- Email: [email protected]
Electrochemical behavior of ellipticine derivatives
J. Electroanal. Chem., 114 ( 1 9 8 0 ) 1 3 9 - - 1 4 6
139
© Elsevier S e q u o i a S.A., L a u s a n n e - - P r i n t e d in T h e N e t h e r l a n d s
ELECTROCHEMICAL BEHAVIOR OF ELLIPTICINE DERIVATIVES PART I. OXIDATION OF 9-HYDROXY-ELLIPTICINE
J. M O I R O U X
Laboratoire de Chimie Analytique (Prof. M.B. Fleury *), Universitd Rend Descartes (Paris V), Facultd des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris Cedex 05 (France) A.M. A R M B R U S T E R
Laboratoire de Biochimie et Enzymologie, L.A. 147 du C.N.R.S., Groupe 140 I.N.S.E.R.M., Institut Gustave Roussy, 94800 Villejuif (France) ( R e c e i v e d 7 t h M a r c h 1 9 8 0 ; in revised f o r m 2 9 t h April 1 9 8 0 )
ABSTRACT T h e e l e c t r o c h e m i c a l o n e - e l e c t r o n o x i d a t i o n o f 9 - h y d r o x y - e l l i p t i c i n i u m c a t i o n s at a platin u m e l e c t r o d e has b e e n e x a m i n e d w i t h p a r t i c u l a r a t t e n t i o n t o t h e t h e r m o d y n a m i c r e d o x pot e n t i a l a n d t o t h e d i m e r i z a t i o n r a t e o f t h e radical species p r o d u c e d . B o t h t h e r e a c t a n t a n d t h e p r o d u c t of t h e e l e c t r o c h e m i c a l r e a c t i o n are s t r o n g l y a d s o r b e d a t t h e s o l u t i o n / e l e c t r o d e interface. T h e initial step o f t h e a n o d i c process is a reversible e l e c t r o n t r a n s f e r a c c o m p a n i e d b y a fast d e p r o t o n a t i o n ; E °' = 9 0 0 - - 53 p H m V vs. NHE. T h e r e s u l t i n g n e u t r a l radical dimerizes, t h e r a t e c o n s t a n t o f t h e surface d i m e r i z a t i o n b e i n g ca. 2.5 X 109 mo1-1 c m 2 s -1.
INTRODUCTION
Ellipticine (E) 5,11
* T o w h o m all c o r r e s p o n d e n c e s h o u l d b e addressed.
140
[ El
E - DNA~
- - +
DNA
g OH E-DNA
~
in vivo conversion
Cyt, P450 ~ 90H-E~... Peroxidase -llke rnonooxygenases (~-~.. ~ . peroxides .)-" 9 O-E or oxygen - -
+ DNA
~ DNA breaks or covalent binding (?)
of dimerization of the radical 9"O--E when adsorbed at the electrode surface. Experiments were made with 9 OH--E and with 2-methyl, 9-hydroxy-ellipticinium (9 OH--E+--2 CH3) which is presently under clinical trial [4]. CH3 H
O
~
N 9
N
H
O~ " ' ~ ' ~ ~N~+
CH3
OH- E
H 9
CH3 T C
H
3
CH3
OH-E + - 2 CH3
These compounds give an anodic voltammetric signal at potentials too positive to allow the use of mercury electrodes, and electrodes o f such solid materials as carbon or platinum must be used. Both the reactant and the product of the electrochemical reaction adsorb strongly at the electrode surface. As a part of a systematic study of the redox characteristics of the ellipticine derivatives, the electrochemical oxidation of 9 0 H - - E and 9 OH--E÷--2 CH3 were examined in buffer aqueous media at various pH in the range 2--12. The excitation and measurement techniques used included single- and multiplesweep voltammetry at stationary and rotated disk electrodes. EXPERIMENTAL
Materials The 9 OH--E and 9 OH--E+--2 CH3 acetate were generously provided by SANOFI, and were used w i t h o u t further purification. Reagent-grade chemicals were obtained from Prolabo. The buffered background solutions used were: 0.5 M acetate buffer for the 3--6 pH range; 0.1 M phosphate buffer for the 6--12 pH range.
Electrodes The working electrode was a platinum ferrule for Tacussel EDI electrodes. The effective electrode area, determined by measurement of the anodic ferrocyanide cyclic voltammetric peak (n = 1, D = 6.32 X 10 -6 cm 2 s -1) [13] was A = 0.031 cm 2. The reference electrode was a mercury/calomel/saturated KC1 Tacussel C 10 electrode to which all potentials cited are referred unless otherwise specified. The counter electrode was a Tacussel Pt 33/88 electrode.
141
Apparatus The cell was a Tacussel CPRA water-jacketed cell whose temperature, unless otherwise specified, was 25 ° C. Voltammetric measurements using rotating~lisk working electrodes (Tacussel EDI electrode plus a CONTROVIT servocontrol electronic amplifier) were made with a Tacussel EPLI recorder and a Tacussel TIPOL polarograph plug-in (potential scan rate v = 2 mV s-l). For cyclic voltammetry at stationary working electrodes, a Tacussel PRT 30-01 rapid response potentiostat with a Tacussel TP-PRT plug-in generating a time-dependent pilot signal and a Tacussel ADTP differential amplifier were used. Current--potential curves were recorded either with a Tektronix R 564B cathode ray oscilloscope with suitable modules and camera or with a Sefram TGM 164 X--Y recorder with suitable modules. Controlled potential electrolysis were carried o u t using a three-compartment water-jacketed cell; a Tacussel PRT 200-IX potentiostat, a Tacussel IG5-N electronic integrator and a general-purpose milliammeter were included in the circuit. The working electrode was a large platinum disk (diameter: 8 cm).
