Electrochemical Oxidation of NADH Analogs W. J. Blaedel and Ronald G . Haas’ Department of Chemistry, University of Wisconsin, Madison, Wis. 53706 The oxidation of the 1-(2,6-dichlorobenzyI)-, 1-methyl-, 1-benzyl-, and l-(n-propyl)-1,4-dihydronicotinamides is examined in acetonitrile at platinum and carbon electrodes. In both basic and unbuffered acetonitrile (AN), the dihydronicotinamides are oxidized to the corresponding pyridinium salts (RN+). In basic AN, RN+ is formed quantitatively. In unbuffered AN, an acid decomposition reaction involving RNH accompanies the electrode reaction, and the RN+ yield is only 40%. In unbuffered media, the oxidation is resolvable into at least two steps, the first of which involves the loss of a single electron. This is followed by one or more steps involving proton transfer and the loss of an additional electron to give RN+. Resolution of these steps is achieved by using oxygen as a radical scavenger in the electrode reaction. An alternative mechanism involving a direct two-electron oxidation to RN+ is also considered and excluded on the basis of additional experiments. THE PYRIDINE COENZYMES, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) participate in a large number of enzymecatalyzed reactions in which they undergo a two-electron reduction to form the corresponding reduced forms, NADH and NADPH. An important biological function of the pyridine coenzymes is their involvement in the electron-transport system for reduction of oxygen to water. In this coupled enzymic reaction chain, NADH is oxidized by riboflavincontaining compounds which are known to form stable semiquinone intermediates (1). Thus NADH appears to be oxidized at a site in the electron-transfer chain at which transition from one-electron to two-electron control occurs. Accordingly, much effort has been directed toward establishing the mechanism of hydrogen transfer in the redox reactions of the pyridine coenzymes ( 2 , 3). Because the redox chemistry of the pyridine nucleotides is due largely to changes occurring in the nicotinamide portion of the molecule, the use of simple nicotinamide compounds as models has been of value in mechanistic studies. A good model compound is one that contains the nicotinamide ring, the known site of hydrogen transfer, in conjunction with a simple aliphatic side chain to replace the complex ribose-pyrophosphate-ribose-adenine moiety. Application of model compounds to studies of pyridine coenzymes is well known and dates back to the thirties with the work of Karrer ( 4 ) who was able to elucidate the nonenzymic reduction of NAD+ using simple l-alkylnicotinamides. In this work, the electrochemistry of the NADH analogs, 1-(2,6-dichlorobenzyl)-l, 4-dihydronicotinamide (ClBzNH)
and 1-methylnicotinamide (Me”) are examined in acetonitrile using platinum and carbon electrodes. The l-benzyland l-n-propyl-l,4-dihydronicotinamides (BzNH and PrNH) are also examined briefly. A novel mechanism is presented in which a transition from a one-electron to a direct twoelectron oxidation can be effected by addition of a suitable proton acceptor. Strong chemical evidence indicates that the oxidation proceeds through the initial formation of a protonated radical intermediate. The oxidation of the NADH analogs is also accompanied by their acid catalyzed decomposition in unbuffered acetonitrile, and additional studies of this reaction are conducted to determine its role in the oxidation pathway. Previous electrochemical investigations have been restricted largely to NAD+ and its analogs. Underwood and coworkers have successfully elucidated the reduction of NAD+ and its analogs (5-8) and also NADP+ (9). The adsorption behavior of these compounds (10) was also examined. Underwood et a/. (6,9) reported that at very positive potentials, NADH and NADPH are oxidizable to NAD+ and NADP+, respectively. The NADH analogs employed in the present study have been investigated extensively in homogeneous solution (3, 11), but no previous electrochemical studies have been reported. EXPERIMENTAL
1 Present address, Laboratory, Memorial Hospital, Kenosha, Wis. 53140
Apparatus. The electrolysis cell employed for both coulometry and voltammetry consisted of an Exax outside taper weighing bottle, size H (E. H. Sargent Co.) which was sealed in a Plexiglas water jacket. The top of the weighing bottle, cover was removed on a glass-cutting saw, and a polyethylene disk, with appropriate holes for the electrodes and gas dispersion tube, was sealed onto the weighing bottle cover with silicone adhesive. The reference electrode was a silver wire in a solution consisting of 0.01M AgC104 and 0.1M tetraethylammonium perchlorate (TEAP) in acetonitrile (AN), separated from the sample solution by two porous glass junctions. The reference electrode made electrical contact with the sample solution by a bridging compartment containing 0.1M TEAP and a polyethylene capillary probe which contained either 0.1M TEAP or the sample solution. The probe tip was positioned within several millimeters of the working electrode. For coulometry, the working electrode was a semicircular platinum gauze electrode (4 cm X 7 cm), positioned concentrically against the wall of the electrolysis cell. A platinum coil counter electrode was isolated during electrolysis by a double-junction salt bridge consisting of an ultrafine porosity frit and a porous glass disk, and was filled with 0.1M TEAP. For voltammetry, the counter electrode was an exposed platinum flag.
(1) H. Beinert and G. Palmer, Aduan. Enzymol. Relat. Subj. Biochem., 27, 105 (1965). (2) H. Sund, “Biological Oxidations,” T. P. Singer, Ed., Interscience, New York, 1968, p 603. (3) K. Wallenfels, “Steric Course of Microbiological Reactions,” Ciba Foundation Study Group No. 2, G. W. E. Wolstenholme and C. M. O’Connor, Ed., Churchill, London, 1959, p 10. (4) P. Karrer, G. Schwarzenbach, F. Benz, and U. Solrnsenn, Hela. Chim. Acta, 19, 811 (1936).
( 5 ) J. N. Burnett and A. L. Underwood, Biochem., 4,2060 (1965). (6) J. N. Burnett and A. L. Underwood, J. Org. Chem., 30, 1154 (1965). (7) R. W. Burnett and A. L. Underwood, Biochem., 7, 3328 (1968). (8) A. J. Cunningham and A. L. Underwood, ibid., 6,266 (1967). (9) A. J. Cunningham and A. L. Underwood, Arch. Biochem. Biophys., 117, 88 (1966). (10) A. M. Wilson and D. G. Epple, Biochem., 5, 3170 (1966). (11) R. F. Hutton and F. H. Westheimer, Tetrahedron, 3, 73 (1958).
