perkin–Elmer Paragon
1000 FT-IR spectrophotometer. Ligand field spectra of
the solids (300–2000 nm, diffuse reflectance) were taken
on a Perkin–Elmer 330 spectrophotometer equipped
with a data station. 1H and 13C NMR spectra were
recorded on a JNM-FX 200 spectrometer. Electron
paramagnetic resonance (EPR) measurements were per-
2.2.1.3.
[Cu(biap)(NO3)2].
Anal.
Calc.
for
C17H29CuN7O6: C, 41.6; H, 5.95; Cu, 12.9; N, 20.0.
Found: C, 41.53; H, 5.81; Cu, 13.0; N, 20.21%. IR
(KBr, cm−1) 3209 (s), 3156 (vs), 3075 (s), 2972 (vs),
2936 (s), 2878 (m), 1756 (w), 1712 (w), 1648 (m), 1548
(s), 1481 (vs, br), 1350 (vs, br), 1285 (vs, br), 1091 (s),
1040 (m), 1008 (s), 993 (w), 958 (w), 916 (m), 852 (w),
824 (w), 811 (m), 788 (w), 740 (w), 573 (w), 355 (w).

M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
43
2.2.2. [Cu(biap)(NCS)2] and [Cu(biap)(NO2)2]
complex in a 10 cm path-length cuvette at 2590.1°C.
The high affinity binding constants for N3− and NO2−
were obtained by analyzing the variation of the ab-
sorbance according to the method described previously
[7].
Copper tetrafluoroborate hexahydrate (1 mmol) was
dissolved in 10 ml of ethanol. The ligand biap (1 mmol)
was dissolved in 10 ml of ethanol. The two solutions
were heated to boiling before mixing. A solution of
ammonium thiocyanate (2 mmol) in 2 ml of water, or
sodium nitrite in 5 ml of water, respectively, was then
added. The hot reaction mixture was filtered through a
paper filter in order to remove any solid impurities, and
the complex crystallized on cooling or after standing
for several days (slow evaporation of the solvent).
Yields varied from 40 to 60%.
2.4. Nitrite reductase acti6ity
Since attempts to isolate the Cu(I) complex [Cu-
(biap)](PF6) gave an extremely air-sensitive material,
which was difficult to handle, the nitrite reductase
activity was studied preparing the complex in situ, from
biap and [Cu(MeCN)4]PF6. The reactions were per-
formed at −14°C, in a 1/1 (v/v) methanol–acetonitrile
mixture, which allows good solubility of all reagents,
under anaerobic conditions using Schlenk techniques,
as we did before for related Cu(I) systems [7]. The
progress of the reaction was followed by monitoring the
development of the d“d bands of the copper(II) com-
plex produced during the reduction of NO2−. The
absorbance of the solution was measured using a cus-
tom-designed fiber-optic quartz probe (0.5 cm path-
length) directly immersed in the reaction solution. The
experiments were performed keeping the concentration
of [Cu(biap)]+ constant at 1.5 mM, while the molar
ratio of NO2−/Cu(I) was varied from 5 to 50. The
reactions were started with the addition of a few micro-
liters (2 H+ equivalents with respect to Cu(I)) of a
degassed and concentrated solution of HBF4 (used as
approximately 50% in diethyl ether) in methanol. In
order to reduce the noise in the measurements, the
difference between the absorbance at 626 nm (where the
Cu(II) d“d band develops) and 500 nm (where the
spectral variations are minimal) were observed. The
reaction rates were obtained from the data in the first
few seconds of the reactions. Transformation of the
rate data from absorbance/s to s−1 was carried out
using the molar absorbance of the copper(II)–nitrite
adduct, which is the product of the reaction in the
presence of excess nitrite (obtained by extrapolating the
absorbance data to infinite time), and the initial con-
centration of the copper(I) complex. The dependence of
the initial rate of nitrite reduction on the copper(I)
concentration, studied at constant nitrite (75 mM) and
HBF4 (3 mM) concentration and varying that of [Cu-
(biap)](PF6) (from 0.7 to 2.2 mM), as expected, gave a
linear behavior.