Electrode pretreatment In the present study, the working electrode was pretreated before each voltammogram unless otherwise specified. The electrode pretreatment consisted of sanding the electrode vigorously with the Tacussel BA 03 abrasive and of applying to the rotated disk electrode (RDE) a negative potential corresponding to a cathodic current of 6 pA in the background solution for 2 min and then a positive potential corresponding to an anodic current of 6 pA, and repeating the cycle twice. At the end of the pretreatment the working electrode was removed and immersed in the sample solution. For RDE voltammetric experiments the electrode was held at the initial potential until the current reached a steady state before recording the current--potential curve. For measurements using a stationary electrode, the pretreated electrode was held stationary at the initial potential for a time tl before potential scanning. RESULTS AND DISCUSSION
Rotating-disk voltammetry The 9 OH--E+--2 CH3 shows a single anodic wave over the whole pH range. On repeated potential scanning, without pretreatment of the working electrode, the half-wave potential E l n shifts positively while the limiting current il decreases. The E~n shift and the i~ decrease are enhanced with increasing 9 OH--E÷--2 CH3 bulk concentration c. The E~n shift and the i~ decrease are probably due to the formation of a polymeric film, as already reported during the anodic detection of phenolic compounds [14]. This film would make the mechanical and the electrochemical pretreatment necessary in order to obtain reproducible results, the electrochemical pretreatment alone being inefficient. The production o f a passivating polymeric film can also explain other aspects
142
of the abnormal behavior of hydroxy:ellipticines at the platinum RDE such as the non-linear dependences of il upon c and of il upon fin (f = revolutions per second of the disk electrode) respectively. At fixed c (0.2 mM) and f (10 rps), the shift of E l n with pH is linear over the whole pH range and E l n = 660--56 pH mV while i~ remains constant. With 9 OH--E an identical behavior is observed as long as this compound is protonated in solution, i.e. at pH < 7.5. During the titration of 9 OH--E+--2 H with dilute sodium h y d r o x y d e , a pK of 7.2 was found, for the following acidbase equilibrium: CH 3
CH 3
+
H
CH 3
9 OH - E + - 2
H H
N+
(1)
CH 3
9 OH - E
Above pH 7.5 the anodic wave disappears and the voltammogram does not exhibit any other wave. At the same time the solubility of the hydroxyellipticine decreases sharply as shown b y UV spectrometry, and the disappearance of the wave may result from the decrease in solubility.
Controlled potential electrolysis Electrolysis o f 9 OH--E+--2 H at pH = 4.6 and at 550 mV results in the formation of a black deposit on the working electrode, corroborating the behavior at the RDE and numerous reports concerning the electrochemical oxidation of phenolic c o m p o u n d s [14]. The film o f deposit gradually insulates the electrode causing the current to drop and vanish after a while. The electrode is then removed and the film is scraped away and stored. The final stage of the cleaning and reactivation of the electrode consists in immersing the electrode in concentrated nitric acid which dissolves the remaining deposit. The electrode is then rinsed with distilled water, wiped with a paper tissue and reset in the cell, after which the electrolysis can be pursued. This cleaning procedure is repeated as often as necessary until the current after cleaning falls to background level. When the electrolysis is complete, the analysis of the coulometric data gives a total faradaic n value of 1.0 + 0.1 electron per molecule of hydroxy-ellipticine initially introduced in the solution. The exhaustively electrolyzed solution does not show any anodic wave. The black deposit is insoluble in acetone, dioxane, dimethylformamide and dimethylsulfoxide and it has been impossible to obtain any information about its structure.
Cyclic voltammetry at a stationary electrode On the forward scan, a dilute solution of 9 OH--E+--2 CH3 (c = 0.05 mM) shows an anodic peak (Fig. 1) which has a symmetrical shape when the delay time tl (time elapsed between the immersion of the working electrode and the starting of the potential sweep, the electrode being held at the initial potential during this delay time) is n o t t o o long (tl = 2 s). The peak height ip is roughly
143
B (I1
1 /
760 E/mv
.<
Z / / /
"-
2
,/ i
0.1
c/rnM
().2
Fig. 1. V o l t a m m e t r i c curves f o r 9 OH--E÷--2 CH3 at s t a t i o n a r y e l e c t r o d e c o r r e c t e d for b a c k g r o u n d c u r r e n t ; c = 0.05 m M ; tl = 2 s; p H = 3.25. (A) v -~ 0.2 V s - 1 ; ( B ) v = 0.5 V s -1. Fig. 2. V o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f t h e electric charge Q corres p o n d i n g to t h e a n o d i c peak area w i t h t h e ellipticinium bulk c o n c e n t r a t i o n c; tl = 2 s; pH = 3.25; v = 0.5 V s -1.