918
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
For voltammetry, a glassy carbon disk electrode (area 0.138 cm2) was prepared from stock glassy carbon rod (Grade GC-20, 4-mm diameter, International Carbon Corp., 245 Park Ave., New York, 10017). A 1.0-1.5 cm section was cut from the rod using a glass-cutting wheel. The end of the rod was then polished under water first with medium and then with extra-fine grade diamond cloth (3M Company, Diamond Products Div., St. Paul, Minn., 55101). A mirrorlike surface was achieved by polishing finally with moistened, Fez03-impregnated filter paper. The section was then cleaned by treating it with warm solutions of 1 :1 HCl, 1 :1 "Os, and water. After air drying, the polished section was press-fitted into a 1-inch length of heat-sealable Teflon tubing flexible expanded natural TFE, l/s-inch i.d., 0.030-inch wall (Pennsylvania Fluorocarbon Company, Clifton Heights, Pa.). The Teflon tubing protruding from the unpolished end of the section was then press-fitted onto a length of glass tubing (5-mm 0.d.). The assembly was then heated by slowly rotating in a flame until the Teflon tubing shrank and became transparent. After cooling, the Teflon tubing was trimmed to extend 0.5-1.0 mm beyond the polished face of the carbon disk, Electrical contact was made with a column of mercury. The dc resistance of the electrode was about 1 ohm. Electrodes prepared in this manner were used without any further polishing or machining. Occasionally, it was necessary to soak an electrode for 30-60 seconds in cleaning solution (H2SO4-Na2Cr2O7)to remove Teflon decomposition products produced during heat-sealing. For fast-sweep voltammetry, a platinum disk working electrode (area, 0.001-0.002 cm2) was prepared by sealing a platinum wire coaxially into the end of a soft glass tube. The wire was then cut and polished until it was flush with the glass. For polarography, a dropping mercury electrode (DME) with a drop time of 4.0 i 0.2 sec was used. Electrochemical experiments were conducted using a Heath polarographic system consisting of an EUW-19A Operational Amplifier System, and EUW-19-4 Stabilizing Unit, and an EUW-19-2 Polarographic Module. For voltammetry, current readout was obtained with either a Sargent Model SR recorder with a 10 inch/min chart speed or a Tektronix Model 564 Oscilloscope equipped with a Type C-12 Camera. For cyclic and fast-sweep voltammetry, a Wavetek Model 112 Function Generator (FG ; Carter Electronics, Inc., 7201 S. Western Ave., Chicago, 60636) was employed. For coulometry, an additional Heath stabilized booster amplifier was used. The current was monitored with a recorder and also integrated using a Baird Atomic Model 530 Scaler-Timer and the F G as a voltage-to-frequency converter. The conversion linearity of the F G was within 0.4% over the range 0.1 to 20 mA, and the drift was less thanO.l % per hour. The temperature of the electrolysis cell was maintained at 25.0 0.5 "C using a Sargent S-84860 Water Bath and S-71590 Liquid Circulating Pump. A Cary Model 14M recording spectrometer was used to obtain ultraviolet spectra. Reagents. Acetonitrile was prepared by purification of technical grade material (Matheson, Coleman & Bell, Downers Grove, Ill.) according to method D or D-1 of Coetzee e t al. (12) using a 75-cm distillation column packed with porcelain burl saddles. The water content measured by Karl Fischer titration was less than one millimolar. All aqueous solutions were prepared from deionized, distilled water. Stock 95% ethanol was used to prepare aqueous ethanol solutions. Nitrogen (Union Carbide Driox bulk grade) for cell deaeration was passed through an all-glass train consisting of a 10-inch drying column filled with alternating layers of calcium sulfate and magnesium perchlorate, followed by a presaturation column containing the appropriate solvent. Oxy-
*
(12) J. F. Coetzee, G. P. Cunningham, D. K. McGuire, and G. R. Padmanabhan, ANAL.CHEM., 34, 1139 (1962).
gen (National Cylinder Gas Co., USP grade) for saturating AN solutions was also dried and presaturated with solvent. Tetraethylammonium perchlorate (Eastman) was recrystallized from ethanol-water, dried at 110 "C for 10 hours in vucuo, and stored over magnesium perchlorate. Reagentgrade pyridine was dried by refluxing over barium oxide and then distilling. Tertiary butylamine was dried by distillation in a nitrogen atmosphere. l-(2,6-Dichlorobenzyl)nicotinamidechloride was prepared from nicotinamide and 2,6-dichlorobenzyl chloride (Eastman) according to Kim and Chaykin (13). 1-Methylnicotinamide iodide was prepared according to Holman and Wiegand (14). The perchlorate salts (MeN+C104- and C1BzN+C1O4-) of the nicotinamides were obtained by treatment with silver perchlorate (15). MeNH was prepared by the dithionite reduction of 1methyl nicotinamide iodide according to previously described procedures ( 4 , 15, 16). The resulting crystalline product was dried over phosphorus pentoxide in uacuo and was stored in a freezer in evacuated glass ampoules, one or more of which were used for each subsequent experiment-mp, 84-85 "C [84 "C reported (16)]; emax, 7.06 X l o a [7.00 X l o 3 reported (17)]. Upon exposure to air, MeNH decomposed slowly to form a red viscous residue which could be purified to recover the remaining undecomposed product by dissolution in deaerated ethyl acetate, filtration, and evaporation of the solvent under aspirator suction. ClBzNH was prepared and purified according to Kim and Chaykin (13). The product was stored over phosphorus pentoxide in a vacuum desiccator-mp, 159-162 "C [162 "C reported (13)]. The molar absorptivity of ClBzNH was observed to decrease by about 5 % over a 2-3 month period, and it was necessary to recrystallize the product at regular intervals-e,,,, 7.38 X l o 3 [7.51 X l o 3reported (IS)]. PrNH and BzNH were prepared from the procedures of Anderson and Berkelhammer (19). Procedure. In acetonitrile, the indifferent electrolyte was 0.1M TEAP. AN solutions were prepared in a glove bag containing phosphorus pentoxide and were exposed to the atmosphere as briefly as possible. Dihydronicotinamide solutions were prepared by adding known quantities of sample and electrolyte to the cell. Alternatively, a weighed amount of sample was added to a previously deaerated solution to reduce sample deaeration time. In both cases, concentrations were determined spectrophotometrically. All voltammetric experiments were completed within 2-2 hours after preparation of the sample solution. For coulometry, sample volumes of 40-68 ml were used. Vigorous stirring was maintained throughout the electrolysis by a continuous flow of the sparging gas (either nitrogen or oxygen) in addition to magnetic stirring. Because the maximum current output of the potentiostat was limited to 20 mA, all coulometric experiments were initiated in the potentiallimiting region of the current-voltage curve. The potential was then increased gradually to the maximum value as specified in each experiment. For voltammetry, both carbon and platinum electrodes were pretreated before each series of scans. For the GCE, pretreatment consisted of immersing the electrode in concentrated sulfuric acid for 20-30 sec. The electrode was (13) C. S. Y . Kim and S. Chaykin, Biochemistry, 7 , 2339 (1968). (14) W. I. M. Holman and C. Wiegand, Biochem. J., 43, 423
(1948).
(15) Ronald G. Haas, Ph.D. Thesis, University of Wisconsin,
Madison, Wis., 1970.
