An approach to model the active site of peptidglycine-α-hydroxylating monooxygenase (PHM)

Article abstract of DOI:10.1002/zaac.201300066

The copper(I) and copper(II) complexes [Cu((TMG_et) ₂N_etSEt)]BPh₄ (1·BPh₄) and [Cu((TMG_et)₂N_etSEt)Cl]Cl (2·Cl) with (TMG_et)₂N_etSEt = ((Me₂N) ₂C=NCH₂CH₂)₂NCH₂CH ₂SEt were synthesized and structurally characterized as a model system for the copper enzyme PHM, a monooxygenase involved in the activation of peptide hormones and neuropeptides. The reaction of the copper(I) complex 1·BPh₄ with dioxygen has been studied using low temperature stopped-flow methods. However, in contrast to PHM no formation of an end-on copper superoxido complex could be observed. Instead an equilibrium between a bis-μ-oxo and a side-on peroxide complex was detected spectroscopically. Copyright

Full text of DOI:10.1002/zaac.201300066

ARTICLE
DOI: 10.1002/zaac.201300066
An Approach to Model the Active Site of Peptidglycine-α-hydroxylating
monooxygenase (PHM)
[
a]
[b]
[a]
[c]
Tobias Hoppe, Patrick Josephs, Natascha Kempf, Christoph Wölper,
[a]
[b]
[b]
Siegfried Schindler,* Adam Neuba,* and Gerald Henkel
Keywords: Copper; Dioxygen activation; Model compounds; Guanidine ligands; Crystal structure
Abstract. The copper(I) and copper(II) complexes [Cu((TMGet
etSEt)]BPh (1·BPh ) and [Cu((TMGet etSEt)Cl]Cl (2·Cl) with
TMGet etSEt = ((Me N) C=NCH CH NCH CH SEt were syn-
thesized and structurally characterized as a model system for the cop-
per enzyme PHM, a monooxygenase involved in the activation of pept-
ide hormones and neuropeptides. The reaction of the copper(I) com-
)
2
-
4
plex 1·BPh with dioxygen has been studied using low temperature
N
(
4
4
)
2
N
stopped-flow methods. However, in contrast to PHM no formation of
an end-on copper superoxido complex could be observed. Instead an
equilibrium between a bis-μ-oxo and a side-on peroxide complex was
detected spectroscopically.
)
2
N
2
2
2
2
)
2
2
2
Introduction
3 for a detailed discussion on the according copper chemistry).
Mainly to the lability of iron and copper “dioxygen adduct”
complexes it took a long time before one of these complexes
could be finally fully characterized. It was 1988 when Karlin
and co-workers could structurally characterize a dinuclear cop-
per peroxido complex.[ In contrast related complexes of co-
balt have been described much earlier. Based on previous work
by Fremy, Alfred Werner had already investigated in 1898 the
reaction of cobalt(II) complexes with dioxygen and correctly
assigned the formation of the dinuclear cobalt peroxido com-
Selective oxidations of organic substrates using air (di-
oxygen) as an oxidant belong to a very important class of
chemical reactions in industry. Due to the fact that nature can
perform this task easily using metalloenzymes (mainly with
iron or copper ions in the active site) under ambient conditions,
efforts are in progress to use according model compounds as
catalysts instead.[ Prior to the reaction with an organic sub-
strate dioxygen needs to be activated by binding to a metal ion
that in general leads in a first step to an end-on binding of
dioxygen according to the following equation (L = ligand,
charges are omitted):
4]
1]
4+ [5|
plex [(NH ) Co–O–O–Co(NH ) ] .
3
5
3 5
It had been suggested for quite a long time that dioxygen
can coordinate in some copper enzymes as an end-on superox-
ido ligand. However, it was only in 2004 when Prigge et al.
finally could solve the crystal structure of peptidglycine-α-hy-
droxylating monooxygenase (PHM) actually demonstrating
this binding mode.[ PHM is a precatalytic copper enzyme
complex involved in the activation of peptide hormones and
neuropeptides.
However, this complex is usually only a transient intermedi-
6]
ate that can further react to e.g. side-on superoxido, oxido or
peroxido complexes.[2] All of these species can either react
further or initiate the oxidation of the substrate (see reference
Shortly after that report on PHM the first model complex
[
7]
for this way of binding could be structurally characterized.
*
*
Prof. Dr. S. Schindler
Fax: +49-641-99-34149
To avoid immediate consecutive reactions of the mononuclear
end-on superoxido copper complex the ligand had to provide
the exact electronic and steric properties. Here a tren-guanidin
E-Mail: siegfried.schindler@anorg.chemie.uni-giessen.de
Dr. A. Neuba
Fax: +49-5251-602165
E-Mail: adam.neuba@uni-paderborn.de
ligand proved (TMG tren, Figure 1) to be a perfect match and
3
[
[
a] Institut für Anorganische und Analytische Chemie
Justus-Liebig-Universität
with this ligand a quite stable end-on superoxido copper com-
plex formed that could be investigated in great detail for the
Heinrich-Buff-Ring 58
35392 Gießen, Germany
[7,8]
first time.
