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Reduction of the pyridine ring of niazid and isoniazid on mercury electrodes. Comparison with other NAD+ model compounds
303
J. EkctroanaL Chem, 348 (1993) 303-315 Elsevier Sequoia S.k, Lausanne
JEC 02503
Reduction of the pyridine ring of niazid and isoniazid on mercury electrodes. Comparison with other NAD+ model compounds M. Angulo, R. Marin Galvfn, M. Ruiz Montoya and J.M. Rodriguez Mellado
l
Lkpartamento de Quhica l%ica y Termodindmica Aplicada, Facultad de Ckwcias, Universidad de C&Ma, MW4-Ckkioba (Spain) (Received 15 June 1992; in revised form 29 September 1992)
The electrochemical behaviour of niazid and isoniazid on mercury electrodes was studied by dc and differential pulse polarography and, in the case of isoniazid, by cyclic voltammetry in neutral and basic media. The effect of pH, reactant concentration and drop time on the polarographic and kinetic parameters is studied. Mechanisms for the reduction of pyridine rings to dihydropyridines consistent with the experimental results are proposed. The results obtained here and those corresponding to other models of NAD+ coenzyrne reported in the literature are thoroughly discussed. A parallelism in the electrochemical behaviour of these compounds is made evident: reversible electron transfers are associated with para-substituted model compounds and electron transfers are associated with meta-substituted compounds. This behaviour is compared with that of NAD+ itself.
INTRODUCI’ION
In enzymatic redox reactions involving NAD+ and NADP+ coenzymes and pyruvate, malate and lactate dehydrogenases, an electron and a hydrogen atom are transferred from the substrate to position 4 of the pyridine ring of the coenzymes to yield NADH or NADPH. The NAD+/NADH redox system has been studied extensively [1,21. Though most authors have reported one cathodic polarographic wave, two reduction waves have been found in buffered media containing tetraall&nmonium salts [3,4l. The second wave merges with the background discharge and the results of the electrolysis at the potentials of this wave are equivocal. Addition of cationic surfactants and ultrasonic agitation increase the
* To whom correspondence 0022-0728/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
304
rate of NADH formation [5]. The participation of the adenine moiety in the reduction of NAD+ to NADH in acidic media has been confirmed by spectrophotometric investigations and enzymatic assessment of the products of large-scale electrolysis [6]. The oxidation of NADH and NADPH in aqueous media [7], acetonitrile [S] and dimethylsulphoxide [9] are bielectronic processes, but evidence of second-order reactions has been reported [9,103. Direct and mediated electrochemical oxidation of NADH has been extensively reviewed [ll]. Several model compounds have been used to study the redox properties of the NAD+/NADH system [1,2,7,12-301. Most of these models are 3- and 4-pyridine carboxamides, though other related molecules were also used. The amide group is reduced either to aldehyde or hydroxyl groups in acidic media, whereas in neutral and basic media the pyridine ring is reduced. The corresponding hydrazides, niazid and isoniazid, can also be used as model compounds of the coenzymes. A comparative study of the polarographic behaviour of these two compounds [31] and detailed studies of their reduction mechanisms in acidic media [32,33] have been reported. The reduction processes occurring at the potentials corresponding to the first waves are controlled by the second one-electron transfer, which is irreversible, except for isoniazid in strongly acidic media, for which a protonation reaction occurring after the electron transfers is the rate-determining step [32]. The end-products of these reactions are the corresponding amides, which are reduced at the potentials corresponding to the second waves. Nevertheless, macroscale electrolysis in basic media, performed at potentials corresponding to the limiting current of the four-electron wave found in the reduction of isoniazid, gave dihydroisonicotinic amide and ammonia as reduction products [34]. CONHNH, CONHNH, Isoniazid ‘I Niazid ‘I i,‘N CT ‘N The aim of this paper was first to complete the study of the kinetic behaviour of niazid and isoniazid in basic media and second to carry out a comparative study of the reduction of the pyridine ring of amides and hydrazides in those media to show the change in influence on the electrochemical parameters. EXPERIMENTAL
AI1 reagents used were of Merck p.a. grade, with the exception of the hydrazides which were obtained from Aldrich. A buffer solution consisting of 0.05 M boric acid and sodium bicarbonate was used as the supporting electrolyte. The pH was adjusted with solid NaOH and the ionic strength was adjusted to 0.3 M with NaCl. The working concentration of hydrazides was 1 x 10m4 M, except in experiments in which the influence of this variable was studied where the range of concentrations used was 1 x 10p5-5 X 10e3 M. All solutions were purged with purified nitrogen and the temperature was kept at 25 f O.l”C.
305
An INELECSA computerized assembly equipped with a PDC 1212 potentiostat and a GOT 1018 function generator was used for polarographic (dc and differential pulse (DP)) and voltammetric measurements. A dropping mercury electrode with m = 1.052 mg s-l, t = 4.21 s (open circuit) in 0.1 M HClO, and h = 50 cm was used for the dc and DP measurements. The drop time was mechanically controlled at 1 s. The pulse amplitude for DP polarograms was 10 mV and the pulse duration was 20 ms. A Metrohm EA-290 hanging-drop mercury electrode was used for voltammetric measurements; the area of the drop was 0.018 cm’. UV absorption spectra were recorded on a computerized Perkin-Elmer spectrophotometer (model Lambda 3B) with 1 cm quartz cuvettes at 25°C and at a hydrazide concentration of 6 x 10 -5 M. From the variation of absorbances with pH, measured at 290 nm for niazid and 300 nm for isoniazid, pK values of 11.5 f 0.2 and 11.0 f 0.2 were obtained for the basic dissociation of the corresponding hydrazide groups. Both electrochemical and spectroscopic data were obtained as ASCII computer files which were read using adequate software developed in Microsoft Quick Basic language. This software is interactive, with graphical outputs of the data to either the screen or the printer. Thus the contribution of the supporting electrolyte to dc and DP polarograms was eliminated by linear extrapolation of the data obtained at potentials prior to the reduction of the reactant. DP polarograms were analysed using a curve-fitting method and the equations described previously [35-371. Logarithmic analyses of dc polarograms were made in the form of log[Za/(Z, - Z)] vs. E plots, where II depends on the mechanism considered [38]. Tafel curves were obtained at potentials corresponding to the foot of the dc polarographic waves, i.e. at Z I 0.051, [38]. RESULTS AND DISCUSSION
Zsoniazid The first reduction wave observed below pH 9 corresponds to the two-electron reduction to isonicotinamide [31,33]. Figure 1 shows the dependences with pH of the peak potentials, in DP polarography, of the waves corresponding to isoniazid and isonicotinamide. The slope of this plot is around - 83 mV/decade for the first wave. The half-wave potentials behave similarly. The plots of E vs. log[Z/(Z, - Z)] for the first wave (logarithmic analyses) were linear with slopes close to -38 mV/decade. These values are independent of both the isoniazid concentration and the pH. The shapes of the DP polarograms were analysed using the following equation derived for first-order processes [35]: I= 41p
L (1+L)2
306
v -1.0
-1..