(16) P. Karrer and F. Blumer, Helc. Chim. Acta, 30, 1157 (1947). (17) G. W. Rafter and S. P. Colowick, J . Biol. Chem., 209, 773 (1954). (18) K. Wallenfels and H. Schuly, Ann., 621, 106 (1959). (19) A. G. Anderson and G. Berkelhammer, J . Amer. Chern. SOC., 80, 992 (1958). ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
919
sa w
a
3
8 f
I
I
-0.4
I
0.0
I
I
I
I
0.4 0.8 E ( V VS. Aq-AN)
I
I
*
1.2
I
I6
Figure 1. Voltammetric behavior of ClBz" at the GCE in unbuffered and basic acetonitrile
then rinsed with distilled water, wiped dry, and allowed to come to equilibrium in the sample solution at some initial potential. Two GCE's, given no other cleaning treatments, were used routinely for over two years with no significant increase in the background current. A similar procedure was used with platinum electrodes. With platinum, however, it was also necessary to cycle the potential of the electrode by rapidly switching or scanning the applied potential within the region of interest (usually -0.6 to 1.4 V). Potentials for experiments in AN are referred to a silver electrode in 0.01M AgC104-0.1M TEAP in AN (abbreviated Ag-AN), which has a potential of 0.30 V against the SCE.
pattern. The heights of the secondary waves relative to Wave I varied with each compound. With Me", for example, Wave I11 was several-fold higher, while Wave I1 was only about one fourth as large, compared to the corresponding waves for ClBzNH. As with ClBzNH, Wave I1 for each compound was reversible. Addition of pyridine or t-butylamine to the AN solution gave in each case a single wave whose height was approximately double that of Wave I. The peak potentials and relative enhancement in the peak current (ip) upon addition of pyridine for each of the analogs are summarized in Table I. Comparison of the limiting currents with the RPE in the potential region of Wave I for both MeNH and ClBzNH with those for the one-electron reduction of the respective NAD+ analogs (MeN+C104- and C1BzN+C1O4-), indicated that Wave I corresponded roughly to a one-electron process, whereas in the presence of base, two-electron behavior was indicated. A shift in the peak potential to less positive values was also noted upon addition of a large excess of base to the unbuffered solution. For 1-3 m M MeNH or ClBzNH at the stationary GCE this amounted to a 70-100 mV shift for 0.01M pyridine or t-butylamine. TWO-ELECTRON OXIDATIONOF REDUCEDANALOGSTO PYRIDINIUM SALTSIN BASICAN. Two coulometric oxidations of MeNH (1-2 mM) at f0.30 V in the presence of 0.05M tbutylamine gave n-values of 1.92 and 1.96 Faraday per mole of MeNH oxidized. In each case, the UV spectrum of the electrolyzed solution was identical to that of authentic MeN+ c104-. Based on the observed absorbances, the MeN+ yield was estimated at 96 and 101 %. Closely similar results were obtained with ClBzNH, indicating that in basic AN, the dihydronicotinamides (RNH) undergo a two-electron oxidation to form the corresponding pyridinium salt (RN+):
I
RESULTS AND DISCUSSION
Preliminary Investigation. GENERALVOLTAMMETRIC BEHAVIOR OF REDUCED ANALOGS.A voltammogram of ClBzNH at the stationary GCE is indicated in Figure 1. In unbuffered' AN at low scan rates, three anodic waves are observed. The first wave (I) is irreversible. The second wave (11) corresponds to a reversible process, as evidenced by the reduction wave obtained upon reversing the scan at a potential anodic of Wave 11. If a slight excess of a base such as pyridine or t-butylamine is added to the same solution, the secondary waves (11) and (111) disappear (Figure l), and a single wave is obtained whose height is approximately double that of Wave 1. Similar results were obtaned for Me", PrNH, and BzNH. In unbuffered solution, each compound exhibited a three-wave
R
(RNH)
I
R+
w+)
Presentation of Electrochemical Oxidation Mechanism for Reduced Analogs in Unbuffered AN. In attempting to elucidate the oxidation mechanism in unbuffered AN, it was found early in the work that an acid-catalyzed decomposition of RNH occurred concurrently with the electrochemical oxidation, so the over-all reaction consisting of both the electron transfer and acid decomposition steps was examined
Table I. Voltammetric Behavior of NADH Model Compounds in Acetonitrile 0.1M TEAP (unbuffered) or 0.1M TEAP with 4mM pyridine (basic); stationary GCE (0.138 cm2);scan rate, 2.0 V/min
Compound ClBzNH
920
Concn, mM
Me"
1.05 1.23
PrNH BzNH
1.47 1.67
Peak currents i,- basic/ i,, PA Concn mM &-unbuffered 20.9 21.8 22.4 23.4
1.99 1.85 1.78 1.87
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
Peak potential (V Wave I 0.30 0.23 0.57 0.30
us.
Ag-AN) in unbuffered solution Wave 111 Wave I1 0.74 0.73 1.02 0.69
1.25 1.22 1.56 1.22
E., V VS. AQ-AN
first. Experiments described in the following section of this paper led to the following mechanism: 2RNH
-+
RN+ 4- H(RNH)+ -t 2e
+I5 -'
I
I
I
I
BASIC/!
(2)
\I
\
+IO1
UNBUFFERED
nonabsorbing 4RXH I products products L _________________------
H ( R y ) t ~ 2 9nm0 I absorbing
+
8'
(3b)
I
In this scheme, the electron transfer step (Equation 2) is represented over-all as a one-electron process (Le., one electron per RNH transformed). A proton is liberated which attaches to RNH in the aprotic AN medium to give the product H(RNH)+, which is nonelectroactive at the potential at which RNH is oxidized. The decomposition of H(RNH)+ is complicated (Equation 3) and gives several products. Some of the products (Equation 3b) are uncharacterized and are distinguished primarily by their ultraviolet absorption behavior. The decomposition reaction is also catalytic with respect to protons liberated, which can recycle by attacking more of the RNH substrate. A side reaction (Step 3a) also occurs, producing a small amount of the pyridinium salt, with only about 6-9 % of the substrate RNH reacting by this pathway. Additional work described in the last part of this paper served to verify that the over-all electron transfer step (Equation 2) was a one-electron process, rather than the two-electron process usually observed in chemical oxidations of the dihydronicotinamides. The Over-All Reaction in Unbuffered AN Solution. VOLTTypical current-voltage curves from AMMETRIC EXPERIMENTS. cyclic scans of Me" at the stationary GCE are shown in Figure 2. Both scans in this figure were obtained under otherwise identical conditions except for the presence of 2mM pyridine during one of the scans. A cathodic wave is evident on the reverse scan when base is present. This wave is due to the one-electron reduction of the oxidation product MeN+. The peak potential for this wave (E, = - 1.40 V) is in good agreement with that measured (in an additional experiment) for the reduction of authentic MeN+C104- (E, = -1.41 V) at the stationary GCE. In unbuffered solution, a cathodic wave is also observed on the reverse cycle at the same potential as that observed in basic medium, indicating that the pyridinium salt is formed as an electrolysis product in unbuffered as well as in basic acetonitrile. An additional wave on the reverse cycle is observed at more cathodic potentials in both basic and unbuffered solution. This wave presumably arises from the two-electron reduction of MeN+ to Me". In the presence of pyridine, this second wave could also involve the concurrent reduction of the protonated pyridine species CjHjN:H+ (20). The ratio (ipc/ipu) of the peak cathodic (ipc) and peak anodic (ipu)currents in unbuffered solution compared to the ratio observed in pyridine under otherwise identical conditions, gives an estimate of the moles of MeN+ generated per Faraday of oxidation current: moles MeN+
QIF Here, Q is the number of coulombs consumed on the forward, anodic cycle and F is the Faraday. For this comparison to (20) M. S. Spritzer, J. M. Costa, and P. J. Elving, ANAL.CHEM., 37, 211 (1965).
