Electrochemical oxidation of 4-thiouracil to bis(4-thiouracil)disulfide and chemical transformations of the disulfide

199

Bioelectrochemistry and Bioenergetics, 10 (1983) 199-212 A section of J. Electroanal. Gem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

563-ELECTROCHEMICAL OXIDATION OF 4-THIOURACIL TO BIS(4-THIOURACIL)DISULFIDE AND CHEMICAL TRANSFORMATIONS OF THE DISULFIDE

K.P. HOLZER

and MONIKA

Department (Poland)

of Biophysics,

(Manuscript

received

SUMMARY

Z. WRONA

Institute

November

l

of Experimental

Physics,

University

of Warsaw,

02-089

Warsaw

10th 1982)

l

The electrochemical oxidation of 4-thiouracil (4TU) and 4-thiouridine (4TUr), which give one voltammetric peak at the pyrolytic graphite electrode (p.g.e.), has been investigated by linear sweep and cyclic voltammetry, macroscale electrolysis and product analysis. Oxidation of 4TU and 4TUr is an irreversible 1 e-, 1 Ht process leading to formation of free radicals which rapidly dimerize to bis(4-thiouracil)disulfide (DIS) or its riboside analog (rDIS). These disulfides can be readily electrochemitally reduced back to the parent compounds. They also undergo spontaneous transformations in aqueous solution to various products, including the corresponding derivatives of 4TU, uracil, sulfenic acid and thiosulfonate, depending on pH and the presence of oxygen. The possible biological meaning of the results reported is discussed.

INTRODUCTION

Previous reports from this laboratory have been concerned with the electrochemical reduction of biologically important thiopyrimidines [l-5]. Thiopyrimidines are minor constituents of various tRNA molecules, but their biological function and the mechanism of metabolic breakdown are unknown. Some thiopyrimidines also possess pharmacological activity [6]. All these facts have stimulated our interest in the redox chemistry of thiopyrimidines. Reported here is the mechanism of the electrochemical oxidation of 4-thiouracil and 4-thiouridine at the pyrolytic graphite electrode (p.g.e.). 4-Thiouracil is the most interesting and widely investigated compound among thiopyrimidines. It has been shown to be present in several tRNA’s and is always located in position 8 from the

To whom reprint requests and other correspondence should be addressed. * Abbreviations: 4TU, 4-thiouracil; 4TUr. 4-thiouridine; DIS, bis(4thiouracil)disulfide; rDIS, bis(4thiouridine)disulfide; 4-SOHU, uracil-4-sulfenic acid; 4-SOHUr, uridine-4-sulfenic acid; 4-SO,HU, uracil-4-sulfinic acid. l l

0302-4598/83/$03.00

0 1983 Elsevier Sequoia

S.A.

200

5’ terminus. Only tyrosine tRNA of Escherichia coli has two adjacent 4-thiouracil moieties at positions 8 and 9. The biological function of 4-thiouracil in tRNA is not clear. It has been found that selective chemical or photochemical modification of 4-thiouracil in the tRNA molecule does not affect the extent, of tRNA aminoacylation and/or ribosomal binding. However, the rate of recognition of tRNA by aminoacylo-tRNA-synthetase and the resulting rate of polypeptide chain elongation are reduced [7,8]. These results suggest that 4-thiouracil is involved in the enzyme recognition process; however, it is not clear whether the enzyme recognizes a definite base sequence containing 4-thiouracil or a definite conformation of tRNA controlled by the presence of 4-thiouracil. In any case, the reports suggest that the function of 4TU is regulatory in nature. In fact, the thione substituent is relatively easily removed by chemical or photochemical reduction of 4-thiouracil [lo- 121. Under polarographic conditions 4-thiouracil and 4-thiouridine undergo an irreversible 4 e-, 4 H+ reduction involving the elimination of the sulphur substituent [2] according to the mechanism presented in equation (1). S