proportional to the sweep rate v. This behavior is typical of an adsorptioncontrolled process in which both the reactant and the product of the electrochemical process are strongly adsorbed [15]. (1) Effect of c. With v and tl being held constant, the electric charge Q corresponding to the area of the anodic peak is proportional to c at low c and becomes independent of c when c is sufficiently high (Fig. 2). (2) Effect of tl. Here, Q is proportional to t]/2 at small values of t~ (Fig. 3). Figures 3 and 4 show that the saturated coverage o f the electrode surface corresponds to Q = ca. 0.84 pC. (3) Effect o f v. Besides the approximately linear dependence of the anodic
1 /=
0 Ct
/'
/"
/"
/
/
%n s
2
i
t ~/2/,s,/2
4
_
t
-1
i
Iog(v/Vs") 0
Fig. 3. V o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f the electric charge Q corresponding t o t h e area of the a n o d i c p e a k w i t h d e l a y t i m e Q. S o l u t i o n o f 9 OH--E÷--2 CH3: c = 0.05 m M ; p H = 3 . 2 5 ; v = 0.5 V s -1. Fig. 4. Cyclic v o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f the peak c u r r e n t ratio ipc/ipa w i t h scan rate v in V s -1. The 9 OH--E÷--2 CH3 c o n c e n t r a t i o n c = 0.05 m M ; p H = 4.6; t l = 2 s. The c o n t i n u o u s curve is c o m p u t e d using t h e w o r k i n g curve given in ref. 18 for k = 2.6 × 1 0 9 t o o l - l c m 2 s -1.
144
peak current ipa u p o n v, it appears that the anodic peak potential Epa remains practically constant over the explored range of v, and that the peak width at mid-height ~ slightly decreases below 90 mV with decreasing v. On the backward scan a cathodic peak exhibiting a symmetrical shape is observed (Fig. 1). Its peak potential Epc is close to Epa and its height/pc compared to ipa shows a great dependence on v (Fig. 4). The ratio ipc/ipa slightly varies when the switching potential is modified'; for example ipc/ipa increases from 0.6 to 0.8 when the switching potential is shifted from 710 to 560 mV, v being equal to 0.5 V s -1, the other experimental conditions being c = 0.05 mM, pH = 4.6, tl = 2 s. (4) Effect of pH. With 9 OH--E÷--2 CH3 the only effect of pH concerns the peak potential Epa or Epc. The peak potential shifts linearly with pH and Ep = (650--53) mV. The peak characteristics do not change with pH. The behavior o f solutions of 9 0 H - - E is identical to the behavior of solutions of 9 OH--E + 2 CH3 as long as 9 OH--E is protonated in solution yielding 9 OH--E÷--2 H, i.e. as long as the pH is <7. No peak appears on the voltammogram above pH 7, a result similar to that observed with a RDE.
Reaction pathway Comparing the experimental results obtained b y means of cyclic voltammetry to the diagnostic criteria given in the literature concerning the elucidation of the mechanism of chemical surface reactions using linear sweep voltammetry [16--18], it appears that a DIM I--1 reaction scheme, i.e.:
H
O
t.
~
.
~
~
CH3 %
+/CH 3
O~
~
CH3 JL ~
~
+ CH3
rl
(If)
('Q O'-E+- 2 CH3oF H)ocls'
( 9 0 F I - E + - 2 CH3OP H)ods,
2(90"--E+--2CH3 or H)ads-
k
>
(dimer)ads
(III)
i~eversible
can account for the observed electrochemical behavior of 9 OH--E+--2 CH3 (or H) at a platinum electrode. As predicted for the occurrence of a DIMI--1 process [16,17], the peak width at mid-height 8 is 90 mV for sufficiently high values of v, or less for lower values of v; the ratio ipc/ipa markedly changes with v and slightly depends on the switching potential. Coulometric data obtained b y means of controlled potential electrolysis confirm the occurrence of a one-electron process. However, the production of a black polymeric film, also observed during RDE experiments, shows that dimerization is only a first step in the oxidative coupling of 9-hydroxy-ellipticinium cations. The structure of the dimer which is likely to be produced b y reaction (III) has n o t been determined. In addition, 9 OH--E+--2 CH3 (or H) and 9 O'--E+--2 CH3 (or H) can exist in resonant forms.
