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Chloranil as a Catalyst for the Electrochemical Oxidation of NADH to NAD+.

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The IR-spectrum of (I) indicates that terminal hydrido hydrogen atoms are absent. In the 'H-NMR spectrum (80
MHz, [De]toluene) two sharp singlets, among other signals,
appear at 6= - 10.36 and - 13.80 (2H each), which can be
assigned to two groups of k2-hydrido bridges, which on the
basis of the C,-symmetry of the cluster are chemically nonequivalent. Figure 2 shows the resulting structural scheme of
(1). When the temperature is increased, the hydrido signals
broaden, collapse together at CQ. 45 "C and eventually form a
sharp signal at 6= - 12.07; the other parts of the spectrum
are essentially unchanged. At higher temperatures, therefore,
a dynamic intramolecular exchange of hydrido hydrogen
atoms, involving pairs of non-equivalent H-atoms, occurs.
The protons of the propyne unit in (1) can be recognized
in the NMR spectrum by two sharp singlets at 6= 3.06 (3 H)
and 5.72 (1 H). These signals are absent in the spectrum of
the product of the reaction of [Ir(COD)Cl]2 and iC3D7MgBr-evidence that the propyne unit originates from
the Grignard reagent.
is one of the few orThe complex (I), H4(COD)41r4C3H4,
ganometallic clusters which does not contain CO-ligands and
to our knowledge is, until now, the only closed hydridometal
cluster with exclusively =-bonded olefinic ligands. In view of
this, its thermal stability is surprizingly high; crystals of (1)
do not decompose under N2-atmosphere at temperatures up
to 230 "C and can be vaporized into the mass spectromer at
CQ. 200 "C. It is therefore expected that further complexes of
this type are synthesizable.
Procedure
All operations are performed under an inert gas atmosphere. The solution of the Grignard reagent prepared from
Mg (65 mg, 2.67 mmol) and i-C3H7Br(0.3 cm') in 20 cm'
ether, is dropped over 15 min into a suspension of
[Ir(COD)C1I2 (500 mg, 0.74 mmol) in 20 cm' ether at
- 50 "C.The mixture is allowed to warm-up to room temperature and the dark brown solution irradiated for 5.5 h with
UV light from an Hg high pressure lamp. (Type Q 81, supplied by Heraeus, Hanau.) The solvent is then completely removed, the residue eluted with 100 cm3 hexane and filtered
over 3 cm Al2O3/5%H20. The solution is concentrated and
allowed to stand in the refrigerator, upon which (1) precipitates as a dark yellow powder; this is purified by recrystallization from hexane or a little toluene under refrigeration and
produces orange red crystals. Yield 55 mg (0.044 mmol;
12%).
Received: October 15, 1980 [Z 742 IE]
German version: Angew. Chem. 93, 407 (1981)
[I]J. Miiller, W. Holzinger, H. Menig, Angew. Chem. 88, 768 (1976); Angew.
Chem. Int. Ed. Engl. 15, 702 (1976); J. Muller, H. Menrg, P. V. Rinre, J. Organomet. Chem. 181, 387 (1979).
[2] J. Miiller, H. Menig, G. Huftner, A. Frank, J. Organomet. Chem. 18s. 251
(1980).
[3] G. Winkhaus, H. Singer, Chem. Ber. 99, 3610 (1966).
141 Triclinic, Pi, 2=4; a= 1549.8(4), b=2186.8(7), c=1138.3(3) pm.
a= 98.82(5), p= 92.86(5), y= 76.40(4)". Four-circle diffractometer Syntex
P2,, MoK,; of 7164 independent reflections with 28540", 3342 having
1 2 2u(I) were used for the structural determination; R=0.125 (Ir anisotropic,
C isotropic). The asymmetric unit contains two independent cluster molecules of which only one is described here; the other is, in principle, similarly
constructed.
IS] L. F Dahl, D. L. Smith, J. Am. Chem. Soc.84, 2450 (1962).
[6] R. Mason, K. M. Thomas, J. Organomet. Chem. 43, C39 (1972).