ij
REVERSE SCAN
+---10
6
-20
3
u
-30
i
\
I
I
Figure 2. Cyclic voltammetry of Me" acetonitrile 0.9 m M Me";
in
scan rate, 4.8 V/rnin
be valid, it is necessary that the peak potentials for the anodic and cathodic processes be the same is both the presence and absence of base. This condition is approximately satisfied at low base concentrations and at a constant scan rate. It is also necessary that the same anodic switching potential be used for both scans. If it is assumed under these conditions that the relative loss of MeN+ by diffusion is the same in both basic and unbuffered solution, then the current ratios can be used as a measure of the MeN+ yield per Faraday. For the experiment shown in Figure 2, a total of five scans was conducted in both the presence and absence of pyridine. ratio of 0.274 was obIn unbuffered solution an average ipclipa tained compared to 0.285 in the presence of base. The relative standard deviations of the ratios were around 5%. Additional experiments at MeNH concentrations in the range 0.3-3mM also gave ratios which remained constant (to within 10-15%) upon addition of base. The constancy of the cathodic-to-anodic peak current ratio for MeNH upon addition of base indicates that in both basic and unbuffered solution, the MeN+ yield per Faraday is roughly the same. Since MeN+ is known to be formed quantitatively upon electrolysis in basic solution, it is evident that MeN+ must also be the principal electrolysis product in unbuffered solution. A corollary of this conclusion is that the secondary waves (I1 and 111, Figure 1) in unbuffered AN cannot be due to oxidation products formed in the initial electron-transfer step. Rather, these products arise from the chemical reaction (Equation 3b) of the substrate with the electro-generated proton. Similar results were obtained with the dichlorobenzyl analog. Cyclic scans were conducted with MeNH and ClBzNH at a platinum electrode at scan rates up to 200 Vjsec in the potential region from -0.5 to 0.7 V. No evidence was observed for a cathodic wave upon reversing the potential in the diffusion-limiting region of Wave I, indicating that Wave I is irreversible in nature. The effect of scan rate of the relative heights of Waves I and I1 at the stationary platinum disk electrode is shown qualitaANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
e
921
n
II I-
, 260
Curve A .
tively in Figure 3 for ClBzNH. The current-voltage curves for this figure were obtained directly from oscilloscope traces. which is The ordinate represents the current function i/o related to the observed oscilloscope deflection, D, by the equation i/v112 = ( E / R V ’ / ~ ) D (4) E is the oscilloscope sensitivity in volts per unit division, R is the load resistance, and i is the observed current, uncorrected for background. To permit comparison of the traces, R was adjusted at each scan rate such that the product E/Rv112was constant for all scans in the figure. Current traces recorded in this manner were thus “precompensated” for the v112-proportionality observed for a simple, diffusion-controlled electrode reaction. The diffusion-compensated current-voltage curves of Figure 3 reveal a systematic decrease in i/u1i2 for Wave I1 relative to that for Wave I, as scan rate increases. The decrease in i/v1I2 for Wave I1 with increasing scan rate is characteristic of an ECE (electrochemical-chemical-electrochemical) mechanism (21) of the type - n’e Wave I1
(5)
where a chemical step giving C is interposed between two electrochemical steps (Waves I and 11). The current-voltage curves of Figure 3 clearly indicate that Wave I1 is due to the oxidation of a product C, which is formed as a result of one or more chemical reactions following the initial charge-transfer step. A similar trend in i / u 1 / 2 for Wave I1 was observed for Me”. Additional scans with Me“ over a greater potential range indicated that the species responsible for Wave I11 was also formed in a chemical step following one of the charge-transfer reactions. An anodic shift in the peak potential of Wave I is also observed (Figure 3) with increasing scan rate for ClBzNH. A similar trend was observed with Me”. The magnitude of this shift is consistent with that expected for a kineticallycontrolled charge transfer, although it could also arise from protons liberated in the reactions occurring at the electrode surface, since the over-all reaction is pH-dependent. UV ABSORPTIONSTUDIE~OF ELECTROLYZED MeNH SOLUTIONS.The spectrum obtained after exhaustive elec(21) R. S . Nicholson and I. Shain, ANAL.CHEM., p 178. 922
Spectrum obtained from the electrolysis of unbuffered 1.2 m M MeNH-15 ml of electrolysate diluted to 100 ml with buffer Curve B . Spectrum of 1.2 mM MeNH in AN acidified with 1.0 mM HC10a-7.5-ml sample 7.5 ml of AN diluted to 100 ml with buffer Curve C. Hand-plotted difference between curves A and B Curve D. Authentic MeN+CIOn-in buffer containing 15 vol % AN
+
2.0 mM ClBzNH; scan rate, V, in V/sec as indicated
k
“A
solutions
0 0.5 1.0 E.,V VS. Aq-AN Figure 3. Scan rate dependence of Waves I and 11 for ClBzNH in unbuffered acetonitrile -0.5
-ne-
280
290 WAVELENGTH, Figure 4. UV spectra of electrolyzed and acidified MeNH
5 0
A - -Wave - - - - It B + C - D
270
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
trolysis of Me” in unbuffered AN at potentials in the diffusion-limited region (0.30-0.40 V) of Wave I is indicated in Figure 4 (Curve A ) . A large, broad maximum in the region of 290 nm is observed in addition to some absorption at shorter wavelengths. The appearance of absorption in the 290-nm region is commonly associated with products formed by the acid treatment of dihydronicotinamides (3, 13, 19). The presence of this band in the electrolyzed solution indicated that R N H had undergone reaction with a proton liberated in the electrochemical step (Equation 2). Some experimental evidence in support of this conclusion was obtained by examining acidified Me” in acetonitrile. In order to approximate the anhydrous conditions present during electrolysis, concentrated acid (HC104/2.3 HzO) was added directly to an AN solution of Me” with a microliter syringe. The mixture was then allowed to react for a time comparable to that for an electrolysis (20-30 min). Finally, after quenching the reaction with an excess of 0.1M p H 9 tris-perchlorate buffer, the spectrum was recorded. By appropriate dilution, the spectrum of the acidified solution (Curve B, Figure 4) could be made coincidental with that for the electrolyzed solution in the region of 290 nm. Therefore, the absorption at 290 nm appears to be due to products formed by the acid decomposition of Me” in acetonitrile. Additional qualitative evidence for the formation of MeN+ in the electrolysis was obtained by plotting the difference of Curves A and B. The resulting spectrum (Curve C) was in good agreement with that for authentic MeN+C104-(Curve D). Thus, the absorption at 265-275 nm in the spectrum of the electrolysate appears to be due to MeN+. ANALYSISOF ELECTROLYZED ClBzNH SOLUTIONS.Additional qualitative evidence for the formation of ClBzN+ in the electrolysis of ClBzNH in unbuffered AN was obtained by chromatographic analysis of the electrolysate on silica gel plates (Brinkman Pre-Coated TLC sheets, Silica Gel F-254 with fluorescent indicator) using a solvent mixture (22) of (22) “Thin-layer Chromatography with CHROMAGRAM Sheet and Developing Apparatus,” Brochure from Distillation Products Industries, Eastman Kodak Company, Rochester, N. Y., 14603, p 3.
