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Evidence for a radical intermediate in the anodic oxidation of reduced nicotinamide adenine dinucleotides obtained by electrogenerated chemiluminescence
Analytica Chimica Acta, 209 (1988) 69-78 Elsevier Science Publishers B:V., Amsterdam -
69 Printed in The Netherlands
EVIDENCE FOR A RADICAL INTERMEDIATE IN THE ANODIC OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDES OBTAINED BY ELECTROGENERATED CHEMILUMINESCENCE
J. LUDViK and J. VOLKE* J. Heyrovsk$ Institute of Physical Chemistry and Electrochemistry, Czechoslovak Academy of Sciences, 182 23 Praha 8, Dolejskova 8 (Czechoslovakia) (Received 25th August 1987)
SUMMARY Electrogenerated chemiluminescence is used to show that the radicals NAD’ and NADP’ are intermediates in the electro-oxidation of NADH and NADPH at a platinum anode in anhydrous or partly aqueous (up to 15% v/v) dimethyl sulfoxide. An ECE mechanism seems to predominate. The use of dimethyl sulfoxide proved to be very convenient, with the advantage of enabling electrogenerated chemiluminescence to be obtained in partly aqueous media even with ionic substances as substrates. The method is useful in proving the existence of unstable radical intermediates in redox processes, even for relatively large molecules like NADH and NADPH.
In recent years, many papers have been published concerning the electrochemical behaviour of the reduced nicotinamide adenine dinucleotides, NADH or NADPH, at solid electrodes, particularly in aqueous media. The aim of these investigations has been to elucidate, by electrochemical techniques, the mechanism of redox processes in biological media in which the NADH/NAD+ coenzyme couple participates, and to find suitable conditions for lowering the oxidation overvoltage of NADH when variously modified electrodes are used to regenerate the coenzyme NAD+, or if need be, for analytical purposes [l-
71.
The investigations devoted to the mechanism proper [8-121 of this overall a-electron oxidation of NADH involved both aqueous and non-aqueous media. Kinetic meaasurements [8-121 and analogies with the results of electrochemical investigation of model 1,4dihydropyridine derivatives in such solvents [ 13-161 suggest the prevalence of the following ECE mechanism: NADH - -’ NADH+’ -
NADH+’
k
0003-2670/88/$03.50
(1)
NAD*+H+
0 1988 Elsevier Science Publishers B.V.
(21
NAD’ - -’
NAD+
(3)
It is, however, still an open question which is the rate-determining step, and, consequently, there is the possible participation of a disproportionation mechanism, in the overall course of the process: NAD’+NADH+‘-
NAD+ +NADH
(4)
The EEC mechanism [8,12] (reactions 1, 5 and 6) also has not yet been excluded: NADH+’ - -’
NADH2+
(5)
NADH2+ -
NAD++H+
(6)
Moreover, there is a strong dependence on the electrode material and on the pretreatment of the electrode surface [ 171. There is still no real evidence, however, for the role of the radical intermediates in the reaction sequence to be studied, which would prove their participation in the mechanism. Based on earlier experience with model compounds, it was possible to make use of electrochemically generated chemiluminescence [ 13-161. Instead of acetonitrile or its mixture with toluene, dimethyl sulfoxide (DMSO) was used, this solvent proved to be more convenient with respect to the solubility of the participating compounds. Platinum was chosen as the electrode material because its surface is not blocked to such a degree by the adsorbed product NAD+ as is a carbon electrode. From the many possible luminescent compounds 1,3-di-p-anisyl-4,7-diphenylisobenzofuran (DADIF) was chosen, both because of its suitable oxidation potential and because of its low triplet energy. In order to confirm the connection between the electrooxidation mechanisms in the model compounds and in the reduced coenzyme, a series of related compounds was taken for the experiments; this series forms a transition from one of the Hantzsch 1,4_dihydropyridines (I) studied in preceding papers [ 13,14,16], via a 1-substituted-1,4-dihydronicotinamide (II) [ 181, to NADH (III) and NADPH (IV). For proving the validity of the considerations and conclusions following the electrogenerated chemiluminescence results, further luminescent compounds were examined: rubrene (R), 9,lOdiphenylanthracene (DPA), 1-anisyL3,5diphenylpyrazoline ( ADP) and, in place of the dihydropyridine derivative, bis (1,2,3-trimethyl-1,2-dihydrobenzimidazolyl-2)) B2, (V); the analogous oxidation mechanism and radical formation of compound V has been reported [ 191.