6
8
10
PH
Fig. 1. DP polarography. Peak potential of isoniazid (0) compared with that of isonicotinamide (0) in neutral and basic media (depolarizer concentration, 1 X 10m4 M): (a) first wave; (b) second wave.
where I = AZ/AE and L = exp[ -(E - E,)/b]. I, and E, are the peak intensity and peak potential respectively, and b is a term which coincides with the logarithmic analysis slope in dc polarography. Values of b obtained from the best fittings of eqn. (1) to the experimental polarograms (see Fig. 2) are close to -38 mV/decade and - 30 mV/decade for the first and second peaks respectively. The Tafel slopes, obtained at potentials corresponding to the foot of the waves, have a mean value of - 39 mV/decade and the reaction order with respect to the H+ ion is 2. On the basis of these results we can conclude that the process corresponding to this wave must consist of the reversible uptake of two H+ ions and one electron followed by an irreversible one-electron transfer, which is the rate-determining step. The unstable product formed finally gives rise to isonicotinamide and ammonia, as occurs in acidic media [33].
301
Fig. 2. DP polarography. Comparison of theoretical (. . .I and experimental (0) data for the reduction of 1 X 10e4 M isoniazid at pH 7.00.
Above pH 9.5 the two waves merge into a four-electron wave. The logarithmic analysis of this wave is linear with a slope of -33 mV/decade (Figure 3(a)). Moreover, at low concentration values (I 5 X 10T5 M), the DP polarograms closely follow eqn. (1) with a logarithmic analysis slope of -32 mV/decade. Both the half-wave and peak potentials in dc and DP polarography shift around -57 mV/pH unit (Fig. 1). Moreover, the variation of the half-wave potential with the logarithm of the drop time is linear with a slope of around 13 mV/decade. One reduction peak was obtained in cyclic voltammetry, but no oxidation peaks were observed until the scan rate reached 20 V s-l. The E, - EP,*value is around - 32 mV. The log Z-E plots at the foot of the polarographic wave were linear. The Tafel slopes, which are independent of the isoniazid concentration, have an average value of -31 mV/decade. The reaction order with respect to the reactant concentration is close to unity, and is independent of the pH and of the potential at which it is measured. The reaction order with respect to the H+ ion is 2. In order to investigate the influence of proton donors other than the H+ ion on the reduction mechanism, HCO; solutions at pH 10.30 were used. No appreciable influence of this species on the Z-E curves was observed. Taking into account all these results and the literature data it can be concluded that the rate-determining step is a chemical reaction occurring after two one-electron transfers. In strongly basic media, a two-electron reduction of isoniazid to yield dihydroisonicotinic hydrazide was observed [34]. Since the dissociation pK of the hydrazide group is around 11, this means that the anionic form of this group is not reducible, at least at the potentials prior to the background discharge. Thus it is reasonable to assume that the reduction of isoniazid in the pH range 9.5-12 occurs via the formation of the dihydroisonicotinic hydrazide and the subsequent reduction of the hydrazide group to yield the amide:
308
+ 2e-
+ H+
+ 2e-
+ 2H+
(2)
e
-
+m3
It is easy to show that the polarographic, voltammetric with this scheme. The Z-E-r relationship is IL E=E,,,+
g
In 7 (
z
and kinetic results agree
(5)
1
where E 1,2 = E, + g
ln(k,c$)
+ g
ln
(6)
Hence the slope of the E,,, vs. pH plot predicted by this equation should be in agreement with the experimental results. - 2.303 RT/F = -59 mV/decade, The logarithmic analyses, in the form of E vs. log[Z/(Z, - Z)] plots, should yield straight lines with slopes of -29.6 mV/decade. Moreover, eqn. (5) leads to eqn. (1) under the conditions of DP polarography [351 with the same b value. The predicted values of aE,,,/alog t and of E, - Ep,2 (linear sweep voltammetry) are 15 mV/decade and 29.6 mV [39] respectively. As can be seen, the experimental values agree with the predictions. The results obtained at the foot of the wave confirm the above scheme. Thus the Z-E relationship in this potential region is Z = 2FK,k,K’c&,
exp( -2FE/RT)
(7)
where K’ = exp(2FAt&.,/RT) and A& is the potential of the reference electrode. K, and k, are the equilibrium and rate constants of reactions (2) and (3) respectively, and the rest of the symbols have their usual meanings.
309
-1100 (0)
-1200
-I 300
E/mV
- 1400 (bl
-1500
- 1600
-1700
ElmV
Fig. 3. Logarithmic analyses of the reduction dc waves of 1 x 10e4 M hydrazides at 25°C: (a) isoniazid (pH from left to right: 9.0, 9.7, 10.0, 10.5, 11.1, 11.5); (b) niazid (pH from left to right: 8.4, 9.0, 9.4, 9.9, 10.5, 11.4).