b] Department Chemie
Fakultät für Naturwissenschaften
Universität Paderborn
Warburgerstr. 100
However, while there is a perfect match of the model com-
pound for the end-on superoxido coordination the other ligands
in PHM are different. As discussed previously by Itoh and co-
workers, the copper ion in the active site in PHM is coordi-
nated by only three ligands, two histidines and one methio-
nine.[ Therefore, we decided to match the active site of the
3
3098 Paderborn, Germany
[
c] Anorganische Chemie
Universität Duisburg-Essen
Universitätsstr. 5–7, S07 S03 C30
9]
45117 Essen, Germany
1
504
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 639, (8-9), 1504–1511

Approach to Model the Active Site of Peptidglycine-α-hydroxylating monooxygenase
The tripodal thioether bisguanidine (TMG ) N SEt was ob-
et 2 et
tained in four synthetic steps according to the following reac-
tion scheme:
Scheme 1. Synthesis of (TMGet
BrCH CN, iPr NEt; iii: THF/LiAlH
CH CCl
)
2
N
etSEt: i: EtOH/KOH, CH
3
CH
2
I; ii:
N,
, conc. H SO ; iv: MeCN/Et
2
4
3
3 2
Figure 1. The ligands TMG tren and (TMGet) NetSEt.
2
2
4
(
3
)
4
N
2
2
.
Starting from 2-aminoethanethiol the corresponding ethyl-
enzyme a bit more closely by replacing one of the guanidine
groups with a sulfur donor unit leading to the new ligand
thioether was prepared. In the next step the amino function
was alkylated with two equivalents of bromoacetonitrile under
basic conditions. After reduction of the nitrile groups with
AlH3 the bisamine was reacted with two equivalents of
N,N,NЈ,NЈ-tetramethylchloroformamidinium chloride to the
thioether-bisguanidine.[
(
TMG ) N SEt (= ((Me N) C=NCH CH ) ·NCH –CH SEt
et 2 et 2 2 2 2 2 2 2
shown in Figure 1). Here we now describe the synthesis of the
according copper(I) complex and its reactivity towards di-
oxygen.
14]
The copper(I) and copper(II) complexes using this thioether
bisguanidine ligand could be prepared according to Scheme 2.
Results and Discussion
Reaction of (TMG ) N SEt with CuCl in a mixture of THF
et 2 et
During the last years copper thiolate and copper nitrogen and MeCN (ratio 1:1) led to a yellow solution. After addition
systems with different N/S- as well as guanidine ligands were of NaBPh and fractional crystallization slight blue crystals of
4
synthesized to mimic the structural, physical and functional [Cu((TMG ) N SEt)]BPh (1·BPh ) could be obtained. The
et 2 et
4
4
characteristics of active sites in metalloproteins, e.g. cyto- copper(II) complex [Cu((TMG ) N SEt)Cl]Cl (2·Cl) was
et 2 et
[10,11]
chrome-c oxidases or tyrosinases.
Recently, the ligand synthesized by reaction of CuCl2 in the presence of
design was additionally extended towards sulfur guanidine- (TMG ) N SEt in MeCN as dark green colored crystals after
et 2 et
type systems possessing basic Nimine donor functions similar slow diffusion of Et O into the mother liquor.
2
to the imidazole group of the amino acid histidine. This basic-
The molecular structure of 1 is shown in Figure 2. All rel-
ity mimics the naturally occurring coordination properties evant crystallographic data and details of the data collections
much better than aliphatic amines or Schiff bases. Further- are listed in Table 1 and Table 2.
I
more, the thioether donor function of sulfur guanidines re-
In 1 the Cu atom has a pseudo-trigonal bipyramidal ar-
sembles the side chain of the amino acid methionine. Based rangement (N S donor set). The tertiary amine atom N(7) oc-
3
on established synthetic protocols several thioether-guanidines cupies the apical (ap) position whereas the donor atoms N(1),
and copper-thiolate/thioether complexes have successfully N(4) and S(1) complete the equatorial (eq) positions around
been synthesized previously.[
12,13]
the Cu(1) atom. The angle sum around Cu(1) is 358.9° with
Scheme 2. Synthesis of [Cu((TMGet
Z. Anorg. Allg. Chem. 2013, 1504–1511
)
2
N
etSEt)]BPh
4 4 2
(1·BPh ) and [Cu((TMGet) NetSEt)Cl]Cl (2·Cl).
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1505

T. Hoppe, P. Josephs, N. Kempf, C. Wölper, S. Schindler, A. Neuba, G. Henkel
ARTICLE
N3S
tripodal N S ligands iPrSPy and L
(Figure 3) related to
3
2
[
16,17]
(
TMG ) N SEt) have been reported previously.
et 2 et
Both complexes have a trigonal-pyramidal arrangement. The
Table 2. Selected bond lengths /pm and angles /° for 1 and 2.
Atoms
Bond length
Atoms
Angle
1
Cu(1)–N(1)
Cu(1)–N(4)
Cu(1)–N(7):
Cu(1)–S(1)
N(1)–C(1)
N(2)–C(1)
N(2)–C(2)
N(4)–C(10)
203.9(1)
203.1(1)
219.6(1)
226.0(1)
130.4(2)
138.4(2)
145.4(2)
130.7(2)
137.5(2)
137.6(2)
N(4)–Cu(1)–N(1):
N(4)–Cu(1)–N(7)
N(1)–Cu(1)–N(7)
N(4)–Cu(1)–S(1)
N(1)–Cu(1)–S(1)
N(7)–Cu(1)–S(1)
120.17(4)
84.94(4)
83.80(4)
124.20(3)
114.51(3)
90.71(13)
Figure 2. Molecular structure of 1 in the crystalline state.