71 HI-I
cONH2
tH3
tH3 v
OCH3 9CH3
00
00
0
0:
0000
000
00
0
58 00
@%
6CH3
DADIF
R
6CH3
DPA
EXPERIMENTAL
Chemicals 2,6-Dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (I) and l-p-methoxybenzyl-1,4-dihydronicotinamide (II) [ 181 were synthesized in the Department of Organic Chemistry, Institute of Chemical Technology, Prague, by Professor Kuthan and his co-workers and used as received. NADH disodium salts3H,O (III; Fluka) and NAD*3H20 (Serva) were chemically pure; NADPH tetrasodium salt (IV; Serva) was an analytical-grade reagent. Rubrene and 9,10-diphenylanthracene (both Aldrich) were also used as received. Bis( 1,2,3trimethyl-1,2-dihydrobenzimidazolyl-2) (V), 1,3-di-p-anisyl-4,7-diphenylisobenzofuran (DADIF) and 1-anisyl-3,5-diphenyl-2-pyrazoline (ADP) were provided by Dr. Pragst, Humboldt University (Berlin, G.D.R. ) . Dimethyl sulfoxide (puriss, Lachema) was fractionally distilled twice on a column; toluene (analytical grade, Lachema) was twice-distilled from sodium. A 0.05 mol 1-l tetrabutylammonium hexafluorophosphate (TBAHFP ) solution was used as supporting electrolyte. Instrumentation All measurements were made in a 2-ml cell with a planar bottom having a separated SCE as reference (Fig. la). A rotating platinum disk electrode of 1 mm diameter (Laboratorni p?istroje, Prague) served as the working electrode.
L
Fig. 1. Electrogenerated luminescence instrumentation. (a) Cell: (1) rotating disk electrode; (2) inlet both for the reference electrode and for filling the cell with the solution to be investigated; (3 ) inert gas inlet; (4) auxiliary electrode; (5) inert gas outlet; (6) level for 2 ml of solution. (b ) Black box: (1) photomultiplier tube; (2) valve controlled from outside; (3) internal blackened tubing for inlet and outlet of the inert gas; (4) side openings for spectral measurements (shielded by movable covers); (5) electrical connectors; (6) position of the rotating electrode and the cell in the box; (7) black rubber seal; (8) cover (metal plate) with screw-fastened grips. (c) Experimental arrangement; ( 1) polarograph, (2 ) cell; (3 ) black box; (4) photomultiplier tube; (5,6) XY recorders.
The auxiliary electrode was formed by a platinum wire sealed in the bottom. The whole cell with the rotating electrode was placed in a black box just above the photomultiplier tube (Fig. lb). The voltammetric curve (Mehrzweckpolarograph GWP 673, Akademie der Wissenschaften der DDR, ZWG, Berlin) and the luminescence curve (i.e., intensity of electrogenerated luminescence vs. potential) were recorded simultaneously by making use of two X-Y re-
73
corders (Fig. lc). The ECL equipment was constructed on the basis of instrumentation introduced by Dr. Pragst and with his kind permission. For each experiment, an appropriate amount of the substance was weighed on a microbalance (MGF, Berlin) corresponding to a concentration of about 2-5 x 10e4 mol 1-l and dissolved directly in a solvent in the cell. Procedures Before each experiment, 2 ml of 0.05 M TBAHFP in dimethyl sulfoxide was deaerated in the cell for 10 min by a stream of pure argon (electrogenerated luminescence is very sensitive to quenching by dissolved dioxygen). First, the baseline was recorded; the luminophore L alone was then dissolved and both the voltammetric and the luminescence curves were recorded. The luminescence curve (the blank) in no case showed any emission even at the maximum sensitivity of the photomultiplier. The studied compound was then dissolved and, under the same conditions, the voltammetric and luminescence curves were recorded, the latter in all cases gave an emission peak (cf. Table 1 and Fig. 2) at the oxidation potential Ey’j, (L) of the luminophore. RESULTS AND DISCUSSION
Principles of electrogenerated luminescence application At a sufficiently positive potential, the compound to be investigated is oxidized at the electrode: NADH
-%-I++
h NAD+
E:;, (NADH)
(7)
and simultaneously the luminophore (L) is transformed: -e L ( +e
L+.