S

3’ ?AN

+2e-

5

H\N

+2H+ +A

H

ii

i

A

+2e-

+2n+

(1) I

SW

L

0

H H

4

H

- H,S

However, such a process requires a relatively high reduction potential of - 1.52 V z)ersw s.c.e. at pH 7 [2] and it is unlikely that it plays a significant role in biological transformations, including catabolism of 4-thiouracil. From a biological view point the oxidation of 4-thiouracil appears to be of particular interest, because of the possibility of disulfide bond formation which might play a role in the structure of tRNA similar to its role in proteins. Furthermore, the known reversible nature of disulfide bond formation might be essential for a regulatory function of Cthiouracil. Although the presence of bis(4-thiouridine)disulfide has not yet been demonstrated in native tRNA, it can be artificially formed by iodine oxidation [ 13,141. Formation of disulfide bonds has been reported during chemical transformation of 4-thiouracil derivatives with iodine [ 151, nitrous acid [ 161 and with near U.V. irradiation of an aqueous solution of 4-thiouridine under aerobic conditions [ 171. The limited stability of bis(4&iouridine)disulfide, which depends on pH and/or on the presence of

201

various reducing agents, has been reported [ 14-16,181. The present communication describes the mechanism of the electrochemical oxidation of 4-thiouracil and 4-thiouridine, leading to the formation of the corresponding disulfide molecule, and further chemical and electrochemical transformations of the latter. Compared to previous studies [ 14-16,181, we have observed a more complicated pathway and the formation of new compounds during spontaneous transformations of bis(4-thiouracil) and bis(Cthiouridine)disulfides. EXPERIMENTAL

Chemicals 4-Thiouracil and 4-thiouridine were synthesized by the methods described in the literature [19,20]. Uracil was obtained from Reanal (Hungary) and uridine from Fluka (Switzerland). Uracil4-sulfonic acid was synthesized according to the procedure of Hayatsu [21]. Phosphate buffers were prepared from reagent grade chemicals and had an ionic strength of 0.5 M, with the exception of buffers of ionic strength 0.01 M used in some macroscale electrolyses. Apparatus Linear sweep voltammograms were obtained using a Radelkis OH-105 (Hungary) polarograph. Cyclic voltammetry was carried out with an instrument based on a conventional operational amplifier design [22]. A three-electrode system was employed for all voltammetric experiments and contained a saturated calomel reference electrode (s.c.e.), a platinum counterelectrode and a pyrolytic graphite working electrode with a geometric area of ca. 0.03 cm2. The p.g.e. was resurfaced by polishing the surface on a 600-grit silicon carbide paper, then washed with a spray of deionized water and gently dried with tissue paper. Cyclic voltammograms were recorded on a Hewlett-Packard Model 7015A X-Y recorder. Macroscale electrolyses were conducted using a Radelkis OH 404A potentiostat with a three-electrode system. The graphite working electrode used in electrolysis experiments had a surface area of ca. 35 cm2. All potentials are referred to the s.c.e. at 25°C. Ultraviolet spectra were obtained on a Zeiss Specord UV-VIS (G.D.R.) spectrophotometer. All I.R. Spectra were obtained in a KBr matrix on a Zeiss 75 I.R. spectrophotometer. Chromatographic

procedures

Solvent systems A: n-butanol/water in panol/water/conc. NH,OH in a V/ V/V on cellulose F254 from Merck (F.G.R.) and Whatman No. 3MM paper. Liquid chromatography separations were

a V/V ratio of 86/14, and B: isoproratio of 170/30/2.6 were used in t.1.c. in ascending paper chromatography on conducted on a 12

X

120 mm Dowex 50

202

W-X2 (H+) column. After application of the sample the column was eluted with several volumes of distilled water until the eluent absorbance at 254 nm was less than 0.01. The column was then eluted with 1 M NH,OH. The same column was also used with H+ ions exchanged by NH: ions. The eluent was water. RESULTS AND DISCUSSION

Linear and cyclic voltammetry 4-Thiouracil and 4-thiouridine exhibit a single oxidation peak (peak I,) over the pH range 3-10.5 and at a slow sweep rate. The variations of the peak potential, U,, with pH are given in Fig. 1 and Table 1. The peak current was independent of pH. The break in the Up versus pH plot occurring around pH 9, is close to pK,, of the compounds examined and reflects changes in the ionization state of the depolarizers. However, the break occurring at acidic pH values is probably associated with the pK, values of the corresponding oxidation products. A typical cyclic voltammogram of 4TU is shown in Fig. 2. On the first anodic sweep a single irreversible peak I, is observed (Fig. 2A). After scanning this peak on the subsequent cathodic sweep two reduction peaks I, and II, appear. If the initial scan at a clean electrode is toward negative potentials only reduction peak II, is detected (Fig. 2B). Thus, in order to observe cathodic peak I, the oxidation peak I, must first be scanned. This indicates that the oxidation product of 4TU derivatives is subsequently reduced at the potentials of peak I,. The potentials and properties of peak II, are identical to those previously reported [2] for the polarographic reduction wave of 4TU derivatives (equation 1). Voltammograms of 4TU recorded at a sweep rate > 100 mV s-l and at concentrations higher than 1 mM exhibit further anodic peaks II, and III, at more positive potentials than peak I,. Peak II, may be observed at lower sweep rates and concentrations; however, under such conditions it appears as a shoulder. Here, U, of peak II, is pH-dependent in a similar way to U, of peak I, (Fig. 3). Above pH 7.8 peak III, appears, its Up being independent of pH