N /S–Cu–N angles from 114.51(3) to 124.20(3)° and a mean
eq
eq
value of 119.6° (ideal-typical value is 120°). The Cu–Neq bond
lengths (203.9(1) and 203.1(1) pm) are nearly the same and N(5)–C(10)
N(6)–C(10)
shorter than the Cu–N /Cu–S bond lengths with 219.6(1) and
ap
226.0(1) pm. The C=N bond lengths are similar with a mean
2
value of 130.5 pm. The N –Cu–Neq angles are little smaller
than the ideal-typical value of 90° [84.94(4) and 83.80(4)°]. In Cu(1)–N(2)
ap
Cu(1)–N(1)
205.4(2)
195.8(2)
196.4(2),
223.8(1)
131.0(3)
132.3(3)
136.4(3)
135.2(3)
135.4(3)
136.5(3)
N(2)–Cu(1)–N(3)
N(2)–Cu(1)–N(1)
N(3)–Cu(1)–N(1)
N(2)–Cu(1)–Cl(1)
N(3)–Cu(1)–Cl(1)
N(1)–Cu(1)–Cl(1)
142.02(7)
85.44(7)
84.86(7)
99.35(5)
106.77(5)
150.88(5)
Cu(1)–N(3)
Cu(1)–Cl(1)
N(2)–C(5)
N(3)–C(6)
contrast the N –Cu–S angle measures 90.71(13)°. Conse-
ap
quently, the copper atom in 1 is slightly axially distorted, being
localized below the equatorial plane defined by the guanidine
nitrogen and sulfur atoms. In the reduced form of PHM and
N(4)–C(5)
DβM, the Cu–S distances were estimated to be 2.27 and N(5)–C(5)
.25 Å.[15]
Thus 1 has structural relevance to the reduced
N(6)–C(6)
N(7)–C(6)
2
form of the copper enzymes. Copper complexes with the
Table 1. Crystal data for [1·BPh
·BPh
61BCuN
4
] and [2·Cl].
1
4
2·Cl
Empirical formula
Empirical weight
Crystal size/mm
Temperature/K
Crystal system
Space group
Unit cell dimensions /Å,°
a
C
42
H
7
S
C
18
H
41CuN
7 2
SCl
770.39
0.50 ϫ 0.45 ϫ 0.36
100(2)
522.08
0.28 ϫ 0.08 ϫ 0.03
100(1)
Monoclinic
1
P2 /c
Triclinic
¯
P1
11.1039(7)
12.2233(8)
15.8202(10)
103.3980(10)
90.2490(10)
96.5610(10)
2074.0(2)
2
1.234
0.615
Bruker SMART
Graphite
0.71073
12.2614(16)
9.1216(12)
22.918(3)
b
c
α
β
98.698(7)
γ
Volume /Å3
2533.7(6)
4
1.369
1.174
Z
Density (calcd.) /g·cm–
Absorption coeff. /mm–1
Data collection
Monochromator
Wavelength /Å
Theta range /°
Index ranges hkl
Reflections collected
Independent reflections
Absorption correction
3
[
19]
APEX CCD
Bruker D8 KAPPA series II with APEX 2 CCD
Triumph Goebel mirror
1.32 to 27.88
Ϯ14, –15/16, Ϯ 20
19851
9711 [R(int) = 0.0110]
1.68 to 27.21
–15/13, Ϯ11, Ϯ29
22995
5586 [R(int) = 0.0306]
Semi-empirical from equivalents BRUKER AXS APEX 2 Vers. 3.0–2009
Semi-empirical from equivalents
[
19]
(SADABS
)
2
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
R1 [I Ͼ 2σ(I))
Full-matrix least-squares an F
9711 / 0 / 479
1.052
4862 / 2 / 261
1.025
0.0339
0.0302
wR2 (all Data)
0.0845
–0.34/0.52
0.0909
–0.81/1.24
min/max ΔF /e·Å–3
1
506
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 1504–1511

Approach to Model the Active Site of Peptidglycine-α-hydroxylating monooxygenase
Cu–Neq and Cu–Nap bond lengths are nearly the same as in 1.
I
N3S 2+
Only the Cu–Nap bond length in [Cu (L )]2 is slightly
longer (225.5(1) pm) while the Cu–S bond lengths are again
of similar length (Table 3).
[
16]
Figure 3. Schematic drawings of the tripodal ligands iPrSPy
N3S [17].
2
and
L
I
Table 3. Selected bond lengths /pm of 1 and reported Cu complexes
Figure 4. Molecular structure of 2 in the crystalline state.
3 4
with tripodal N S/N ligands.
[Cu (PMDTA)Cl]+ are 202.8/204.9 (mean values) and
222.1(1)/222.0(1) pm, respectively. These values are in good
agreement with the structural parameters of 2 with the average
distance of 199.2 pm for the Cu–N bond lengths and 223.8(1)
pm for the Cu–Cl bond length.