Eci% (L)
(8)
If the radical NAD’ is assumed to be an intermediate in Reaction 7, its oxidation potential E $ (NAD’ ) is much more negative than E TT2(NADH ) because this oxidation is the back-reaction of the reversible process +e NAD+ -_e
NAD’
E’lj, (NAD’ )
(9)
Consequently, if this system gives rise to luminescence, it provides evidence for the formation in the solution of a strong electron donor as intermediate (here NAD’ ) which will react with the simultaneously formed L+’ to produce energy. Only this electron transfer can lead to the formation of an excited (triplet) state: L+‘+NAD’
-
3L+NAD+
(IO)
74
This is valid if it is assumed that the fundamental energetic condition for the electrochemical generation of luminescence is satisfied, i.e., if Reaction 10 produces sufficient energy to exceed the triplet energy ET(L): -dI5’=E$
(L) -EgZ
(NAD’) -TdSa,?&
(L)
(11)
Triplet-triplet annihilation (Eqn. 12 ) gives an excited singlet state leading to the emission process (Eqn. 13) [ 201. 23L -
‘L*+L
(12)
IL* -
L+hv,
(12)
The entropy term TAS in eqn. 11 is usually given [ 211 as 0.12 0.1 eV. Evidently, the choice of a luminophore with a suitable oxidation potential and a sufficiently low triplet energy is important. Elucidation of the reactions As a link to the preceding papers [ 13-161, the electrogenerated luminescence of Hantzsch-type 1,4_dihydropyridine derivatives was investigated in different solvents (acetonitrile, dimethylformamide, propionitrile, dimethylacetamide and dimethyl sulfoxide) using the various luminophores (DPA, rubrene, ADP, DADIF). The results fully agreed with the conclusions of the preceding papers on model compounds [ 13-161; they again confirmed the observation that in order to obtain a higher luminescence intensity it is necessary to choose a luminophore which is oxidized at potentials sufficiently more positive than those of the substrate (the substance to be investigated) because, with decreasing difference between Ey’j, (L) and E& of the substrate, the inTABLE 1 Half-wave potentials and electrogenerated chemiluminescence luminophores Substance
Solvent
E l/Z
properties of NADH analogues and
Luminescence”
Luminophore
09 I I II II III IV V
DMSOb DMSO/toluene’ DMSO DMSO/toluene” DMSO DMS0/5% H,O DMSO
0.76 0.76 0.48 0.51 0.49 0.50 - 0.44
Type
G/2
DADIF Rubrene DADIF Rubrene DADIF DADIF DADIF
0.73 0.92 0.73 0.94 0.74 0.71 0.74
W)
Ep,,
0.70 0.92 0.70 0.93 0.68 0.67 0.69
W)
1
25 80 100 400 80 40 2x104
“Potential corresponding to half the peak height on the luminescence intensity/potential curve (analogous to Ep12in cyclic voltammetry). The intensity (I) is given in arbitrary units. bDimethyl sulfoxide. “2: 1 (v/v) DMSO/toluene, because of the solubility of rubrene.
75
-1.5
-1.0
-0.5
0
0.5
1.0
E(V)
Fig. 2. Voltammetric (-) and electrogenerated luminescence (ECL) (----) curves as a function of potential (E) vs. SCE (0.05 M TBAHFP in dimethyl sulfoxide ) . (a) ( 1) Blank, (2 ) compound I (5 x 10m4M); (3) DADIF (2 x 10e4M); (4) ECL cuNe corresponding to cuNe 3. (b) (1) Blank; (2) DADIF (3x10-*M); (3) compound11 (2X10e4M); (4) ECL cuNe corresponding to curve 3. (c) (1) Blank; (2) DADIF (3.5x10w4M); (3) compound III (4.5x10m4M); (4) NAD+ (4x10m4M); (5) ECL curve corresponding to curves 3 and 4. (d) (1) Blank; (2) DADIF (3x10m4M); (3) compoundIV (2x10e4M); (4) ECLcuNecorrespondingtocuNe3. (e) (1) Blank; (2) DADIF (2x10W4M); (3) compoundV (4x10e4M); (4) ECLcuNecorrespondingto curve 3.
tensity of the electrogenerated luminescence decreases [ 14 ] (Table 1). When dimethyl sulfoxide was used with DADIF, I was chosen because of its low oxidation potential; the other Hantzsch-type compounds which were available are oxidized at more positive potentials and, consequently, would lead to lower luminescence signals, often almost indistinguishable from the noise. Compound II, which represents a structural transition from the Hantzsch 1,4dihydropyridines to NADH (with respect to the presence of an amide group at position 3) had not previously been investigated with respect to electrogenerated luminescence; the result was analogous to that obtained with compound I, even with regard to the intensity.