0.4 -

I 2

I 3

# 4

I 5

PH 6

7

8

1 9

11 10

11

I_ 12

Fig. 1. Variation of UP with pH for oxidation peak I, of 1 mM 4TU mV s-‘.

(X)

and 4TUr (0). Sweep rate: 6.67

203 TABLE

1

Peak potential and 4TUr Compound

4TU

4TUr

a

oersus pH relationships

for voltammetric

oxidation

peak I, and reduction

peak I, of 4TU

PK, b

Peak

pH range

8.00

Ia c

3.00- 4.05 4.05- 9.00 9.00- 10.55

I Ed

3.00- 7.90 7.90- 10.55

Ia e

1.50- 2.70 2.70- 8.50 8.50-10.50

0.88 1.05 - 0.062 pH 0.52

I Ed

1.50- 6.30 6.30- 10.50

- 0.18 - 0.098 pH - 0.80

8.40

q (V) (0ersu.s s.c.e.) 0.78 1.05 - 0.067 pH 0.45 -0.17-0.078 - 0.785

pH

a Data obtained at a concentration of 1 mM. b Taken from Ref. 23. ’ Sweep rate 6.67 mV s- ‘. d Data taken from cyclic voltammograms at a sweep of 100 mV s- ‘. e Sweep rate 16.7 mV s- ’ (at lower sweep rate peak I, is poorly defined).

(Fig. 3). Peak III, overlaps peak I, and II,, making the resolution of the three oxidation peaks difficult. The conditions where peaks II, and III, appear seem to indicate their adsorption character. In order to gain additional insights into the peak I, and II, processes, the effects of sweep rate and 4TU concentration were studied. The results are presented in Table 2A, B. The ratio of the peak II, current to the peak I, current increases with increasing sweep rate (Table 2A). This ratio is also concentration dependent and decreases with increasing 4TU concentration below 0.3 mM. The trend is reversed for higher concentrations (Table 2B). These results are consistent with the conclusion that peak II, is an adsorption post-peak and indicates strong adsorption of the reactant [24]. This is further supported by the similarity of the U, versus pH plot for peaks I, and II, (Fig. 3). The absence of a break near the pK, of 4TU in the Up ne7.rU.spH plot for peak II, could be the result of the existence of adsorbed 4TU in the neutral form at basic pH. The appearance of peak III, at pH values close to the pK, of 4TU and the lack of dependence of its U, on pH (Fig. 3) suggests that peak III, results from the oxidation of the adsorbed monoanionic form of 4TU. Further confirmation that peaks II, and III, are caused by adsorption phenomena is supplied by electrolysis experiments. Electrolysis of 4TU at potentials corresponding to peaks I,, II, or III, and higher gives the same product. Furthermore, it has been found that the peak current function (1,/u’/‘) increases for peak I, and II, with increasing sweep rate (0). This result, considered together with the effect of 4TU concentration on the

, U (V vs s.c.e.)

y,, IIC -1.5

-1.0

0

-0.5

,

,

0.5

1.0

Fig. 2. Cyclic voltammograms at the p.g.e. of 0.5 mM 4TU in phosphate buffer pH 7. Scan rate 200 mV s-‘. (A) scan pattern: 0.0 V+ + 1.0 V+ - 1.6 V + 0.0 V; (B) scan pattern: 0.0 V --f - 1.6 V + + 1.0 v + 0.0 v.

0.8 0.7 -_ 0.6-

E fi 1

0.5 -2 9 o.4-a 0.3

I

I

2

3

Fig. 3. Variation

PH I,

4

I

5

6

I

7

I,

8

I

9

10

I

11

I 12

with pH of UP of 1 m&f 4TU peak I, (X), II, (A) and III, (0). Scan rate: 100 mV SK’.

