Less symmetrical dicopper(II) complexes as catechol oxidase models - An adjacent thioether group increases catecholase activity

Article abstract of DOI:10.1002/chem.200400768

Three new unsymmetrical compartmental dinucleating ligands, 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(1-piperidyl)ethyl}aminomethyl] phenol (HL1), 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(morpholin-4-yl) ethyl}aminomethyl]phenol (HL2), and 4-

Full text of DOI:10.1002/chem.200400768

FULL PAPER
Less Symmetrical Dicopper(ii) Complexes as Catechol Oxidase Models—An
Adjacent Thioether Group Increases Catecholase Activity
Michael Merkel,[a] Niclas Mçller,[a] Manuel Piacenza,[b] Stefan Grimme,*[b]
Annette Rompel,[a] and Bernt Krebs*[a]
Dedicated to Professor Arndt Simon on the occasion of his 65th birthday
Abstract: Three new unsymmetrical
compartmental dinucleating ligands, 4-
bromo-2-(4-methylpiperazin-1-ylmeth-
yl)-6-[{2-(1-piperidyl)ethyl}aminome-
thyl]phenol (HL1), 4-bromo-2-(4-meth-
ylpiperazin-1-ylmethyl)-6-[{2-(morpho-
lin-4-yl)ethyl}aminomethyl]phenol
(HL2), and 4-bromo-2-(4-methylpiper-
azin-1-ylmethyl)-6-[{2-(thiomorpholin-
4-yl)ethyl}aminomethyl]phenol (HL3),
have been synthesized in order to
model the active site of type 3 copper
proteins. The dicopper(ii) complexes of
these ligands give first hints about the
influence of a thioether group close to
the metal site. The bromophenol-based
ligands have one piperazine arm and
one other bidentate arm in positions 2
and 6 of the phenolic ring, respectively.
With each ligand a dinuclear copper(ii)
complex was prepared and structurally
characterized. The copper ions were
found to have square pyramidal envi-
ronments and a mixture of endogenous
phenoxo and exogenous acetate bridg-
ing. The influence of a heteroatom in
one arm of the ligand on catecholase
activity and speciation in solution was
studied by UV/Vis spectroscopy, ESI-
MS experiments and, DFT calculations.
Keywords: bioinorganic chemistry ·
catechol oxidase · copper · enzyme
models · structure–property rela-
tionships
Introduction
nase, which catalyzes the hydroxylation of phenols to cate-
chols (cresolase activity) and the oxidation of catechols to
quinones (catecholase activity),[4] and catechol oxidase (CO)
that exhibits only catecholase activity. CO was first isolated
by Kubowitz in 1937 from potatoes.[5,6] Since then COs have
been purified from different sources, among which are pota-
toes, spinach, apples, grape berries,[7] lychee fruit,[8] beans,[9]
bananas,[10] opium plants,[11] coffee plants,[12] black poplars,[13]
and gypsy wort.[13]
A new insight into the catalytic mechanism of catechol
oxidases was gained with the determination of the crystal
structure of the enzyme from sweet potatoes (Ipomoea bata-
tas; ibCO).[14] Even though the first coordination sphere of
each copper center consists of three histidines and one m-
OH bridge, both copper ions were found to be nonequiva-
lent. The most striking asymmetric feature is a covalent thio-
ether bond formed between the Ce atoms of His109 and
Cys92. A cysteinyl–histidinyl bond has also been reported
for other type 3 copper proteins, like tyrosinase from Neuro-
spora crassa[15] and hemocyanin from Helix pomatia[16] and
Octopus dofleini.[17] A thioether bond between cysteine and
tyrosine is also present in the mononuclear copper enzyme
galactose oxidase.[18] Biomimetic models mimicking this fea-
ture were presented by Wieghardt and co-workers.[19]
Transport, activation, and metabolism of dioxygen are very
important processes in many living organisms. Metallopro-
teins containing one or more copper centers are often re-
sponsible for these functions.[1] The so-called type 3 copper
proteins have an antiferromagnetically coupled dicopper
core with three histidine ligands on each copper ion and m-
hydroxo bridging in the met-CuII–CuII form, which results in
an EPR-silent active site. Well-known representatives of
type 3 copper proteins are hemocyanin, the dioxygen carrier
and transport protein for arthropods and molluscs,[2,3] tyrosi-
[a] Dr. M. Merkel, Dr. N. Mçller, Dr. A. Rompel, Prof. Dr. B. Krebs
Institut fꢀr Anorganische und Analytische Chemie der Westfꢁlischen
Wilhelms-Universitꢁt
Wilhelm-Klemm-Strasse 8, 48149 Mꢀnster (Germany)
Fax : (+49)251-8338-366
E-mail: krebs@uni-muenster.de
[b] Dr. M. Piacenza, Prof. Dr. S. Grimme
Organisch-Chemisches Institut der Westfꢁlischen
Wilhelms-Universitꢁt
Corrensstrasse 40, 48149 Mꢀnster (Germany)
Fax : (+49)251-8336-515
E-mail: grimmes@uni-muenster.de
A wide range of work has been done in the field of bio-
mimetic model compounds for type 3 copper proteins. It has
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 1201 – 1209
DOI: 10.1002/chem.200400768
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1201

Physical measurements: 1H and 13C NMR spectra were recorded on a
Bruker WH 300 instrument; all chemical shifts are reported relative to
an internal standard of tetramethylsilane. Elemental analyses were per-
formed on a Heraeus CHN-O-RAPID or an ELEMENTAR Vario El III
analyzer. Electronic spectroscopy was carried out with a Hewlett–Pack-
ard 8453 diode array spectrometer by using quartz cuvettes (1 cm, metha-
nolic solutions, 5ꢄ10ꢀ4 m). IR spectra (in KBr or Nujol) were recorded
on a Bruker IF 48 Fourier transform spectrometer; FIR spectra (in poly-
ethylene) were recorded on a Bruker IF 113v instrument. ESI-MS meas-
urements were performed on a Micromass Quattro LC instrument.