The electrochemical reaction orders with respect to isoniazid and the H+ ion and the value of the Tafel slope derived from this equation (1, 2 and -29.6 mV/decade respectively) agree with the experimental values. Niazid The reduction process of niazid is more complex than that proposed for isoniazid. At a concentration of 1 X lop4 M and pH > 6, single dc waves are observed. The variation of E1,2 with pH is not linear, but resembles that of the peak potential shown in Fig. 4. DP polarograms show an additional peak or shoulder at more negative potentials in the pH range 9-10.5. As can be seen in Fig. 3(b), the logarithmic analyses show deviations from linearity at pH I 11, with the curves being linear at pH 2 11.5; the slope in this last case is around -42 mV/decade. At the lowest pH values studied (pH < 81, the wave splits into two components when the niazid concentration is increased as is shown in Fig. 5. This indicates that the process is complicated by electrodimerization reactions, as in the case of nicotinamide [27]. Above pH 9.5 an increase in the niazid concentration causes a deformation of the wave, but no separate waves or peaks were obtained. The DP polarograms are symmetrical only at pH 2 11.5 and low concentration values (Fig. 6). In these conditions the polarograms correspond to a first-order process with a logarithmic analysis slope of - 40 mV/decade. As in the preceding case, the evolution of the limiting current from the height corresponding to a six-electron process (at low pH values) to that corresponding to a four-electron process (at high pH values) must be attributed to the transition between the reduction of the hydrazide group and that of the pyridine ring. Cyclic voltammograms do not show anodic peaks accompanying the cathodic peak at any pH value
310 Ep& -1.5
-1.6
-1.:
-1.t
11
9
7
PH
Fig. 4. DP polarography. Peak potential of niazid (0) compared with that of nicotinamide (0) in neutral and basic media (depolarizer concentration 1 x 10m4 M): (a) first wave; (b) second wave/shoulder.
in neutral and basic media. The Tafel slopes and reaction orders with respect to niazid and the H+ ion concentration were -43 mV/decade, 1 and close to 2 respectively.
Ep/v -1.3. Ic-'/Ak
1
0 -1.1 (a)
-12
-1.3
-1.4
-4.5
E/V
-3.5
-2.5 lOg(c/M)
(b)
Fig. 5. Effect of niazid concentration on the DP polarograms at pH 6.2. (a) DP polarograms normalized by dividing the intensity by the concentration (start potential, - 1125 mV): (1) 2.6 X 10W3 M; (2) 7.3 x 10e5 M. (b) Variation of peak potentials with concentration: (1) single peak observed; (2) first peak; (3) second peak.
311
I/)rA 0.45.
!
*=x
t
i
l +
.:: i ?’ t
0.30.
0.15.
i L . ,./
* . ‘. :*
I’ ‘t
l
2
*..
-1750
-1850
*.a.
E/mV
Fig. 6. DP polarography. Comparison of theoretical of 5 X low5 M niazid at pH 12.00.
(. . .) and experimental (0) data for the reduction
Taking into account these results and those corresponding to the reduction of isoniazid, the following scheme is proposed for the pH range 9.5-12: CONHNH, + e-
+ 2H+
(9
CONHNH, + e-
CONHNH,
+ 2e-
In this case the Z-E-f
(9
CONH, + 2H+
+ NH,
(10)
relationship is [38]
E = E1,2 +
(11)
with E 1,2 = E; + -
RT
2aF
RT
In k”,cL + 4aF
(12)
where k”, is the rate constant of reaction (9) at potential zero. The logarithmic analysis predicted by eqn. (11) in the form of E vs. log[Z/(Z, Z)] plots should yield straight lines with slopes of - 39 mV/decade, provided that (in = 1.5 [38].
312
On the basis of the above scheme, the Z-E relationship sponding to the foot of the wave can be expressed as I =
exp[ -
2FK,k”,K”c~c,
(’+RpT)FE]
for potentials corre-
(13)
where K” = exp[ - (1 + P)FA&,/RTl. Assuming p = 0.5 the theoretical values of the Tafel slope (-39 mV/decade) and the reaction orders derived from eqn. (13) agree with the experimental ones. Comparative study The overall reactions corresponding to the two-electron reductions of the pyridine rings of the model compounds can be explained on the basis of irreversible processes. This irreversibility may be due to either the electron transfer itself or the occurrence of a chemical reaction (namely a protonation) occurring after the electron transfers [21-301. In all cases 1,4-dihydroderivatives were the main reduction products, as occurs for the two-electron reduction of NAD+ itself [401. Taking into account the results obtained here and those reported in the literature, the reduction mechanisms of the pyridine ring of the model compounds can be represented by the following reaction schemes: mechanism (a) PY +mH++
e-+
PY’+ e-+
PYH,
PYH,
PY’ (m=0,1,2)
+ (2 - m)H++
PYH,
mechanism (b) PY + mH++ 2e-+
PY”
PY” + H++ PYH,,, PYIL+,
+ (1 - m)H++
(m = 0,l) PYH,
In the case of 1-alkyl-3,5-di(alkylcarbamoy1) pyridinium ions (ACPY+) the formation of the dihydro compounds in the pH range 6-9 follows an EEC mechanism 1291. Since the influence of pH on the wave is weak, i.e. m = 0, and taking into account the processes observed for the rest of the model compounds, it seems reasonable to think that the rate-determining step for this compound must be the second one-electron transfer. This assumption is supported by the reversible or quasi-reversible character of the first one-electron transfer [29,30]. A study of the electrochemical reduction of 1-benzyl-3-carbamoyl pyridinium chloride (BCP+) in aqueous media to form the dihydroderivative has been reported in the literature 1171.In this case, the proposed sequence of reactions also consists of two consecutive one-electron transfers followed by a protonation