205 TABLE

2

Effect of sweep rate and concentration

on the peak &/peak

A

ration

for 4TU at the p.g.e.

B

Sweep rate (n-IV s-l)

Peak II, current Peak I, current

10 20 50 100 200

0.20 0.27 0.33 0.32 0.36

a Data obtained b Data obtained

I, current

0

Concentration of 4TU (m&f)

Peak II, current Peak I, current

0.07 0.28 0.56 1.10 2.21

0.42 0.27 0.33 0.40 0.54

b

at pH 6.6 and a 4TU concentration of 0.56 mM. at pH 6.6 and a sweep rate of 100 mV s-‘.

A

P la

Fig. 4. Cyclic voltammograms at the p.g.e. of 0.5 mA4 4TUr in phosphate buffer pH 7. Scan rate 100 mV s-‘. (A) scan pattern: 0.0 V+ +l.O V-, -1.1 V-+0.0 V; (B) scan pattern: 0.0 V+ -I.]* +I.0 V + 0.0 V. Potential span limited to the first reduction peak.

206

ratio of the peak II, current to the peak I, current (Table 2B), points to strong adsorption of the reactant and relatively weak adsorption of the oxidation product [24]. Such a qualitative description may only be applied to the case under consideration, where both product and reactant are adsorbed in an irreversible electrode reaction, because of the number of parameters involved. 4TUr exhibits very similar voltammetric behavior to the parent 4TU (Figs. 1 and 4, Table 1). Macroscale

electrolysis and product analysis

Between pH 3 and 10.5 electrolysis of 4TU and 4TUr at peak I, potentials leads to the formation of a single product, identified as DIS or rDIS respectively, on the basis of the following evidence. Spectroscopic evidence (Table 3) The electrooxidation products of 4TU and 4TUr exhibit U.V. spectra identical to those reported [ 15,16,25] for rDIS and its methyl analog. Electrochemical evidence The oxidation products of 4TU and 4TUr undergo reduction at potentials of peak I,, regenerating the starting compounds, i.e. 4TU or 4TUr, respectively. The regeneration is quantitative, provided that the electroreduction is carried out under anaerobic conditions in order to avoid formation of H,O, which readily oxidizes 4TU derivatives [26]. The reduction electrolysis has to be conducted immediately after completion of the electrooxidation reaction because the oxidation product slowly decomposes, spontaneously regenerating, among others, the starting compounds. It was because of this problem that coulometric measurements for the peak I, process as well as for the peak I, process were unsuccessful. Thus, pH-dependent peak I, (Table 1) is due to reduction of the putative disulfides DIS or rDIS, respectively, formed upon the oxidation of 4TU or 4TUr in the peak I, process.

TABLE 3 Ultraviolet absorption spectra of the oxidation products of 4-thiouracil derivatives at pH 7 Compound

x ,,,/nm

Xmzn/nm

x moxl/nm

%WXI Emax*

DIS a 1-methyl DIS * rDIS a,c TSU

305 308 310 306

279 280 280 250

260 260 260 233

1.3 1.5 1.4 2.9

a Obtained by electrochemical oxidation of 4TU. ’ Data taken from Ref. 15. ’ Data taken from Refs. 15, 16, 26.