isopropanol/ammonia/water (9 :1 :2). Examination of the plates under ultraviolet light indicated a component having an R f value of 0.38-0.39. Authentic C1BzN+C104- in 0.1M TEAP when chromatographed under identical conditions gave an R f value of 0.38. The UV spectrum of the ClBzNH electrolysate gave a maximum in the 270-277 nm region, which did not agree well with that observed around 267 nm for authentic CIBzN+C104-. In order to obtain more definitive spectral data, the mixture obtained after a preparative electrolysis was separated using an aqueous extraction procedure (15). One of the fractions obtained in the extraction procedure was found to give a spectrum in reasonable agreement with that for authentic C1BzN+C1O4-. Two additional fractions were obtained, one of which contained species which absorbed in the 284-290 nm region, indicating the presence of one or more acid decomposition products. A nonabsorbing product, which gave two irreversible anodic waves at 0.75 and 1.36 V in AN at the stationary GCE, was also isolated from the remaining fraction. The properties of this material were similar to those observed for the dimeric, nonabsorbing decomposition products isolated by other workers in the acid decomposition (19) of MeNH (23) and l-methyl-3-acetyl-l,4-dihydropyridine and suggested that this material was also a product formed in the acid decomposition reaction (Equation 3b) rather than a product formed in the electron-transfer reaction. This conclusion is also in accord with the results of cyclic voltammetry and coulometry (see below) which indicate that ClBzNf is the only oxidation product formed in the electron-transfer reaction (Equation 2) associated with Wave I. Anodic voltammograms of electrolyzed ClBzNH and MeNH solutions indicated the presence of several ill-defined, irreversible anodic waves in the region from 0.75 to 1.5 V. The reversible wave (11, Figure 1) observed during voltammetry prior to electrolysis was no longer present, indicating that Wave I1 is due to a transient species, C (Equation 5) which is formed by a rapid chemical reaction, and which is also consumed in a slower chemical step occurring during the time required for bulk electrolysis. Additional spectral and voltammetric experiments were conducted in acidified AN solutions of ClBzNH to gain some insight into the intermediates and products responsible for Waves I1 and 111, but the results were inconclusive. COULOMETRY OF MeNH AND ClBzNH. Six exhaustive electrolyses of 1-3mM MeNH solution in unbuffered AN in the diffusion-limited region of Wave I (0.30-0.40 V) gave coulometric'n-values ranging from 0.63 to 0.72 Faradays per mole of Me" oxidized. Four additional electrolyses of 1-2 m M ClBzNH at 0.40-0.50 V gave n-values ranging from 0.60 to 0.79. The observed, nonintegral n-values of less than unity suggested that the accompanying decomposition reaction (Equation 3) might be catalytic with respect to hydrogen ion. This hypothesis was tested by studying the decomposition reaction under conditions where a small quantity of acid was added to a large excess of the dihydronicotinamide. In one experiment, perchloric acid was added to a ClBzNH solution (2.7mM), and the decrease in the ClBzNH content was measured spectrophotometrically. After a reaction time of 30 minutes, 89% of the original ClBzNH was destroyed by a quantity of acid equivalent to 20% of the substrate present initially. Thus, about 4.4 moles of ClBzNH were irreversibly consumed per mole of hydrogen ion added. Additional experiments with ClBzNH gave values ranging from 3-4.5 de(23) H. L. Ammon and L. H. Jensen, Acta Cryst., 23,805 (1967).
pending on the initial ClBzNH and acid concentrations. The acid-addition experiments support the conclusion that the decomposition reaction is catalyzed by hydrogen ion. Because of this catalysis, a substantial fraction of RNH is consumed in excess of the actual quantity of protons liberated in the electrochemical step, resulting in n-values which are less than unity. In six of the electrolyses with Me" and CIBzNH, the yield of the pyridinium salt was measured polarographically with a DME. In each instance, it was found that more RN+ was generated than could be accounted for electrochemically according to Equation 2. Approximately 6 - 9 z of the RNH was converted to RN+ by some nonelectrochemical process. To establish the source of this nonelectrochemically generated RN+, the acid decomposition reaction was again examined by acidifying solutions of either MeNH or ClBzNH with perchloric acid. Subsequent polarographic analysis of the reaction mixtures for RN+ in three separate experiments indicated that 7-10z of the RNH was converted to RN+ in a side-reaction associated with the decomposition reaction. Thus the 7-10% yield of RN+ in the acid-addition experiments accounts well for the 6-9 % yield of nonelectrochemically generated RN+ (Equation 3a) obtained during electrolysis. The results of coulometry thus support the conclusion that RN+ is the only stable oxidation product (Equation 2) formed in the electrochemical step associated with Wave I. Mechanism of Electron Exchange Step. ONE-ELECTRON us. TWO-ELECTRON OXIDATION MECHANISM.The electrochemical oxidation of the dihydronicotinamides in unbuffered AN (Equation 2) could occur through either a one-electron or a two-electron exchange mechanism, which are the simplest mechanisms consistent with the observed electrochemical behavior. The one-electron hypothesis involves loss of a single electron to form a protonated pyridinyl radical RNH. + (Equation 6) which is nonelectroactive at the potential at which RNH is oxidized. In the aprotic AN, the protonated pyridinyl radicals disproportionate (Equation 7), which is a common reaction of free radical species, and which leads to formation of RN+ and the protonated form of the substrate, H(RNH)+ : RNH 2~".
+
RNH . e- (Wave I) one-electron + + RN+ + H(RNH)+ mechanism
+
+
}
(6)
(7)
The two-electron hypothesis involves a direct two-electron oxidation to give the pyridinium salt (Equation 8). The electrogenerated proton may then undergo a rapid, parallel reaction (Equation 9) with RNH to form the transient nonelectroactive species H(RNH)+. If it is assumed that the rate of protonation is faster than the time scale of a voltammetric experiment, then an apparent one-electron wave should be observed: RNH RNH
RN+
+ 2e + H+
two-electron
+ H+ $ H(RNH)+ } m ~ h a n i s m
+
(8)
(9)
Both mechanisms give the same over-all electron exchange reaction (Equation 2). Further, both mechanisms can account for the behavior in basic AN. Thus, for the one-electron mechanism, it may be assumed that the protonated radical intermediate RNH. undergoes rapid reaction with the base B to give the pyridinyl radical R N . (Equation 10). Cunningham and Underwood (8) have shown that R N . is formed in the reversible reduction of RN+ at very cathodic potentials. Consequently, at the potential at which RNH is oxidized, the pyridinyl radical would undergo immediate electro+
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
923
10 . my ClBzNH
I
-0.4
0.0
I
I
0
I
I
I
I
0.5 TIME, MIN
I
I
12
0.4 0.8 E (V VS. Aq-AN) I
I
I .o
Figure 5. Voltammetry of ClBzNH in the presence of oxygen chemical oxidation (Equation 11) resulting in an over-all reaction identical to Equation 1. R N H . + + e (0.2 to 0.5 V) one-electron + B e R N . + HBf mechanism R N . e RN+ + e (-1.0 to -1.4 V) in basic AN RNH + B - + R N + + HB+ + 2e RNH
+
RNH*+
(6) (10) (11) (1)
In the two-electron mechanism, base would react with the protons liberated in Equation 8, preventing the formation of H(RNH)+, and giving an over-all reaction identical to Equation l. VOLTAMMETRY IN PRESENCE OF OXYGEN.Early in this study, it was noted that oxygen had a considerable effect on the height of Wave I1 during voltammetry. This effect is indicated in Figure 5 which gives current-voltage curves at the stationary GCE before and after admission of oxygen to a previously deaerated solution of ClBzNH. For the experiment of Figure 5 , an initial scan was conducted in deaerated solution. The background current was then obtained by generating a potential waveform which was clamped to a constant value upon reaching a predetermined potential in the diffusion-limited region of Wave I. Under these conditions, the current for Wave I exhibits a normal l/tl’Z decay in the region of Wave 11. The solution was then sparged with pure oxygen for 10 minutes, and another scan was made (Figure 5). A 43z reduction in the height of Wave I1 was observed under these conditions. Significantly, Wave I was not affected, indicating that oxygen interacted only with the products of the electrode reaction and not with R N H itself. Subsequent deaeration of the solution for 15 minutes restored Wave I1 to 88 of its original value, indicating that the effect was largely reversible. Voltammetry with MeNH in the presence of oxygen gave results similar to those observed with CIBzNH. With Me”, however, Wave I1 disappeared entirely upon admission of oxygen. As before, Wave I was unaffected by oxygen, and Wave I1 was restored by again purging the solution with nitrogen. It was previously established that Wave I1 was due to the oxidation of some species C (Equation 5 ) which is formed by the reaction of R N H and electrogenerated protons. Since oxygen is an excellent radical scavenger, the voltammetric study of Figure 5 suggested that oxygen was reacting with a radical intermediate in a manner such that the yield of C was diminished. The decreased height of Wave I1 in the presence of oxygen thus suggested that C might be a product derived from the reaction of a radical intermediate.