2.2.2.1. [Cu(biap)(NCS)2]. Anal. Calc. for C19H29-
CuN7S2: C, 47.2; H, 6.05; Cu, 13.2; N, 20.3. Found: C,
45.81; H, 5.83; Cu, 13.1; N, 20.61%. IR (KBr, cm−1
)
3150 (vs, multiple peaks), 2966 (s), 2933 (m), 2875 (w),
2080 (vs, br), 1642 (m), 1541 (m), 1466 (s), 1376 (m),
1347(w), 1324(w), 1287 (m), 1254(w), 1233 (w),
1141(w), 1089 (s), 1062 (w), 1039 (s), 988 (m), 952 (m),
916 (m), 854 (m), 772 (s), 642 (w), 601 (w), 560 (m), 477
(m).
2.2.2.2. [Cu(biap)(NO2)2]. Anal. Calc. for C17H29-
CuN7O4: C, 44.48; H, 6.37; N, 21.4. Found: C, 43.23;
H, 6.08; N, 20.94%. IR (KBr, cm−1) 3430 (m, br), 3194
(m), 3137 (s), 3065 (m), 2970 (vs), 2934 (s), 2875 (m),
1641 (m), 1547 (m), 1470 (vs), 1394 (s), 1380 (m), 1330
(m), 1267 (s), 1225 (vs,br), 1123, vs, 1090 (s), 1058 (s),
990 (w), 956 (w), 927 (w), 862 (w), 827 (w), 788 (w), 759
(w), 487 (w), 331 (m).
2.2.3. [Cu(biap)(H2O)x](ClO4)2(H2O)4−x
This complex was prepared by adding a 0.056 M
solution of copper(II) perchlorate hexahydrate in etha-
nol (3.18 ml) to a solution of biap (54.1 mg) dissolved
in the same solvent (20 ml). The green solution thus
obtained was stirred for 0.5 h and then concentrated to
a small volume under vacuum. To complete precipita-
tion, a small amount of diethyl ether was added and the
mixture was kept in a refrigerator for several hours.
The blue–green precipitate was collected by filtration
and dried under vacuum (Yield 85%). Anal. Calc. for
C17H29Cl2CuN5O8·4H2O: C, 32.00; H, 5.85; N, 10.98.
Found: C, 31.66; H, 5.69; N, 11.24%. IR (Nujol mull,
cm−1) 3595 (m), 3305 (m), 1649 (m), 1542 (w), 1322
(w), 1287 (w), 1090 (s), 995 (w), 960 (m), 933 (w), 918
(w), 854 (w), 843 (w), 722 (m), 622 (m).
2.5. Crystal structure determinations
2.3. Nitrite and azide binding to
[Cu(biap)(H2O)x](ClO4)2
The reflection intensities were measured and collected
on a four-circle Enraf-Nonius CAD-4 diffractometer
with monochromatized Mo Ka radiation (u=0.71073
The complexation of nitrite and azide to the cop-
per(II) complex of biap was studied by observing the
spectral changes upon addition of a concentrated solu-
tion of the ligands to a dilute methanol solution of the
,
A). Even though we had crystals of a number of
compounds, only two of these had good enough dif-
fraction properties allowing high-resolution structure

44
M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
Table 1
3. Results and discussion
Crystal and refinement data for the structures [Cu(biap)Br2] (1) and
[Cu(biap)(NO2)2] (2)
3.1. Syntheses
1
2
Imidazole-thioether ligands, a large variety of which
have been investigated extensively in our group, are
obtained through a simple one-pot condensation reac-
tion of a thiol and (substituted) 4-hydroxymethylimida-
zole in acetic acid [10]. Unfortunately, amines do not
condense with 4-hydroxymethylimidazole. A route to
the synthesis of this type of chelating imidazole-amine
ligands appeared to be a method based on the Mannich
reaction [11]. In such cases the C5 position needs to be
substituted in order to prevent reaction of both the C4
and C5 positions resulting in the formation of other-
wise interesting cyclic ligands [12,13]. The preparation
of the ligand biap is simple and straightforward result-
ing in the precipitation of the pure ligand in reasonable
yield [6a].
The synthesis of transition metal coordination com-
pounds with the ligand biap is straightforward, but the
isolation of the coordination complexes in many cases
appeared to be tedious. Many solvents have been used
in order to obtain reasonable yields of products [6a]. In
many cases the complexes are very soluble and only
crystallize from very concentrated solutions. However,
the compounds are well defined, often resulting in
(micro-) crystalline material. In contrast with the other
transition metals, the copper complexes with the ligand
biap readily crystallize from ethanolic solutions.