207

Chemical evidence It has been reported [ 14- 16,271 that rDIS is unstable under both basic and acidic conditions. However, there is some controversy concerning both the mechanism of rDIS hydrolysis and the products formed. Uziel [27] reported that hydrolysis of rDIS with a base leads to the formation of two major products, 4TUr and an unidentified compound X, which he postulated to be uridine4-sulfenic acid, 4SOHUr. In contradiction to this observation, Lipsett [ 141 reported the formation of a single product T, also unidentified, as result of basic hydrolysis of rDIS. She also found that either rDIS or compound T were quantitatively reduced to 4TUr with mercaptoethanol, thiosulfate or 1,6dithiothreitol[ 141. Pal et al. [ 151 have established that rDIS and its methyl analog are hydrolyzed almost quantitatively in alkali to the corresponding thiones and sulfenic acids in a 1 : 1 molar ratio. In alkaline solution the sulfenic acid is very slowly transformed to uridine; the rate of this transformation is higher under aerobic conditions [ 151. In the same study Pal et al. [ 151 reported that the cleavage of rDIS in acidic solution leads to the quantitative formation of 4TUr and uridine. No data concerning the behavior of rDIS in neutral solution have been reported. The disulfides formed upon electrooxidation of 4TU or 4TUr are unstable over the pH range 1- 13. At pH < 5 they are quantitatively hydrolyzed to the corresponding thiones and uracil. The rate of S-S bond cleavage increases with decrease in pH. At pH = 12 both disulfides instantaneously hydrolyze to the corresponding thiones and sulfenic acids. After neutralization of the solution the sulfenic acids were detected by their characteristic U.V. absorption band around 363 nm and by means of cyclic voltammetry. Both 4-SOHU and its nucleoside give an oxidation peak at 0.24 and 0.21 V respectively, and a reduction peak at - 1.10 and - 1.05 V respectively, at pH 7 and at a sweep rate of 100 mV SC’. The electrochemical processes were not studied in detail, but cyclic voltammetry permitted certain conclusions to be drawn. Electrochemical reduction of these sulfenic acids leads to formation of the corresponding thiones. Electrooxidation of sulfenic acids gives a species reducible at - 0.67 V for the base and at - 0.75 V for the nucleoside at pH 7. The chemical identity of this species is unknown, but a compound with similar electrochemical characteristics is observed as an intermediate in the neutral hydrolysis of the sulfenic acids. Here, 4-SOHU has been found to be unstable in neutral solution and it decomposes with a first-order rate constant of 1.8 X lop5 s-‘. The ultimate product of this reaction is uracil. However, a transitory accumulation of an intermediate adsorbing around 310 nm has been observed. This intermediate was not identified. However, on the basis of our studies of the oxidation of 4TU with H,O, [26] we postulate that it is uracil-4-sulfinic acid, 4-SO,HU. Interestingly, an unidentified intermediate species, absorbing around 310 nm, has been observed [28] during the photochemical transformation of an aqueous solution of 4TUr. Possibly, this is 4-SqHUr. Ochiai and Shibata [ 171 have also postulated the formation of 4-SO,HUr upon photochemical oxidation of 4TUr in aerobic, aqueous solution. In agreement with earlier data by Pal et al. [15], we have found that in neutral solution 4-SOHUr hydrolyzes very slowly to uridine.

208

Subsequently, we have found that both DIS and rDIS are also unstable in neutral solution. However, the rate of their hydrolysis is significantly lower than in acidic or basic solution. Hydrolysis of DIS at pH 7 leads to the formation of a third product in addition to 4TU and uracil. This product will be designated TSU. We have found that TSU is formed by transformation of DIS over the pH range 5- 11. However, the amount of TSU in the solution increases with increasing pH and depends on the oxygen concentration. When the reaction in neutral solution was carried out in the presence of nitrogen the formation of TSU was reduced by one-third, as compared with the results of the experiment carried out in an oxygen-saturated solution. Here, TSU is stable in aqueous solution as well as in the solid state and may be separated by liquid chromatography for identification. Ultraviolet spectra of TSU are shown in Table 3; TSU has two pK, values, pK, = 2.5 and pK, = 10.4. Electron-impact ionization mass spectra of TSU are not very clear. However, ions at m/e: 282 (OS%), 281 (0.9%), 280 (1%) are the highest molecular weight fragments in the mass spectrum of TSU. This may imply that TSU has the structure of a thiosulfonate, e.g.

R-S-S-R

c

0

Quite firm evidence that R-SO,-S-R is the correct structure of TSU has been obtained from a comparison of I.R. spectra of TSU and DIS (Table 4). The most characteristic absorption bands of DIS are observed in the I.R. spectrum of TSU. However, the latter exhibits two additional bands at 1332 and 1304 cm- ’ which may be assigned to the asymmetric vibration of the -SO2 group observed in the solid state [29]. The symmetric vibration of the -SO, group may be represented by the band at 1126 cm-‘. Two characteristic lower frequency bands at 695 and 589 cm-‘, which are not observed in the I.R. spectrum of DIS, further support the presence of an - SO, group in TSU [29]. There are two other facts which confirm that TSU is a thiosulfonate (V, equation 2). First, its stability is characteristic of structures of type