313 TABLE 1 List of the model compounds and the corresponding’ reduction mechanisms of the pyridine rings Mechanism
Compound
m
Reference 27 24 28 25 29 17 20 This work This work
Nicotinamide N-methyl nicotinamide Nicotinamide N-oxide Isonicotinamide ACPY + BCP+ MPP+ N&id Isoniazid
reaction (e,e,H+), with the second electron transfer being the rate-determining step, as in the preceding case. Finally, the two-electron reduction of the pyridine ring of 1-methyl-4-phenyl pyridinium (MPP+) in dimethylformamide, acetonitrile and dimethyl sulphoxide has been shown to occur via the following scheme [20]:
MPP+ +MPP*>
-e
MPP- %MPPH+OH-
i.e. the mechanism is of type (b). The reduction mechanisms corresponding to each compound considered here are listed in Table 1. As can be seen, there is a great variety of mechanisms, which indicates that the reduction to 1,4-dihydropyridine is strongly affected by the nature and position of the substituents. Therefore substituents in the pm-a position with respect to the heterocyclic nitrogen (isonicotinamide, isoniazid and MPP+) give rise to reduction processes of type (b), whereas substituents in the meta position (the rest) imply type (a) mechanisms. Moreover, the reductions of these last compounds take place at potentials over 400-500 mV more negative than those corresponding to the para-compounds. Thus reversible electron transfers are associated with isonicotinamide and isoniazid and irreversible transfers with the rest. In addition, mechanism (a) is complicated by dimerization reactions at neutral pH values, which indicates that the radicals formed after the first one-electron transfers are more stable than those corresponding to the compounds following mechanism (b), which never show a tendency to dimerization. The degree of irreversibility associated with the meta position decreases as the pH increases (in neutral and basic media) when the heterocyclic nitrogen is bonded to an alkyl group, as occurs in the case of N-methyl nicotinamide [24]. In this last case the half-wave, or peak, potential is affected by changes in the pH of the medium, which indicates that the protonation of pyridinyl radicals must play a significant role in the reduction. In the same way, the smah dependence of the reduction potential of BCP+ on the supporting electrolyte [17] could be attributed
314
to the participation of proton donors in the rate-determining step, in addition to adsorption effects. These results agree with those found in the reduction of NAD+ [41]. Thus the free-radical NAD. is not reduced in aprotic media and the protonation of this species is necessary for its subsequent reduction [42]. Moreover, in this protonation reaction proton donors other than the H30+ ion must be involved [42,43] in a similar manner to that found in the electrochemical behaviour of some model compounds [21,25,27,28]. Mechanistic studies of the oxidation of NADH and model compounds have also been made [8,11,44-481. Diagnostic tests over a wide range of concentrations showed that the oxidation consists of two consecutive one-electron transfers together with an acid-base reaction. The following scheme was proposed Bl: NADH 2
NADH.-
5
NAD+
NAD+ fast
As can be seen, this scheme corresponds to mechanism (a) with the sequence of steps placed in inverted order and the acid-base reaction being the inverse of that proposed for the NAD. reduction. ACKNOWLEDGEMENT
The authors wish to express their acknowledgement the financial support of this work.
to Junta de Andalucia for
REFERENCES
7 8 9 10 11 12 13 14 15 16 17 18 19 20
B. Janik and P.J. Elving, Chem. Rev., 68 (1968) 295. J.N. Burnett and A.L. Underwood, J. Org. Chem., 30 (1965) 1154. C. Carruthers and V. Suntzeff, Arch. Biochem. Biophys., 56 (1955) 441. B. Ke, Biochim. Biophys. Acta, 20 (1956) 547. J.N. Burnett and A.L. Underwood, Biochemistry, 7 (1968) 3328. B. Czochralska, E. Bojarska, P. Valenta and H.W. Niirnberg, Bioelectrochem. Bioenerg., 14 (1985) 503. A.J. Cunningham and A.L. Underwood, Biochemistry, 6 (1967) 266. H. Jaegfeldt, J. Electroanal. Chem., 110 (1980) 295. H. Jaegfeidt, A. Tortensson and G. Johansson, Anal. Chim. Acta, 97 (1978) 221. Z. Samec, W.T. Bresnaham and P.J. Elving, J. Electroanal. Chem., 133 (1982) 1. L. Gorton, J. Chem. Sot. Faraday Trans. I, 82 (1986) 1245. S.J. Leach, J.H. Baxendale and M.G. Evans, Aust. J. Chem., 6 (1953) 395. C.O. Schmakel, K.S.V. Santhanam and P.J. Elving, J. Electrochem. Sot., 121 (1974) 345. P.J. Elving, C.O. Schmakel and K.S.V. Santhanam, CRC Crit. Rev. Anal. Chem., (May 1976) 1. D. Thevenot and R. Buvet, J. Electroanal. Chem., 39 (1972) 447. R. Raghavan and R.T. Iwamoto, J. Electroanal. Chem., 92 (1978) 101. I. Care& M.E. Cardinali and F.M. Moracci, J. Electroanal. Chem., 107 (1980) 391. S. Kashti-Kaplan, J. Hermolin and E. Kirowa-Eisner, J. Electrochem. Sot., 128 (1981) 802. J. Moiroux, S. Deycard and T. Malinski, J. Electroanal. Chem., 194 (1985) 99. M.A. Oturan, P. Dostart, M.S. Benedetti, J. Moiroux, A. Anne and M.B. Fleury, J. Electroanai. Chem., 242 (1988) 171.