145 Electrode coverage
Using the value of 0.84 pC (Figs. 2 and 3) found for the electric charge Q corresponding to the area of the anodic peak in the case of a saturated coverage of the electrode surface, it is possible to calculate t h e corresponding superficial concentration of adsorbed ellipticinium cations Fo (m) = Q / F A = 2 . 8 × 1 0 - 1o mol cm -2 = 17 × 1013 molecules cm -2. Assuming t h a t adsorbed ellipticinium molecules form a monolayer, the area occupied by one molecule is ca. 6 0 / ~ . As 9 OH--E+--2 CH3, based on its crystal structure [19], has an area of 100/~2 when in the planar conformation, and an area of 50/~2 in the perpendicular conformation, the largest dimension o f the molecule being parallel to the electrode surface, or an area of 25/~2 in the perpendicular conformation, the largest dimension of the molecule being perpendicular to the electrode surface, it is fair to conclude that ellipticinium cations form a monolayer of molecules perpendicular to the electrode surface. Thermodynamic
redox potential and dimerization rate constant
In the present work, Epa does n o t shift appreciably in the narrow range of potential scan rate explored, a result which is normal since a cathodic peak always appears in this range of v [16]. Then Epa and Epc are roughly equal to the standard apparent surface redox potential E °' at the corresponding pH for the redox couple 9 OH--E+--2 CH3 (or H)/9 O'--E*--2 CH3 (or H). When the ratio ipe/ipa tends towards unity, Epa = Epc = E °' [16]. Therefore, we can conclude t h a t E °' = (650 -- 53 pH) mV, i.e. E °' = (900 -- 53 pH) mV vs. NHE for the one-electron oxidation of 9-hydroxy-ellipticinium cations. The peak current ratio ipi/ipa vs. l o g [ F v / R T k F o ( t ,)] working curve given in the literature [18] enables the determination of k and computation o f the peak current ratio vs. log v curve as drawn in Fig. 4, for which the theoretical curve fits well the experimental plot. For the surface dimerization of 9 O'--E*--2 CH3 (or H), k was found to be (2.5 + 1) × 109 mo1-1 cm 2 s -1 (mean and standard deviation for five experiments). When v was sufficiently high to o u t r u n the dimerization (v > 1 V s -1) no second peak corresponding to a further oxidation of the radical 9 O'--E÷--2 CH3 (or H) was observed. The pH dependence of E °' and the pH independence of k show that the deprotonation accompanying the electron transfer (reaction II) is fast. ACKNOWLEDGEMENT
The authors t h a n k the Association de la Recherche sur le Cancer (Villejuif) which helped support this work. REFERENCES 1 L . K . D a l t o n , S. D e m e r a c , B.C. E l m e s , J.W. L o d e r , J . M . S w a n . a n d T. Teitei, Aust. J. C h e m . , 2 0 ( 1 9 6 7 ) 2715. 2 J . B . L e P e c q , N. D a t X u o n g , C. G o s s e a n d C. P a o l e t t i , P r o c . N a t l . A c a d . Sci. U . S . A . , 7 1 ( 1 9 7 4 ) 5 0 7 8 . 3 G. M a t h e , M. H a y a t , F. De Vassal, L. S c h w a r z e n b e r g , M. S c h n e i d e r , J . R . S c h l u m b e r g e r , C. Jasmin and C. R o s e n f e l d , R e v . E u r . E t u d . Clin. Biol., 1 5 ( 1 9 7 0 ) 5 4 1 .
146
4 P. J u r e t , A. T a n g u y , A. G i r a r d , J . y . L e T a l a e r , J.S. A b b a t u c c i , N. D a t X u o n g , J . B . Le P e c q a n d C. P a o l e t t i , Eux. J. C a n c e r , 1 4 ( 1 9 7 8 ) 2 0 5 . 5 J . B . L e P e c q , C. G o s s e , N. D a t X u o n g a n d C. P a o l e t t i , C . R . A c a d . Sci. (Paris), S~r. D, 2 8 1 ( 1 9 7 5 ) 1365. 6 C. P a o l e t t i , J.B. Le P e c q , N. D a t X u o n g , P. L e s c a a n d P. L e c o i n t e , C u r . C h e m o t h e r , ( 1 9 7 8 ) 1 1 9 5 . 7 B. F e s t y , J . P o i s s o n a n d C. P a o l e t t i , F . E . B . S . L e t t . , 1 7 ( 1 9 7 1 ) 3 2 1 . 8 C. P a o l e t t i , P. L e c o i n t e , P. L e s c a , S. C r o s , D. M a n s u y a n d N. D a t X u o n g , B i o c h i m i e , 6 0 ( 1 9 7 8 ) 1 0 0 3 . 9 P. L e s c a , P. L e c o i n t e , C. P a o l e t t i a n d D. M a n s u y , B i o c h i m i e , 6 0 ( 1 9 7 8 ) 1 0 1 1 . 1 0 P. Lesca, E. R a f i d i n a x i v o , P. L e c o i n t e a n d D. M a n s u y , C h e m . Biol. I n t e r a c t i o n s , 2 4 ( 1 9 7 9 ) 1 8 9 . 1 1 C. A u c l a i r , A. G o u y e t t e a n d C. P a o l e t t i , u n p u b l i s h e d r e s u l t s . 1 2 J.W. L o w n , H . H . C h e n , S.K. Sire a n d J . A . P l a m b e c k , B i o o r g . C h e m . , 8 ( 1 9 7 9 ) 1 7 . 1 3 M. y o n S t a c k e l b e r g , M. P i l g r a m a n d V. T o o m e , Z. E l e k t r o c h e m . , 5 7 ( 1 9 5 3 ) 3 4 2 . 1 4 R . C . K o i l e a n d D . C . J o h n s o n , A n a l . C h e m . , 51 ( 1 9 7 9 ) 7 4 1 , a n d r e f e r e n c e q u o t e d t h e r e i n . 1 5 E. Lavi~on, Bull. S o c . C h i m . F r . , ( 1 9 6 7 ) 3 7 1 7 . 1 6 E. L a v i r o n , E l e c t r o c h i m . A c t a , 16 ( 1 9 7 1 ) 4 0 9 . 1 7 E. Lavixon, C h e m . I n g . T e c h n o l . , 4 4 ( 1 9 7 2 ) 1 8 3 . 1 8 E. L a v i r o n , J. E l e c t r o a n a l . C h e m . , 3 4 ( 1 9 7 2 ) 4 6 3 . 1 9 C. C o u r s e i l l e , Th~se d e D o c t o r a t d ' E t a t , Universit~ d e B o r d e a u x I, T a l e n c e , 1 9 7 7 . 2 0 J. Koryt~i, C o l l e c t . C z e c h . C h e m . C o m m u n . , 1 8 ( 1 9 5 3 ) 2 0 6 .