z
924
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
Several ESR experiments were conducted with both MeNH and ClBzNH using the internal generation technique. Results with both analogs were negative and indicated that if a radical species were produced in the oxidation, its half-life was probably much less than the minimum half-life necessary for convenient detection, estimated at 0.1 to 1 second based on ESR studies of the short-lived (24), electrochemically-generated radical derived from triethylenediammine. ELECTROLYSIS OF ClBzNH IN PRESENCE OF OXYGEN.Since the yield of C (Equation 5), a product derived from the reaction of R N H and an electrogenerated proton, was reduced significantly upon admission of oxygen, it was of interest to determine what effect oxygen would have on the yield of the principal oxidation product, the pyridinium salt. Clearly, if both RN+ and C were derived from the reaction of a common radical intermediate as postulated in the one-electron mechanism (Equation 6), then oxygen might be expected to have a significant effect on the yield of RN+ as well. Alternatively, if the two-electron mechanism were operative, little change in the RN+ yield would be anticipated in an electrochemical experiment conducted in the presence of oxygen. Thus, use of the scavenging ability of oxygen might allow discrimination between the alternative mechanisms. In order to employ oxygen as a scavenger in the electrode reaction, it was necessary to establish whether ClBzNH itself was stable in oxygen-saturated AN. The lack of any significant change in the height of Wave I upon admission of oxygen (Figure 5 ) strongly suggested that ClBzNH was not readily oxidizable under such conditions. Additional evidence was obtained from spectral and polarographic studies. In one experiment, a 2.0mMClBzNH solution was deaerated, and the polarographic background was determined in the cathodic region where ClBzN+ is electroactive. The solution was then sparged with oxygen for 22 minutes, again deaerated, and finally, the RN+ content was determined by polarography after buffering with pyridine. The increase in the cathodic background corresponded to a ClBzN+ concentration lower than 0.02mM, indicating that negligible oxidation of the substrate had occurred. In an additional experiment, it was found that the characteristic ultraviolet absorption for ClBzNH was unaffected upon purging a 2.0mM solution with oxygen for 30 minutes. To determine the effect of oxygen on the ClBzN+ yield in the electrode reaction, a series of bulk electrolyses was conducted in a ClBzNH solution which was previously saturated with oxygen and which was kept saturated with the pure gas throughout the electrolysis, Results are summarized in Table 11. An experiment in deaerated solution is also included for comparison, After completion of electrolysis, the CIBzN+ content was determined by ultraviolet spectrophotometry and also in several experiments by polarography. For the spectrophotometric procedure, an aliquot of the electrolysate was diluted with methanol and the peak absorbance was measured at 267.5 nm. For the polarographic method, the electrolysate was evaporated to dryness under aspirator suction. The remaining residue was then heated in a boiling water bath for 10 minutes in vacuo using a vacuum pump and liquid ethanol containing nitrogen cold trap, dissolved in 48 vol 0.1M tris-perchlorate at pH 8.4, and measured by standard addition of authentic C1BzN+C104 at -1.05 V us. SCE by polarography with a DME. The electrolyses of Table I1 reveal several significant differences in the behavior of ClBzNH in oxygen-saturated AN
z
(24) T. M. McKinney and D. G. Geske, J. Amer. Chem. Soc., 87, 3014 (1965).
Table 11. Electrolysis of ClBzNH in Oxygen-Saturated Acetonitrile Oxygen-saturated 0.1M TEAP at 0.45 V us. Ag-AN
Micromoles ClBzN+ formed PolaroSpectral graphic analysis analysis
Original ClBzNH solution n-Value Concn, mM pmoles 0.98 55.5 1.03 53.4 0.97 89.6 1.52 88.0 ... 133 1.91 130 0.96 148 2.13 145 0.98 140 2.06~ 140 ... 0.68 1.65 (NO02) 104 Original solution also contained 20 mM H20.
...
... 133
...
139 40
0
compared to that observed in deaerated media. First of all, n-values are essentially equal to unity during electrolysis with oxygen; whereas in deaerated solution, values in the range of 0.6 to 0.8 are obtained, The integral n-values indicate that during electrolysis with oxygen, the catalytic decomposition reaction (Equation 3) is no longer occurring. Furthermore, in deaerated AN, less than 40% of the substrate was converted to the pyridinium salt, the remainder being consumed in the decomposition step (Equation 3). During electrolysis with oxygen, however, the ClBzNH in each experiment in Table I1 was quantitatively converted to ClBzN+. Thus, in the presence of oxygen, no acid decomposition products were formed. Also supporting this conclusion was the excellent agreement between the polarographic and spectrophotometric methods of analysis for ClBzN+. Had 290-nm absorbing products been formed during electrolysis with oxygen, they would have interfered with the spectrophotometric method and would have caused disagreement with the polarographic method. The absence of 290-nm absorbing products in the oxygensaturated electrolysate indicated that no protons were liberated freely to the solution in the overall reaction. Since a proton is necessarily lost in the conversion of RNH to RN+, it was evident that this proton was converted to some relatively nonacidic form such as water or hydrogen peroxide. Accordingly, a spot test (25) was conducted to check for the (25) F. Feigl, “Spot Tests, Inorganic Applications,” Elsevier, New York, 1954, p 326.