76
NADH (III) has a considerably smaller diffusion coefficient [ 81, which leads to a lower height of the observed voltammetric wave as well as to a less intense luminescence signal. The emission, however, is quite reproducible. The investigation of NADPH (IV) was complicated by its low solubility in dimethyl sulfoxide; for this reason, water was gradually added. It turned out that with as little as 5-10% (v/v) water, NADPH was fully dissolved and gave luminescence signals during electro-oxidation. This emission was detectable even with water concentrations up to 15%. This is the first observation of this type of electrogenerated emission in partly aqueous solutions. In order to check the correctness of the interpretation presented here for the electrogenerated luminescence results, the following additional experiments were done. Instead of DADIF, the substituted pyrazoline (ADP) was used as the luminophore in an experiment with NADH. Repeated experiments gave no emission. Thus the energetic condition in Eqn. 11 was not satisfied because Ey;2 (ADP) = +0.64 V, E;“P, (NAD+) =J?+ (NAD’) = -1.06 V (values obtained from the voltammetric reduction of authentic NAD+ in the given solvent system as shown in Fig. 2c) and E,(P) = 1.88 eV, so that the difference between the two potentials (1.70 V) did not exceed the triplet energy. When the same calculation was done for DADIF (ET= 1.60 eV, ET’j, = +0.73 V), then the energy released in the reaction of DADIF +* with NAD’ (1.79 eV) clearly exceeds ET (DADIF). This series of compounds was enlarged and made more general by including the compound Bz (V), for which a radical intermediate in the ECE mechanism has been detected before [ 191. The luminescence observed even in this case again confirmed the validity of the above theoretial explanation of the conditions necessary to obtain the luminescence. Moreover, the very intense emission (about 2 orders higher than that obtained with compounds I-IV) indicated that a direct, more effective (but energetically more demanding) singlet mechanism (Eqns. 14-16) participates in the mechanism in addition to the indirect triplet mechanism (Eqns. 7-13): L+‘+B’
-
‘L*+B+
[if -dHO>&(L)]
(14)
or B’+LL-‘+L+’ IL* -
B++L-’ -
(15)
‘L*+L
(16)
L+hv,
Here Es(L) is the E$(L=DADIF)=-1.95V.
(13) singlet
excitation
energy,
E’;?, (B’) = - 1.98 V and
Conch5ions By using electrogenerated luminescence, the radicals NAD’ and NADP’ which result from the anodic oxidation of NADH and NADPH at a platinum
II
electrode were detected and their existence proved in non-aqueous or partly aqueous media. When the mechanism of such an oxidation is considered, an EEC mechanism is the least probable if the radical NAD’ is formed; the high reactivity of this radical at positive potentials as well as the instability of the radical cation NADH+’ indicate that subsequent bimolecular reactions between them are very improbable. Moreover, the possible disproportionation reaction (Eqn. 4) competes with Reaction 10, which, since the radical cation L+’ is several orders of magnitude more stable, is much more probable. Because relatively weak electrochemiluminescence is still observed, it cannot be assumed that the electro-oxidation (Eqn. 7) takes place mainly by the disproportionation mechanism. All this indicates that in the electro-oxidation of NADH and NADPH at a platinum electrode, an ECE mechanism predominates, i.e., the starting material partly adsorbed on the electrode (or closely adjacent to the electrode) deprotonates so rapidly after the loss of the electron that during this chemical step only a few molecules diffuse back into the bulk solution and the resulting secondary radical also loses its second electron chiefly at the electrode. The results obtained do not allow an unambiguous decision about the degree to which adsorption or the presence of platinum oxides at the electrode affects the mechanism. As regards the electrogenerated luminescence method, this is the first case in which dimethyl sulfoxide has been used as solvent; it proved to be very suitable. Further, a partly aqueous (up to 15% ) solution does not measurably decrease the oxidation-type luminescence intensity. This finding offers ample possibilities to extend such luminescence to further ionic compounds. Based on control experiments, it was confirmed that the above method is generally applicable for the proof of existence of even extremely unstable radical intermediates in redox mechanisms not only for small molecules but also with relatively large molecules of biological significance such as NADH and NADPH. This paper is dedicated to Professor A.A. VlEek, Prague, on the occasion of his 60th birthday.
REFERENCES H. Huck and H. L. Schmidt, Angew. Chem., 93 (1981) 421. L. Falat and H. Y. Cheng, J. Electroanal. Chem., 157 (1983) 393. W. J. Albery and P. N. Bartlett, J. Chem. Sot., Chem. Commun.,(1984) 234. J.M. Laval, C. Bourdillon and J. Moiroux, J. Am. Chem. Sot., 106 (1984) 4701. L. Gorton, A. Torstensson, H. Jaegfeldt and G. Johansson, J. Electroanal. Chem., 161 (1984) 103. N. K. Cenas, J. J. Kanapieniene and J. J. Kulys, J. Electroanal. Chem., 189 (1985) 163. Y. Kimura and K. Niki, Anal. Sci., 1 (1985) 271.
