Polarographic investigations of 7-methylguanosine

J. Electroanal. Chem., 63 (1975) 207-219 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

207

POLAROGRAPHIC INVESTIGATIONS OF 7-METHYLGUANOSINE

J. M. SEQUARIS and J. A. REYNAUD*

Centre de Biophysique MolOculaire, 45045 OrlOans Cedex (France) (Received 4th February 1975; in revised form 2nd April 1975)

ABSTRACT

The mechanism of reduction of 7-methylguanosine (7-MeGuo) in buffered solution was studied by electrochemical methods. The polarographic reduction involves complex processes due to the presence of maxima (maximum of the first kind and maximum of catalytic reduction of protons). Consequently other techniques are required such as u.v. and n.m.r, spectroscopies, and chromatography for the identification of the reduction product. A mechanism of reduction is suggested in acid medium, the reduction saturates the imidazolium ring by fixation of two electrons and one proton. INTRODUCTION

The alkylation of guanosine has been the subject of many biological and chemical studies in recent years, and has been reviewed by Lawley 1. Natural processes of alkylation may occur such as the methylation of 7-methylguanosine which occurs in different tRNAs 2. We have previously reported the polarographic behaviour of methylated deoxyribonucleic acid (DNA-CH3) in N7 of guanine 3'4. To elucidate this polarographic signal, it was necessary to study the electrochemical properties of 7-methyl deoxyguanosine, or, as we did hereafter, of the 7-methylguanosine since the glycosidic moiety of the 7-methyldeoxyguanosine is not directly involved in the mechanism of reduction (Fig. 1 lc). MATERIALS AND METHODS

(I) Chemicals 7-Methylguanosine (7-MeGuo) was purchased from Sigma Chemical Co. The buffer solutions were prepared from analytical reagent-grade Merck. Argon U was bubbled through the solutions tO eliminate oxygen. We used doubly distilled water for preparing the solutions. * To whom correspondence should be sent.

208

J. M. SEQUARIS, J. A. R E Y N A U D

( II ) Polarography The polarographic curves were recorded on a Tacussel polarograph of PRG 3 type, using three dropping mercury electrodes having the following characteristics in open circuit in NaC1 molar solution. (1) m = l . 9 mg s -1 with a drop time t=2.84 s (2) m=0.4 mg s -1 used with a drop time t = 3 s (3) m=0.3 mg s -1 with a drop time t=8.5 s. The mercury column was 50 cm high, and the temperature of the polarographic cell was kept constant at 25~C. The potential was measured L,ersus a calomel electrode saturated in KCI. All pH measurements were performed with a Radiometer pH meter. The current-time (I-t) curves were measured at constant potentials with a horizontal capillary (m=0.16 mg s-1, t = 18 s, h = 50 cm in NaC1 molar solution). The curves were recorded via an amplifier on tektronic oscilloscope.

(IlI ) Electrolysis at controlled potential We used for these electrolyses a cell of 20 ml volume whose anodic compartment is separated from the cathodic compartment by a gelose bridge saturated with KC1. The control of the electrolysis potential was provided by a Tacussel potentiostat, and we used an electronic coulometric integrator for measuring the quantity of electricity passing through the electrolysis cell. The cathode is a mercury pool of 7 cm 2 area whose surface was constantly renewed by a spinning magnetic stirrer. There was a constant flow of argon U through the cell, throughout the whole experiment. The various electrolysis potentials were determined from the polarographic curves by selecting the potential corresponding to half of the maximum polarographic current. These potential values are respectively: at p H = 3 , - 1 . 3 8 V; at p H = 5 . 4 - 1 . 6 0 V; at p H = 8 , - 1 . 7 0 V.

(I) Concentration of the reagents. Coulometry: concentration in 7-MeGuo: 5 X 10 -4. M ; concentration of the phosphate, acetate and Tris buffer: 10 2 M ; concentration of the NaC1 electrolyte support: 1 M. (2) Preparation of the reduction product. The complete reduction of the 7-MeGuo at the various pH values was controlled by u.v. spectrophotometry. Concerning the n.m.r, spectroscopy and the chromatography performed on a thin layer of silica gel, the solutions, previously frozen in liquid nitrogen, were concentrated by lyophilization. The possibility of reoxidation of the reduction product was limited by using special apparatus fitted with a tap. This proceeding makes it possible to handle the reduction product safe from any contact with the air. Vacuum can be broken later on by introducing inert gases such as argon U. ( I V ) U.v. spectroscopy U.v. spectra were obtained with a Cary 14 spectrophotometer.