been summarized in a number of articles.[20–33] Model com-
pounds that seize the idea of asymmetry might especially
help to elucidate important questions concerning the bind-
ing of the substrate in the natural enzyme: for example,
does the catecholate bridge the two copper(ii) centers[1,34] or
is it only bound to one metal center?[35] Dinuclear copper
complexes employing the model substrate tetrachlorocate-
cholate have proven that both binding modes can be realiz-
ed.[36,37]
Ligands: The to-date-unknown compartmental ligands 4-bromo-2-(4-
Some dinuclear copper compounds from compartmental
ligands have already been reported.[38–44] In this work, we
have prepared three new dinucleating N4O donor ligands
(Scheme 1) to mimic the unsymmetric active site in catechol
methylpiperazin-1-ylmethyl)-6-[{2-(1-piperidyl)ethyl}aminomethyl]phenol
(HL1),
4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(morpholin-4-
yl)ethyl}aminomethyl]phenol (HL2), and 4-bromo-2-(4-methylpiperazin-
1-ylmethyl)-6-[{2-(thiomorpholin-4-yl)ethyl}aminomethyl]phenol (HL3)
were synthesized according to a route
described by Fenton and co-work-
ers.[46] The first step in the synthesis of
all three ligands is a Mannich reaction
of
5-bromo-2-hydroxybenzaldehyde
with N-methylpiperazine and parafor-
maldehyde to produce 4-bromo-2-
formyl-6-(4-methylpiperazin-1-ylme-
thyl)phenol.[47] N-(2-aminoethyl)piper-
idine and N-(2-aminoethyl)morpholine
were obtained from commercial sour-
ces, whereas N-(2-aminoethyl)thiomor-
pholine was synthesized following
literature procedure.[48]
a
General
procedure:
4-Bromo-2-
formyl-6-(4-methylpiperazin-1-ylme-
thyl)phenol (3.74 g, 12 mmol) was dis-
solved in ethanol (40 mL). One equiv-
alent of the corresponding amine
(1.54 g of N-(2-aminoethyl)piperidine
Scheme 1. Structures of the compartmental phenol-based ligands HL1–HL3.
for HL1, 1.56 g of N-(2-aminoethyl)-
morpholine for HL2, and 1.76 g of N-
(2-aminoethyl)thiomorpholine for HL3, respectively) was added and the
reaction mixture was heated to reflux for 3 h. At 08C, sodium borohy-
dride (1.21 g, 36 mmol) was carefully added. The resulting suspension
was stirred overnight at room temperature and once again heated to
reflux for 1 h. To destroy excess borohydride, hydrochloric acid was
added to the reaction mixture, which was then stirred vigorously for
30 minutes. Afterwards, the pH value was adjusted to 8 by addition of
sodium hydroxide solution and the resulting precipitate was removed by
filtration. The filtrate was concentrated under reduced pressure and the
residue was taken up in chloroform and dried over sodium sulfate. Filtra-
tion and removal of the solvent yielded the product as a yellowish, highly
viscous oil.
HL1: Yield: 3.82 g (7.67 mmol, 64%); 1H NMR (300 MHz, CDCl3): d=
1.49 (m, 4H; CH2), 1.66 (m, 2H; CH2), 2.25 (s, 3H; CH3), 2.55 (t, 4H;
CH2), 2.56 (brs, 4H; CH2), 2.63 (brs, 4H; CH2), 2.74 (t, 2H; CH2), 2.95
(t, 2H; CH2), 3.68 (s, 2H; CH2), 3.96 (s, 2H; CH2), 7.12 (s, 1H; CHar),
7.29 (s, 1H; CHar), 8.09 (brs, 1H, OH) ppm; 13C NMR (75 MHz, CDCl3):
d=23.8 (CH2), 25.3 (CH2), 44.0 (CH2), 45.8 (CH3), 48.1 (CH2), 52.5
(CH2), 54.2 (CH2), 54.8 (CH2), 55.6 (CH2), 56.7 (CH2), 110.9 (Car), 123.3
(Car), 124.0 (Car), 131.5 (Car), 132.0 (Car), 155.7 (Car) ppm; IR (Nujol): n˜ =
3498 (s), 2939 (m), 2849 (m), 2798 (m), 2448 (w), 1608 (m), 1457 (s), 1398
(w), 1367 (m), 1350 (m), 1299 (m), 1284 (m), 1233 (m), 1160 (m), 1135
(m), 1092 (m), 1051 (m), 1008 (m), 986 (m), 920 (w), 867 (m), 819 (m),
781 (w), 754 (m), 728 (w), 660 (w), 624 (w), 559 (w), 542 (w), 491 (w),
442 (w) cmꢀ1; MS (ESI): m/z: 425–430 [M+ꢀH isotope cluster]; elemen-
tal analysis: calcd (%) for C20H33BrN4O (425.4): C 56.47, H 7.82, N 13.17;
found: C 55.98, H 7.94, N 12.34.
oxidase, 4-bromo-2-(4-methylpiperazin-1-ylmethyl)-6-[{2-(1-
piperidyl)ethyl}aminomethyl]phenol (HL1), 4-bromo-2-(4-
methylpiperazin-1-ylmethyl)-6-[{2-(morpholin-4-yl)ethyl}-
aminomethyl]phenol (HL2), and 4-bromo-2-(4-methylpipera-
zin-1-ylmethyl)-6-[{2-(thiomorpholin-4-yl)ethyl}aminome-
thyl]phenol (HL3). These compartmental ligands build dif-
ferent coordination surroundings for two copper ions in
close proximity. This proximity is important, since EXAFS
ꢀ
data suggest a Cu Cu distance of 2.9 ꢃ for the met-form of
the enzyme.[45] Furthermore, one arm of the ligand is modi-
fied from piperidine (L1) to morpholine (L2) and finally thio-
morpholine (L3). The effects of these variations, including
the introduction of the thioether group in HL3, on catecho-
lase activity and speciation in solution were studied. For the
adjacent thioether group, we observed an involvement in
metal coordination. Thus, its function in these metal com-
pounds is different from that in type 3 copper proteins,
where metal coordination of the thioether group is not
likely.