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8 9
10 11 12 13 14 15 16 17 18 19 20 21
P. Leduc and D. Thevenot, Bioelectrochem. Bioenerg., 1 (1974) 96. P. J. Elving, W. T. Breesnahan,J. Moiroux and Z. Samec, Bioelectrochem. Bioenerg., 9 (1982) 365. J. Moiroux and P. J. Elving, J. Am. Chem. Sot., 102 (1980) 6533. R. D. Braun, K. S. V. Santhanam and P. J. Elving, J. Am. Chem. Sot., 97 (1975) 2591. R. L. Blankespoor and L. L. Miller, J. Electroanal. Chem., 171 (1984) 231. J. Ludvlk, J. Volke and J. Klfma, Electrochim. Acta, 32 (1987) 1063. J. Ludvfk, J. Volke and F. Pragst, J. Electroanal. Chem., 215 (1986) 179. V. Skala, J. Volke, V. Ohtika and J. Kuthan, Collect. Czech. Chem. Commun., 42 (1977) 292. F. Pragst, B. Kaltofen, J. Volke and J. Kuthan, J. Electroanal. Chem., 119 (1981) 301. L. Gorton, G. Johansson and A. Torstensson, J. Electroanal. Chem., 196 (1985) 81. F. Pavllkova-Raclovi and J. Kuthan, Collect. Czech. Chem. Commun., 48 (1983) 1408. J. Ludvik, F. Pragst and J. Volke, J. Electroanal. Chem., 180 (1984) 141. F. Pragst, Z. Chem., 18 (1978) 41. S. M. Park and D. A. Tryk, Rev. Chem. Intermed., 4 (1981) 43.
69 Printed in The Netherlands
EVIDENCE FOR A RADICAL INTERMEDIATE IN THE ANODIC OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDES OBTAINED BY ELECTROGENERATED CHEMILUMINESCENCE
J. LUDViK and J. VOLKE* J. Heyrovsk$ Institute of Physical Chemistry and Electrochemistry, Czechoslovak Academy of Sciences, 182 23 Praha 8, Dolejskova 8 (Czechoslovakia) (Received 25th August 1987)
SUMMARY Electrogenerated chemiluminescence is used to show that the radicals NAD’ and NADP’ are intermediates in the electro-oxidation of NADH and NADPH at a platinum anode in anhydrous or partly aqueous (up to 15% v/v) dimethyl sulfoxide. An ECE mechanism seems to predominate. The use of dimethyl sulfoxide proved to be very convenient, with the advantage of enabling electrogenerated chemiluminescence to be obtained in partly aqueous media even with ionic substances as substrates. The method is useful in proving the existence of unstable radical intermediates in redox processes, even for relatively large molecules like NADH and NADPH.
In recent years, many papers have been published concerning the electrochemical behaviour of the reduced nicotinamide adenine dinucleotides, NADH or NADPH, at solid electrodes, particularly in aqueous media. The aim of these investigations has been to elucidate, by electrochemical techniques, the mechanism of redox processes in biological media in which the NADH/NAD+ coenzyme couple participates, and to find suitable conditions for lowering the oxidation overvoltage of NADH when variously modified electrodes are used to regenerate the coenzyme NAD+, or if need be, for analytical purposes [l-
71.
The investigations devoted to the mechanism proper [8-121 of this overall a-electron oxidation of NADH involved both aqueous and non-aqueous media. Kinetic meaasurements [8-121 and analogies with the results of electrochemical investigation of model 1,4dihydropyridine derivatives in such solvents [ 13-161 suggest the prevalence of the following ECE mechanism: NADH - -’ NADH+’ -
NADH+’
k
0003-2670/88/$03.50
(1)
NAD*+H+
0 1988 Elsevier Science Publishers B.V.
(21
NAD’ - -’
NAD+
(3)
It is, however, still an open question which is the rate-determining step, and, consequently, there is the possible participation of a disproportionation mechanism, in the overall course of the process: NAD’+NADH+‘-
NAD+ +NADH
(4)
The EEC mechanism [8,12] (reactions 1, 5 and 6) also has not yet been excluded: NADH+’ - -’
NADH2+
(5)
NADH2+ -
NAD++H+
(6)
Moreover, there is a strong dependence on the electrode material and on the pretreatment of the electrode surface [ 171. There is still no real evidence, however, for the role of the radical intermediates in the reaction sequence to be studied, which would prove their participation in the mechanism. Based on earlier experience with model compounds, it was possible to make use of electrochemically generated chemiluminescence [ 13-161. Instead of acetonitrile or its mixture with toluene, dimethyl sulfoxide (DMSO) was used, this solvent proved to be more convenient with respect to the solubility of the participating compounds. Platinum was chosen as the electrode material because its surface is not blocked to such a degree by the adsorbed product NAD+ as is a carbon electrode. From the many possible luminescent compounds 1,3-di-p-anisyl-4,7-diphenylisobenzofuran (DADIF) was chosen, both because of its suitable oxidation potential and because of its low triplet energy. In order to confirm the connection between the electrooxidation mechanisms in the model compounds and in the reduced coenzyme, a series of related compounds was taken for the experiments; this series forms a transition from one of the Hantzsch 1,4_dihydropyridines (I) studied in preceding papers [ 13,14,16], via a 1-substituted-1,4-dihydronicotinamide (II) [ 181, to NADH (III) and NADPH (IV). For proving the validity of the considerations and conclusions following the electrogenerated chemiluminescence results, further luminescent compounds were examined: rubrene (R), 9,lOdiphenylanthracene (DPA), 1-anisyL3,5diphenylpyrazoline ( ADP) and, in place of the dihydropyridine derivative, bis (1,2,3-trimethyl-1,2-dihydrobenzimidazolyl-2)) B2, (V); the analogous oxidation mechanism and radical formation of compound V has been reported [ 191.