( V ) N.m.r. spectroscopy The spectra were recorded on a HFX 90 Mz Bruker instrument. Water was twice sublimed off the reduction products by lyophilisation performed in a D 2 0 solution or in dimethylsulfoxide (DMSO), Chemical shifts are reported from an external HMS (hexamethylsilane) capillary.

POLAROGRAPHY OF 7-METHYLGUANOSINE

209

(VI) Chromatography We used ascending chromatography with a silica gel system which employed chloroform containing solvent as described by Drach and Novack 5. Prescored 20 x 20 plates of silica gel were obtained from Kodak (Eastman Chromatogram sheet). The chloroform containing solvent was prepared by mixing chloroform methanol and 3~o of ammonia and adding 3 parts of absolute methanol to 17 parts of the lower phase. RESULTS

(I) Appearance of the polarographic curves A sharp peak and a round-shaped maxima can be noticed in a highly acid medium, as displayed on the polarographic curves of Fig. 1. Variations in their relative heights along with their shifting to higher negative potentials depend on the range of pH values. When the pH value increases above 8, the sharp peak disappears. It is worth considering that if we change the buffer component or the concentration of the buffer, the current varies markedly for the same pH value. This variation as much as the hydrogen discharge at the dropping mercury electrode are typical of a catalytic hydrogen wave. Besides, this gas discharge disturbs the current voltage curve in acid medium. Evidence for reduction of the 7-MeGuo is easily obtained in u.v. spectroscopy by the variation of the optical density of a previously electrolysed solution.

'/~ 20

1.4 1.5 1.6 1.7 _E / V Fig. 1. D.c. polarograms of 7-MeGuo at various pH. 7-MeGuo 10 -3 M, NaC1 1 M, acetate buffer 2 x 10 -2 M (capillary 1, h = 4 0 cm). (1) pH 3.41, (2) pH 4.36, (3) pH 5.30.

~A 15

10

5

4

7

/ 14

15

16

1-7

-E/V

1£}

Fig. 2. D.c. polarograms of 7-MeGuo at various pH in the presence of gelatin (0.005~o). 7-MeGuo

10 3 M, NaCI 1 M, phosphate and acetate buffers 2 x 10 2 m (capillary 2, h = 50 cm). (1) pH 3, (2) pH 4, (3) pH 4.5, (4) pH 5, (5) pH 5.5, (6) pH 7, (7) pH 8.

210

J. M. SEQUARIS, J. A. R E Y N A U D

( I I ) Identification of the maxima (1) Sharp peak maximum. On studying first the sharp peak maximum we realize its extreme sensitivity to the presence of surface active substances such as gelatin which removes it completely when the concentration of 0.005)o is reached, this is shown on Fig. 2.

7vA 4

2 2

I

I

Fig. 3. Current-time curves of 7-MeOuo at various potentials, 7-MeOuo 5 x 10 -4 M, NaC1 1 M, acetate buffer pH 5. (1) 1.60 V, (2) 1.65 V, (3) 1.70 V. TABLE I S T U D Y O F T H E E X P O N E N T O F T H E l ( t ) C U R V E S AT V A R I O U S P O T E N T I A L S A N D p H F O R 7-MeGuo 7-MeGuo 5 x 10 -4 M in NaCI 1 M, phosphate and acetate buffers 10 2 M. -E/V

n at pH 3

1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75 1.80

1.1 streaming streaming streaming 0.52 0.30 0.27 0.25 0.20

n at pH 5

n at pH 7.5

0.90 1.42 0.88 0.60 streaming streaming streaming 0.30 0.20

1.30 0.84 0.68 0.30 0.21

On the current-time curves of Fig. 3, we can observe that a streaming is accompanying the maximum. This phenomenon is similar to the one described by Heyrovsky and Kuta 6. The exponent of the curves I = k t " is characteristic of the electrochemical processes occurring at the electrode. In Table 1 are displayed several values of n determined in a molar solution of NaC1 in presence of the 7-MeGuo at various potentials and pH values. For any of the pH values, the variation of n versus the potential are identical. At the bottom of the polarographic wave, we find some values ofn above 2/3 and according to Levich et al. 9, a depolarizer adsorption taking