HL2: Yield: 3.79 g (8.88 mmol, 74%); 1H NMR (300 MHz, CDCl3): d=
2.30 (s, 3H; CH3), 2.38 (m, 4H; CH2), 2.44 (t, 2H; CH2), 2.54 (brs), 4H;
CH2), 2.59 (brs, 4H; CH2), 2.66 (t, 2H; CH2), 3.62 (m, 4H; CH2), 3.68 (s,
2H; CH2), 3.99 (s, 2H; CH2), 7.12 (s, 1H; CHar), 7.29 (s, 1H; CHar) ppm;
Experimental Section
Materials: All chemicals were purchased from commercial sources and
used without any further purification.
1202
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www.chemeurj.org
Chem. Eur. J. 2005, 11, 1201 – 1209

Catechol Oxidase Models
FULL PAPER
13C NMR (75 MHz, CDCl3): d=43.8 (CH3), 45.7 (CH2), 48.0 (CH2), 52.3
(CH2), 53.2 (CH2), 54.7 (CH2), 55.5 (CH2), 59.9 (CH2), 66.9 (CH2), 110.8
(Car), 123.1 (Car), 123.5 (Car), 131.5 (Car), 132.0 (Car), 155.6 (Car) ppm; IR
(KBr): n˜ =3439 (m), 2936 (m), 2797 (m), 2076 (w), 1887 (w), 1653 (w),
1606 (w), 1455 (s), 1390 (w), 1367 (w), 1348 (w), 1293 (w), 1226 (w), 1160
(m), 1136 (m), 1095 (m), 1010 (m), 920 (w), 868 (w), 815 (m), 748 (w),
623 (w), 489 (w), 441 (m) cmꢀ1; MS (ESI): m/z: 427–431 [M+ꢀH isotope
cluster]; elemental analysis: calcd (%) for C19H31BrN4O2 (427.4): C 53.40,
H 7.31, N 13.11; found: C 52.67, H 7.44, N 12.81.
HL3: Yield: 4.42 g (9.97 mmol, 83%); 1H NMR (300 MHz, CDCl3): d=
2.30 (s, 3H; CH3), 2.55 (m, CH2), 2.60 (t, 2H; CH2), 2.70 (m, CH2), 2.90
(t, 2H; CH2), 3.70 (s, 2H; CH2), 3.90 (s, 2H; CH2), 7.11 (s, 1H; CHar),
7.31 (s, 1H; CHar) ppm; 13C NMR (75 MHz, CDCl3): d=27.8 (CH2), 44.0
(CH3), 45.6 (CH2), 48.0 (CH2), 52.3 (CH2), 54.6 (CH2), 54.7 (CH2), 55.5
(CH2), 59.9 (CH2), 110.8 (Car), 123.0 (Car), 131.6 (Car), 132.0 (Car), 155.6
(Car) ppm; IR (KBr): n˜ =3393 (m), 2939 (m), 2807 (m), 1608 (w), 1459
(s), 1397 (w), 1365 (w), 1348 (w), 1285 (m), 1233 (m), 1160 (m), 1135
(m), 1087 (w), 1008 (w), 960 (w), 918 (w), 870 (w), 818 (m), 753 (s), 663
(w), 623 (w), 560 (w), 491 (w), 441 (w) cmꢀ1; MS (ESI): m/z: 443–447
[M+ꢀH isotope cluster]; elemental analysis: calcd (%) for
C19H31BrN4OS (443.5): C 51.46, H 7.05, N 12.63; found: C 51.34, H 7.35,
N 12.49.
(m), 928 (m), 904 (w), 887 (m), 842 (m), 809 (m), 770 (m), 659 (m), 634
(m), 612 (w), 595 (w), 576 (w), 519 (w), 465 (w) cmꢀ1; FIR: n˜ =395 (w),
368 (w), 350 (s), 271 (w), 245 (m), 203 (m), 178 (s), 150 (m) cmꢀ1; ele-
mental analysis: calcd (%) for C23H36Cu2BBrF4N4O6 (758.4): C 36.43, H
4.78, N 7.38; found: C 36.36, H 4.69, N 7.43.
[Cu2(L3)(m-OAc)2](BF4)·0.25MeOH·0.75H2O
(3):
Yield:
77 mg
(0.10 mmol, 51%); m.p. 2358C (decomp); IR (KBr): n˜ =3639 (w), 3562
(w), 3423 (w), 3262 (m), 2972 (m), 2923 (m), 2872 (m), 1614 (s), 1463 (s),
1414 (s), 1361 (w), 1338 (m), 1299 (m), 1279 (m), 1240 (m), 1181 (w), 989
(m), 964 (m), 883 (m), 866 (m), 810 (m), 774 (m), 756 (m), 662 (m), 644
(m), 629 (m), 589 (m), 559 (w), 522 (m), 458 (m) cmꢀ1; FIR: n˜ =398 (m),
348 (m), 311 (m), 289 (w), 260 (m), 206 (m), 162 (m), 104 (w) cmꢀ1; ele-
mental analysis: calcd (%) for C23H36Cu2BBrF4N4O5S (774.4; without sol-
vent): C 35.67, H 4.68, N 7.23; found: C 35.54, H 4.61, N 7.31.
X-ray crystal structures: The intensity data of compounds 1 and 3 were
collected on an STOE IPDS diffractometer (MoKa, l=0.71073 ꢃ, graph-
ite monochromator) by using the f-scan technique. Data collection for
compound 2 was performed on a Bruker AXS SMART APEX CCD dif-
fractometer (MoKa, l=0.71073 ꢃ, graphite monochromator) by using the
w-scan technique. All structures were solved by direct methods and re-
fined by full-matrix least-squares methods on F2.[49] Further data collec-
tion parameters are summarized in Table 1. Selected bond lengths and
angles for the dinuclear copper(ii) complexes are reported in Table 2.