P

R-S-S-R

and excludes

R-S-S-R

or

.-f-9_,

compounds

or

of type

P

R-S-S-R

which are very unstable [30,31]. Second, we have found that TSU undergoes electrochemical oxidation. At pH 7 TSU gives a voltammetric oxidation peak A (Fig. 5). After scanning peak A two small reduction peaks B and C are observed, but only for sweep rates >, 100 mV s- ‘. Thus, peaks B and C are caused by very reactive

209

TABLE

4

Infrared

band frequences

of bis(4-thiouracil)disulfide

(DIS) and thiosulfonate

(TSU) in KBr matrices

Compound

crnmtO

DIS

1660 (s, vb); 1607 (s, b); 1538 (w); 1450 (s); 1412 (s); 1236 (s); 1219 (w); 1202 (w); 118 (m); 1086 (s); 994 (s); 935 (m); 780 (s); 710 (m); 596 (w)

TSU

1658 (s, b); 1638 (s, b); 1586 (s); 1537 (w); 1471 (m); 1427 (s); 1332 (m); 1304 (m); 1236 (m); 1215 (m); 1199 (w); 1126 (w); 1112 (w); 1055 (w); 970 (w) 915 (w); 797 (m); 740 (m); 695 (w); 597 (w); 589 (w)

n Notation

of band intensity:

(s) strong;

(w) weak; (m) medium;

(b) broad;

(vb) very broad.

species formed during the peak A oxidation process. The mechanism of electrooxidation of TSU was not studied in detail. However, transitory formation of sulfenic acid 4-SOHU has been detected. This excluded the structure of type 7

7

R-S-S-R A

A

and further confirms the thiosulfonate structure of TSU (equation 2). ~&--~~+

(2)

Formation of the appropriate thiosulfonate has been observed during neutral transformations of rDIS. The riboside analog of TSU does not exhibit a pK, in accordance with the structure in equation (2), because the N, position is blocked by a ribose moiety. Mechanism

Oxidation of 4TU and 4TUr over the pH range 3-10.5 leads to the formation of the disulfide products, DIS or rDIS respectively. Because disulfides spontaneously regenerate the starting compounds coulometric measurements could not be carried out. However, the formation of disulfides requires an uptake of a single electron by 4TU or 4TUr. The dU,/d(pH) slope for peak I, (Table l), ca. - 65 mV, is close to that expected for a reversible electrode process involving the same number of electrons and protons. Peak I, corresponds to an irreversible electrode process; nevertheless the data obtained suggest that oxidation of 4TU and 4TUr is a 1 e-, 1 H+ process leading to the formation of a neutral free radical at pH -CpK, of 4TU or 4TUr, and a one-electron process at pH > pK,. Rapid dimerization of the radicals to disulfide molecules is responsible for the observed irreversibility of the electrochemical reaction. The proposed reaction pathway for 4TU is presented in equation

210

O(vvs s.c.e ) I

-15

1

-1 0

Fig. 5. Cyclic voltammogram

I

I

1

-0.5

0

0.5

I

1.0

at the p.g.e. of 0.3 mM TSU in phosphate

buffer pH 7. Scan rate 100 mV

SC’.

(3). An identical mechanism is proposed for 4TUr. 4TUr and its radical are 8.4 and 2.7 respectively.

However,

the pK,

values

for

pK= 4.0 -H’ *Ii*

(3)

211

Voltammetric peak I, is caused by the reduction of DIS or rDIS to the parent 4TU or 4TUr. The dependence of the peak I, potential on pH (Table 1) suggests the mechanism for electroreduction of DIS shown in equation (4). An identical reaction mechanism is proposed for rDIS.

e- +2H+

(4)

i S

1

2

?I x

“,‘8\ I

Py.0 y:

+H+

CONCLUSIONS

Over the whole pH range 4TU and 4TUr readily undergo electrooxidation leading to the formation of the corresponding disulfides, DIS or rDIS. The oxidation potential of 4TU is relatively low, confirming its strong electron-donor properties, which have been predicated based on calculations of HOMO * energies [32]. The sulfur atom, S(4), which is the electroactive center of the molecule, greatly improves the electron-donor properties of the molecules. Uracil, which instead of S(4) has an oxygen atom, does not undergo electrooxidation and also exhibits a lower HOMO energy than 4TU. Disulfides DIS and rDIS are readily electrochemically reducible back to the parent compounds. These disulfides also undergo spontaneous decomposition over the whole pH range to the products previously reported [ 14-16,271, i.e. the corresponding thiones, uracil and sulfenic acid derivatives. However, a new product, thiosulfonate (equation 2), has been found. The low oxidation potential of 4TU (+ 0.58 V versus s.c.e. at pH 7) compared to its high reduction potential (- 1.52 V versus s.c.e. at pH 7) [2] suggests that the oxidation process is more likely to play a role in the biological transformations of 4TU derivatives. The formation of disulfide products which can be very easily reduced back to the parent compounds or to uracil may also be important from a biological viewpoint for example, reversible control of tRNA structure.