POLAROGRAPHY OF 7-METHYLGUANOSINE

211

place in the region of the drop should account for these abnormally high values, this adsorption would favor the reduction (autocatalysis). Bourdin 1° also noticed such a phenomenon on studying the reduction of the Co 2 ÷ ion. When the potential decreases, n decreases. In the area of the maximum of the first kind, n values approach 2/3, as foreseen by von Stackelberg in his theory. The height of the maximum varies with the concentration of the supporting electrolyte. At constant depolarizer concentration, the maximum increases with increasing concentration up to a limit, then decreases again; whereas there is no sharp peak maximum nor streaming appearing on the polarographic curves of reduction product as can be seen on Fig. 5. All the properties we have determined clearly suggest that this peak is a maximum of the first kind and can be essentially associated with the cation 7methylguanosine, i.e. the acid form of the 7-MeGuo (pK = 7), since it disappears above pH 8*.

(2) The round shaped maximum. The second maximum appears as round shaped in acid medium, but is more or less screened by the maximum of the first kind in neutral or slightly acid medium. It is however possible to differentiate one from the other at pH 7.25, in a medium of low ionic strength. This maximum is characterized as follows: it decreases markedly with increasing concentration of the supporting electrolyte - w h e n the buffer and depolarizer concentrations respectively increase, tending to approach a limiting value, Fig. 4. - evidence for hydrogen gas discharge can be obtained by performing a macroelectrolysis at its potential for an extended period of time. 6

f e ~

2

102 c/mol t" Fig. 4. Variations of the catalytic maximum current at various phosphate buffer concentration. 7-MeGuo 10 3 M, NaC1 1 M, phosphate buffer pH 7.25 (capillary 3, h = 50 cm).

These results are in concordance with the process of catalytic reduction of the proton as described by Mairanovski 8. * Nevertheless, recent investigations carried out by Frumkin e t al. 7 have defined a new maximum, a maximum of the third kind, which appears during the adsorption of some organic compounds. This adsorption at the dropping mercury electrode is followed by the phenomenon of association, as for the 7-MeGuo 4. And the disappearance of the maximum simultaneously with the associated forms of the adsorbed 7-MeGuo in basic medium, might be characteristic also of this type of maximum.

212

J. M. SEQUARIS. J.

A. REYNAUD

( I I I ) Profile qf the polarographic curves of the reduction prodttcts The reduction product was prepared by performing an exhaustive electrolysis in acid medium on a mercury pool acting as cathode. Throughout the whole range of pH values investigated, displayed on Fig. 5, the maximum current varies with the potential and the polarographic curves are round shaped.

~'pA 14 12

10846

a

f

2 1.3

1.5

1.7 - E/V

Fig. 5. D.c. polarograms of the reduction product at ~arious pH. Reduction product of 7-MeGuo 5 x 10 -'~ M, NaC1 1 M, acetate and phosphate buffers 10 2 M (capillary 2, h = 50 cm). (a) pH 3, (b) pH 3.52, (c) p n 4.02, (d) pH 4.52, (e) p n 5.50. (r) p n 6.52, (g) p n 7.50.

/

J 104 c/tool [-~ Fig. 6. Variations of the current of the catalytic maximum at various concentrations of reduction product of 7-MeGuo. NaCI 1 M acetate and phosphate buffer 10 2 ~//, pH 5.55 (capillary 2, h - 50 cm).

On one hand, during the recording time a hydrogen charge can also be detected in the region of the dropping mercury electrode. When plotted against increasing concentrations of reduction product, the currents I,.... draw a curve which again tends towards a limiting value, as we can see in Fig. 6. On the other hand, the maximum of the first kind has vanished from the polarographic curves and

POLAROGRAPHY OF 7-METHYLGUANOSINE

213

the phenomenon of streaming associated with it is missing on the I(t) curves. We again meet the catalytic phenomenon of proton reduction previously observed in the study of 7-MeGuo, but here isolated. Moreover for identical pH values, the polarographic waves of the reduced product start at higher positive potentials than the polarographic curyes of the 7-MeGuo. Thus we can draw the conclusion that, over the range of pH values we investigated, i.e. from pH 3 to pH 9, the reduction of the 7-MeGuo cannot be dissociated from the catalytic reduction of proton and that the reduction product is at the origin of this mechanism.

( I V ) Study of the reduction product We have just seen that the polarographic reduction of 7-MeGuo involves complex processes. In a buffered solution, the catalytic phenomenon and its associated maxima make the mathematical analysis of the polarographic curves so intricate that other techniques are required, such as u.v. and n.m.r, spectroscopies or chromatography, for a further investigation of the reduction product (localization of the bonds involved in the reduction and of the heteroatoms and protons exchanged during this process, etc .... ).