Formula
Mr
Crystal system
Space group
Unit cell dimensions
C17H29Br2CuN5
526.8
monoclinic
P21/c
C17H29CuN7O4
459.01
monoclinic
P21/c
,
a (A)
8.740(6)
25.60(4)
10.04(1)
98.03(5)
2224(4)
4
4.55
293
27.5
8.587(3)
14.374(6)
18.430(6)
100.90(4)
2234(1)
4
1.01
293
,
b (A)
,
c (A)
i (°)
Volume (A )
3
,
Z
v Mo Ka (mm−1
)
T (K)
qmax
29.9
h/k/l
−11, 11/0, 33/0,
12
5075
−11, 12/−20, 20/−25,
0
12908
6374
262
0.052
0.129
−1.35/1.14
Unique reflections
Observed [I\2|(I)] 2084
Pars
R [I\2|(I)]
wR [I\2|(I)]
Dz min./max.
226
0.049
0.047
−1.36/1.49
Table 2
,
Selected bond distances (A), angles (°), and parent atom hydrogen
bond distances (A) for [Cu(biap)Br2] (1)
a
,
Cu1–Br2
Cu1–Br3
Cu1–N13
2.745(5)
2.441(4)
1.965(6)
Cu1–N23
Cu1–N30
1.952(7)
2.151(7)
3.2. Description of the structures
Br2–Cu1–Br3
Br2–Cu1–N13
Br2–Cu1–N23
Br2–Cu1–N30
Br3–Cu1–N13
104.7(1)
95.7(2)
95.0(2)
108.8(2)
95.9(2)
Br3–Cu1–N23
Br3–Cu1–N30
N13–Cu1–N23
N13–Cu1–N30
N23–Cu1–N30
95.8(2)
146.5(2)
161.6(3)
81.5(3)
3.2.1. General
The flexible tridentate ligand biap can coordinate in
either a facial or a meridional fashion to a transition
metal [6a]. In the copper complexes only the meridional
binding mode has been observed. The amine-nitrogen
to imidazole-nitrogen bite angle appears to lie between
80.9(3)
N11···Br2%
3.296(9)
N21···Br2¦
3.369(9)
a Symmetry operations: %=2−x, 1−y, −z; ¦=x, 1/2−y, 1/2+z.
Table 3
,
Selected bond distances (A), angles (°), and parent atom hydrogen
a
,
bond distances (A) for [Cu(biap)(NO2)2] (2)
determinations. For both the structures of [Cu(-
biap)Br2] (1) and [Cu(biap)(NO2)2] (2) a selection of the
crystal data and additional parameters are presented in
Table 1. Intensities were corrected for Lorentz and
polarization effects. The structures were solved with
automated Patterson techniques, using DIRDIF92 [8].
Refinement was accomplished using XTAL [9] by full-
matrix least-squares fitting. Hydrogen atoms were in-
troduced on calculated positions and refined riding on
the atoms they are attached to. Selected bond distances
and bond angles are given in Tables 2 and 3. The
estimated standard deviations in the distances and an-
gles were calculated from the full correlation matrix.
Cu1–N13
Cu1–N23
Cu1–N30
1.958(6)
1.964(6)
2.143(5)
Cu1–O4
Cu1–O7
Cu1–O3
2.012(5)
2.348(6)
2.648(6)
O4–Cu1–O7
O4–Cu1–O3
O4–Cu1–N13
O4–Cu1–N23
O4–Cu1–N30
O7–Cu1–O3
O7–Cu1–N13
O7–Cu1–N23
91.3(2)
50.6(2)
97.3(2)
96.1(2)
165.5(2)
141.4(2)
102.4(2)
87.9(2)
O7–Cu1–N30
N13–Cu1–O3
N13–Cu1–N23
N13–Cu1–N30
N23–Cu1–O3
N23–Cu1–N30
N30–Cu1–O3
103.1(2)
90.0(2)
162.9(2)
81.8(2)
90.4(2)
82.6(2)
114.9(2)
N11···O6%
2.905(8)
N21···O7¦
2.912(7)
a Symmetry operations: %=1−x, 2−y, 1−z; ¦=1−x, 1−y, 1−z.