171 G. F. Stuntr, J. R. Shapley, C. G. Pierponf, Inorg. Chem. 17, 2596 (1978).
402
0 Verlog Chemie GmbH, 6940 Wernheim, 1981
Chloranil as a Catalyst
for the Electrochemical
Oxidation of NADH to NAD ["I
+
By Horst Huck and Hanns-Ludwig Schmidt[']
An "optical test" for the specific determination of substrates by means of NAD +-dependent dehydrogenases is not
suitable for the analysis of turbid solutions or for continuous
monitoring of concentrations; for these purposes an electrochemical process should be used. A prerequisite for the development of the corresponding "enzyme electrodes" or a
preparative method for the specific electrochemical dehydrogenation of substrates is the conception of NADH-oxidizing
electrodes. Such electrodes must, in particular, catalyze the
dehydrogenation of NADH in an analogous way to the natural process via two electron steps, since one electron transitions can lead to decomposition of the coenzymes. Carbon or
platinum electrodes are unsuitable because of the correspondingly high overpotentials which could lead to interference from other oxidizable materials present in the sample.
A number of investigations to reduce the magnitude of this
overpotential have been carried out e.g. by oxidative pretreatment of the carbon electrodes (resulting in reduction of
the overpotential by CQ. 200-250 mV)Iil, as well as by the
use of dissolved or covalently bound o-quinones12].In both
cases the oxidative peak potential could be reduced from 420
to 250 mV relative to an Ag/AgCl-electrode in 0.010 M KCl
(from 370 to 200 mV/SCE) in cyclic voltammetry investigations. However, the activity of the bound quinones only remained constant for a few cycles. For our purposes, we considered water-insoluble redox catalysts of sufficiently high
activity, more advantageous. Tetrachloro-p-benzoquinone
(chloranil) seemed to be particularly suitable: it has a standard redox potential of 100 mV/SCE at pH=7, is stable and
spontaneously reacts with dihydro nitrogen heterocycles via
hydrogen abstraction''].
Conducting electrodes of this type, consisting of pressed
chloranil and graphite powder are already
However, in our investigations, we treated the basal surfaces of
laterally insulated 6 mm diameter graphite electrodes with a
solution of chloranil in ether and then removed the solvent
by evaporation. The activity of the electrodes was investigated via cyclic voltammetric (Fig. 1) and potentiostatic
measurements (Fig. 2) of stirred aqueous NADH solutions.
The cyclic voltammograms of the NADH oxidation using an
unmodified graphite electrode show peak potentials at 320
and 335 mV/SCE for the first (Fig. 1, curve al) and second
(Fig. 1, curve a2) sweeps respectively; the peak height remains constant after the second sweep. Voltammogram b in
Figure 1 was obtained in an NADH-free solution using a
graphite electrode, which had been treated with 20 kl of a
0.2 mM solution of chloranil(4 nmol): c1and c2are the curves
obtained from the first and second sweeps, respectively, in
the presence of NADH; here again the height of the peak
maximum was constant after the second sweep. The peak
maximum at 90 mV/SCE in the anodic sweep is more intense in the chloranil catalyzed anodic NADH oxidation;
this can be attributed to superposition of the anodic chloranil
and anodic NADH peaks. The anodic NADH peaks ob-
['I
["I
Prof. Dr. H.-L. Schmidt, Dr. H. Huck
Lehrstuhl fur Allgemeine Chemie und Biochemie
der Technischen Universitat Miinchen
D-8050 Freising-Weihenstephan (Germany)
This work was supported by the Bundesministerium fur Forschung und
Technologie.
0570-0833/81/0404-0402 $02.50/0
Angew. Chem. Inf. Ed. Engl. 20 (1981) No. 4
CV
l
-02
-01
0
01
02
03
04
05
06
07
08 V/SCE
Fig. 1. Cyclic voltammograms for the investigation of the NADH oxidation in a
stirred electrolyte. Starting potential - 200 mV/SCE, sweep rate 100 mV/s, sensitivity 1 mA/full recorder scale. Electrolyte: 1 mM NADH, 0.1 M phosphate buffer pH 7.1 M NaCI. a, unmodified graphite electrode (0 6 mm) at the first, a2 at
the second sweep. With 4 nmol chloranil modified graphite electrode in absence
of N A D H (b) and in presence of NADH at the first (c,) and the second (c2)sweep
under stationary conditions.
100 mV/SCE, 2 h), in conjunction with the concentration
decrease of NADH determined photometrically at 340 nm,
show that a two electron transfer occurs with both the modified and unmodified electrodes. No deactivation of the electrodes by fouling was observed in the treated, at 100 mV/
SCE, and untreated electrodes, at 300 mV/SCE, after a 2 h
investigation. However, fouling occurred with the unmodified electrode at a potential of 100 mV/SCE. Unspecific responses can be eliminated using a differential circuit of an
enzyme-chloranil electrode versus a chloranil electrode.