(I ) U.v. spectroscopy 7-MeGuo was reduced at various pH values: pH 3, pH 5.4, pH 8, while the reduction product proceeding from every electrolysis was simultaneously studied through u.v. spectroscopy. (a) pH = 3. In Fig. 7 are displayed the u.v. spectra obtained after the passage of increasing quantities of electricity through the electrolysis cell. We can notice that the adsorption maximum of 7-MeGuo is lowered at OD e

1.0"

a

0.8.

,i02

0.6"

ab

0.4-

250 . . . .

300'

n'miT,

350'

Fig. 7. U.v. spectra of 7-MeGuo in function of the percentage of reduction. 7-MeGuo 5 x 10-4 M in NaCI 1 M, acetate buffer 10 z M at pH 3, (a) 0 mcb, (b) 300 mcb, (c) 900 mcb, (d) 24000 mcb, (e) 5000 mcb.

214

J. M. SEQUARIS, J. A. R E Y N A U D

257 nm while a new peak due to the reduction product appears around 270 nm. The presence of three isosbestic points is characteristic of the coexistence in solution of two compounds, 7-MeGuo and its reduced form (curves a, b, c, d). After a long period of electrolysis (30 min approximately) the disappearance of the isosbestic points implies the appearance in solution of ~tdditional compounds most probably resulting from the slow process of break of the glycosidic linkage 1. The determination of the number n of electrons involved in the charge transfer step results in values which are always much above two (from 3 to 6). These high values are due to the catalytic hydrogen reduction caused by the accumulation of the reduced product in solution. In fact, the hypothetical value of n in the reduction of a carbocation compound is equal to 2. (b) pH = 5.4. In this case, the examination of the electrolysed solution shows that the adsorption maximum of 7-MeGuo lowers at 258 nm while a new peak characteristic of the reduction product appears around 305 nm, Fig. 8. The spectra pass through one isosbestic point (289 nm). We have followed the variations of optical density at 305 nm at the different pH values from pH 3 to pH 8 (scale of pH where the reduction product is stable) and we have determined by u.v. spectroscopy the pK value of the reduction product as 3.7. We can localize the site of protonation as being N 3 by referring to the study of the isocytosine derivatives 12. There must be another pK above pH 9 only detectable through a catalytic hydrogen reduction which cannot be performed because of the extreme instability of the product, as 7-MeGuo, at this pH 13. (c) pH=8. The characteristics of the u.v. spectra obtained after electrolysis carried out at pH 8 differ somewhat Trom those of the u.v. spectra obtained in acid medium. The isosbestic point disappears more rapidly. Electrolysis yields charge number n ranging from 1 to 2. Considering that the catalytic hydrogen reduction a

°Dt 1.O

d

0.8.

0.6.

0.40.2 2 .

.

.

.

.

'

'

.

.

.

.

.

Fig. 8. U.v. spectra of 7-MeGuo in function of the percentage of reduction. 7-MeGuo 5.35 x 10 -4 M in solution NaCI 1 M. Acetate buffer 10 2 M at pH 5.4. (a) 0 mcb, (b) 900 mcb, (c) 5.600 mcb, (d) 800 mcb.

POLAROGRAPHY OF 7-METHYLGUANOSINE

215

takes place, the reduction of 7-MeGuo might either involve the addition of one electron or imply the formation in solution of several reduction products. We have summarized in Table 2 the different u.v. spectroscopic properties of the products obtained by electrolysis of 7-MeGuo. It is worth noticing that the characteristics of the products resulting from the electrolysis performed at pH 8 and pH 5.4, differ when compared at a same pH value. This already lead us to assume the existence of different forms of the reduction product.

(2) N.m.r. spectroscopy The n.m.r, of 7-MeGuo and of its reduction product were recorded in the dimethylsulfoxide (DMSO) and in D20. In the DMSO solvent, we have recorded the chemical shifts of the exchangeable protons. In the D 2 0 solvent we have excluded the hydroxylic protons of the ribose from the recorded spectra by operating a deuterium exchange, so that we can more easily define the peaks in the interesting part of the spectra.

5.65 5.60 5.55

4.65 4.60 ppm

Fig. 9. N.m.r. spectrum of the reduction product of the 7-MeGuo in DMSO. Only the sections of the spectrum involving the protons CH2 andiH] are shown.