315 21 R. Marin Galvin and J.M. Rodriguez Mellado, J. Electroanal. Chem., 250 (1988) 399; Electrochim. Acta, 33 (1988) 1795. 22 R. Marin Galvin, J.M. Rodriguez Mellado and F. Garcia-Blanco, J. Electroanal. Chem., 251 (1988) 163. 23 R. Marin Gahin and J.M. Rodriguez Mellado, J. Electroanal. Chem., 260 (1989) 101. 24 R. Marin Galvin and J.M. Rodriguez Mellado, J. Electroanal. Chem., 265 (1989) 195. 25 J.M. Rodriguez Mellado and R. Marin Galvin, J. Electroanal. Chem., 267 (1989) 221. 26 R. Marin Gab&t and J.M. Rodriguez Mellado, Electrochim. Acta, 34 (1989) 1425. 27 R. Marin Galvin and J.M. Rodriguez Mellado, J. Electroanal. Chem., 283 (1990) 337. 28 M. Ruiz Montoya, J.M. Rodriguez Mellado and R. Marin Galvin, J. Electroanal. Chem., 293 (1990) 185. 29 J.-C. Lepr&tre, D. Liiosin, G. Pierre, P. Chautemps, G. Gellon and J.L. Pierre, J. Electroanal. Chem., 286 (1990) 63. 30 J.-C. Leprstre, D. Limosin and G. Pierre, J. Electroanal. Chem., 324 (1992) 115. 31 J.M. Rodriguez Mellado and R. Marin Galvfn, Bull. Electrochem., 7 (1991) 142. 32 J.M. Rodriguez Mellado and R. Marin Galvin, Electrochim. Acta, 37 (1992) 1147. 33 R. Marin Galvin and J.M. Rodriguez Mellado, Cell. Czech. Chem. Commun., in press. 34 H. Lund, Acta Chim. Stand., 17 (1963) 1077. 35 J.M. Rodriguez Mellado, M. Bl&zquez, M. Dominguez and J.J. Ruiz, J. Electroanal. Chem., 195 (1985) 263. 36 J.M. Rodriguez Mellado, M. Bl&quez and M. Dominguez, J. Electroanal. Chem., 241 (1988) 291. 37 J.M. Rodriguez Mellado, M. BHzquez and M. Dominguez, Comput. Chem., 12 (1988) 257. 38 J.M. Rodriguez Mellado, M. Blazquez, L. Camacho and J.J. Ruiz, J. Electroanal. Chem., 190 (1985) 47. 39 R.S. Nicholson and I. Shain, Anal. Chem., 36 (1964) 706. 40 H. Jaegfeldt, Bioelectrochem. Bioenerg., 8 (1981) 355. 41 P.J. Elving, W.T. Bresnaham, J. Moirouz and Z. Samec, Bioelectrochem. Bioenerg., 9 (1982) 365. 42 K.S.V. Santhanam and P.J. Elving, J. Am. Chem. Sot., 95 (1973) 5482. 43 Y. Ohmishi, Y. Kikuchi and M. Kitani, Tetrahedron Lett., 32 (1979) 3005. 44 W.J. Blaedel and R.G. Haas, Anal. Chem., 42 (1979) 918. 45 Z. Samec and P.J. Elving, J. Electroanal. Chem., 144 (1983) 217. 46 R.L. Blankespoor and L.L. Miller, J. Electroanal. Chem., 171 (1984) 231. 47 P. Leduc and D. Thhenot, Bioelectrochem. Bioenerg., 1 (1974) 96. 48 R.D. Braun, K.S.V. Santhanam and P.J. Elving, J. Am. Chem. Sot., 97 (1975) 2591.
J. EkctroanaL Chem, 348 (1993) 303-315 Elsevier Sequoia S.k, Lausanne
JEC 02503
Reduction of the pyridine ring of niazid and isoniazid on mercury electrodes. Comparison with other NAD+ model compounds M. Angulo, R. Marin Galvfn, M. Ruiz Montoya and J.M. Rodriguez Mellado
l
Lkpartamento de Quhica l%ica y Termodindmica Aplicada, Facultad de Ckwcias, Universidad de C&Ma, MW4-Ckkioba (Spain) (Received 15 June 1992; in revised form 29 September 1992)
The electrochemical behaviour of niazid and isoniazid on mercury electrodes was studied by dc and differential pulse polarography and, in the case of isoniazid, by cyclic voltammetry in neutral and basic media. The effect of pH, reactant concentration and drop time on the polarographic and kinetic parameters is studied. Mechanisms for the reduction of pyridine rings to dihydropyridines consistent with the experimental results are proposed. The results obtained here and those corresponding to other models of NAD+ coenzyrne reported in the literature are thoroughly discussed. A parallelism in the electrochemical behaviour of these compounds is made evident: reversible electron transfers are associated with para-substituted model compounds and electron transfers are associated with meta-substituted compounds. This behaviour is compared with that of NAD+ itself.
INTRODUCI’ION
In enzymatic redox reactions involving NAD+ and NADP+ coenzymes and pyruvate, malate and lactate dehydrogenases, an electron and a hydrogen atom are transferred from the substrate to position 4 of the pyridine ring of the coenzymes to yield NADH or NADPH. The NAD+/NADH redox system has been studied extensively [1,21. Though most authors have reported one cathodic polarographic wave, two reduction waves have been found in buffered media containing tetraall&nmonium salts [3,4l. The second wave merges with the background discharge and the results of the electrolysis at the potentials of this wave are equivocal. Addition of cationic surfactants and ultrasonic agitation increase the
* To whom correspondence 0022-0728/93/$06.00
should be addressed.