139
© Elsevier S e q u o i a S.A., L a u s a n n e - - P r i n t e d in T h e N e t h e r l a n d s
ELECTROCHEMICAL BEHAVIOR OF ELLIPTICINE DERIVATIVES PART I. OXIDATION OF 9-HYDROXY-ELLIPTICINE
J. M O I R O U X
Laboratoire de Chimie Analytique (Prof. M.B. Fleury *), Universitd Rend Descartes (Paris V), Facultd des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris Cedex 05 (France) A.M. A R M B R U S T E R
Laboratoire de Biochimie et Enzymologie, L.A. 147 du C.N.R.S., Groupe 140 I.N.S.E.R.M., Institut Gustave Roussy, 94800 Villejuif (France) ( R e c e i v e d 7 t h M a r c h 1 9 8 0 ; in revised f o r m 2 9 t h April 1 9 8 0 )
ABSTRACT T h e e l e c t r o c h e m i c a l o n e - e l e c t r o n o x i d a t i o n o f 9 - h y d r o x y - e l l i p t i c i n i u m c a t i o n s at a platin u m e l e c t r o d e has b e e n e x a m i n e d w i t h p a r t i c u l a r a t t e n t i o n t o t h e t h e r m o d y n a m i c r e d o x pot e n t i a l a n d t o t h e d i m e r i z a t i o n r a t e o f t h e radical species p r o d u c e d . B o t h t h e r e a c t a n t a n d t h e p r o d u c t of t h e e l e c t r o c h e m i c a l r e a c t i o n are s t r o n g l y a d s o r b e d a t t h e s o l u t i o n / e l e c t r o d e interface. T h e initial step o f t h e a n o d i c process is a reversible e l e c t r o n t r a n s f e r a c c o m p a n i e d b y a fast d e p r o t o n a t i o n ; E °' = 9 0 0 - - 53 p H m V vs. NHE. T h e r e s u l t i n g n e u t r a l radical dimerizes, t h e r a t e c o n s t a n t o f t h e surface d i m e r i z a t i o n b e i n g ca. 2.5 X 109 mo1-1 c m 2 s -1.
INTRODUCTION
Ellipticine (E) 5,11
* T o w h o m all c o r r e s p o n d e n c e s h o u l d b e addressed.
140
[ El
E - DNA~
- - +
DNA
g OH E-DNA
~
in vivo conversion
Cyt, P450 ~ 90H-E~... Peroxidase -llke rnonooxygenases (~-~.. ~ . peroxides .)-" 9 O-E or oxygen - -
+ DNA
~ DNA breaks or covalent binding (?)
of dimerization of the radical 9"O--E when adsorbed at the electrode surface. Experiments were made with 9 OH--E and with 2-methyl, 9-hydroxy-ellipticinium (9 OH--E+--2 CH3) which is presently under clinical trial [4]. CH3 H
O
~
N 9
N
H
O~ " ' ~ ' ~ ~N~+
CH3
OH- E
H 9
CH3 T C
H
3
CH3
OH-E + - 2 CH3
These compounds give an anodic voltammetric signal at potentials too positive to allow the use of mercury electrodes, and electrodes o f such solid materials as carbon or platinum must be used. Both the reactant and the product of the electrochemical reaction adsorb strongly at the electrode surface. As a part of a systematic study of the redox characteristics of the ellipticine derivatives, the electrochemical oxidation of 9 0 H - - E and 9 OH--E÷--2 CH3 were examined in buffer aqueous media at various pH in the range 2--12. The excitation and measurement techniques used included single- and multiplesweep voltammetry at stationary and rotated disk electrodes. EXPERIMENTAL
Materials The 9 OH--E and 9 OH--E+--2 CH3 acetate were generously provided by SANOFI, and were used w i t h o u t further purification. Reagent-grade chemicals were obtained from Prolabo. The buffered background solutions used were: 0.5 M acetate buffer for the 3--6 pH range; 0.1 M phosphate buffer for the 6--12 pH range.
Electrodes The working electrode was a platinum ferrule for Tacussel EDI electrodes. The effective electrode area, determined by measurement of the anodic ferrocyanide cyclic voltammetric peak (n = 1, D = 6.32 X 10 -6 cm 2 s -1) [13] was A = 0.031 cm 2. The reference electrode was a mercury/calomel/saturated KC1 Tacussel C 10 electrode to which all potentials cited are referred unless otherwise specified. The counter electrode was a Tacussel Pt 33/88 electrode.
141
Apparatus The cell was a Tacussel CPRA water-jacketed cell whose temperature, unless otherwise specified, was 25 ° C. Voltammetric measurements using rotating~lisk working electrodes (Tacussel EDI electrode plus a CONTROVIT servocontrol electronic amplifier) were made with a Tacussel EPLI recorder and a Tacussel TIPOL polarograph plug-in (potential scan rate v = 2 mV s-l). For cyclic voltammetry at stationary working electrodes, a Tacussel PRT 30-01 rapid response potentiostat with a Tacussel TP-PRT plug-in generating a time-dependent pilot signal and a Tacussel ADTP differential amplifier were used. Current--potential curves were recorded either with a Tektronix R 564B cathode ray oscilloscope with suitable modules and camera or with a Sefram TGM 164 X--Y recorder with suitable modules. Controlled potential electrolysis were carried o u t using a three-compartment water-jacketed cell; a Tacussel PRT 200-IX potentiostat, a Tacussel IG5-N electronic integrator and a general-purpose milliammeter were included in the circuit. The working electrode was a large platinum disk (diameter: 8 cm).