presence of hydrogen peroxide in the electrolysate. This test was based on the ability of peroxide to reduce ferricyanide to ferrocyanide, which in the presence of iron(II1) gives an intensely colored precipitate, Prussian Blue. The electrolysate gave a positive spot test, whereas a control solution containing 1mM ClBzN+C104- in 0.1M TEAP-AN gave a negative test. Polarographic studies were also consistent with the formation of hydrogen peroxide during electrolysis in the presence of oxygen. Polarograms of the electrolysate residue in basic ethanol-water indicated the presence of an additional wave cathodic to the ClBzN+ reduction wave which was enhanced upon addition of authentic hydrogen peroxide. All work on electrolysis in the presence of oxygen is consistent with the one-electron mechanism, which involves the formation of the protonated pyridinyl radical RNH. + (Equation 6). All data can be explained by postulating that R N H . + undergoes preferential reaction with oxygen instead of disproportionation according to Equation 7 : RNH.+ f
l/202
5 RN+ 4-
l/2H202
(14)
The quantitative recovery of the pyridinium salt and the absence of 290-nm absorbing products indicate that the rate of radical scavenging by oxygen is much greater than the rate of disproportionation. ACIDDECOMPOSITION OF ClBzNH IN PRESENCE OF OXYGEN. Acid decomposition experiments offer another way to test for the two-electron mechanism, Oxidation of the reduced analog to the pyridinium salt is quantitative, and no 290-nm absorbing products are formed on electrolysis in the presence of oxygen. Therefore, if the two-electron mechanism is operative, it is necessary to postulate that the rate of oxidation of H(RNH)+ is much faster than the rate of the acid decomposition process. This postulate was tested by adding acid to an oxygen-saturated solution of ClBzNH. The results of a series of experiments of this type are given in Table 111, along with a previously-reported experiment in deaerated solution for comparison. For each experiment in Table 111, concentrated perchloric acid (11.79M) was added, usually in increments, or continuously (Experiment 4), to better approximate the conditions under which free protons would be generated during electrolysis. The total amount of added acid relative to the initial ClBzNH content is given in Column 3. The fraction of ClBzNH that reacted (Column 4) during a 30-minute period was measured spectrophotometrically by the decrease in the absorbance at 350 nm. The yield of ClBzN+ relative to the initial
Table 111. Acid Decomposition of ClBzNH in Oxygen-Saturated Acetonitrile (1) (2) (3) (4) (5) (6) (7) (8) Absorbance at 267.5 nm Concn of Moles HCIOp Fraction of Moles ClBzN+ Calcd from Expt . ClBzNH, added/mole orig ClBzNH formed/mole Observed Observed for polarog detd mM ClBzNH reacted orig ClBzNH peak, mm Rx. Products” ClBzN+ No. 0.07 292-295 0.531 0.067 0.21b 0.89 1 2.7 274-279 0.565 0.219 0.78 0.22 2 2.7 0.230 278-287 0.737 0.112 0.96 0.19 0.49d 3 2.7 0.15 283-295 0.514 0.136 0.53e 0.93 4 2.1 268-277 1.117 0.701 0.81 0.10-0.15 0.34g 5J 1.2 a Corrected for unreacted ClBzNH and solvent absorption. b Deaerated solution, for comparison. c Added 43 of total acid initially, and added remainder 9 min later. d Added 35 of acid at 5-min intervals. 8 Added acid continuously over a 16-min interval using a syringe pump. f Solution also contained 1.3mM CIBzN+CIOa-initially. Added 50 of acid initially, and added remainder 5 min later.
z z
ANALYTICAL CHEMISTRY, VOL. 42, NO. 8, JULY 1970
925
Table IV. Electrolysis of Me" in Oxygen-Saturated and Deaerated Acetonitrilea
Medium Deaerated AN Oxygensaturated AN
MeNH in original sample Concn, mM Micromoles
MeN+ Yield? n-Value
z
1.2 2.3 2.7 31
61.5 115 148 172
0.69 0.63 0.65 0.66
41 42
1.0
66.7 81.6 74.0 135 138
0.91 0.86 0.94 0.88 0.92
...
1.3 1.1 2.0 2.0
39 39
..
87 85 87
Electrolysis in 0.1MTEAP at 0.3-0.4 V us. Ag-AN. yield = moles MeN+ formed per mole of MeNH in original sample. F,MeN+
ClBzNH content (Column 5) was determined polarographically, by a procedure similar to that employed following electrolysis in oxygen-saturated AN, Reference to Table I11 indicates that only 10-22z (Column 5 ) of the original ClBzNH was converted to the pyridinium salt in the presence of oxygen. The two-electron mechanism requires quantitative conversion under these conditions, Although some enhancement in the ClBzN+ content (Column 5) is observed upon sparging with oxygen, it is far too small to account for the quantitative conversion observed during electrolysis with oxygen. The two-electron mechanism does not adequately explain the observed behavior of ClBzNH during electrolysis under oxygen saturated conditions. The solution obtained after electrolysis with oxygen gave a single maximum at 267.5 nm, characteristic of C1BzN+C1O4-. In addition, the concentration as measured spectrophotometrically was in good agreement with that measured by polarography. The acid-addition experiments indicate, however, that 290-nm absorbing products are formed in each case. These products exhibit an absorption maximum (Column 6) at longer wavelengths than that for authentic C1BzN+C1O4-. They would also interfere with an analysis for the ClBzN+ by spectrophotometry. The extent of this interference is indicated in Columns 7 and 8 where the absorbance observed at 267.5 nm is compared with that calculated from the ClBzN+ content as measured by polarography. In each experiment, great differences exist between the calculated and observed absorbances. The presence of 290-nm absorbing species in the acidified reaction mixture thus excludes the two-electron mechanism and lends further support to the one-electron mechanism (Equations 6 and 7), which does not require the liberation of protons concurrent with the electron-transfer step, and which would permit oxygen to block the acid decomposition reaction. Experiment 5 (Table 111) also indicates that the ClBzN+ yield is insignificantly affected by the presence of additional CIBzNf, rendering unlikely any mechanism which might involve a direct two-electron oxidation followed by a disproportionation equilibrium (26) between RN+ and H(RNH)+ giving the readily oxidizable radical RNH . +. EFFECTOF WATER. Kim and Chaykin (13) have recently characterized a 290-nm absorbing product in aqueous studies of the acid decomposition of ClBzNH, formed by the addi(26) T. Kuwana and J. W. Strojeck, J . Electroanal. Chem., 16, 471 (1968).
926
ANALYTICAL C H E M I S T R Y , VOL. 42, NO. 8, J U L Y 1970
tion of water across the 5,6-double bond of ClBzNH. In the acid-addition experiments of Table 111, the water content of the perchloric acid was about 2.3 moles per mole of HC104. The possibility thus existed that this amount of water might introduce a side-reaction in acid-addition experiments with oxygen, giving rise to 290-nm absorbing products which would not ordinarily be formed under the more anhydrous conditions present during electrolysis with oxygen. To check this possibility, coulometry was conducted in a ClBzNH solution (2.lmM, Table 11) containing about 20 m M water. As before, quantitative conversion of ClBzNH to ClBzN+ was obtained, and no 290-nm absorbing products were formed in the electrolysate, indicating that the electrode reaction is not sensitive to trace quantities of water. Thus in the presence of oxygen, acid-addition gives rise to considerable 290-nm absorption, whereas electrolysis gives none at all, even when a wet solvent is employed. ELECTROLYSIS OF MeNH IN PRESENCE OF OXYGEN.The effect of oxygen on the electrode reaction for MeNH was examined by conducting a series of electrolyses in oxygensaturated AN. The MeN+ content of the electrolysate was determined for each experiment by polarographic analysis in aqueous 0.1M tris-perchlorate (pH S.l), using the general procedure described for the determination of ClBzN+. Results for oxygen-saturated as well as deaerated AN are summarized in Table IV. A control experiment (without electrolysis) consisted of passing pure oxygen through a 2.8mM MeNH solution for 26 minutes. No change in the characteristic UV absorption of MeNH was observed, indicating that MeNH is stable in oxygen-saturated AN. With the methyl analog in oxygen-saturated AN, an increase in the n-value is observed (Table IV), but it is less than the value of unity obtained with ClBzNH under similar conditions. Also, polarographic analysis of the oxygen-sparged electrolysate indicates that the MeN+ yield is enhanced considerably; about twofold relative to the yield in deaerated solution. The spectrum of the oxygen-sparged electrolysate was also examined and was found to agree well with that for authentic MeN+C104- in the 250-270 nm region. In the vicinity of 290 nm, however, a small peak superimposed on the spectrum of the pyridinium salt was apparent. This peak indicates the formation of acid-decomposition products in the electrolysis of MeNH in oxygen-saturated AN. The behavior of MeNH is similar to that of ClBzNH in that both an enhancement in the n-value and the yield of RN+ is observed. However, the lack of quantitative conversion to the pyridinium salt and the presence of some 290-nm absorption indicates that the rate of disproportionation of Me". is sufficiently rapid to compete successfully with the radical scavengingreaction of oxygen. It is of interest to compare the relative concentrations of oxygen and the proposed radical intermediate during a typical electrolysis. The oxygen concentration in a solution saturated with the pure gas is about 7-8mM, based on Henry's law and the reported concentration (27) in air-saturated 0.1 M TEAPAN. The maximum concentration of the intermediate at the electrode surface, Le., at a current of 20 mA, is in the range of 0.6 to 0.9 mM. The scavenging effectiveness of oxygen is thus due, in part, to its relatively high concentration, which facilitates the reaction of the intermediate with oxygen rather than with itself by disproportionation. +
(27) J. F. Coetzee and 1. M. Kolthoff, J . Amer. Chem. Soc., 79, 6110 (1957).