The chloranil electrode can also be used with a covering
dialysis membrane. However, in order to limit the decrease
in sensitivity resulting, the membrane and electrolyte films
must be as thin as possible.
The extraordinary activity of chloranil with respect to oxidation of NADH is probably due to formation of a chargetransfer complex. In contrast benzoquinone, whose redox potential is only 30 mV lower, is catalytically inactive. The systematic search for other catalysts for NADH-oxidation,
which can be irreversibly adsorbed on carbon electrodes, has
led to several heterocyclic compounds151.
Received Apnl 28, 1980,
supplemented December 15, 1980 (Z 746 IE]
German version: Angew. Chem. 93. 421 (1981)
CAS Registry numbers:
NADH, 58-68-4; NAD +,865-054; chloranil. 118-75-2
[I] W. J. Blaedel, R. A . Jenkins, Anal. Chem. 47, 1337 (1975).
[2] D. Chi-Sing, Th. Kuwana, Anal. Chem. 50, 1315 (1978)
[3] E. A . Braude, J. Hannah, R. Linsiead, J. Chem. SOC.1960, 3249.
141 H. Ali, H. Binder, A . Kohling, G. Sandsrede, Angew. Chem. 83, 502 (1971);
Angew. Chem. Int. Ed. Engl. 10, 514 (1971).
[5] H. Huck, H.-L. Schmidi, unpublished results.
Axially Unsymmetrical Osmium(i1)-Porphyrins
with Sulfur and Nitrogen Donors
as Models for Cytochrome cf"]
0"
01
02
Fig. 2. Stationary current-voltage curves of stirred electrolytes (as in Fig. 1).
Curve a, untreated graphite electrode, curve b graphite electrode treated with
120 nmol chloranil.
served at higher potentials are completely absent in the presence of chloranil.
The catalytic effect is particularly conspicuous in the stationary current-voltage curves obtained using controlled
electrode potentials (Fig. 2). Here, the electrodes were
treated with a large excess of chloranil(20 p.1 of a 6 m u solution, corresponding to 120 mmol). Measurements of the current were performed 2 min after each adjustment of the potential. The plot of the unmodified electrode (Fig. 2a) corresponds to the exponential course of an irreversible reaction,
while that of the modified electrode (Fig. 2b) falls steeply
after passing the standard redox potential of chloranil. This
characteristic curve-form can be accounted for by the dependence of the potential on the surface concentration of the
oxidized form of the redox catalyst, which can be reproduced
using the Nernst equation. The limiting current of the plateau is caused by diffusion.
Coulometric data obtained by integrating the area under
the potentiostatic current-time curves (5 cm3 1 mM NADH,
Angew. Chem. Inr. Ed. Engl. 20 (1981) No. 4
By Johann Walter Buchler and Wolfgang Kokischf''
Dedicated to Professor Hans Herloff Inhoffen on the
occasion of his 75th birthday
Reduced cytochrome c is an electron transporting heme
protein in which the Fe" ion is coordinated axially unsymmetrically by sulfur and nitrogen donors of the protein chain
(methionine 80 and histidine 18 respectively)[''. The central
N-Fe-S
unit can only be prepared from the protein-free
Fe"-porphyrin if, as in the "tail-porphyrin" of Reed et al.[*I,
at least one of the donors is covalently fixed to the periphery
of the porphyrin by a side chain; otherwise only the axially
symmetrical complex with two nitrogen or sulfur donors is
isolated because of the kinetic lability of the heme group. In
cytochrome c the N-Fe-S
unit is stabilized by a double
chelate effect. The inherently more stable ligand-osmium
bond allowed the synthesis of the axially unsymmetrical bisligand-osmium(I1) porphyrins (1) and (2) ("osmochromes"),
which are stable without any chelate-effect.
[*I Prof. Dr. J. W. Buchler, Dr. W. Kokisch
Fachbereich Anorganische Chemie und Kernchemie der Technlschen
Hochschule
Hochschulstrasse4, D-6100 Darmstadt (Germany) (address for correspondence)
and Institut fur Anorganische Chemie der Technischen Hochschule
D-5100 Aachen (Germany)
[**I
Metal Complexes with tetrapyrrole ligands, Part 25. This work was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der
Chemischen 1ndustrie.-Part 2 4 1. W. Buchler, K.-L. Lay, H. Sroppa, Z .
Naturforsch. B 35. 433 (1980).
0 Verlag Chemie GmbH, 6940 Weinheim, 1981
0570-0833/81/0404-0403 S 02.50/0
403
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