Electrode pretreatment In the present study, the working electrode was pretreated before each voltammogram unless otherwise specified. The electrode pretreatment consisted of sanding the electrode vigorously with the Tacussel BA 03 abrasive and of applying to the rotated disk electrode (RDE) a negative potential corresponding to a cathodic current of 6 pA in the background solution for 2 min and then a positive potential corresponding to an anodic current of 6 pA, and repeating the cycle twice. At the end of the pretreatment the working electrode was removed and immersed in the sample solution. For RDE voltammetric experiments the electrode was held at the initial potential until the current reached a steady state before recording the current--potential curve. For measurements using a stationary electrode, the pretreated electrode was held stationary at the initial potential for a time tl before potential scanning. RESULTS AND DISCUSSION
Rotating-disk voltammetry The 9 OH--E+--2 CH3 shows a single anodic wave over the whole pH range. On repeated potential scanning, without pretreatment of the working electrode, the half-wave potential E l n shifts positively while the limiting current il decreases. The E~n shift and the i~ decrease are enhanced with increasing 9 OH--E÷--2 CH3 bulk concentration c. The E~n shift and the i~ decrease are probably due to the formation of a polymeric film, as already reported during the anodic detection of phenolic compounds [14]. This film would make the mechanical and the electrochemical pretreatment necessary in order to obtain reproducible results, the electrochemical pretreatment alone being inefficient. The production o f a passivating polymeric film can also explain other aspects
142
of the abnormal behavior of hydroxy:ellipticines at the platinum RDE such as the non-linear dependences of il upon c and of il upon fin (f = revolutions per second of the disk electrode) respectively. At fixed c (0.2 mM) and f (10 rps), the shift of E l n with pH is linear over the whole pH range and E l n = 660--56 pH mV while i~ remains constant. With 9 OH--E an identical behavior is observed as long as this compound is protonated in solution, i.e. at pH < 7.5. During the titration of 9 OH--E+--2 H with dilute sodium h y d r o x y d e , a pK of 7.2 was found, for the following acidbase equilibrium: CH 3
CH 3
+
H
CH 3
9 OH - E + - 2
H H
N+
(1)
CH 3
9 OH - E
Above pH 7.5 the anodic wave disappears and the voltammogram does not exhibit any other wave. At the same time the solubility of the hydroxyellipticine decreases sharply as shown b y UV spectrometry, and the disappearance of the wave may result from the decrease in solubility.
Controlled potential electrolysis Electrolysis o f 9 OH--E+--2 H at pH = 4.6 and at 550 mV results in the formation of a black deposit on the working electrode, corroborating the behavior at the RDE and numerous reports concerning the electrochemical oxidation of phenolic c o m p o u n d s [14]. The film o f deposit gradually insulates the electrode causing the current to drop and vanish after a while. The electrode is then removed and the film is scraped away and stored. The final stage of the cleaning and reactivation of the electrode consists in immersing the electrode in concentrated nitric acid which dissolves the remaining deposit. The electrode is then rinsed with distilled water, wiped with a paper tissue and reset in the cell, after which the electrolysis can be pursued. This cleaning procedure is repeated as often as necessary until the current after cleaning falls to background level. When the electrolysis is complete, the analysis of the coulometric data gives a total faradaic n value of 1.0 + 0.1 electron per molecule of hydroxy-ellipticine initially introduced in the solution. The exhaustively electrolyzed solution does not show any anodic wave. The black deposit is insoluble in acetone, dioxane, dimethylformamide and dimethylsulfoxide and it has been impossible to obtain any information about its structure.
Cyclic voltammetry at a stationary electrode On the forward scan, a dilute solution of 9 OH--E+--2 CH3 (c = 0.05 mM) shows an anodic peak (Fig. 1) which has a symmetrical shape when the delay time tl (time elapsed between the immersion of the working electrode and the starting of the potential sweep, the electrode being held at the initial potential during this delay time) is n o t t o o long (tl = 2 s). The peak height ip is roughly
143
B (I1
1 /
760 E/mv
.<
Z / / /
"-
2
,/ i
0.1
c/rnM
().2
Fig. 1. V o l t a m m e t r i c curves f o r 9 OH--E÷--2 CH3 at s t a t i o n a r y e l e c t r o d e c o r r e c t e d for b a c k g r o u n d c u r r e n t ; c = 0.05 m M ; tl = 2 s; p H = 3.25. (A) v -~ 0.2 V s - 1 ; ( B ) v = 0.5 V s -1. Fig. 2. V o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f t h e electric charge Q corres p o n d i n g to t h e a n o d i c peak area w i t h t h e ellipticinium bulk c o n c e n t r a t i o n c; tl = 2 s; pH = 3.25; v = 0.5 V s -1.