The disordered solvent molecules in 1 and 3 were refined with isotropic
displacement parameters and without hydrogen atoms, whereas all other
Metal complexes
General procedure: A solution of copper(ii) tetrafluoroborate hydrate
(47 mg, 0.2 mmol in acetonitrile/methanol (1:1; 2 mL)) and a solution of
copper(ii) acetate hydrate (40 mg,
0.2 mmol in acetonitrile/methanol (1:1;
2 mL)) were added to a stirred so-
Table 1. Crystallographic data and experimental details.
lution of the ligand (0.2 mmol; HL1:
85 mg, HL2: 86 mg, or HL3: 89 mg, re-
spectively) in acetonitrile/methanol
(1:1; 2 mL), then triethylamine (eight
drops) was added. The color of the so-
lution changed from green to green-
blue. After being stirred for 10 min,
the reaction mixture was filtered and
vapor diffusion of diethyl ether into
the complex solution afforded dark
green cuboid shaped crystals of
1
2
3
empirical formula
Mr
C25H40.5BBrCu2F4N4O5.25
774.91
213(2)
MoKa, 0.71073
green cuboid
0.32ꢄ0.24ꢄ0.12
triclinic
C23H36BBrCu2F4N4O6
758.36
153(2)
MoKa, 0.71073
green cuboid
0.28ꢄ0.25ꢄ0.13
triclinic
C23.25H38.5BBrCu2F4N4O6S
795.94
213(2)
MoKa, 0.71073
green cuboid
0.36ꢄ0.24ꢄ0.24
triclinic
temperature [K]
radiation, l [ꢃ]
crystal shape
crystal size [mm]
crystal system
space group
a [ꢃ]
b [ꢃ]
c [ꢃ]
a [8]
b [8]
¯
¯
¯
P1 (no. 2)
P1 (no. 2)
P1 (no. 2)
14.282(3)
16.612(3)
16.914(3)
62.43(3)
81.36(3)
68.14(3)
3300(2)
4
1.522
2.562
1578
8.66–52.20
ꢀ17ꢁhꢁ16
ꢀ20ꢁkꢁ20
ꢀ20ꢁlꢁ20
26123
10.410(5)
10.594(5)
14.245(5)
78.330(5)
69.750(5)
75.050(5)
1413(2)
2
1.783
2.991
768
3.08–56.54
ꢀ13ꢁhꢁ13
ꢀ13ꢁkꢁ14
ꢀ18ꢁlꢁ18
14766
12.930(3)
16.088(3)
17.367(3)
87.52(3)
74.82(3)
78.34(3)
3415(2)
4
1.548
2.534
1616
8.78–52.06
ꢀ15ꢁhꢁ15
ꢀ19ꢁkꢁ19
ꢀ21ꢁlꢁ21
27120
[Cu2(L1)(m-OAc)2](BF4)
[Cu2(L2)(m-OAc)2](BF4)
(1),
or
(2),
[Cu2(L3)(m-OAc)2](BF4) (3), respec-
tively.
g [8]
[Cu2(L1)(m-OAc)2](BF4)·0.25Et2O (1):
Yield: 68 mg (0.09 mmol, 47%); m.p.
2408C (decomp); IR (KBr): n˜ =3420
(m), 3256 (m), 2931 (m), 2868 (m),
1607 (s), 1462 (m), 1446 (m), 1415 (s),
1361 (w), 1340 (w), 1297 (w), 1276 (w),
1242 (m), 1182 (w), 1082 (s), 950 (w),
931 (w), 893 (w), 876 (w), 810 (m), 773
(m), 757 (m), 662 (m), 645 (w), 630
(w), 615 (w), 586 (w), 561 (w), 522
(w), 464 (w) cmꢀ1; FIR: n˜ =371 (w),
350 (m), 284 (s), 259 (m), 231 (m), 208
(s), 164 (m), 133 (w), 85 (m) cmꢀ1; ele-
mental analysis: calcd (%) for
C24H38Cu2BBrF4N4O5 (756.4; without
V [ꢃ3]
Z
1calcd [g cmꢀ3
]
m [mmꢀ1
F(000)
]
scan range 2q [8]
index ranges
reflections collected
unique reflections
reflections I>2s(I)
Rint
data/restraints/parameters
goodness-of-fit on F2
final R indices (I>2s(I))
R1
12088
5739
0.1290
10902/0/739
1.016
6957
5660
0.0247
6957/0/370
0.966
12442
7159
0.0974
12439/0/740
0.952
solvent):
C 38.11, H 5.06, N 7.41;
found: C 37.82, H 5.11, N 7.45.
0.0717
0.0391
0.0857
[Cu2(L2)(m-OAc)2](BF4) (2): Yield:
83 mg (0.11 mmol, 53%); m.p. 2058C
(decomp); IR (KBr): n˜ =3435 (m),
3275 (m), 3069 (w), 2971 (m), 2930
(m), 2901 (m), 2869 (m), 2817 (w),
1603 (s), 1420 (s), 1394 (s), 1358 (w),
1338 (w), 1306 (w), 1279 (m), 1241
(m), 1180 (w), 1057 (s), 988 (s), 953
wR2
0.1495[a]
0.1019[b]
0.2132[c]
R indices (all data)
R1
0.1689
0.0488
0.1449
wR2
0.1880[a]
0.1057[b]
0.2401[c]
largest diff. peak/hole [e ꢃꢀ3
]
0.697/ꢀ0.639
1.169/ꢀ0.627
1.081/ꢀ1.008
[a] w=1/[s2(Fo2)+(0.0698P)2+0.0090P] with P=(F2o+2Fo2)/3. [b] w=1/[s2(F2o)+(0.0607P)2] with P=(Fo2+2F2o)/3.
[c] w=1/[s2(Fo2)+(0.0751P)2+20.5428P] with P=(Fo2+2F2o)/3.
Chem. Eur. J. 2005, 11, 1201 – 1209
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ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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S. Grimme, B. Krebs et al.
Table 2. Selected lengths [ꢃ] and angles [8] in complexes 1–3.
Results and Discussion
1
Crystal structures of 1–3: All
95.1(2) three compounds reported in
ꢀ
Cu(1)···Cu(2)
3.304(2)
1.950(6)
1.965(6)
2.137(7)
2.042(7)
2.025(8)
1.965(5)
2.232(7)
1.937(6)
1.994(7)
2.107(7)
O(1)–Cu(1) O(2)
O(1)-Cu(1)-O(4)
O(1)-Cu(1)-N(1)
O(1)-Cu(1)-N(2)
O(2)-Cu(1)-O(4)
O(2)-Cu(1)-N(1)
O(2)-Cu(1)-N(2)
O(4)-Cu(1)-N(1)
O(4)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
97.7(3)
95.7(3)
91.7(3)
164.1(3)
99.7(3)
149.7(3)
94.3(3)
107.9(3)
92.4(3)
72.8(3)
O(1)-Cu(2)-O(3)
O(1)-Cu(2)-O(5)
O(1)-Cu(2)-N(3)
O(1)-Cu(2)-N(4)
O(3)-Cu(2)-O(5)
O(3)-Cu(2)-N(3)
O(3)-Cu(2)-N(4)
O(5)-Cu(2)-N(3)
O(5)-Cu(2)-N(4)
N(3)-Cu(2)-N(4)
Cu(1)-O(1)-Cu(2)
92.2(2)
ꢀ
Cu(1) O(1)
ꢀ
Cu(1) O(2)
91.4(3)
170.0(3)
this work crystallize in the cen-
ꢀ
Cu(1) O(4)
¯
trosymmetric space group P1.