II
Compound
Cu–Neq
*
Cu–Nap
Cu–S
I
etSEt)]+
[16]
[
[
[
[
Cu ((TMGet
)
2
N
203.5
200.8
203.9
205.2
219.6(1)
217.3(9)
225.5(1)
219.3(3)
226.0(1)
224.9(3)
220.2(1)
I
Cu (iPrSPy
2
)](ClO
4
)
I
N3S
[17]
2 6 5 4 2
Cu (L )] [B(C H ) ]
I
[18]
3
Cu (TMG tren)]Cl
*
Average bond length of the equatorial nitrogen atoms to metal
While the molecular structure of 1 is quite similar to the
atom.
analogous copper(I) complex of TMG tren as discussed above,
3
its reaction behavior towards dioxygen was quite different.
Furthermore it is interesting to compare
1
with
tren)]+ reacts reversibly with dioxygen to
I
+
[18]
While [Cu(TMG
form the reported end-on superoxido copper complex
3
[
Cu (TMG tren)] reported by Raab et al.
There are no sig-
3
[
7,8]
this
nificant differences in coordination geometry and structural pa-
rameters (see Table 3). The average of the C=N bond lengths
129.5 pm) is the same as in 1 (130.5 pm) but the axial distor-
was not the case for 1. Analogous low temperature stopped-
flow measurements did not allow the detection of such an in-
termediate in different solvents and at different temperatures.
As an example, time-resolved UV/Vis spectra for the reaction
of 1·OTf in acetone at –76 °C are presented in Figure 5 (triflate
as an anion was used for the spectroscopic measurements as
described in the experimental section).
(
tion of the copper atom shows a small difference (20.5 pm in
I
+
[
Cu (TMG tren)] , 12.8 pm in 1).
3
The molecular structure of 2 is presented in Figure 4. All
relevant crystallographic data and details of the data collec-
II
tions are listed in Table 1 and Table 2. In 2 the Cu atom pos-
sesses a distorted square planar arrangement (N Cl donor set)
3
without coordination of the thioether sulfur atom. The bite
angles N(1)–Cu(1)–Cl(1) and N(3)–Cu(1)–N(1) of 99.35(5)
and 84.86(7)° would fit good into a square-planar metal coor-
dination, but the planes containing these angles are twisted
against each other by 42.5° in order to minimize steric strain.
The ideal-typical value for a tetrahedron is 90° and for a
square-planar coordination 0°. Hence, the observed coordina-
tion in 2 has intermediate character between the square-planar
and the tetrahedral situation. The Cu–Ngua and Cu–Ntert bond
lengths (196.4(2), 195.8(2) and 205.4(2) pm) are slightly
shorter than 1 (Cu–Ngua: 203.5 pm (mean value), Cu–Ntert
:
219.6(1)). For C=N bond lengths in 1 and 2 no significant
differences are observed.
In the literature only a few CuII complexes with a
II
N X donor set are reported: [Cu (L)Cl]ClO (L = bis(2-
3
4
[20]
fluorobenzylaminoethyl)amine) from Dong et al.
Cu (PMDTA)Cl]PF (PMDTA = pentamethyldiethylenetri-
amine) from Margraf et al. . Both complexes exhibit nearly
and Figure 5. Time-resolved UV/Vis spectra of the reaction of O
1·OTf in acetone at –76 °C temperature (Δt = 15 s, overall time =
55 s).
2
with
II
[
6
2
[21]
regular square-planar coordination geometry. The Cu–N as
From previous reports by us and others we can assign the
II
+
well as Cu–Cl bond lengths in [Cu (L)Cl]
and spectral changes to the formation of at least two species, a
Z. Anorg. Allg. Chem. 2013, 1504–1511
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.zaac.wiley-vch.de 1507

T. Hoppe, P. Josephs, N. Kempf, C. Wölper, S. Schindler, A. Neuba, G. Henkel
ARTICLE
dinuclear side-on peroxide (maximum at 360 nm) and a bis-μ- way as described earlier by the same authors for [(tmpa)Cu-
oxido complex (maximum at 425 nm).[ The equilibrium for O-O-Cu(tmpa)] (tmpa = tris(2-pyridylmethyl)amine), the all
23]
2+
N3S
these two species according to the following equation (charges nitrogen analogue. However, with L
as a ligand it was
are omitted) is well known.[
23a,b]
found that in the molecular structure of the according copper(I)
complex the sulfur donor atom did not coordinate to the same
copper ion as the rest of the ligand. Instead it coordinated to
the copper ion of a second complex thus leading to a dinuclear
copper(I) complex [{(L )Cu}2] bridged over two sulfur
arms. Obviously in solution the situation is different demon-
strating again that molecular structures only should give some
ideas about the situation in solution.
N3S
2+
A transient end-on superoxido copper intermediate will form
here as well prior to the formation of these dinuclear com-
plexes. However, so far it turned out that this species could
not be observed for this type of reactions. This can be easily
explained by the kinetics of these reactions: the consecutive
reactions are much faster than the formation of the end-on su-
peroxido complex and therefore there is no build-up of this
intermediate to be detected.[
Conclusions
23c,e]
We could develop a synthetic protocol for introducing a
Kinetic data fitting turned out to be complicated and so far thioether group into a tren guanidine system to allow the syn-
was not possible in contrast to our previous investigations on thesis of copper complexes that allow more closely to model
related systems.[
23e]
Some decomposition is observed (insert in the natural analogue PHM. While we have not been successful
Figure 5) and there might be other complexes involved as well. to obtain a more closer functional model of PHM using the
However, we could observe that the dinuclear side-on peroxido ligand (TMG ) N SEt our results are interesting in regard to
et 2 et
complex is more stable at higher temperatures than the bis-μ- a general better understanding of the reactivity of copper(I)
oxido complex, a finding that goes along with previous reports complexes towards dioxygen. Furthermore, we could show
in the literature. Due to the fact that the reaction of 1 with how sensitive these systems react towards changes in its ligand
dioxygen turned out to be complicated and especially did not environment. That it is not easy to develop a good functional
allow us to obtain a stable end-on superoxido complex we did model system for PHM has been demonstrated by the reports
not investigate this reaction further.