proportional to the sweep rate v. This behavior is typical of an adsorptioncontrolled process in which both the reactant and the product of the electrochemical process are strongly adsorbed [15]. (1) Effect of c. With v and tl being held constant, the electric charge Q corresponding to the area of the anodic peak is proportional to c at low c and becomes independent of c when c is sufficiently high (Fig. 2). (2) Effect of tl. Here, Q is proportional to t]/2 at small values of t~ (Fig. 3). Figures 3 and 4 show that the saturated coverage o f the electrode surface corresponds to Q = ca. 0.84 pC. (3) Effect o f v. Besides the approximately linear dependence of the anodic
1 /=
0 Ct
/'
/"
/"
/
/
%n s
2
i
t ~/2/,s,/2
4
_
t
-1
i
Iog(v/Vs") 0
Fig. 3. V o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f the electric charge Q corresponding t o t h e area of the a n o d i c p e a k w i t h d e l a y t i m e Q. S o l u t i o n o f 9 OH--E÷--2 CH3: c = 0.05 m M ; p H = 3 . 2 5 ; v = 0.5 V s -1. Fig. 4. Cyclic v o l t a m m e t r y at a s t a t i o n a r y e l e c t r o d e . Variation o f the peak c u r r e n t ratio ipc/ipa w i t h scan rate v in V s -1. The 9 OH--E÷--2 CH3 c o n c e n t r a t i o n c = 0.05 m M ; p H = 4.6; t l = 2 s. The c o n t i n u o u s curve is c o m p u t e d using t h e w o r k i n g curve given in ref. 18 for k = 2.6 × 1 0 9 t o o l - l c m 2 s -1.
144
peak current ipa u p o n v, it appears that the anodic peak potential Epa remains practically constant over the explored range of v, and that the peak width at mid-height ~ slightly decreases below 90 mV with decreasing v. On the backward scan a cathodic peak exhibiting a symmetrical shape is observed (Fig. 1). Its peak potential Epc is close to Epa and its height/pc compared to ipa shows a great dependence on v (Fig. 4). The ratio ipc/ipa slightly varies when the switching potential is modified'; for example ipc/ipa increases from 0.6 to 0.8 when the switching potential is shifted from 710 to 560 mV, v being equal to 0.5 V s -1, the other experimental conditions being c = 0.05 mM, pH = 4.6, tl = 2 s. (4) Effect of pH. With 9 OH--E÷--2 CH3 the only effect of pH concerns the peak potential Epa or Epc. The peak potential shifts linearly with pH and Ep = (650--53) mV. The peak characteristics do not change with pH. The behavior o f solutions of 9 0 H - - E is identical to the behavior of solutions of 9 OH--E + 2 CH3 as long as 9 OH--E is protonated in solution yielding 9 OH--E÷--2 H, i.e. as long as the pH is <7. No peak appears on the voltammogram above pH 7, a result similar to that observed with a RDE.
Reaction pathway Comparing the experimental results obtained b y means of cyclic voltammetry to the diagnostic criteria given in the literature concerning the elucidation of the mechanism of chemical surface reactions using linear sweep voltammetry [16--18], it appears that a DIM I--1 reaction scheme, i.e.:
H
O
t.
~
.
~
~
CH3 %
+/CH 3
O~
~
CH3 JL ~
~
+ CH3
rl
(If)
('Q O'-E+- 2 CH3oF H)ocls'
( 9 0 F I - E + - 2 CH3OP H)ods,
2(90"--E+--2CH3 or H)ads-
k
>
(dimer)ads
(III)
i~eversible
can account for the observed electrochemical behavior of 9 OH--E+--2 CH3 (or H) at a platinum electrode. As predicted for the occurrence of a DIMI--1 process [16,17], the peak width at mid-height 8 is 90 mV for sufficiently high values of v, or less for lower values of v; the ratio ipc/ipa markedly changes with v and slightly depends on the switching potential. Coulometric data obtained b y means of controlled potential electrolysis confirm the occurrence of a one-electron process. However, the production of a black polymeric film, also observed during RDE experiments, shows that dimerization is only a first step in the oxidative coupling of 9-hydroxy-ellipticinium cations. The structure of the dimer which is likely to be produced b y reaction (III) has n o t been determined. In addition, 9 OH--E+--2 CH3 (or H) and 9 O'--E+--2 CH3 (or H) can exist in resonant forms.