0 1993 - Elsevier Sequoia S.A. All rights reserved
304
rate of NADH formation [5]. The participation of the adenine moiety in the reduction of NAD+ to NADH in acidic media has been confirmed by spectrophotometric investigations and enzymatic assessment of the products of large-scale electrolysis [6]. The oxidation of NADH and NADPH in aqueous media [7], acetonitrile [S] and dimethylsulphoxide [9] are bielectronic processes, but evidence of second-order reactions has been reported [9,103. Direct and mediated electrochemical oxidation of NADH has been extensively reviewed [ll]. Several model compounds have been used to study the redox properties of the NAD+/NADH system [1,2,7,12-301. Most of these models are 3- and 4-pyridine carboxamides, though other related molecules were also used. The amide group is reduced either to aldehyde or hydroxyl groups in acidic media, whereas in neutral and basic media the pyridine ring is reduced. The corresponding hydrazides, niazid and isoniazid, can also be used as model compounds of the coenzymes. A comparative study of the polarographic behaviour of these two compounds [31] and detailed studies of their reduction mechanisms in acidic media [32,33] have been reported. The reduction processes occurring at the potentials corresponding to the first waves are controlled by the second one-electron transfer, which is irreversible, except for isoniazid in strongly acidic media, for which a protonation reaction occurring after the electron transfers is the rate-determining step [32]. The end-products of these reactions are the corresponding amides, which are reduced at the potentials corresponding to the second waves. Nevertheless, macroscale electrolysis in basic media, performed at potentials corresponding to the limiting current of the four-electron wave found in the reduction of isoniazid, gave dihydroisonicotinic amide and ammonia as reduction products [34]. CONHNH, CONHNH, Isoniazid ‘I Niazid ‘I i,‘N CT ‘N The aim of this paper was first to complete the study of the kinetic behaviour of niazid and isoniazid in basic media and second to carry out a comparative study of the reduction of the pyridine ring of amides and hydrazides in those media to show the change in influence on the electrochemical parameters. EXPERIMENTAL
AI1 reagents used were of Merck p.a. grade, with the exception of the hydrazides which were obtained from Aldrich. A buffer solution consisting of 0.05 M boric acid and sodium bicarbonate was used as the supporting electrolyte. The pH was adjusted with solid NaOH and the ionic strength was adjusted to 0.3 M with NaCl. The working concentration of hydrazides was 1 x 10m4 M, except in experiments in which the influence of this variable was studied where the range of concentrations used was 1 x 10p5-5 X 10e3 M. All solutions were purged with purified nitrogen and the temperature was kept at 25 f O.l”C.
305
An INELECSA computerized assembly equipped with a PDC 1212 potentiostat and a GOT 1018 function generator was used for polarographic (dc and differential pulse (DP)) and voltammetric measurements. A dropping mercury electrode with m = 1.052 mg s-l, t = 4.21 s (open circuit) in 0.1 M HClO, and h = 50 cm was used for the dc and DP measurements. The drop time was mechanically controlled at 1 s. The pulse amplitude for DP polarograms was 10 mV and the pulse duration was 20 ms. A Metrohm EA-290 hanging-drop mercury electrode was used for voltammetric measurements; the area of the drop was 0.018 cm’. UV absorption spectra were recorded on a computerized Perkin-Elmer spectrophotometer (model Lambda 3B) with 1 cm quartz cuvettes at 25°C and at a hydrazide concentration of 6 x 10 -5 M. From the variation of absorbances with pH, measured at 290 nm for niazid and 300 nm for isoniazid, pK values of 11.5 f 0.2 and 11.0 f 0.2 were obtained for the basic dissociation of the corresponding hydrazide groups. Both electrochemical and spectroscopic data were obtained as ASCII computer files which were read using adequate software developed in Microsoft Quick Basic language. This software is interactive, with graphical outputs of the data to either the screen or the printer. Thus the contribution of the supporting electrolyte to dc and DP polarograms was eliminated by linear extrapolation of the data obtained at potentials prior to the reduction of the reactant. DP polarograms were analysed using a curve-fitting method and the equations described previously [35-371. Logarithmic analyses of dc polarograms were made in the form of log[Za/(Z, - Z)] vs. E plots, where II depends on the mechanism considered [38]. Tafel curves were obtained at potentials corresponding to the foot of the dc polarographic waves, i.e. at Z I 0.051, [38]. RESULTS AND DISCUSSION
Zsoniazid The first reduction wave observed below pH 9 corresponds to the two-electron reduction to isonicotinamide [31,33]. Figure 1 shows the dependences with pH of the peak potentials, in DP polarography, of the waves corresponding to isoniazid and isonicotinamide. The slope of this plot is around - 83 mV/decade for the first wave. The half-wave potentials behave similarly. The plots of E vs. log[Z/(Z, - Z)] for the first wave (logarithmic analyses) were linear with slopes close to -38 mV/decade. These values are independent of both the isoniazid concentration and the pH. The shapes of the DP polarograms were analysed using the following equation derived for first-order processes [35]: I= 41p
L (1+L)2
306
v -1.0
-1..
6
8
10
PH
Fig. 1. DP polarography. Peak potential of isoniazid (0) compared with that of isonicotinamide (0) in neutral and basic media (depolarizer concentration, 1 X 10m4 M): (a) first wave; (b) second wave.
where I = AZ/AE and L = exp[ -(E - E,)/b]. I, and E, are the peak intensity and peak potential respectively, and b is a term which coincides with the logarithmic analysis slope in dc polarography. Values of b obtained from the best fittings of eqn. (1) to the experimental polarograms (see Fig. 2) are close to -38 mV/decade and - 30 mV/decade for the first and second peaks respectively. The Tafel slopes, obtained at potentials corresponding to the foot of the waves, have a mean value of - 39 mV/decade and the reaction order with respect to the H+ ion is 2. On the basis of these results we can conclude that the process corresponding to this wave must consist of the reversible uptake of two H+ ions and one electron followed by an irreversible one-electron transfer, which is the rate-determining step. The unstable product formed finally gives rise to isonicotinamide and ammonia, as occurs in acidic media [33].
301
Fig. 2. DP polarography. Comparison of theoretical (. . .I and experimental (0) data for the reduction of 1 X 10e4 M isoniazid at pH 7.00.