species RNH .+may be an important intermediate in some of these reactions. More recently, some definitive evidence favoring a radical process was reported by Gutman et al. (33) in a kinetic study of the reduction of iron(II1) by NADH in aqueous solution at pH 3-4. After making corrections for the concurrent acid decomposition of NADH, they found that NADH behaved as a one-electron donor in the redox reaction, whereas with a large excess of iron(II1) or in the presence of a mediator such as flavin adenine dinucleotide, normal twoelectron behavior was observed. To account for their data, a mechanism involving the cation radical NADH .+ was proposed. Thus, the behavior upon electrolysis in unbuffered AN is in accord with the findings of several other workers, and indicates that under appropriate conditions the dihydronicotinamides can readily function as one-electron transfer agents.
ALTERNATIVE MECHANISMS. The results of this study are consistent with a mechanism involving the formation of a protonated radical intermediate. However, the actual fate of this intermediate once it is formed in deaerated solution has not been established with certainty. Thus a simple disproportionation is one pathway by which the pyridinium salt may be formed ; obviously, other, more complex pathways involving additional intermediates may also be formulated. The possibility of a mechanism involving a proton exchange reaction between RNH .+ and RNH cannot be excluded. Wawzonek and McIntyre (28) have postulated an exchange reaction similar to that shown in the oxidation of aniline in acetonitrile. RNH
RNH
+
RNH.+
+ e-
+ R N H . + * (RNH-RNH.+) * RN.
2 RNH
-
RN. RNf
e RN+ + e-
+ H(RNH)+ + 2e-
(6)
+ H(RNH)+
(15) (12)
SUMMARY AND CONCLUSIONS
(2)
In the electrochemical oxidation of reduced NAD analogs (RNH), all experimental work is consistent with the formation of a protonated pyridinyl radical (RNH .+) by a one-electron transfer reaction (Equation 6). RNH . + is nonelectroactive at the potential at which RNH is oxidized. The RNH then undergoes one or more additional reactions involving proton or electron transfer, depending on conditions. In the absence of base in the aprotic AN, the RNH + disproportionates (Equation 7) to form the oxidized product pyridinium salt (RN+) and the protonated form of the substrate [H(RNH)+], which undergoes further chemical reactions to give a variety of products (Equation 3). The n-values observed in such electrolyses are less than 1 Faraday per mole of RNH oxidized. In the presence of a base, the protonated pyridinyl radical intermediate reacts to form the pyridinyl radical (RN .) which undergoes immediate electrochemical oxidation to RN+ (Equations 10 and 11). The disproportionation and aciddecomposition reactions that occur in base-free AN are blocked, and n-values of 2 Faradays per mole of RNH consumed are observed in the presence of base. Conversion to RNf is quantitative. Oxygen also reacts (Equation 14) with RNH . + to block the disproportionation and acid-decomposition reactions that occur in base-free AN. For ClBzNH, blocking is quantitative: no acid-decomposition products are observed, conversion to the pyridinium salt is quantitative, and n-values of unity are observed. For Me”, blocking is not quantitative. Experimental results clearly exclude oxidation of RNH by a two-electron mechanism.
As is evident from the overall reaction, a mechanism of this type would also give rise to an apparent one-electron wave. Also, if the rate of proton exchange were slow relative to the rate of radical scavenging by oxygen (Equation 14) an enhancement in the RN+ content and a decrease in aciddecomposition products (Equation 3) would also be expected upon electrolyzing in the presence of oxygen. LITERATURE SUPPORT FOR ONE-ELECTRON MECHANISM. Wallenfels (3) and Sund (2) have examined the chemical oxidation reactions of the dihydronicotinamides, and have concluded that they occur principally by a so-called hydride ion transfer involving the simultaneous loss of a proton and two electrons. The results of the present study indicate that this mechanism is not operative in acetonitrile and that the electrochemical oxidation is resolvable into at least two distinct steps, the first of which involves the transfer of a single electron (Equation 6). This is then followed by one or more steps involving proton transfer and the loss of an additional electron to give the electrolysis product, the pyridinium salt. Although the dihydronicotinamides have been considered to be primarily hydride ion donors, several other studies indicate that they are also capable of functioning as one-electron donors. Schellenberg and Hellerman (29), for example, observed that NADH reacted rapidly with several one-electron transfer agents, and only slowly with oxidants considered to be obligatory two-electron acceptors. Similarly, Westheimer and coworkers (30, 31) have also reported that a radical process may be operative in the oxidation of the 1-alkyl-1,4dihydronicotinamides. Kosower (32) has proposed that the (28) S. Wawzonek and T. W. McIntyre, J . Electrochem. Soc., 114, 1025 (1967). (29) K . A. Schellenberg and L. Hellerman, J. Biol. Chem., 231, 547 (1958). (30) F. H. Westheimer in “Advances in Enzymology,” F. F. Nord, Ed., Interscience, New York, 1962, Vol. 24, pp 469-482. (31) J. L. Kurz, R. Hutton, and F. H. Westheimer, J. Amer. Chem. SOC.,83, 584 (1961). (32) E. M. Kosower, in “Progress in Physical Organic Chemistry,” S. G. Cohen, A. Streitweiser, Jr., and R. W. Taft, Ed., Interscience, N. Y . , 1965, Vol. 3, p 136.
e +
a
RECEIVED for review March 19, 1970. Accepted May 6, 1970. Material presented in part at the 1969 Great Lakes Regional Meeting, ACS, De Kalb, Ill., June 1969. This work was supported in part by the United States National Institutes of Health Grant Nos. GM-14310 and GM-12795 and Atomic Energy Commission Grant No. AT(11-1)-1082. (33) M. Gutman, R. Margalit, and A. Schejter, Biochemistry, 7 , 2778 (1968).
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