ꢀ
Cu(1) N(1)
96.9(3)
94.4(3)
96.8(3)
166.7(3)
88.3(3)
83.5(3)
115.1(3)
While the triclinic unit cell of 2
contains two formula units, the
cells of 1 and 3 contain four for-
mula units. The most likely
reason for this is a reduction of
the symmetry by the partly oc-
cupied solvent molecules in 1
and 3. Since the two crystallo-
graphically independent com-
plex cations in 1 and 3, respec-
tively, are chemically equivalent
and show broad similarities,
only one of them will be dis-
cussed for each compound. El-
lipsoid plots of the cations in 1–
ꢀ
Cu(1) N(2)
ꢀ
Cu(2) O(1)
ꢀ
Cu(2) O(3)
ꢀ
Cu(2) O(5)
ꢀ
Cu(2) N(3)
ꢀ
Cu(2) N(4)
2
ꢀ
Cu(1)···Cu(2)
3.263(2)
1.972(2)
2.163(2)
1.938(2)
2.018(2)
2.076(3)
1.954(2)
1.933(2)
2.278(2)
1.997(2)
2.063(2)
O(1)-Cu(1) O(3)
91.5(1)
100.2(1)
92.4(1)
162.8(1)
107.9(1)
94.9(1)
98.4(1)
153.5(1)
90.2(1)
72.9(1)
O(1)-Cu(2)-O(4)
O(1)-Cu(2)-O(6)
O(1)-Cu(2)-N(3)
O(1)-Cu(2)-N(4)
O(4)-Cu(2)-O(6)
O(4)-Cu(2)-N(3)
O(4)-Cu(2)-N(4)
O(6)-Cu(2)-N(3)
O(6)-Cu(2)-N(4)
N(3)-Cu(2)-N(4)
Cu(1)-O(1)-Cu(2)
96.1(1)
88.8(1)
91.0(1)
175.2(1)
102.9(1)
155.5(1)
88.1(1)
100.6(1)
88.0(1)
86.1(1)
ꢀ
Cu(1) O(1)
O(1)-Cu(1)-O(5)
O(1)-Cu(1)-N(1)
O(1)-Cu(1)-N(2)
O(3)-Cu(1)-O(5)
O(3)-Cu(1)-N(1)
O(3)-Cu(1)-N(2)
O(5)-Cu(1)-N(1)
O(5)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
ꢀ
Cu(1) O(3)
ꢀ
Cu(1) O(5)
ꢀ
Cu(1) N(1)
ꢀ
Cu(1) N(2)
ꢀ
Cu(2) O(1)
ꢀ
Cu(2) O(4)
ꢀ
Cu(2) O(6)
ꢀ
Cu(2) N(3)
ꢀ
Cu(2) N(4)
112.4(1) 3 are depicted in Figures 1–3,
whereas selected bond lengths
and angles are listed in Table 2.
3
Cu(1)···Cu(2)
Cu(1) O(1)
Cu(1) O(2)
Cu(1) O(4)
Cu(1) N(1)
Cu(1) N(2)
Cu(2) O(1)
Cu(2) O(3)
3.299(2)
1.952(7)
1.935(8)
2.130(7)
2.028(9)
2.050(9)
1.974(6)
2.173(8)
1.917(7)
1.981(9)
2.128(8)
O(1)-Cu(1)-O(2)
O(1)-Cu(1)-O(4)
O(1)-Cu(1)-N(1)
O(1)-Cu(1)-N(2)
O(2)-Cu(1)-O(4)
O(2)-Cu(1)-N(1)
O(2)-Cu(1)-N(2)
O(4)-Cu(1)-N(1)
O(4)-Cu(1)-N(2)
N(1)-Cu(1)-N(2)
99.4(3)
94.0(3)
91.5(3)
163.2(3)
104.7(3)
150.9(3)
91.4(4)
101.4(3)
95.7(3)
73.1(4)
O(1)-Cu(2)-O(3)
O(1)-Cu(2)-O(5)
O(1)-Cu(2)-N(3)
O(1)-Cu(2)-N(4)
O(3)-Cu(2)-O(5)
O(3)-Cu(2)-N(3)
O(3)-Cu(2)-N(4)
O(5)-Cu(2)-N(3)
O(5)-Cu(2)-N(4)
N(3)-Cu(2)-N(4)
Cu(1)-O(1)-Cu(2)
91.2(3)
95.1(3)
90.9(3)
173.0(3)
100.2(3)
95.2(3)
94.4(3)
163.3(3)
The two copper ions in all
ꢀ
three dinuclear [CuII(L)(m-
ꢀ
OAc)2]+ complexes are m-phe-
ꢀ
ꢀ
noxo-bridged by a deprotonat-
ed ligand. Furthermore, two
exogenous acetate bridges are
ꢀ
ꢀ
ꢀ
ꢀ
Cu(2) O(5)
Cu(2) N(3)
Cu(2) N(4)
87.8(3) found in each complex. This
ꢀ
84.6(3)
114.3(3)
ꢀ
bridging motif leads to Cu(1)
Cu(2) distances of 3.304(2) (1),
ꢀ
non-hydrogen atoms were refined anisotropically with ligand hydrogen
atoms riding on ideal positions.[50]
Kinetic measurements: Catechol oxidase activities of complexes 1–3 were
measured at 258C by time-dependent UV/Vis spectroscopy. 3,5-Di-tert-
butylcatechol (5, 10, 20, 40, 50, 90, or 100 equivalents) dissolved in meth-
anol was added to a sample (1 mL) of 2ꢄ10ꢀ4 m solutions of compounds
1–3 in methanol. The final sample volume was 2 mL and, accordingly, the
concentration of the metal complex was 10ꢀ4 m. During the first 3 minutes
of the reaction, the development of the absorption band at 400 nm was
monitored. The average initial rates over three independent measure-
ments were determined from the slope of the absorption versus time plot
by utilizing the Lambert–Beer equation.