The reason that compound 1 did not react as expected to the (only some selected examples are given in the reference).[25]
end-on superoxido complex can be explained with the molecu- Most likely in our system the ethyl group at the sulfur atom
of other research groups who are working on this topic as well
lar structure of 2. In 2 the sulfur atom is not coordinated any- is sterically too demanding to allow coordination at the same
more to the copper(II) ion. Instead a chloride ion has replaced metal atom. Therefore, we are quite optimistic that substitution
this donor unit. While it is clear that a crystal structure does of the ethyl group in (TMG ) N SEt to a methyl group should
et 2 et
not have to represent the complex environment in solution it allow the preparation of a quite stable copper end-on super-
here points out an obvious possibility. Thus the tetradentate oxido complex. Work is currently in progress to demonstrate
ligand has become a tridentate ligand and the copper(I) com- this, however ligand synthesis of (TMG ) N SMe has turned
et 2 et
plex reacts as one would expect, to the dinuclear side-on per- out to be quite demanding.
oxido or the bis-μ-oxido complexes represented in following
Figure 6.
Experimental Section
It is interesting to compare our results with previous results
by Karlin and co-workers using the related tripodal copper
N3S
[17,24]
Materials and Methods
complex with the ligand L
(Figure 3).
Here the system
followed pretty much the expectations. An end-on peroxido All manipulations were performed under nitrogen (99,996%) dried
N3S
N3S 2+
4
complex, [(L )Cu-O-O-Cu(L )] , formed in the same with P O10 granulate using Schlenk techniques. Solvents were purified
Figure 6. Representation of the dinuclear side-on peroxido and according bis-μ-oxido copper complex (R = CH
2 2
CH SEt).
1
508 www.zaac.wiley-vch.de © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Z. Anorg. Allg. Chem. 2013, 1504–1511

Approach to Model the Active Site of Peptidglycine-α-hydroxylating monooxygenase
according to literature procedures[22] and stored under nitrogen. wards, water (5 mL) was added carefully at 0 °C. The suspension was
N,N,NЈ,NЈ-Tetramethylchlorformamidinium chloride was prepared ac-
filtered through a frit and the collected precipitate was washed with
[
15]
cording to literature procedure.
purchased from Sigma–Aldrich.
2-Aminoethanethiol was used as 400 mL THF. The organic phases were dried with sodium sulfate and
the solvent was removed under reduced pressure to give a green-yel-
1
low oil. Yield: 1.7 g (45%). H NMR (500 MHz, CDCl
3
, 25 °C,
), 2.50 (q, 6 H, CH ),
). C NMR
), 29.3
). CI-MS (m/z (%)): 192.11
δ [ppm]): 1.23 (t, 3 H, CH
3
), 1.42 (s, 4 H, NH
2.61 (d, 2 H, CH ), 2.66 (d, 2 H, CH
2
2
1
3
Physical Measurements
2
2
), 2.72 (t, 4 H, CH
2
(
(
(
125 MHz, CDCl
CH ), 40.0 (CH ), 55.4 (CH
16) [MH ], 56.97 (100). IR (NaCl, ν [cm ]): 667m, 732m, 867w,
3
, 25 °C, δ [ppm]): 14.9 (CH
3 2
), 26.1 (CH
Spectra were recorded using the following spectrometers: NMR:
Bruker Avance (500 and 125 MHz, respectively). The NMR signals
were referenced to residual solvents measured relative to TMS. IR:
Bruker Vertex 70. UV: Varian Cary 50. MS (EI: 200 °C, 70 eV; CI:
2
2
2
), 57.6 (CH
2
+
–1
948m, 1047m, 1097w, 1267m, 1376w, 1454m, 1645s, 2958s, 3359vs.
80 °C, 150 eV): DFS Thermo Scientific.