71 HI-I
cONH2
tH3
tH3 v
OCH3 9CH3
00
00
0
0:
0000
000
00
0
58 00
@%
6CH3
DADIF
R
6CH3
DPA
EXPERIMENTAL
Chemicals 2,6-Dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (I) and l-p-methoxybenzyl-1,4-dihydronicotinamide (II) [ 181 were synthesized in the Department of Organic Chemistry, Institute of Chemical Technology, Prague, by Professor Kuthan and his co-workers and used as received. NADH disodium salts3H,O (III; Fluka) and NAD*3H20 (Serva) were chemically pure; NADPH tetrasodium salt (IV; Serva) was an analytical-grade reagent. Rubrene and 9,10-diphenylanthracene (both Aldrich) were also used as received. Bis( 1,2,3trimethyl-1,2-dihydrobenzimidazolyl-2) (V), 1,3-di-p-anisyl-4,7-diphenylisobenzofuran (DADIF) and 1-anisyl-3,5-diphenyl-2-pyrazoline (ADP) were provided by Dr. Pragst, Humboldt University (Berlin, G.D.R. ) . Dimethyl sulfoxide (puriss, Lachema) was fractionally distilled twice on a column; toluene (analytical grade, Lachema) was twice-distilled from sodium. A 0.05 mol 1-l tetrabutylammonium hexafluorophosphate (TBAHFP ) solution was used as supporting electrolyte. Instrumentation All measurements were made in a 2-ml cell with a planar bottom having a separated SCE as reference (Fig. la). A rotating platinum disk electrode of 1 mm diameter (Laboratorni p?istroje, Prague) served as the working electrode.
L
Fig. 1. Electrogenerated luminescence instrumentation. (a) Cell: (1) rotating disk electrode; (2) inlet both for the reference electrode and for filling the cell with the solution to be investigated; (3 ) inert gas inlet; (4) auxiliary electrode; (5) inert gas outlet; (6) level for 2 ml of solution. (b ) Black box: (1) photomultiplier tube; (2) valve controlled from outside; (3) internal blackened tubing for inlet and outlet of the inert gas; (4) side openings for spectral measurements (shielded by movable covers); (5) electrical connectors; (6) position of the rotating electrode and the cell in the box; (7) black rubber seal; (8) cover (metal plate) with screw-fastened grips. (c) Experimental arrangement; ( 1) polarograph, (2 ) cell; (3 ) black box; (4) photomultiplier tube; (5,6) XY recorders.
The auxiliary electrode was formed by a platinum wire sealed in the bottom. The whole cell with the rotating electrode was placed in a black box just above the photomultiplier tube (Fig. lb). The voltammetric curve (Mehrzweckpolarograph GWP 673, Akademie der Wissenschaften der DDR, ZWG, Berlin) and the luminescence curve (i.e., intensity of electrogenerated luminescence vs. potential) were recorded simultaneously by making use of two X-Y re-
73
corders (Fig. lc). The ECL equipment was constructed on the basis of instrumentation introduced by Dr. Pragst and with his kind permission. For each experiment, an appropriate amount of the substance was weighed on a microbalance (MGF, Berlin) corresponding to a concentration of about 2-5 x 10e4 mol 1-l and dissolved directly in a solvent in the cell. Procedures Before each experiment, 2 ml of 0.05 M TBAHFP in dimethyl sulfoxide was deaerated in the cell for 10 min by a stream of pure argon (electrogenerated luminescence is very sensitive to quenching by dissolved dioxygen). First, the baseline was recorded; the luminophore L alone was then dissolved and both the voltammetric and the luminescence curves were recorded. The luminescence curve (the blank) in no case showed any emission even at the maximum sensitivity of the photomultiplier. The studied compound was then dissolved and, under the same conditions, the voltammetric and luminescence curves were recorded, the latter in all cases gave an emission peak (cf. Table 1 and Fig. 2) at the oxidation potential Ey’j, (L) of the luminophore. RESULTS AND DISCUSSION
Principles of electrogenerated luminescence application At a sufficiently positive potential, the compound to be investigated is oxidized at the electrode: NADH
-%-I++
h NAD+
E:;, (NADH)
(7)
and simultaneously the luminophore (L) is transformed: -e L ( +e
L+.