Above pH 9.5 the two waves merge into a four-electron wave. The logarithmic analysis of this wave is linear with a slope of -33 mV/decade (Figure 3(a)). Moreover, at low concentration values (I 5 X 10T5 M), the DP polarograms closely follow eqn. (1) with a logarithmic analysis slope of -32 mV/decade. Both the half-wave and peak potentials in dc and DP polarography shift around -57 mV/pH unit (Fig. 1). Moreover, the variation of the half-wave potential with the logarithm of the drop time is linear with a slope of around 13 mV/decade. One reduction peak was obtained in cyclic voltammetry, but no oxidation peaks were observed until the scan rate reached 20 V s-l. The E, - EP,*value is around - 32 mV. The log Z-E plots at the foot of the polarographic wave were linear. The Tafel slopes, which are independent of the isoniazid concentration, have an average value of -31 mV/decade. The reaction order with respect to the reactant concentration is close to unity, and is independent of the pH and of the potential at which it is measured. The reaction order with respect to the H+ ion is 2. In order to investigate the influence of proton donors other than the H+ ion on the reduction mechanism, HCO; solutions at pH 10.30 were used. No appreciable influence of this species on the Z-E curves was observed. Taking into account all these results and the literature data it can be concluded that the rate-determining step is a chemical reaction occurring after two one-electron transfers. In strongly basic media, a two-electron reduction of isoniazid to yield dihydroisonicotinic hydrazide was observed [34]. Since the dissociation pK of the hydrazide group is around 11, this means that the anionic form of this group is not reducible, at least at the potentials prior to the background discharge. Thus it is reasonable to assume that the reduction of isoniazid in the pH range 9.5-12 occurs via the formation of the dihydroisonicotinic hydrazide and the subsequent reduction of the hydrazide group to yield the amide:
308
+ 2e-
+ H+
+ 2e-
+ 2H+
(2)
e
-
+m3
It is easy to show that the polarographic, voltammetric with this scheme. The Z-E-r relationship is IL E=E,,,+
g
In 7 (
z
and kinetic results agree
(5)
1
where E 1,2 = E, + g
ln(k,c$)
+ g
ln
(6)
Hence the slope of the E,,, vs. pH plot predicted by this equation should be in agreement with the experimental results. - 2.303 RT/F = -59 mV/decade, The logarithmic analyses, in the form of E vs. log[Z/(Z, - Z)] plots, should yield straight lines with slopes of -29.6 mV/decade. Moreover, eqn. (5) leads to eqn. (1) under the conditions of DP polarography [351 with the same b value. The predicted values of aE,,,/alog t and of E, - Ep,2 (linear sweep voltammetry) are 15 mV/decade and 29.6 mV [39] respectively. As can be seen, the experimental values agree with the predictions. The results obtained at the foot of the wave confirm the above scheme. Thus the Z-E relationship in this potential region is Z = 2FK,k,K’c&,
exp( -2FE/RT)
(7)
where K’ = exp(2FAt&.,/RT) and A& is the potential of the reference electrode. K, and k, are the equilibrium and rate constants of reactions (2) and (3) respectively, and the rest of the symbols have their usual meanings.
309
-1100 (0)
-1200
-I 300
E/mV
- 1400 (bl
-1500
- 1600
-1700
ElmV
Fig. 3. Logarithmic analyses of the reduction dc waves of 1 x 10e4 M hydrazides at 25°C: (a) isoniazid (pH from left to right: 9.0, 9.7, 10.0, 10.5, 11.1, 11.5); (b) niazid (pH from left to right: 8.4, 9.0, 9.4, 9.9, 10.5, 11.4).
The electrochemical reaction orders with respect to isoniazid and the H+ ion and the value of the Tafel slope derived from this equation (1, 2 and -29.6 mV/decade respectively) agree with the experimental values. Niazid The reduction process of niazid is more complex than that proposed for isoniazid. At a concentration of 1 X lop4 M and pH > 6, single dc waves are observed. The variation of E1,2 with pH is not linear, but resembles that of the peak potential shown in Fig. 4. DP polarograms show an additional peak or shoulder at more negative potentials in the pH range 9-10.5. As can be seen in Fig. 3(b), the logarithmic analyses show deviations from linearity at pH I 11, with the curves being linear at pH 2 11.5; the slope in this last case is around -42 mV/decade. At the lowest pH values studied (pH < 81, the wave splits into two components when the niazid concentration is increased as is shown in Fig. 5. This indicates that the process is complicated by electrodimerization reactions, as in the case of nicotinamide [27]. Above pH 9.5 an increase in the niazid concentration causes a deformation of the wave, but no separate waves or peaks were obtained. The DP polarograms are symmetrical only at pH 2 11.5 and low concentration values (Fig. 6). In these conditions the polarograms correspond to a first-order process with a logarithmic analysis slope of - 40 mV/decade. As in the preceding case, the evolution of the limiting current from the height corresponding to a six-electron process (at low pH values) to that corresponding to a four-electron process (at high pH values) must be attributed to the transition between the reduction of the hydrazide group and that of the pyridine ring. Cyclic voltammograms do not show anodic peaks accompanying the cathodic peak at any pH value
310 Ep& -1.5
-1.6
-1.:
-1.t
11
9
7
PH
Fig. 4. DP polarography. Peak potential of niazid (0) compared with that of nicotinamide (0) in neutral and basic media (depolarizer concentration 1 x 10m4 M): (a) first wave; (b) second wave/shoulder.
in neutral and basic media. The Tafel slopes and reaction orders with respect to niazid and the H+ ion concentration were -43 mV/decade, 1 and close to 2 respectively.
Ep/v -1.3. Ic-'/Ak
1
0 -1.1 (a)
-12
-1.3
-1.4
-4.5
E/V
-3.5
-2.5 lOg(c/M)
(b)
Fig. 5. Effect of niazid concentration on the DP polarograms at pH 6.2. (a) DP polarograms normalized by dividing the intensity by the concentration (start potential, - 1125 mV): (1) 2.6 X 10W3 M; (2) 7.3 x 10e5 M. (b) Variation of peak potentials with concentration: (1) single peak observed; (2) first peak; (3) second peak.
311
I/)rA 0.45.
!
*=x
t
i
l +
.:: i ?’ t
0.30.
0.15.
i L . ,./
* . ‘. :*
I’ ‘t
l
2
*..
-1750
-1850
*.a.
E/mV
Fig. 6. DP polarography. Comparison of theoretical of 5 X low5 M niazid at pH 12.00.