2
Ј,2Ј-(((2-(Ethylthio)ethyl)azanediyl)bis(ethane-2,1-diyl))bis-
(
2
1,1,3,3-tetramethyl-guanidine) ((TMGet) NetSEt): A solution of
N,N,NЈ,NЈ-Tetramethylchlorformamidiniumchloride (3.24 g, 0.020
Preparation of Compounds
mol) in dry MeCN (60 mL) was added dropwise under vigorous stir-
1
1
ring to an ice-cooled solution of N -(aminomethyl)-N -(2-(ethylthio)-
ethyl)ethane-1,2-diamine (1.91 g, 0.010 mol) and triethylamine
2-(Ethylthio)ethanamine: To a solution of KOH (5.8 g, 0.1 mol) in
Ethanol (500 mL) was added 2-aminoethanethiol (7.72 g, 0.1 mol) and
stirred at room temperature for 1 h. Iodoethane (16.1 g, 8 mL, 0.1 mol)
was added dropwise at 0 °C to the suspension. Afterwards, the mixture
was heated to reflux for 3 h and after cooling the ethanol was removed
under reduced pressure. The oily solid was treated with water
(
2.02 g, 2.8 mL, 0.020 mol) in dry MeCN (80 mL). After 3 h under
reflux, a solution of NaOH (0.84 g, 0.020 mol) in water (3 mL) was
added. Under vacuum, solvents and NEt were evaporated. In order to
3
deprotonate the bis-hydrochloride, KOH (50 wt.-%, aq, 25 mL) was
added and the free base was extracted into the MeCN phase (3 ϫ
(150 mL) and extracted with dichloromethane (3 ϫ 80 mL). The col-
25 mL). The organic phase was dried with Na
2 4
SO and the solvent
lected organic phases were dried with sodium sulfate and the solvent
was removed under reduced pressure to give a yellow oil. Yield: 6.3 g
evaporated under reduced pressure to give a magenta colored oil. Yield
1
3
.4 g (88%). H NMR (500 MHz, CDCl
H, CH ), 2.48 (q, 2 H, CH ), 2.57 (m, 8 H, CH2, CH
), 2.67 (s, 12 H, CH ), 2.73 (m, 8 H, CH , CH
). 3C NMR (125 MHz, CDCl
, 25 °C, δ [ppm]): 15.1 (CH
), 29.3 (CH ), 38.6 (CH ), 38.9 (CH ), 39.7 (CH ), 48.4 (CH
5.4 (CH ), 57. 6 (CH ), 160.5 (Cgua.) CI-MS (m/z (%)): 388.40 (26)
3
, 25 °C, δ [ppm]): 1.18 (t, 3
), 2.62 (q, 4 H,
), 3.18 (q, 4 H,
), 26.1
),
1
(
60%). H NMR (500 MHz, CDCl
3
, 25 °C, δ [ppm]): 1.07 (t, 3 H,
), 2.35 (q, 2 H, CH ), 2.44 (t, 2 H, CH ), 2.68
). C NMR (125 MHz, CDCl , 25 °C, δ [ppm]): 14.8
), 25.5 (CH ), 35.8 (CH ), 41.1 (CH ). EI-MS (m/z (%)): 106,11
6) [M ], 105(84), 76,05 (100). IR (NaCl, ν˜ [cm ]): 850vs, 1002m,
3
2
3
CH
3
), 1.18 (s, 2 H, NH
t, 2 H, CH
2
2
2
CH
CH
2
3
2
3
13
(
(
(
2
3
1
2
3
3
CH
3
2
2
2
(CH
2
2
3
3
3
2
+
–1
5
[
2
2
1072w, 1228w, 1261s, 1375m, 1452s, 1591m.
+
–1
MH ], 56.97 (100). IR (NaCl, ν [cm ]): 748w, 782w, 914m, 991m,
1
2
060m, 1133vs, 1234m, 1365vs, 1452m, 1496s, 1616vs, 1648s,
875m.
2
,2Ј-((2-(Ethylthio)ethyl)azanediyl)diacetonitrile:
2-(ethylthio)-
ethylamine (29.15 g, 0.275 mol) and diisopropylethylamine (77.5 g,
05.5 mL, 0.6 mol) were placed in a three neck Schlenk flask fitted
1
[Cu((TMGet
)
2
NetSEt)]BPh
4 4 2
(1·BPh ): The reaction of (TMGet) -
with a condenser and magnetic stir bar. At 0 °C two equivalents of
bromoacetonitrile (66.0 g, 38.3 mL, 0.555 mol) was added dropwise
to the solution. Afterwards the mixture was heated to 60 °C for 3 h.
During this time the mixture solidified and became brown. After cool-
ing the oily solid was treated with water (150 mL) and extracted with
dichloromethane (3 ϫ 80 mL). The collected organic phases were
dried with sodium sulfate and the solvent was removed under reduced
pressure. The brown oily residue was extracted (3 ϫ 250 mL) with an
alloy of hexane and ethyl ether (ratio 5:1). The collected hexane/di-
ethyl ether phases were dried with sodium sulfate and the solvent was
N
etSEt (407 mg, 1.05 mmol) with CuCl (99 mg, 1 mmol) suspended in
abs. MeCN/THF (5 mL, ratio 1:1) led to a yellow solution. The reac-
tion mixture was stirred for a period of 30 min at room temperature.
Then NaBPh
stirred for further 24 h. Filtration of the suspension and slow diffusion
of Et O into the filtrate afforded slightly blue crystals of 1·BPh in
small yield suitable for X-ray diffraction. IR (KBr, ν˜ = [cm ]): 574w,
75w, 898w, 1072w, 1159m, 1232w, 1394m, 1560s, 2931m, 3427m.