DFT calculations: All calculations were performed on a parallel LINUX-
PC cluster by using the TURBOMOLE 5.3[51] program suite. All DFT
calculations were carried out with the B3-LYP[52,53] density functional,
and an m3 numerical quadrature grid was applied in all DFT calculations.
The resolution of identity (RI) approximation was used for the MP2[54]
calculations. The basis sets were taken from the TURBOMOLE basis set
library.[55] Basis sets of split-valence-double-z and split-valence-triple-z
quality with polarization functions SV(d)[56] and TZV(d,p)[57] have been
used. To simplify the calculations, the bromo substituent on the phenol
spacer was treated as a hydrogen atom. The calculated energies refer to
the corresponding complexes with two acetate bridges as the zero point.
Figure 1. Representation of the cation in 1 (50% probability); the hydro-
gen atoms are omitted for clarity.
3.263(2) (2), and 3.299(2) ꢃ (3). The remaining equatorial
positions on each copper ion are occupied by nitrogen
donors derived from the compartmental ligand, a fact result-
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Chem. Eur. J. 2005, 11, 1201 – 1209

Catechol Oxidase Models
FULL PAPER
corner. The t values[58] are t=0.24 and 0.06 for Cu(1) and
Cu(2) in 1, t=0.16 and 0.33 for Cu(1) and Cu(2) in 2, and
t=0.21 and 0.16 for Cu(1) and Cu(2) in 3, respectively. Fur-
thermore, the coordination polyhedra are stretched. This be-
ꢀ
comes evident in different apical and equatorial Cu O bond
lengths. The latter are about 0.2–0.3 ꢃ larger in all cases.
Calculations of the least-squares planes around Cu(1) and
Cu(2) that contain the equatorial donor atoms reveal a dis-
placement of the copper ions towards the apical ligands. In
1 Cu(1) is 0.305 ꢃ and Cu(2) is 0.174 ꢃ out of plane, where-
as in 2 Cu(1) is 0.288 ꢃ and Cu(2) is 0.173 ꢃ out of plane,
and in 3 Cu(1) is 0.309 ꢃ and Cu(2) is 0.182 ꢃ out of plane.
All these deviations towards the apical donor lead to en-
ꢀ
ꢀ
larged Oapical Cu X angles. The average values for these
angles are 98.7 8 for Cu(1) and 95.2 8 for Cu(2). The pheno-
late oxygen atom is a shared edge of the two pyramids in all
ꢀ
ꢀ
reported compounds with Cu(1) O(1) Cu(2) angles of
115.1(3) (1), 112.4(1) (2), and 114.3(3)8 (3), respectively. The
polyhedra are twisted against each other, which results in
Figure 2. Representation of the cation in 2 (50% probability); the hydro-
gen atoms are omitted for clarity.
ꢀ
ꢀ
ꢀ
Oapical Cu(1) Cu(2) Oapical torsion angles of 93.8(2) (1),
128.5(1) (2), and 102.8(4)8 (3), respectively.
The intermolecular distances in compounds 1–3 suggest
one (1 and 2) or two (3) hydrogen bonds between the tetra-
fluoroborate counter ions and the secondary amine N(3)
atom with donor acceptor distances of 2.94(1)–3.23(1) ꢃ.
Electronic spectra: In general, two kinds of transitions are
expected for copper(ii) complexes like 1–3, d–d bands and
ligand-to-metal charge transfer (LMCT) bands. In the pre-
sented compounds, the latter can either be phenolate–cop-
per(ii) or acetate–copper(ii) transitions, which usually
appear in the range from 300–340 nm.[59–62] However, in
some cases these transitions were observed at wavelengths
of up to 418 nm.[63] The phenolate-to-copper(ii) charge trans-
fer transition is particularly affected by the substituents on
the phenolate and the electron density of the copper ions.[64]
Finally, the intensity of these LMCT transitions is dependent
Figure 3. Representation of the cation in 3 (50% probability); the hydro-
gen atoms are omitted for clarity.
ing in a nonequivalent environment for the metal centers of
each dinuclear unit. Cu(1) is, in all cases, coordinated by the
two tertiary amines of the piperazine group (N(1) and N(2),
respectively) that is found to be in its boat conformation,
whereas Cu(2) is ligated by the secondary amine N(3) and
the tertiary amine N(4) of the piperidine, morpholine or thio-
morpholine group (in 1–3, respectively). The piperazine ni-
trogen atoms are part of two five-membered chelate rings,
while the nitrogen donors on Cu(2) are embedded in only
one ring. The limited bite of these constellations leads to
significantly narrowed angles between the corresponding
donor atoms (N(1)-Cu(1)-N(2)=72.8(3)/72.9(1)/73.1(4)8 (1/
2/3) and N(3)-Cu(2)-N(4)=83.5(3)/86.1(1)/84.6(3)8 (1/2/3)).
The coordination polyhedra around the copper ions can be
regarded as distorted square pyramidal with a shared
Figure 4. Electronic spectra of 1–3.
Chem. Eur. J. 2005, 11, 1201 – 1209
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ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1205

S. Grimme, B. Krebs et al.
ꢀ ꢀ
on the Cu O Cu angle, which correlates with the overlap
The three model compounds presented in this work differ
only in the nature of the heteroatom at position 4 of the
ligand piperidine moiety. The different turnover numbers
for the oxidation of 3,5-di-tert-butylcatechol give a first hint
at the function of sulfur in these systems. In catalytic sys-
tems it is important to have free or labile coordination sites
for substrate binding. In the solid states of compounds 1–3,
exogenous acetate bridges occupy these sites. Thus, research
must be aimed at developing strategies to dissociate these
bridges. A higher affinity of the heteroatom in the piperi-
dine ring seems to weaken the copper–acetate bond and
result in a higher turnover number.
of the phenolate p-orbital and the copper d-orbital.[61,65]
The electronic spectra of complexes 1–3 are depicted in
Figure 4. Bands and extinction coefficients are summarized
in Table 3. The absorption bands below 400 nm can be as-
signed to LMCT transitions, but it is not possible to distin-
Table 3. Summary of the UV/Vis spectroscopy data of 1–3.