M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
45
2. Relevant bond distances and bond angles are given
in Table 2. The Addison [15] structural index ~, with
~=(i−h)/60 and h and i being the two largest coor-
dination angles, equals zero for a perfect square pyra-
midal geometry and becomes unity for a perfect
trigonal bipyramidal geometry. For [Cu(biap)Br2], ~
equals 0.25. So, the copper(II) ion is in a square-pyra-
midal environment, which is only slightly distorted
towards trigonal bipyramidal, with Br2 in the apical
position and the three nitrogens of the ligand and the
other bromide anion in the equatorial plane. The cop-
,
per(II) ion lies at 0.431(1) A above the least-squares
plane of N11, N30, N23, and Br3. Hydrogen bonding
between bromide and the imidazole-NH atoms (see
Table 2) tie the individual molecules into an infinite
two-dimensional network.
3.2.3. Structure of [Cu(biap)(NO2)2]
The asymmetric unit consists of one molecule of
[Cu(biap)(NO2)2]. A PLUTON [14] projection and the
atomic labeling are given in Fig. 3. Selected bond
distances and angles, and hydrogen bond distances are
given in Table 3. At first view the copper(II) ion is in a
square pyramidal environment with the two imidazole
(N13 and N23) and the one amine (N30) biap nitrogens
together with a nitrite oxygen (O4) in the equatorial
plane and an oxygen of the other nitrite (O7) at the
apex. The Addison [15] structural index t is almost
equal to zero, which should be indicative for a very
regular tetragonal pyramid; however, a closer inspec-
tion of the coordination angles shows that the geometry
could also be described as distorted octahedral. The
oxygen at the apex leans over towards one side, with
the O7–Cu1–O4 and the O7–Cu1–N23 angles (respec-
tively 91.3(2)° and 87.9(2)°) significantly smaller than
the O7–Cu1–N13 and the O7–Cu1–N30 angles (re-
spectively 102.4(2)° and 103.1(2)°). Moreover the two
nitrite-anions are oriented differently, with O6 of the
apical nitrite pointing away from Cu1, whereas O3 of
the equatorial nitrite is pointing towards Cu1. In the
latter case one should regard the Cu1–O3 distance of
Fig. 2. Pluton projection of [Cu(biap)Br2] (1).
Fig. 3. Pluton projection of [Cu(biap)(NO2)2] (2).
,
2.648(6) A as semi-coordinating, in which case the
80° and 83°. The meridional binding of the biap results
in a flattening of the ligand, as can be deduced from the
trans imidazole angles in the range of 161–163°, which
is very close to the sum of the amine-imidazole bite
angles. Furthermore, the calculated least-squares plane
through both the imidazole rings and the copper ion
results in deviations of the constituting atoms from this
coordination environment of the copper(II) ion can be
described as (very) distorted octahedral. As the copper
ion lies well above the least-squares plane defined by
,
N13, N30, N23, and O4 (at 0.206(1) A), O3 is unlikely
to play an important role in the coordination.
Both monodentate and didentate coordinating nitrito
groups have been reported; the semi-didentate coordi-
nation of the nitrito group is also quite common [7,16].
Interestingly, copper nitrate compounds of bis(2-benz-
imidazolylmethyl)amines have been reported, in which
one of the nitrate anions is binding chelating diden-
tately, with the other anion residing in the crystal
lattice, while the sixth coordination position is occupied
either by a methanol molecule, or by the already bound
nitrate thus forming bridged dinuclear compounds [17].
,
plane of less than 0.06 A. The observed distortions
from ideal coordination geometries can therefore be
attributed chiefly to this restriction of the ligand bite
angles.
3.2.2. Structure of [Cu(biap)Br2]
A
PLUTON [14] projection of [Cu(biap)Br2], also
showing the atomic numbering scheme, is given in Fig.