4
(342 mg, 1 mmol) was added and the mixture was
2
4
–
1
7
[
Cu((TMGet
407 mg, 1.05 mmol) with CuCl
mL of abs. MeCN led to a deep green solution. The reaction mixture
was stirred for a period of 30 min at room temperature. Filtration and
slow diffusion of Et O into the filtrate afforded green crystals of 2·Cl
in small yield suitable for X-ray diffraction. UV/Vis (MeCN,
)
2
NetSEt)Cl]Cl (2·Cl): The reaction of (TMGet
2
) NetSEt
removed under reduced pressure to give a yellow oil. Yield: 36.4 g
(
3
2
(134 mg, 1 mmol) suspended in
1
(
72%). H NMR (500 MHz, CDCl
3
, 25 °C, δ [ppm]): 1.17 (t, 3 H,
), 2.59 (t, 2 H, CH ), 2.80 (t, 2 H, CH ), 3.60
). C NMR (125 MHz, CDCl , 25 °C, δ [ppm]): 14.7
), 26.1 (CH ), 28.9 (CH ), 42.3 (2 CH ), 52.9 (CH ), 114.5
quat.). EI-MS (m/z (%)): 183.09 (18) [M ], 42.0 (82), 61.0 (42), 89.0
CH
3
), 2.48 (q, 2 H, CH
s, 4 H, CH
2
2
2
13
(
(
(
(
2
3
2
CH
3
2
2
2
2
+
C
–1
–1
λ
max [nm], ε [m ·cm ]): 222 (40000), 275 (14700). IR (KBr,
–1
25), 108.0 (86). IR (NaCl, ν [cm ]): 792.s, 867.vs, 9712s, 1060m,
124vs, 1263s, 1319m, 1427s, 1747w, 2239w, 2833w, 2966m.
–1
ν˜ = [cm ]): 661m, 703s, 842w, 1047m, 1141m, 1259m, 1386s, 1512s,
581ws, 2925m, 3421w.
1
1
1
1
N -(Aminomethyl)-N -(2-(ethylthio)ethyl)ethane-1,2-diamine: To
LiAlH (4.55 g, 0.120 mol) suspended in ice-cooled dry THF (200 mL)
placed in a three neck Schlenk flask fitted with a condenser and mag-
netic stir bar was added carefully conc. H SO (6.12 g, 3.3 mL, 0.060 All relevant crystallographic data and details of the data collections
mol). After the addition was complete the suspension was stirred for are listed in Table 1. Full crystallographic data (excluding structure
4
Crystal Structure Analyses
2
4
further 30 min at 0 °C. Afterwards, 2,2Ј-((2-(Ethylthio)ethyl)azane-
diyl)diacetonitrile (3.66 g, 0.020 mol) in dry THF (60 mL) was added
dropwise to the suspension. The mixture was stirred at room tempera-
ture for 24 h. During this time the suspension became yellow. After-
factors) have been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication no. CCDC-922662 for
1·BPh
4
and 921947 for 2·Cl. Copies of the data can be obtained free
of charge on application to CCDC, 12 Union Road, Cambridge CB2
Z. Anorg. Allg. Chem. 2013, 1504–1511
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1509

T. Hoppe, P. Josephs, N. Kempf, C. Wölper, S. Schindler, A. Neuba, G. Henkel
ARTICLE
1
EZ, UK (Fax: +44-1223-336-033; E-mail: deposit@ccdc.cam.ac.uk).
[8] a) M. Schatz, V. Raab, S. P. Foxon, G. Brehm, S. Schneider, M.
Reiher, M. C. Holthausen, J. Sundermeyer, S. Schindler, Angew.
Chem. 2004, 116, 4460–4464; Angew. Chem. Int. Ed. 2004, 43,
Hydrogen atoms were refined using a riding model or rigid methyl
groups. In 2·Cl methyl carbon atom C(18) refined isotropic and disor-
dered over two sites with occupanc factors 2/3 and 1/3 together with
the riding hydrogen atoms. As a result hydrogen atoms H(17a, 17b)
are also disordered over two sites with occupancies factors 2/3 and
4360–4363; b) D. Maiti, D.-H. Lee, K. Gaoutchenova, C.
Würtele, M. C. Holthausen, A. A. N. Sarjeant, J. Sundermeyer, S.
Schindler, K. Karlin, Angew. Chem. 2008, 120, 88–91; Angew.
Chem. Int. Ed. 2008, 47, 82–85; c) J. S. Woertink, L. Tian, D.
Maiti, H. R. Lucas, R. A. Himes, K. D. Karlin, F. Neese, C.
Würtele, M. C. Holthausen, E. Bill, J. Sundermeyer, S. Schindler,
E. I. Solomon, Inorg. Chem. 2010, 49, 9450–9459.
1/3. The distances between C(17) and C(18, 18A) are restraint to a
target value of 1.542 (1)
[
9] a) S. Itoh, Curr. Opin. Chem. Biol. 2006, 10, 115–122; b) A.
Kunishita, M. Kubo, H. Sugimoto, T. Ogura, K. Sato, T. Takui,
S. Itoh, J. Am. Chem. Soc. 2009, 131, 2788–2789.
Low Temperature Stopped-flow Measurements
[
10] a) S. Herres-Pawlis, P. Verma, R. Haase, P. Kang, C. T. Lyons,
E. C. Wasinger, U. Flörke, G. Henkel, T. D. Stack, J. Am. Chem.
Soc. 2009, 131, 1154–1169; b) S. Herres-Pawlis, S. Binder, A.
Eich, R. Haase, B. Schulz, G. Wellenreuther, G. Henkel, M.
Rübhausen, W. Meyer-Klaucke, Chem. Eur. J. 2009, 15, 8678–
8682.