Compound
lmax [nm]
e [mꢀ1 cmꢀ1
]
lmax [nm]
e [mꢀ1 cmꢀ1
]
1
2
3
395
397
1575
2254
-
675
680
677
331
533
307
325-355 (sh)
To support this thesis, DFT calculations were performed
to determine the different reaction energies ([LCu2(OAc)2]+
![LCu2(OAc)]2+ +OAcꢀ) for the piperidine, morpholine,
and thiomorpholine monocations (1A–3A) converting into
the corresponding monoacetato-bridged dications in their
boat (1B–3B) and armchair (1C–3C) conformations
(Scheme 2). All nine structures and a free acetate anion
guish between phenolate- and acetate-derived bands. Al-
though the three compounds resemble each other, the spec-
trum of 3 differs from the others. The LMCT transition is
shifted to higher energies and appears as a shoulder rather
than as a distinct maximum. A possible explanation for this
behavior would be an exchange
of one or both acetate bridges
by methanolates or hydroxides
in solution. In the region of d–d
transitions, all three complexes
show a broad absorption with a
maximum intensity at 675–
680 nm that is indicative of
their square-pyramidal geome-
tries.[66,67]
Kinetic investigations: Air-satu-
rated methanol solutions of 1–3
(10ꢀ4 m) were treated with
50 equivalents of 3,5-di-tert-bu-
tylcatchol. No base was added
to the solutions in order to sup-
press oxidation of the substrate
Scheme 2. Structures [Cu2(L)(OAc)2]+ and the boat and chair conformations of [Cu2(L)(OAc)]2+ (with X=
CH2 (1), O (2), and S (3)).
by base. The first apparent result, while the reaction was
monitored by UV/Vis spectroscopy, was a significant activi-
ty, namely, a formation of a band at about 400 nm, which is
indicative of an oxidation from 3,5-di-tert-butylcatchol to
3,5-di-tert-butyl-o-quinone. Consequently, the catecholase
activity of all complexes was studied and saturation kinetics
at high substrate concentrations were found for all com-
pounds. Complex 3 was found to have the highest turnover
number, with a rate of catalysis kcat. =40.0ꢂ0.3 hꢀ1. The ini-
tial rates, as well as the Lineweaver–Burk plots are depicted
in Figures S1–S6 in the Supporting Information. The results
of the kinetic investigations are summarized in Table 4.
have been optimized at the B3-LYP/SV(d) level of theory.
The resulting relative energies (DE), the bond lengths of the
ꢀ
coordinating Cu X linkage of the boat conformers (RCu-X),
and the dihedral angle between the two piperazine nitrogen
atoms and the two coordinating oxygen atoms of the left
copper center (aNNOO), which is a measure for the pla-
narity of the ligand sphere of this metal center, are shown in
Table 5.
For the thiomorpholine system, the isomer with a boat
conformation of the subunit (3B) is found to be
5.5 kcalmolꢀ1 more stable than the corresponding armchair
conformer (3C). Moreover, the structure of 3B possesses a
linkage of the sulfur atom to one of the copper centers
(RCu-S =2.42 ꢃ), which clearly indicates that the sulfur atom
is capable of displacing the acetate bridge and building a
free coordination site for the substrate, thus yielding an
almost planar surrounding (aNNOO=2.98) of the lower-
coordinated (left) copper center.
Table 4. Summary of the kinetic data of 1–3.
Complex
kcat. [hꢀ1
]
KM[a][molLꢀ1
]
1
2
3
10.7ꢂ0.1
28.9ꢂ0.1
40.0ꢂ3
(5.2ꢂ0.3)ꢄ10ꢀ3
(6.7ꢂ0.3)ꢄ10ꢀ4
(3.4ꢂ0.4)ꢄ10ꢀ3
For the morpholine compound, the boat (2B) and arm-
chair (2C) structures are rather close in energy. 2C is found
[a] KM =Michaelis constant.
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Chem. Eur. J. 2005, 11, 1201 – 1209

Catechol Oxidase Models
FULL PAPER
Table 5. Relative energies and structural parameters of the model complexes optimized at the B3-LYP/SV(d)
level of theory.
ed piperidine (4), morpholine
(5), and thiomorpholine (6)
subunits are highly desirable
(Scheme 3). Although this topic
has been previously investigat-
ed[68,69] for 4 and 5, these results
were calculated by quantum-
chemical methods of different
quality and are difficult to com-
pare for the distinct structures.
Moreover, it seems necessary to
apply the same methods as
those used for 1–3 to be able to
System
X
Ligand conformation
RCu-X [ꢃ]
aNNOO [8]
DE [kcalmolꢀ1
]
1A: [LCu2(OAc)2]+
CH2
CH2
CH2
O
O
O
S
S
S
0.0
196.1
185.7
0.0
203.6
202.2
0.0
1B: [LCu2(OAc)]2+ + OAcꢀ
1C: [LCu2(OAc)]2+ + OAcꢀ
2A: [LCu2(OAc)2]+
boat
armchair
3.12
1.3
25.4
2B: [LCu2(OAc)]2+ + OAcꢀ
2C: [LCu2(OAc)]2+ + OAcꢀ
3A: [LCu2(OAc)]2+
boat
armchair
2.27
2.42
3.0
25.4
3B: [LCu2(OAc)]2+ + OAcꢀ
3C: [LCu2(OAc)]2+ + OAcꢀ
boat
armchair
2.9
25.3
195.5
201.0
to be only 1.4 kcalmolꢀ1 lower in energy than 2B. It is also
noteworthy in this case that the subunit with the boat con-
formation is capable of establishing a coordinative bond to-
wards one of the copper centers (RCu-O =2.27 ꢃ), again re-
sulting in an almost planar coordination (aNNOO=3.08)
of the other metal center. The methylene group of the piper-
idine ring in 1 is not able to undergo binding interactions
(RCu-CH2 =3.12 ꢃ) with the copper ion and, as a result, the
chair conformation (1C) is significantly stabilized (by
10.4 kcalmolꢀ1) compared to the boat conformation (1B) of
the piperidine subunit.