46
M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
Table 4
Ligand field and EPR data of the copper biap complexes
Compound (color)
LF (solid state, ×103 cm−1
14.0
13.3
14.1
16.1
16.1; 11.8 (sh)
15.7
)
EPR (solid state, 293 K) EPR (dmso solution, 77 K, A in Gauss)
g// 2.25; gÞ 2.06 g1 2.26; g2 2.09; g3 2.06; A// 143
g1 2.24; g2 2.21; g3 2.09 (see text)
Cu(biap)Cl2 (light green)
Cu(biap)Br2 (light green)
Cu(biap)(NCS)2 (green)
Cu(biap)(NO3)2 (blue)
Cu(biap)(NO2)2 (dark green)
[Cu(biap)(H2O)x](ClO4)2
(blue–gray)
g1 2.27; g2 2.11; g3 2.03 g// 2.25; gÞ 2.09; A// 145
g// 2.23; gÞ 2.05
g1 2.23; g2 2.08; g3 2.04 g1 2.25; g2 2.10; g3 2.06; A// 145
g// 2.28; gÞ 2.06 g1 2.28; g2 2.09; g3 2.05; A// 146
g1 2.28; g2 2.10; g3 2.05; A// 139
Hydrogen bridges from the hydrogen atoms of the
imidazole-nitrogen atoms N(11) and N(21) to the coor-
dinating nitrite oxygen atom (O7) and the non-coordi-
nating oxygen O6 of other molecules link the molecules
together, thus forming a one-dimensional chain.
these groups, the position of the ligand field absorption
is intermediate. The X-ray determinations of the com-
pounds [Cu(biap)Br2] and [Cu(biap)(NCS)2] show that
the copper ions are in distorted square pyramidal envi-
ronments, whereas the complex [Cu(biap)(NO2)2] may
also be described as distorted octahedral. From similar-
ities of the various compounds and the elemental analy-
ses there is little doubt to suggest that the coordination
environments of the copper ions in [Cu(biap)Cl2] and
[Cu(biap)(NO3)2] are five-coordinate and octahedral,
respectively. However, the IR spectra of [Cu(biap)Cl2]
and [Cu(biap)Br2] are not completely identical, which
may originate from different hydrogen bond interac-
tions and packing effects.
3.2.4. Unrefined structure of [Cu(biap)(NCS)2]
Single crystals of this compound were also subjected
to X-ray diffraction. The quality of the crystals ap-
peared to be rather poor, therefore the structure could
not be fully refined1. The molecular structure of this
compound is very similar to the structure of [Cu-
(biap)Br2], whereby the thiocyanate anions coordinate
through the nitrogen atoms to the copper(II) ion.
All copper(II) compounds are paramagnetic at r.t. At
first sight, only solid [Cu(biap)Cl2] shows a distinctly
axial EPR spectrum, whereas the spectra of [Cu-
(biap)(NO3)2] and [Cu(biap)(H2O)x](ClO4)2 are nearly
isotropic, with only a faint indication of an axial g
value. The EPR spectra of the other copper compounds
are distinctly rhombic. However, even the spectra
3.3. Spectroscopy and analyses
Elemental analyses of the copper complexes with
coordinating anions revealed that all compounds are
1:1 in metal to ligand ratio, and that neither solvents of
crystallization, nor additional coordinating solvent
molecules are present. Unlike the transition metals
cobalt, nickel and zinc [6a], the use of copper salts with
non-coordinating anions did not result in a complex
with a 1:2 metal to ligand ratio. According to elemental
analysis, the reaction of copper perchlorate with biap in
a 1:1 ratio resulted in a copper complex that precipi-
tated with four molecules of water. In the solid state
two or three of these water molecules will probably be
coordinated to the copper ion in order to complete its
coordination sphere. The spectroscopic data of the
copper coordination compounds obtained with biap are
summarized in Table 4. The ligand field spectra of the
copper(II) complexes, apart from the nitrite compound,
show a single band, from which no detailed conclusions
regarding the geometry of the copper ions can be
drawn. The positions of the absorption bands suggest
that the geometry of the copper ions in the complexes
of the (pseudo-) halides on the one hand, and the
nitrate and nitrite anions on the other hand are similar.
The perchlorate compound does not belong to either of
of
[Cu(biap)Cl2],
[Cu(biap)(NO3)2]
and
[Cu-
(biap)(H2O)x](ClO4)2 show some very weak underlying
rhombicity, which is more clearly expressed in their
dmso frozen solution spectra. The EPR spectra have
been simulated [18] satisfactorily using rhombic g val-
ues and substantial line broadening parameters for one
of the g values. The solution spectra of all compounds
are all very similar, rhombic with a hyperfine splitting
of approximately 140 Gauss. The spectra are seemingly
axial with an apparent gÞ of 2.08, but the simulated
spectra suggest that this really is the average of two g
values at about 2.10 and 2.06. The copper bromide
complex in dmso solution only showed broad signals.
