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Electrochemical reduction mechanism of 2-thiouracil derivatives
Bioelectrochemist~ and Bioenergetics, 10 (1983) 169-183 A section of J. Electroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
550-ELECTROCHEMICAL DERIVATIVES
REDUCTION
MECHANISM
169
OF 2-THIOURACIL
MONIKA Z. WRONA University of Warsaw, Institute of Experimental Physics, Department of Biophysics, 93 Zwirki & Wigury, 02-089 Warszawa (Poland) (Manuscript received October 2nd 1982)
SUMMARY * The mechanism of the polarographic reduction of 2-thiouracil and its S-methyl and ethoxy derivatives with stabilized 2-thiol-4-keto or 2-thione-4-enol structure has been studied. 2-Thiouracil is not electroreducible in aqueous solutions. However, it exhibits a strong tendency to adsorption and association on the mercury electrode. 2-Thio-4-ethoxypyrimidine undergoes a 4 e-, 4 H + reduction which involves 2 ereduction of the 3,4 N = C bond, elimination of the ethoxy substituent to form 2-thiopyrimidine and 2 e-, 2 H + reduction of the latter to 3,4-dihydroderivative. For 2-thiomethyluracil, a model compound of the 2-thiol-4-keto form, the electroactive centre is shifted to C-2 and in a 2 e-, 2 H + reduction process the sulphur substituent undergoes elimination, to give pyrimidone-4; the latter can be further reduced at more negative potentials to tetrahydroderivatives (4 e -process). Biological implications of these results have been discussed.
INTRODUCTION F o r several y e a r s this l a b o r a t o r y has b e e n i n v e s t i g a t i n g the e l e c t r o c h e m i c a l p r o p e r t i e s of t h i o p y r i m i d i n e d e r i v a t i v e s [1-5] in the e x p e c t a t i o n t h a t k n o w l e d g e of t h e r e d o x c h e m i s t r y of t h e s e c o m p o u n d s m i g h t l e a d to an u n d e r s t a n d i n g of their biological significance. T h i o p y r i m i d i n e s are m i n o r c o n s t i t u e n t s of v a r i o u s t R N A species [6]. H o w e v e r , t h e i r f u n c t i o n still r e m a i n s a l a r g e l y u n k n o w n a n d i n t r i g u i n g p r o b l e m . T h e e l e c t r o c h e m i c a l r e d u c t i o n m e c h a n i s m of 2 - t h i o p y r i m i d i n e [ 3 - 5 ] a n d 4 - t h i o u r a c i l d e r i v a t i v e s [1,2] has b e e n i n v e s t i g a t e d b y us p r e v i o u s l y . I n this p a p e r the e l e c t r o r e d u c t i o n m e c h a n i s m of 2 - t h i o u r a c i l d e r i v a t i v e s is p r e s e n t e d . If 2 - t h i o u r a c i l o r its d e r i v a t i v e s are p r e s e n t in t R N A t h e y are u s u a l l y l o c a t e d in t h e first p o s i t i o n (5' e n d ) of the a n t i c o d o n o f t h e t R N A m o l e c u l e [7 10]. T h e r e is a h y p o t h e s i s t h a t 2 - t h i o u r a c i l d e r i v a t i v e s p l a y the c o n t r o l l i n g r o l e in the b i n d i n g of
* Abbreviations: ~p, pseudouridine; polyU, poly(uridylic acid); poly s2U, poly(2-thiouridyfic acid). 0302-4598/83/$03.00
© 1983 Elsevier Sequoia S.A.