By a closer inspection of the molecular structures it can
be seen that all isomers carrying a subunit in the boat con-
formation provide a planar surrounding of the lower-coordi-
nated copper center. The corresponding dihedral angles are
found to be 1.3–3.08, whereas for the corresponding arm-
chair isomer this angle is about 258. This observation, in
combination with the knowledge of the relative stabilities of
the isomers in question, provides an explanation for the dif-
ferent reaction rates observed in the kinetic studies. The
discuss the results in the whole context. For these reasons,
we decided to repeat the calculations for the various con-
formers by using density functional theory and perturbative
methods together with larger AO basis sets. For each arm-
chair and boat conformer there exist two possible orienta-
ꢀ
tions of the N H hydrogen atom, either in an equatorial or
an axial position. But only two of these conformers, that is,
the ones which correspond to the subligand orientation in
the model complexes (1–3), are of relevance for this study:
ꢀ
a boat isomer with the N H in the equatorial position and
ꢀ
an armchair isomer with the N H in the axial position
(Scheme 3). The results are shown in Table 6. From the data
Table 6. Relative energies of the investigated piperidine, morpholine,
and thiomorpholine conformers with respect to their individual armchair
(equatorial) conformers [kcalmolꢀ1].
Compound
B3-LYP/
SV(d)
B3-LYP/
TZV(d,p)
MP2[a]
TZV(d,p)
/
MP2[a]
/
TZV(2df,2pd)
4: piperidine
A: boat (eq)
B: armchair
(ax)
7.4
0.9
7.3
0.6
7.5
0.8
7.7
0.9
ꢀ
Cu X linkage leads for 3, and to a lesser extent also for 2,
to an unusual stabilization of the boat conformation of the
coordinated ligand subunits, whereas for 1 this additional
coordinative bond cannot be established. The additional
linkage has a more planar uncoordinated metal center as a
consequence. This type of coordination geometry clearly en-
hances the reactivity of this center. With regard to the rela-
tive energies, this effect is strongly pronounced with the thi-
omorpholine ligand (in 3). For the complex with the mor-
pholine structure (2) an equilibrium mixture of both isomers
can be expected, whereas in the case with piperidine (1) this
equilibrium is clearly shifted towards the less-reactive boat
conformer, thus providing an explanation for the experimen-
tally observed turnover numbers of the complexes.
5: morpholine
A: boat (eq)
B: armchair
(ax)
6: thiomorpho-
line
A: boat (eq)
B: armchair
(ax)
9.6
1.1
9.6
0.8
10.2
1.1
10.2
1.0
10.0
0.6
10.0
0.5
10.4
0.9
10.2
0.9
[a] B3-LYP/TZV(d,p)-optimized geometries.
one can immediately see that the armchair conformations
are strongly stabilized compared to the boat conformations
for all molecules. The results show only a weak dependency
on the applied method and basis set.
ꢀ
To estimate the strength of the Cu X linkages, knowledge
of the relative energies of different conformers of the isolat-
The energy differences are large enough to allow a quali-
tative discussion, even at the B3-LYP/SV(d) level of theory.
For piperidine (4) the energetic difference between both iso-
mers is, at 6.5 kcalmolꢀ1, somewhat less than that for the
large complexes. This might be due to steric reasons. For
morpholine (5), the calculation yields an energetic differ-
ence of 8.5 kcalmolꢀ1, but in the metal complex this gap is
only 1.4 kcalmolꢀ1. This leads to an approximate binding
Scheme 3. Piperidine, morpholine, and thiomorpholine conformers.
Chem. Eur. J. 2005, 11, 1201 – 1209
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ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1207

S. Grimme, B. Krebs et al.
energy of the coordinative bond of 7 kcalmolꢀ1. In the case
of the thiomorpholine, the energetic order is reversed. For
the free ligand (6) the energetic difference is 9.4 kcalmolꢀ1
in favor of the armchair conformation, whereas for the com-
plex (3) the boat conformer is 5.5 kcalmolꢀ1 lower in energy
than the armchair conformer. The approximated stabiliza-
tion in the complex amounts to 15 kcalmolꢀ1. This large en-
ergetic difference in the gas phase should remain for the
complex in solution but probably to a lesser extent. This sta-
bilizing effect and the subsequent influence upon the molec-
ular structure of the model complexes provide a sound ex-
planation for the different reaction rates of the three differ-
ent compounds.
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In summary, we have presented the synthesis and characteri-
zation of three new compartmental ligands and their dinu-
clear copper(ii) complexes. The utilized ligands were penta-
dentate m-phenoxo-bridged and differ in one donor arm that
was changed from (N-(2-aminoethyl)piperidine to N-(2-ami-
noethyl)-morpholine or N-(2-aminoethyl)thiomorpholine.
The crystal structures of these three new compounds re-
vealed broad resemblances. Kinetic investigations based on
the Michaelis–Menten model showed that the turnover
numbers of the complexes increases in the order 1<2<3.
DFT calculations and ESI-MS experiments support the
thesis that a higher affinity of the group in the position 4 of
the piperidine ring of the ligand to the copper ion enhances
removal of one acetate bridge. The resulting free coordina-
tion site can be used for substrate binding, thereby resulting
in higher turnover numbers for the catechol oxidation. The
presented compounds are excellent models for the active
site of catechol oxidase that mimic the short copper–copper
distance of approximately 3 ꢃ as well as the dissimilar envi-
ronment of the metal ions, including a thioether bond in 3.
The electronic spectra of all three compounds show typical
features of copper(ii) complexes.
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Financial support from the Deutsche Forschungsgemeinschaft (Program
SFB 424—Molecular Orientation and its Functions in Chemical Systems)
and the Fonds der Chemischen Industrie is gratefully acknowledged.
1208
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 1201 – 1209

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Received: July 27, 2004
Published online: December 27, 2004
Chem. Eur. J. 2005, 11, 1201 – 1209
www.chemeurj.org
ꢂ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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