Table 5
Cyclic voltammetry data of copper biap complexes
Compound
Epc (V)
Epa (V)
DEp (V) E1 (V)
2
Cu(biap)Cl2
Cu(biap)(NCS)2
Cu(biap)(NO3)2
−0.07
−0.06
−0.07
+0.03
+0.04
+0.04
+0.11
0.10
0.10
0.11
0.19
−0.02
−0.01
−0.01
+0.02
1 X-ray data for [Cu(biap)(NCS)2]: C19H29CuN7S2, M=483.15,
monoclinic, space group P21, Z=2, a=9.675(7), b=14.544(5), c=
[Cu(biap)(H2O)x](ClO4)2 −0.08
3
,
,
8.861(3) A, i=102.24(6)°, V=1218.5(8) A , serious disorder.

M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
47
Fig. 4. Difference spectra taken during titration of [Cu(biap)]2+ with nitrite in dilute methanol solution. The inset shows the fitting of the plot
of absorbance at 360 nm versus nitrite concentration.
tion potentials suggest that the copper(I) biap complex
should be relatively stable. However, it was found that
the isolated copper(I) compound is very air-sensitive
(see Section 2).
3.4. Cyclic 6oltammetry
The data obtained in electrochemical measurements
of some of the copper complexes in acetonitrile solu-
tions are presented in Table 5. When scanning towards
negative potentials, the complexes [Cu(biap)Cl2], [Cu-
(biap)(NCS)2], [Cu(biap)(NO3)2], and [Cu(biap)(H2O)x]-
(ClO4)2 all show a quasi-reversible reduction wave at a
potential between −20 to +20 mV versus Ag/AgCl.
The peak separations of approximately 100 mV are
quite large for a one-electron reduction, however, these
are in the same order as was found for the Fc/Fc+
couple in the same circumstances. Only the perchlorate
complex [Cu(biap)(H2O)x](ClO4)2 has an even larger
peak separation of 190 mV. The reproducibility of the
voltammogram of [Cu(biap)(H2O)x](ClO4)2 is also less
satisfactorily than those of the other complexes. This
poor reproducibility and the larger peak separation are
probably due to the presence of varying amounts of
water. The reduction potentials are relatively high com-
pared to those of copper complexes with chelating
triamines [19]. The values of the reduction potential as
found for the copper–biap systems are in the same
range as those found for copper complexes of the
similar bis(pyrazolylethyl)amine and bis(benzimidazoly-
lalkyl)amine ligands [7], as well as what was found for
a dinuclear copper compound of a ligand containing a
similar bis(4-imidazolylmethyl)amine moiety [20]. In
contrast with this, the reduction of copper complexes
with tripod tris(imidazolylmethyl)amine ligands occur
at considerably lower potentials [21]. The high reduc-
3.5. Nitrite and azide binding to [Cu(biap)]2+
Since the structure of the biap ligand is correlated
with those of several other tridentate ligands containing
nitrogen heterocycles studied previously [7,16], it is of
interest to compare the binding behavior of nitrite to
the corresponding copper(II) complexes in solution.
The affinity of nitrite to [Cu(biap)]2+ was estimated by
spectrophotometric titration of the anion to the per-
chlorate salt of the complex in dilute methanol solution,
following the development of the near-UV charge
transfer band at 362 nm (m 1000 M−1 cm−1), probably
LMCT in origin. The difference spectra obtained from
the titration are shown in Fig. 4, together with the
fitting of the data corresponding to a simple association
equilibrium. The binding of a single nitrite molecule, in
the strong equatorial coordination position, occurred
using a limited excess of ligand and gave a high affinity
constant K=1.4×106 M−1. For comparison purposes,
a parallel experiment performed using azide in the same
conditions (LMCT band at 386 nm, m 2700 M−1 cm−1
(difference spectra in Fig. 5) gave K=1.9×106 M−1
)
,
confirming the parallel behavior of these two ligands
towards Cu(II). The slightly larger K value observed for
azide, with respect to nitrite, agrees with our previous
observations on the behavior of related systems [7],
since nitrite is essentially monodentate in the Cu(II)–

48
M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
Fig. 5. Difference spectra taken during titration of [Cu(biap)]2+ with azide in dilute methanol solution. The inset shows the fitting of the plot of
absorbance at 386 nm versus azide concentration.