(. . .) and experimental (0) data for the reduction
Taking into account these results and those corresponding to the reduction of isoniazid, the following scheme is proposed for the pH range 9.5-12: CONHNH, + e-
+ 2H+
(9
CONHNH, + e-
CONHNH,
+ 2e-
In this case the Z-E-f
(9
CONH, + 2H+
+ NH,
(10)
relationship is [38]
E = E1,2 +
(11)
with E 1,2 = E; + -
RT
2aF
RT
In k”,cL + 4aF
(12)
where k”, is the rate constant of reaction (9) at potential zero. The logarithmic analysis predicted by eqn. (11) in the form of E vs. log[Z/(Z, Z)] plots should yield straight lines with slopes of - 39 mV/decade, provided that (in = 1.5 [38].
312
On the basis of the above scheme, the Z-E relationship sponding to the foot of the wave can be expressed as I =
exp[ -
2FK,k”,K”c~c,
(’+RpT)FE]
for potentials corre-
(13)
where K” = exp[ - (1 + P)FA&,/RTl. Assuming p = 0.5 the theoretical values of the Tafel slope (-39 mV/decade) and the reaction orders derived from eqn. (13) agree with the experimental ones. Comparative study The overall reactions corresponding to the two-electron reductions of the pyridine rings of the model compounds can be explained on the basis of irreversible processes. This irreversibility may be due to either the electron transfer itself or the occurrence of a chemical reaction (namely a protonation) occurring after the electron transfers [21-301. In all cases 1,4-dihydroderivatives were the main reduction products, as occurs for the two-electron reduction of NAD+ itself [401. Taking into account the results obtained here and those reported in the literature, the reduction mechanisms of the pyridine ring of the model compounds can be represented by the following reaction schemes: mechanism (a) PY +mH++
e-+
PY’+ e-+
PYH,
PYH,
PY’ (m=0,1,2)
+ (2 - m)H++
PYH,
mechanism (b) PY + mH++ 2e-+
PY”
PY” + H++ PYH,,, PYIL+,
+ (1 - m)H++
(m = 0,l) PYH,
In the case of 1-alkyl-3,5-di(alkylcarbamoy1) pyridinium ions (ACPY+) the formation of the dihydro compounds in the pH range 6-9 follows an EEC mechanism 1291. Since the influence of pH on the wave is weak, i.e. m = 0, and taking into account the processes observed for the rest of the model compounds, it seems reasonable to think that the rate-determining step for this compound must be the second one-electron transfer. This assumption is supported by the reversible or quasi-reversible character of the first one-electron transfer [29,30]. A study of the electrochemical reduction of 1-benzyl-3-carbamoyl pyridinium chloride (BCP+) in aqueous media to form the dihydroderivative has been reported in the literature 1171.In this case, the proposed sequence of reactions also consists of two consecutive one-electron transfers followed by a protonation
313 TABLE 1 List of the model compounds and the corresponding’ reduction mechanisms of the pyridine rings Mechanism
Compound
m
Reference 27 24 28 25 29 17 20 This work This work
Nicotinamide N-methyl nicotinamide Nicotinamide N-oxide Isonicotinamide ACPY + BCP+ MPP+ N&id Isoniazid
reaction (e,e,H+), with the second electron transfer being the rate-determining step, as in the preceding case. Finally, the two-electron reduction of the pyridine ring of 1-methyl-4-phenyl pyridinium (MPP+) in dimethylformamide, acetonitrile and dimethyl sulphoxide has been shown to occur via the following scheme [20]:
MPP+ +MPP*>
-e
MPP- %MPPH+OH-
i.e. the mechanism is of type (b). The reduction mechanisms corresponding to each compound considered here are listed in Table 1. As can be seen, there is a great variety of mechanisms, which indicates that the reduction to 1,4-dihydropyridine is strongly affected by the nature and position of the substituents. Therefore substituents in the pm-a position with respect to the heterocyclic nitrogen (isonicotinamide, isoniazid and MPP+) give rise to reduction processes of type (b), whereas substituents in the meta position (the rest) imply type (a) mechanisms. Moreover, the reductions of these last compounds take place at potentials over 400-500 mV more negative than those corresponding to the para-compounds. Thus reversible electron transfers are associated with isonicotinamide and isoniazid and irreversible transfers with the rest. In addition, mechanism (a) is complicated by dimerization reactions at neutral pH values, which indicates that the radicals formed after the first one-electron transfers are more stable than those corresponding to the compounds following mechanism (b), which never show a tendency to dimerization. The degree of irreversibility associated with the meta position decreases as the pH increases (in neutral and basic media) when the heterocyclic nitrogen is bonded to an alkyl group, as occurs in the case of N-methyl nicotinamide [24]. In this last case the half-wave, or peak, potential is affected by changes in the pH of the medium, which indicates that the protonation of pyridinyl radicals must play a significant role in the reduction. In the same way, the smah dependence of the reduction potential of BCP+ on the supporting electrolyte [17] could be attributed
314
to the participation of proton donors in the rate-determining step, in addition to adsorption effects. These results agree with those found in the reduction of NAD+ [41]. Thus the free-radical NAD. is not reduced in aprotic media and the protonation of this species is necessary for its subsequent reduction [42]. Moreover, in this protonation reaction proton donors other than the H30+ ion must be involved [42,43] in a similar manner to that found in the electrochemical behaviour of some model compounds [21,25,27,28]. Mechanistic studies of the oxidation of NADH and model compounds have also been made [8,11,44-481. Diagnostic tests over a wide range of concentrations showed that the oxidation consists of two consecutive one-electron transfers together with an acid-base reaction. The following scheme was proposed Bl: NADH 2
NADH.-
5
NAD+
NAD+ fast
As can be seen, this scheme corresponds to mechanism (a) with the sequence of steps placed in inverted order and the acid-base reaction being the inverse of that proposed for the NAD. reduction. ACKNOWLEDGEMENT
The authors wish to express their acknowledgement the financial support of this work.
to Junta de Andalucia for
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