(a) Spectra recorded in DMSO. The n.m.r, spectrum of the reduction product resulting from an electrolysis at pH 5 shows the effect of the saturation of the imidazolium ring of 7-MeGuo. The H'I proton doublet is shifted to higher field. After the electrolysis, Fig. 9, a new peak (4.6 ppm) appears whereas the Ha proton disappears. The NH 2 protons group shifts to lower field and this is due to the variation of aromaticity of the six-membered ring, this increase in resonance may be connected with the bathochromic shift 14 of the long-wave band in the electronic spectra and the value of the pK, i.e. pK(N3)= 3.7. The ratio of the integral value of the new peak to that of the HI proton is approximately equal to 2 ( - 1.90), Fig. 9. We can deduce that the reduction brings in an additional proton and that the reduction product obtained after the electrolysis performed at pH 5 then holds two protons at ~, the imidazolium ring being thus saturated. (b) Spectra recorded in D20. In D 2 0 w e observe that the exchange proton/ deuteron of the NH 2 and CH group of the 7-MeGuo is instantaneous. This swift CH exchange is justified by the positive charge located in the five membered ring

J. M. SEQUARIS, J. A. R E Y N A U D

216

,..d

©

m,,

k)

(-,q

~4

© ¢-~ b'Z

i-,q

¢-q

~q

r, k~

~ +~ ~ Yr z ,< k.-,

© [..., k9 ©

Z ©

o +1£

k)

o

oo~_o ~

_

ez

.~ ¢-q

~('-4

+1 ~

+1

z

< o

i

I I i

o

,7¢-q

< < Z

e r~ ,,6 o6 +1 ez +1 [..

©

<

217

POLAROGRAPHY OF 7-METHYLGUANOSINE

which destabilizes the linkage 15. The n.m.r, spectrum of the reduction product obtained after electrolysis at pH 3, shows no variation in the chemical shift of the two protons in C of the dihydropurine form when compared with the spectrum recorded in DMSO, Fig. 10. H~ ox

H~ red

;

Call2

CH~ red 7 I~

I

I

~

4

~

ppm

Fig. 10. N.m.r. spectrum of 7-MeGuo and its product of reduction in D20 (pD 5) The chemical shifts are measured from a hexamethylsilane capillary. TABLE 3 NUCLEAR MAGNETIC RESONANCE SPECTRAL DATA FOR 7-MeGuo All measurements were obtained with HFX 90 MHz Bruker spectrometer with dimethylsulfoxide D20 and H20 as solvent. The chemical shifts of the proton (in ppm) were calculated with reference to an external hexamethylsilane capillary.

Chemical shift

(~)

Solvent DMSO

CH a 1)IH2 CH H~.

4.25 6.60 9.30 6.05

D20

1-120 pH =8

pD 8.1

pD 4.3

4.33

4.42 7.30 9.20

6.22

6.33

TABLE 4 NUCLEAR MAGNETIC RESONANCE SPECTRAL DATA FOR THE REDUCTION PRODUCTS OBTAINED AT DIFFERENT pH OF ELECTROLYSIS

Chemical shift

(~)

CH3 /~H 2 (~H2 Hi

Electrolysis at pH 5 DMSO "

pH 3 D20 pD 5

pH 8

H20 peak 7.30 4.60 5.60

3.10

3.20

4.60 5.75

4.55 6.05

D20 pD 9

In D 2 0 , the C H 3 proton group and the Hi proton are shifted to higher field when we proceed from 7-MeGuo to the reduction product. This effect must again be connected with the saturation of the imidazolium ring of the 7-MeGuo. The ratio of the integral of the C H 2 protons group value to that of the C H 3

218

J.M. SEQUARIS, J. A. REYNAUD

TABLE 5 THIN LAYER CHROMATOGRAPHY ON SILICA GEL PLATES OF 7-MeGuo AND THE REDUCTION PRODUCTS OBTAINED AT DIFFERENT pH OF ELECTROLYSIS

Compound

R~. value

7 MeGuo Electrolysis at pH 3 pH 5 pH 8

0.17 0.25 0.26 0.24

protons is 1.9/3.1. Besides, we can measure at 4.6 ppm a spin coupling constant I-~ 2 Hz which confirms the presence of geminal protons in C 8. These results corroborate the ideas we had after the experiments carried out in D M S O of the structure of the reduction product obtained at pH 5. The electrochemical reduction of the 7-MeGuo in acid medium implies the addition of two electrons and one proton at the carbon 8 of the imidazolium ring. After electrolysis at pH 8, the ratio of the integral value of the ~ proton to that of the CH a proton, yielded by the reduction product is much below 2/3. As this was expected in our previous study with u.v. spectroscopy, there is at this pH of electrolysis a mixture of several forms of the reduction product. We have summarized in Tables 3 and 4 the whole of the characteristics of the n.m.r, spectra.