Eci% (L)
(8)
If the radical NAD’ is assumed to be an intermediate in Reaction 7, its oxidation potential E $ (NAD’ ) is much more negative than E TT2(NADH ) because this oxidation is the back-reaction of the reversible process +e NAD+ -_e
NAD’
E’lj, (NAD’ )
(9)
Consequently, if this system gives rise to luminescence, it provides evidence for the formation in the solution of a strong electron donor as intermediate (here NAD’ ) which will react with the simultaneously formed L+’ to produce energy. Only this electron transfer can lead to the formation of an excited (triplet) state: L+‘+NAD’
-
3L+NAD+
(IO)
74
This is valid if it is assumed that the fundamental energetic condition for the electrochemical generation of luminescence is satisfied, i.e., if Reaction 10 produces sufficient energy to exceed the triplet energy ET(L): -dI5’=E$
(L) -EgZ
(NAD’) -TdSa,?&
(L)
(11)
Triplet-triplet annihilation (Eqn. 12 ) gives an excited singlet state leading to the emission process (Eqn. 13) [ 201. 23L -
‘L*+L
(12)
IL* -
L+hv,
(12)
The entropy term TAS in eqn. 11 is usually given [ 211 as 0.12 0.1 eV. Evidently, the choice of a luminophore with a suitable oxidation potential and a sufficiently low triplet energy is important. Elucidation of the reactions As a link to the preceding papers [ 13-161, the electrogenerated luminescence of Hantzsch-type 1,4_dihydropyridine derivatives was investigated in different solvents (acetonitrile, dimethylformamide, propionitrile, dimethylacetamide and dimethyl sulfoxide) using the various luminophores (DPA, rubrene, ADP, DADIF). The results fully agreed with the conclusions of the preceding papers on model compounds [ 13-161; they again confirmed the observation that in order to obtain a higher luminescence intensity it is necessary to choose a luminophore which is oxidized at potentials sufficiently more positive than those of the substrate (the substance to be investigated) because, with decreasing difference between Ey’j, (L) and E& of the substrate, the inTABLE 1 Half-wave potentials and electrogenerated chemiluminescence luminophores Substance
Solvent
E l/Z
properties of NADH analogues and
Luminescence”
Luminophore
09 I I II II III IV V
DMSOb DMSO/toluene’ DMSO DMSO/toluene” DMSO DMS0/5% H,O DMSO
0.76 0.76 0.48 0.51 0.49 0.50 - 0.44
Type
G/2
DADIF Rubrene DADIF Rubrene DADIF DADIF DADIF
0.73 0.92 0.73 0.94 0.74 0.71 0.74
W)
Ep,,
0.70 0.92 0.70 0.93 0.68 0.67 0.69
W)
1
25 80 100 400 80 40 2x104
“Potential corresponding to half the peak height on the luminescence intensity/potential curve (analogous to Ep12in cyclic voltammetry). The intensity (I) is given in arbitrary units. bDimethyl sulfoxide. “2: 1 (v/v) DMSO/toluene, because of the solubility of rubrene.
75
-1.5
-1.0
-0.5
0
0.5
1.0
E(V)
Fig. 2. Voltammetric (-) and electrogenerated luminescence (ECL) (----) curves as a function of potential (E) vs. SCE (0.05 M TBAHFP in dimethyl sulfoxide ) . (a) ( 1) Blank, (2 ) compound I (5 x 10m4M); (3) DADIF (2 x 10e4M); (4) ECL cuNe corresponding to cuNe 3. (b) (1) Blank; (2) DADIF (3x10-*M); (3) compound11 (2X10e4M); (4) ECL cuNe corresponding to curve 3. (c) (1) Blank; (2) DADIF (3.5x10w4M); (3) compound III (4.5x10m4M); (4) NAD+ (4x10m4M); (5) ECL curve corresponding to curves 3 and 4. (d) (1) Blank; (2) DADIF (3x10m4M); (3) compoundIV (2x10e4M); (4) ECLcuNecorrespondingtocuNe3. (e) (1) Blank; (2) DADIF (2x10W4M); (3) compoundV (4x10e4M); (4) ECLcuNecorrespondingto curve 3.
tensity of the electrogenerated luminescence decreases [ 14 ] (Table 1). When dimethyl sulfoxide was used with DADIF, I was chosen because of its low oxidation potential; the other Hantzsch-type compounds which were available are oxidized at more positive potentials and, consequently, would lead to lower luminescence signals, often almost indistinguishable from the noise. Compound II, which represents a structural transition from the Hantzsch 1,4dihydropyridines to NADH (with respect to the presence of an amide group at position 3) had not previously been investigated with respect to electrogenerated luminescence; the result was analogous to that obtained with compound I, even with regard to the intensity.