Low temperature stopped-flow measurements were performed as de-
scribed previously,[ however with the difference that we used a ho-
mebuilt stopped-flow instrumentation similar to a Hi-tech SF-3L low
temperature stopped-flow unit (Hi-Tech, Salisbury, UK now TgK Sci-
enctific, Bradford-on-Avon, UK). Measurements performed with this
26]
setup allowed investigations at temperatures as low as –90.0 °C. Data [11] a) J. Schneider, M. Köckerling, R. Kopitzky, G. Henkel, Eur. J.
were collected with a J&M diode array spectrometer (J&M, Aalen,
Germany) and the integrated Kinspec as well as the program Origin
were used for data fitting. For the measurements two solutions in the
syringes of the instrument were mixed. One solution was the solvent
saturated with dioxygen while the other contained the copper(I) com-
plex. The solvent saturated with dioxygen was prepared by bubbling
dry dioxygen gas (grade 5.5, Messer Griesheim, Germany) through the
solvent for about 10 minutes. The dioxygen concentration in a satu-
Inorg. Chem. 2003, 1727–1734; b) G. Henkel, J. Schneider, T.
Meyer, B. Lippold, J. Bioinorg. Chem. 2001, 86, 258–258; c) S.
Pohl, M. Harmjanz, J. Schneider, W. Saak, G. Henkel, J. Chem.
Soc., Dalton Trans. 2000, 3473–3479.
[
12] a) A. Neuba, U. Flörke, G. Henkel, Acta Crystallogr., Sect. E
2011, 67, o2887–o2887; b) A. Neuba, U. Flörke, G. Henkel, Acta
Crystallogr., Sect. E 2007, 63, o4661–o4661; c) A. Neuba, U.
Flörke, G. Henkel, Acta Crystallogr., Sect. E 2007, 63, o4683–
o4683; d) A. Neuba, U. Flörke, G. Henkel, Acta Crystallogr.,
Sect. E 2007, 63, o3476–o3477.
–2
–1
rated acetone solution is approx. 1.02ϫ 10 mol·L at room tempera-
ture.[
27]
[13] a) A. Neuba, U. Flörke, W. Meyer-Klaucke, M. Salomone-Stagni,
The complex solutions for the stopped-flow measurements
[
28]
E. Bill, E. Bothe, P. Höfer, G. Henkel, Angew. Chem. 2011, 123,
were prepared by adding a solution of [Cu(CH
in a small amount of acetone dropwise to a solution of (TMGet
7,5 mg) in acetone (2 mL). The resulting complex solution was di-
luted in a 20 mL volumetric flask to obtain an exact concentration of
3
CN)
4
]OTf
(7.7 mg)
4596–4600; Angew. Chem. Int. Ed. 2011, 50, 4503–4507; b) A.
2
) NetSEt
Neuba, R. Haase, U. Flörke, W. Meyer-Klaucke, G. Henkel, An-
gew. Chem. 2012, 124, 1746–1750; Angew. Chem. Int. Ed. 2012,
(
51, 1714–1718.
–1
1
ϫ 10–3 mol·L . The final concentration in the stopped-flow experi-
[
14] a) S. Herres-Pawlis, A. Neuba, O. Seewald, T. Seshadri, H. Egold,
U. Flörke, G. Henkel, Eur. J. Org. Chem. 2005, 4879–4890; b)
W. Kantlehner, E. Haug, W. W. Mergen, P. Speh, T. Maier, J. J.
Kapassakalidis, H.-J. Bräuner, H. Hagen, Liebigs Ann. Chem.
1984, 1, 108–126.
15] a) S. T. Prigge, A. S. Kolhekar, B. A. Eipper, R. E. Mains, L. M.
Amzel, Nat. Struct. Biol. 1999, 6, 976–983; b) B. J. Reedy, N. J.
Blackburn, J. Am. Chem. Soc. 1994, 116, 1924–1931; c) B. A.
Eipper, A. S. W. Quon, R. E. Mains, J. S. Boswel, N. J. Black-
burn, Biochemistry 1995, 34, 2857–2865.
–4
–1
ment is 5ϫ 10 mol·L after mixing.
Acknowledgments
[
We thank the Deutsche Forschungsgemeinschaft (DFG) and the Bun-
desministerium für Bildung und Forschung (BMBF) for the continuous
support of our work. A. N. is thankful to the University of Paderborn
for financial support. Prof. Sonja Herres-Pawlis is acknowledged for
providing the Vilsmeier salt.
[
16] T. Ohta, T. Tachiyama, K. Yoshizawa, T. Yamabe, T. Uchida, T.
Kitagawa, Inorg. Chem. 2000, 39, 4358–4369.
[
17] a) L. Q. Hatcher, D.-H. Lee, M. A. Vance, A. E. Milligan, R. Sa-
rangi, K. O. Hodgson, B. Hedman, E. I. Solomon, K. D. Karlin,
Inorg. Chem. 2006, 45, 10055–10057; b) D.-H. Lee, L. Q.
Hatcher, M. A. Vance, R. Sarangi, A. E. Milligan, A. A. Nard-
ucci Sarjeant, C. D. Incarvito, A. L. Rheingold, K. O. Hodgson,
B. Hedman, E. I. Solomon, K. D. Karlin, Inorg. Chem. 2007, 46,
6056–6068.
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[
Received: February 1, 2013
Published Online: April 30, 2013
Z. Anorg. Allg. Chem. 2013, 1504–1511
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
1511