l The higher the energy of a highest occupied molecular orbital (HOMO) the better the electron-donor properties of a molecule.

212 ACKNOWLEDGEMENTS

The authors are deeply indebted to Prof. Glenn Dryhurst (University of Oklahoma) for the equipment for cyclic voltammetry which has made these studies possible and for discussion and criticism of the manuscript. This work was supported by the Ministry of Science, Technology and Higher Education through Project MR-I-5. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

M. Wrona and B. Czochralska, J. Electroanal. Chem., 48 (1973) 433. M. Wrona, B. Czochralska and D. Shugar, J. Electroanal. Chem., 68 (1976) 355. M. Wrona, Bioelectrochem. Bioenerg., 6 (1979) 243. M. Wrona and M. Geller, Bioelectrochem. Bioenerg., 6 (1979) 263. M. Wrona, Bioelectrochem. Bioenerg., 10 (1983) 169. P. Langen, Antimetabolites of Nucleic Acid Metabolism, Gordon and Breach, New York, 1975. M. Saneyoshi and S. Nishimura, B&him. Biophys. Acta, 246 (1971) 123. M. Hara, T. Horivchi, M. Saneyoshi and S. Nishimura, Biochem. Biophys. Res. Commun., 38 (1970) 305. M. Yaniv, A. Chestier, F. Gross and A. Favre, J. Mol. Bioll, 58 (1971) 381. J. Fox, J. Am. Chem. Sot., 82 (1960) 486. P. Cerutti, K. Ikeda and B. Witkop, J. Am. Chem. Sot., 87 (1965) 2505. E. Sato and Y. Kanaoka, Chem. Pharm. Bull., 22 (1974) 799. M.N. Lipsett and B.P. Doctor, J. Biol. Chem., 242 (1967) 4072. M.N. Lipsett, J. Biol. Chem., 242 (1967) 4067. B.C. Pal, M. Uziel, D.G. Doherty and W.E. Cohn, J. Am. Chem. Sot., 91 (1969) 3634. S. Iida and H. Hayatsu, B&hem. Biophys. Res. Commun., 43 (1971) 163. H. Ochiai and H. Shibata, Nippon Nogei Kagaku Kaishi, 48 (1974) 643. B.C. Pal and D. Grob Schmidt, J. Org. Chem., 39 (1974) 1466. M. Ikehara, T. Ueda and K. Ikeda, Chem. Pharm. Bull., 10 (1962) 767. E.A. Falco, B.A. Otter and J.J. Fox, J. Org. Chem., 35 (1970) 2326. M. Hayatsu, J. Am. Chem. Sot., 91 (1969) 5693. G. Dryhurst, M. Rosen and P.J. Elving, Anal. Chim. Acta, 42 (1968) 143. A. Psoda, Z. Kazimierczuk and D. Shugar, J. Am. Chem. Sot., 96 (1974) 6832. R.H. Wopschall and I. Shain, Anal. Chem., 39 (1967) 1515. J.J. Fox, D.V. Praag, I. Vempen, I. Doerr, L. Cheong, J.E. Knoll, M.L. Edinoff, A. Bendich and G.B. Brown, J. Am. Chem. Sot., 81 (1959) 178. K. Holzer and M. Wrona, in preparation. M. Uziel, B&hem. Biophys. Res. Commun., 25 (1966) 105. A. Favre, Photochem. Photobiol., 19 (1974) 15. H. Bredereck, A. Wagner, H. Beck and R.-J. Klein, Chem. Ber., 93 (1960) 2738. P.C. Jacelyn, Biochemistry of the SH Groups, Academic Press, London, New York, 1972, p. 108. S. Oae and T. Takata, Chem. Lett., (1981) 845. M. Geller, A. Pohorille and A. Jaworski, B&him. Biophys. Acta, 331 (1973) 1.