76
NADH (III) has a considerably smaller diffusion coefficient [ 81, which leads to a lower height of the observed voltammetric wave as well as to a less intense luminescence signal. The emission, however, is quite reproducible. The investigation of NADPH (IV) was complicated by its low solubility in dimethyl sulfoxide; for this reason, water was gradually added. It turned out that with as little as 5-10% (v/v) water, NADPH was fully dissolved and gave luminescence signals during electro-oxidation. This emission was detectable even with water concentrations up to 15%. This is the first observation of this type of electrogenerated emission in partly aqueous solutions. In order to check the correctness of the interpretation presented here for the electrogenerated luminescence results, the following additional experiments were done. Instead of DADIF, the substituted pyrazoline (ADP) was used as the luminophore in an experiment with NADH. Repeated experiments gave no emission. Thus the energetic condition in Eqn. 11 was not satisfied because Ey;2 (ADP) = +0.64 V, E;“P, (NAD+) =J?+ (NAD’) = -1.06 V (values obtained from the voltammetric reduction of authentic NAD+ in the given solvent system as shown in Fig. 2c) and E,(P) = 1.88 eV, so that the difference between the two potentials (1.70 V) did not exceed the triplet energy. When the same calculation was done for DADIF (ET= 1.60 eV, ET’j, = +0.73 V), then the energy released in the reaction of DADIF +* with NAD’ (1.79 eV) clearly exceeds ET (DADIF). This series of compounds was enlarged and made more general by including the compound Bz (V), for which a radical intermediate in the ECE mechanism has been detected before [ 191. The luminescence observed even in this case again confirmed the validity of the above theoretial explanation of the conditions necessary to obtain the luminescence. Moreover, the very intense emission (about 2 orders higher than that obtained with compounds I-IV) indicated that a direct, more effective (but energetically more demanding) singlet mechanism (Eqns. 14-16) participates in the mechanism in addition to the indirect triplet mechanism (Eqns. 7-13): L+‘+B’
-
‘L*+B+
[if -dHO>&(L)]
(14)
or B’+LL-‘+L+’ IL* -
B++L-’ -
(15)
‘L*+L
(16)
L+hv,
Here Es(L) is the E$(L=DADIF)=-1.95V.
(13) singlet
excitation
energy,
E’;?, (B’) = - 1.98 V and
Conch5ions By using electrogenerated luminescence, the radicals NAD’ and NADP’ which result from the anodic oxidation of NADH and NADPH at a platinum
II
electrode were detected and their existence proved in non-aqueous or partly aqueous media. When the mechanism of such an oxidation is considered, an EEC mechanism is the least probable if the radical NAD’ is formed; the high reactivity of this radical at positive potentials as well as the instability of the radical cation NADH+’ indicate that subsequent bimolecular reactions between them are very improbable. Moreover, the possible disproportionation reaction (Eqn. 4) competes with Reaction 10, which, since the radical cation L+’ is several orders of magnitude more stable, is much more probable. Because relatively weak electrochemiluminescence is still observed, it cannot be assumed that the electro-oxidation (Eqn. 7) takes place mainly by the disproportionation mechanism. All this indicates that in the electro-oxidation of NADH and NADPH at a platinum electrode, an ECE mechanism predominates, i.e., the starting material partly adsorbed on the electrode (or closely adjacent to the electrode) deprotonates so rapidly after the loss of the electron that during this chemical step only a few molecules diffuse back into the bulk solution and the resulting secondary radical also loses its second electron chiefly at the electrode. The results obtained do not allow an unambiguous decision about the degree to which adsorption or the presence of platinum oxides at the electrode affects the mechanism. As regards the electrogenerated luminescence method, this is the first case in which dimethyl sulfoxide has been used as solvent; it proved to be very suitable. Further, a partly aqueous (up to 15% ) solution does not measurably decrease the oxidation-type luminescence intensity. This finding offers ample possibilities to extend such luminescence to further ionic compounds. Based on control experiments, it was confirmed that the above method is generally applicable for the proof of existence of even extremely unstable radical intermediates in redox mechanisms not only for small molecules but also with relatively large molecules of biological significance such as NADH and NADPH. This paper is dedicated to Professor A.A. VlEek, Prague, on the occasion of his 60th birthday.
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