biap complex and, in turn, the nitrite binding mode
(monodentate versus didentate) is ruled by the chelate
ring size formed by the supporting tridentate nitrogen
donor ligand.
after protonation of the bound substrate. The parame-
ters Kp and k can be obtained from the fitting of the
data according to Eq. (4). Kp corresponds to the bind-
ing constant of nitrite to the copper(I) complex. Its
value, Kp=3095 M−1, indicates that [Cu(biap)]+ has
a reduced affinity toward nitrite, comparable to that of
the copper(I) complex with the ligand ddah [7], where
dimethylpyrazole rings replace the alkyl-imidazoles of
biap. The reduced affinity may depend on the fact that
both these Cu(I) complexes contain a ligand bearing
alkyl substituents adjacent to the donor atoms. Com-
plexes with ligands related to biap, but lacking this type
of substituents, exhibit significantly larger affinity for
3.6. Nitrite reductase acti6ity
It has been previously reported [7,16,22] that cop-
per(I) complexes can reduce nitrite to NO in the pres-
ence of protons according to the reaction:
Cu++NO2− +2H+“Cu2+ +NO+H2O
(2)
Kinetic studies on the dependence of the rates on the
concentration of the reactants indicate a first order
dependence on copper(I) and proton concentrations [7].
Instead, the rate follows [NO2−] with a saturation
behavior, and this is true also for [Cu(biap)]+ (Fig. 6).
This behavior indicates that the formation of an adduct
between the copper(I) complex and nitrite is an impor-
tant step in the catalysis. The kinetic observations agree
with the following simplified mechanism:
+ +
H
K
H
p
Cu++NO−2 X [Cu+ꢀNO2−] “ “Cu2+ +NO+H2O
k
obs
(3)
The initial rates can be described with the following
equation:
k
obsKp[NO−2 ]
r=
[Cu+]
(4)
1+Kp[NO−2 ]
where kobs depends linearly on H+ [7]. Thus, the nitrite
reductase reaction likely occurs through an inner sphere
electron transfer between Cu(I) and coordinated nitrite,
Fig. 6. Dependence of the nitrite reductase reaction rate of [Cu-
(biap)]+ on nitrite concentration at constant proton concentration
([H+]/[Cu+]=2).

M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
49
lel development of d–d absorptions occurs. However, it
is noteworthy that, unlike the systems studied before
[7], the characteristic multicomponent band of free
HNO2 here does not appear. This indicates that in the
present system the amount of free nitrite must be
negligible at low temperature. Probably, bound nitrite
is protonated and reduced without dissociation of sig-
nificant amounts of nitrous acid. After a few minutes
the absorbance in the near-UV region begins to in-
crease, due to accumulation of Cu(II). This traps the
available nitrite and stops the reaction after about 50%
nitrite has been reduced.
4. Concluding remarks
Fig. 7. Difference spectra recorded during the anaerobic reduction of
nitrite by [Cu(biap)]+ (1.2×10−3 M) in the presence of H+ (molar
ratio [NO2−]/[Cu]/[H+]=1/2/1) in methanol–acetonitrile 1:1 (v/v) at
−45°C. (1) Difference spectrum on addition of nitrite to Cu(I); (2)
and (3) difference spectra obtained about 1 and 5 min after the
addition of H+; (4) difference spectrum obtained after about 1 h
reaction.
The new, versatile tridentate ligand biap has been
synthesized through a very easy one-pot procedure. The
presence of the imidazole groups makes the ligand biap
suitable for the use in the synthesis of mimics for
histidine-containing active sites of metalloproteins. A
large number of coordination complexes of biap have
been obtained with the divalent first row transition
metals cobalt, nickel, zinc [6a] and copper. The ligand
biap coordinates either didentately, by only using the
imidazole nitrogens, or tridentately, through the two
imidazole and the tertiary amine-nitrogens. In the tri-
dentate mode either a facial or a meridional binding to
a transition metal occurs. With copper(II) only the
tridentate meridional binding mode has been observed
resulting in a flattening of the ligand. Therefore, in the
copper(II) compounds the observed deviations from
ideal coordination geometries are largely due to the
restricted ligand bite angles. Unlike the transition
metals cobalt, nickel and zinc, the use of non-coordi-
nitrite [7]. However, besides the steric hindrance at the
coordination site, the Kp value probably depends on
other factors, including the basicity of the ligand and
the size of the chelate rings formed on coordination to
Cu(I). The value of the kinetic constant kobs is also a
composite parameter since it is connected with the
basicity of nitrite coordinated to copper(I) and the rate
of electron transfer from Cu(I) to the protonated nitrite
bound to the metal. The value obtained for [Cu(biap)]+,
k
obs=8.7 (90.6)×10−3 −1, is slightly larger than
s
that found for [Cu(ddah)]+ and indicates an intermedi-
ate efficiency in the nitrite reductase reaction [7].