145 Electrode coverage
Using the value of 0.84 pC (Figs. 2 and 3) found for the electric charge Q corresponding to the area of the anodic peak in the case of a saturated coverage of the electrode surface, it is possible to calculate t h e corresponding superficial concentration of adsorbed ellipticinium cations Fo (m) = Q / F A = 2 . 8 × 1 0 - 1o mol cm -2 = 17 × 1013 molecules cm -2. Assuming t h a t adsorbed ellipticinium molecules form a monolayer, the area occupied by one molecule is ca. 6 0 / ~ . As 9 OH--E+--2 CH3, based on its crystal structure [19], has an area of 100/~2 when in the planar conformation, and an area of 50/~2 in the perpendicular conformation, the largest dimension o f the molecule being parallel to the electrode surface, or an area of 25/~2 in the perpendicular conformation, the largest dimension of the molecule being perpendicular to the electrode surface, it is fair to conclude that ellipticinium cations form a monolayer of molecules perpendicular to the electrode surface. Thermodynamic
redox potential and dimerization rate constant
In the present work, Epa does n o t shift appreciably in the narrow range of potential scan rate explored, a result which is normal since a cathodic peak always appears in this range of v [16]. Then Epa and Epc are roughly equal to the standard apparent surface redox potential E °' at the corresponding pH for the redox couple 9 OH--E+--2 CH3 (or H)/9 O'--E*--2 CH3 (or H). When the ratio ipe/ipa tends towards unity, Epa = Epc = E °' [16]. Therefore, we can conclude t h a t E °' = (650 -- 53 pH) mV, i.e. E °' = (900 -- 53 pH) mV vs. NHE for the one-electron oxidation of 9-hydroxy-ellipticinium cations. The peak current ratio ipi/ipa vs. l o g [ F v / R T k F o ( t ,)] working curve given in the literature [18] enables the determination of k and computation o f the peak current ratio vs. log v curve as drawn in Fig. 4, for which the theoretical curve fits well the experimental plot. For the surface dimerization of 9 O'--E*--2 CH3 (or H), k was found to be (2.5 + 1) × 109 mo1-1 cm 2 s -1 (mean and standard deviation for five experiments). When v was sufficiently high to o u t r u n the dimerization (v > 1 V s -1) no second peak corresponding to a further oxidation of the radical 9 O'--E÷--2 CH3 (or H) was observed. The pH dependence of E °' and the pH independence of k show that the deprotonation accompanying the electron transfer (reaction II) is fast. ACKNOWLEDGEMENT
The authors t h a n k the Association de la Recherche sur le Cancer (Villejuif) which helped support this work. REFERENCES 1 L . K . D a l t o n , S. D e m e r a c , B.C. E l m e s , J.W. L o d e r , J . M . S w a n . a n d T. Teitei, Aust. J. C h e m . , 2 0 ( 1 9 6 7 ) 2715. 2 J . B . L e P e c q , N. D a t X u o n g , C. G o s s e a n d C. P a o l e t t i , P r o c . N a t l . A c a d . Sci. U . S . A . , 7 1 ( 1 9 7 4 ) 5 0 7 8 . 3 G. M a t h e , M. H a y a t , F. De Vassal, L. S c h w a r z e n b e r g , M. S c h n e i d e r , J . R . S c h l u m b e r g e r , C. Jasmin and C. R o s e n f e l d , R e v . E u r . E t u d . Clin. Biol., 1 5 ( 1 9 7 0 ) 5 4 1 .
146
4 P. J u r e t , A. T a n g u y , A. G i r a r d , J . y . L e T a l a e r , J.S. A b b a t u c c i , N. D a t X u o n g , J . B . Le P e c q a n d C. P a o l e t t i , Eux. J. C a n c e r , 1 4 ( 1 9 7 8 ) 2 0 5 . 5 J . B . L e P e c q , C. G o s s e , N. D a t X u o n g a n d C. P a o l e t t i , C . R . A c a d . Sci. (Paris), S~r. D, 2 8 1 ( 1 9 7 5 ) 1365. 6 C. P a o l e t t i , J.B. Le P e c q , N. D a t X u o n g , P. L e s c a a n d P. L e c o i n t e , C u r . C h e m o t h e r , ( 1 9 7 8 ) 1 1 9 5 . 7 B. F e s t y , J . P o i s s o n a n d C. P a o l e t t i , F . E . B . S . L e t t . , 1 7 ( 1 9 7 1 ) 3 2 1 . 8 C. P a o l e t t i , P. L e c o i n t e , P. L e s c a , S. C r o s , D. M a n s u y a n d N. D a t X u o n g , B i o c h i m i e , 6 0 ( 1 9 7 8 ) 1 0 0 3 . 9 P. L e s c a , P. L e c o i n t e , C. P a o l e t t i a n d D. M a n s u y , B i o c h i m i e , 6 0 ( 1 9 7 8 ) 1 0 1 1 . 1 0 P. Lesca, E. R a f i d i n a x i v o , P. L e c o i n t e a n d D. M a n s u y , C h e m . Biol. I n t e r a c t i o n s , 2 4 ( 1 9 7 9 ) 1 8 9 . 1 1 C. A u c l a i r , A. G o u y e t t e a n d C. P a o l e t t i , u n p u b l i s h e d r e s u l t s . 1 2 J.W. L o w n , H . H . C h e n , S.K. Sire a n d J . A . P l a m b e c k , B i o o r g . C h e m . , 8 ( 1 9 7 9 ) 1 7 . 1 3 M. y o n S t a c k e l b e r g , M. P i l g r a m a n d V. T o o m e , Z. E l e k t r o c h e m . , 5 7 ( 1 9 5 3 ) 3 4 2 . 1 4 R . C . K o i l e a n d D . C . J o h n s o n , A n a l . C h e m . , 51 ( 1 9 7 9 ) 7 4 1 , a n d r e f e r e n c e q u o t e d t h e r e i n . 1 5 E. Lavi~on, Bull. S o c . C h i m . F r . , ( 1 9 6 7 ) 3 7 1 7 . 1 6 E. L a v i r o n , E l e c t r o c h i m . A c t a , 16 ( 1 9 7 1 ) 4 0 9 . 1 7 E. Lavixon, C h e m . I n g . T e c h n o l . , 4 4 ( 1 9 7 2 ) 1 8 3 . 1 8 E. L a v i r o n , J. E l e c t r o a n a l . C h e m . , 3 4 ( 1 9 7 2 ) 4 6 3 . 1 9 C. C o u r s e i l l e , Th~se d e D o c t o r a t d ' E t a t , Universit~ d e B o r d e a u x I, T a l e n c e , 1 9 7 7 . 2 0 J. Koryt~i, C o l l e c t . C z e c h . C h e m . C o m m u n . , 1 8 ( 1 9 5 3 ) 2 0 6 .