(3) Chromatography Chromatography of the reduction products was performed on thin layers of silica gel 5. It takes a short time for the chloroform solvent to migrate, 1 h approximately, which minimizes the risks of reoxydation of the migrating products by the air oxygen 16. Whatever may be the electrolysis pH value, all the polarograms show the

&,,

0 Me H\. 1~......-!

HOCH~ 5 0

U

o

Me

o

.co I ,

.,J" ~'t~'f~1N-CH + O~j~-

"~e

o Mel "N~vIN--H

OH 014 Fig. 11. Reactions of 7-MeGuo. (A) 7-Methylguanine in acid medium. (B) 2-Amino-4-ribosylamino5-methylamino-6-pyrimidone in basic medium. (C) Reduction in acid medium.

POLAROGRAPHY OF 7-METHYLGUANOSINE

219

same aspect. The reduction product migrates into a main spot whose boundaries are clearly outlined and whose R v values remain unchanged when the electrolysis pH varies (Table 5). There is, contiguous to this main spot, a large secondary spot of lesser intensity, characterized by its "tail of comet" shape and which must be imputed to the partial reoxidation of the reduction product. In basic medium, the presence of such a large spot has made it impossible for us to determine the exact R v values of the other reduction products of 7-MeGuo which had been evidenced in u.v. and n.m.r, spectroscopies. DISCUSSION

Considering the results that we have just reported, we can suggest a process of reduction of 7-MeGuo, for the acid media. The .reaction which occurs in 7-MeGuo takes place in the region of imidazolium ring, where the double bond C=N comes to saturation after addition of two electrons and one proton as shown in Fig. 11 (c). The reduction product thus obtained acts as a catalyst for the proton reduction and is identical to the product which would be obtained by a chemical reduction 16 (by NaBH4). In basic medium, the reduction of 7-MeGuo still occurs in the region of the imidazolium ring but here yields a mixture of unknown reduction products, as has been shown by u.v. and n.m.r, spectra. ACKNOWLEDGEMENTS

The authors are indebted to Dr. Dimicoli for several comments on n.m.r. spectra and to Mrs. M. C. Sequaris for her skilful technical assistance. REFERENCES P. D. Lawley, Progr. Nucl. Acid Research, 5 (1966) 89. S. Nishimura, Progr. Nucl. Acid Research, 12 (1972) 49. J. Ramstein, J. A. Reynaud and M. Leng, C.R. Acad. Sci., Paris, 273 (1971) 2643. J. M. Sequaris, ThOse, Orl6ans, 1974. J. C. Drach and J. M. Novack, Anal. Biochem., 52 (1973) 633. J. Heyrovsky and J. Kuta, Principles of Polarography, Academic Press, New York, 1966. A. N. Frumkin, N. V. Fedorovich, B. B. Damaskin, V. Steniwae and V. S. Krylov, J. Electroanal. Chem., 50 (1974) 103. 8 S. G. Mairanovskii, J. Electroanal. Chem., 6 (1963) 77. 9 G. Levich, B. I. Kchaikin and E. D. Belokolos, Electrochemistry, Acad. Sci. U.R.S.S., 1 (1965) 1273. 10 M. Bourdin, ThOse, Montpellier, 1967. 11 M. von Stackelberg and R. Doppelfeld, Advances in Polarography , Vol. 1, Pergamon Press, London, 1960, pp. 68 104. 12 A. R. Katritzky, Physical Methods in Heterocyclic Chemistry, Vol. I, Academic Press, New York, 1963, pp. 8344. 13 L. B. Townsend and R. K. Robins, J. Amer. Chem. Soc., 85 (1963) 242. 14 E. A. Braude, Determination of Organic Structures by Physical Methods, Vol. 1, Academic Press, New York, 1955. 15 A. D. Broom and R. K. Robins, J. Heterocyclic Chem., 1 (1964) 110. 16 Y. Pochon, P. Pascal, P. Pitha and A. M. Michelson, Biochim. Biophys. Acta, 213 (1970) 273. 1 2 3 4 5 6 7