The nitrite reductase reaction was also followed spec-
troscopically at low temperature (−45°C), in the same
conditions used previously with other Cu(I) complexes
with tridentate heterocyclic ligands [7], employing a
molar ratio [Cu(biap)]+/NO−2 =2/1, in order to reduce
the amount of free nitrite in solution. The addition of
nitrite to the Cu(I) solution produced a significant
increase of absorption (Dm:330 M−1 cm−1) in the
near-UV region where [Cu(biap)]+ exhibits a very weak
band (umax 336 nm, m 170 M−1 cm−1). Such increase of
absorption cannot be due to free nitrite, since the
extinction coefficient of free nitrite is one order of
magnitude smaller [16], and therefore it must be associ-
ated with the formation of an adduct between [Cu-
(biap)]+ and nitrite. On addition of H+ (1/1 ratio with
respect to nitrite) the near-UV band initially decreases
in intensity and progressively shifts to the red (up to
umax 346 nm), as it is clearly seen in the difference
spectra shown in Fig. 7. These changes are due to the
progress of the nitrite reductase reaction, since a paral-
nating anions did not result in a complex [Cu(biap)2]2+
.
The [Cu(biap)]2+ moiety exhibits a rather high
affinity for small anionic ligands like Cl−, NO2− and
NCS−. This has been confirmed by determining the
binding constants for the biomimetically significant
NO2− and N3− ions.
The potential relevance of Cu–biap complexes as
active site analogs of copper nitrite reductases [5],
prompted the investigation of the Cu(I) mediated re-
duction of nitrite to nitric oxide. Although [Cu(biap)]+
exhibits reduced affinity towards NO2− and moderate
efficiency in the nitrite reductase reaction, its behavior
at low temperature disclosed some interesting features.
Unlike the systems studied previously [7], in fact, proto-
nation of the [Cu(biap)]+–NO2− adduct does not lead
to the immediate release of nitrous acid. Possibly, the
presence of alkyl substituents adjacent to the donor
atoms slows down this process enabling the intramolec-
ular electron transfer reaction to proceed smoothly.

50
M. Beretta et al. / Inorganica Chimica Acta 310 (2000) 41–50
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wards the transition metals Mn and Fe, and the possi-
ble use of these complexes in oxidation catalysis is
currently under investigation.
[4] J.P. Klinman, D. Mu, Annu. Rev. Biochem. 63 (1994) 299.
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5. Supplementary material
Crystallographic data (excluding structure factors)
for the structures reported in this paper have been
deposited with the Cambridge Crystallographic Data
Centre, CCDC no. 138367 for compound 1, and no.
138368 for compound 2. Copies of this information
may be obtained free of charge from the Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
(fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk
or http://www.ccdc.cam.ac.uk).
Acknowledgements
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[19] P. Zanello, in: I. Bernal (Ed.), Stereochemical Control, Bonding
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181–365.
We are indebted to Mr. S. Gorter for the X-ray data
collection and to Dr. R.A.G. de Graaff for assistance in
data processing. E.M. and L.C. thank the Italian CNR
Target Project on ‘Biotechnology’ for support. M.B.
(University of Pavia, Italy) and L.G.S. (University of
Valencia, Spain) were involved in the project through
the Erasmus student exchange program. B.D. thanks
the EU for TMR support (ERBFMBICT982885). Ex-
change of information and intra-project collaboration
stimulated via a COST action (D8/0015/97) is thank-
fully acknowledged.
[20] M. Kodera, N. Koura, S. Hosohara, M. Nishimura, M. Ohba,
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