170
tRNA to ribosomes [11,12]. It has been demonstrated that selective chemical modification of 2-thiouracil in tRNAL2yS by removing sulphur and the resulting transition of 2-thiouracil into uracil does not affect the extent of aminoacylation of this tRNA [12]. However, the efficiency of ribosomal binding and protein synthesis is markedly reduced. 2-Thiouracil derivatives have also been discovered in the T - ~ - C - G sequences of tRNA of Flavobacterium thermophilium [13] where it has been suggested that it is important for the capacity of tRNA to synthesize protein at high temperature. Further studies have discovered the specific interaction of the T - + - C - - G sequence in tRNA of thermophilic bacteria with ribosomes [13-15]. Thus, it is very likely that 2-thiouracil derivatives are necessary instead of thymine in the majority of thermophilic tRNA for the interaction of the T - + - C - G region with ribosomes at high temperature. Numerous studies have also demonstrated that 2-thiouracil exhibits pharmacological [16], anti-viral [17,18] and anti-tumor activity [19], and is incorporated into bacterial, viral, plant and mammalian RNA replacing uracil [20]. Furthermore, 2-thiouracil has been reported to cause some mutagenic effects [21]. The present communication also describes the electrochemical properties of 2-alkylthiouracil, some derivatives of which specifically inhibit polio-virus multiplication [22]. Contrary to the widely recognized biological activity of 2-thiouracil, relatively little is known about its metabolic activity. The biological properties of 2-thiouracil derivatives mentioned above, as well as the fact that catabolic processes of the normal nucleic acid bases involve redox reaction have stimulated out interest in the redox chemistry of 2-thiouracil derivatives. Only a very few other reports on electrochemical studies of 2-thiouracil have appeared. Zuman and Kuik [23] studied the catalytic activity of 2-thiouracil derivatives in buffer solutions containing cobalt ions. Preliminary studies of polarographic oxidation of 2-thiouracil on the d.m.e, were made by Zuman et al. [24,25]. Because 2-thiouracil (I) is able to exhibit keto-enol and thione-thiol tautomerism this study has been extended to include 2-thio-4-ethoxypyrimidine (II) and 2thiomethyluracil (III), which are the models of 2-thione-4-enol and 2-thiol-4-keto structures respectively (Scheme 1). o
S
S (I)
H (IT)
CH3S (1]I)
S c h e m e 1.
Ultraviolet spectroscopic studies of the 2-thiouracil tautomeric equilibria in aqueous medium suggest that a neutral molecule exists in the 2-thiol-4-keto form I [26,27]. However, recently Ueda et al. [28], on the basis of ultraviolet absorption and magnetic circular dichroism studies have been suggested that 2-thiouracil exists as an
171
equilibrium mixture of thione and thiol forms in solution. 2-Thiomethyluracil(III) can exist in aqueous medium in three tautomeric forms (IIIA-C) (Scheme 2). 0
0
OH
"itrA
~B
11TC
Scheme 2.
Ultraviolet, infrared and dipole moment data [29,30] unequivocally exclude the existence of form IIIC, and suggest that forms IIIA and IIIB are present in a 55 : 45 ratio [31]. EXPERIMENTAL
Chemicals
2-Thiouracil was a product of Fluka A.G. 2-Methylthiouracil, m.p. 200°C, was prepared according to the procedure of Matsukowa and Ochta [32]. 2-Thio-4-ethoxypyrimidine was synthesized by the procedure described by Psoda and Shugar [31]. Pyrimidone-4 was prepared from 2-thiouracil according to the procedure of Brown [33]. All coumpounds were checked for chromatographic homogenity and by comparison of melting temperatures and U.V. absorption spectra with appropriate compounds [34]. Apparatus and procedure
Electrochemical methodology, instruments and methods of calculation of the electrode kinetics parameters have been adequately described elsewhere [4,5]. RESULTS A N D DISCUSSION
2- Thiouracil d.c. and a.c. polarography It has been found that 2-thiouracil is not polarographically reducible in aqueous medium. However, it is strongly adsorbed on the surface of the d.m.e.a.c, polarograms of 2-thiouracil exhibit a marked depression of the base current at potentials where the electrode is positively charged (Fig. 1). Over the pH range 4.3-8.8 and at about the 0.3-0.5 m M concentration level, a very well-defined pit is observed. Further increase of 2-thiouracil concentration causes no further increase in the depth
172
<
~.
4.15
pH4.3
/ 3.6
pH 7.1
3.6 pHlO/ f,,), /- 3.6
/
f~
/ /
I
- 1.O
I
I
-O.6
- 0.2
2.4
//
1.2
"~ i
4.8
48
2.4
/ //iI
j~/ll/i I
k
1.2
1.2 ..~.~-" U(V)
I
!
I
-1.0 - 0.6 -0. 2
i
-- 1.0
-
0.6
2.4
I
- 0.2
Fig. 1. Dependence on p H of the alternating current polarographic behavior of 0.5 m M 2-thiouracil in Britton-Robinson buffer (0.5 M ) at 25°C. ( - - I I ) Background.
of the pit, but the potential range over which the sharp depression occurs increases (Fig. 2A). This behavior is most marked at pH 7, i.e. for a relatively low concentration of 2-thiouracil the pit extends over the largest potential range (Fig. 2B). As the p H is changed the potential range of the pit decreases. Above pH 8 the extent of the base current depression decreases and at pH > 10.5 completely disappears (Fig. 1). Bearing in mind that p K a of 2-thiouracil is 7.74 [26], this behaviour indicates that the depression is caused by adsorption of the neutral form of the molecule. Above
A
/"
3.0
/I
/
- 0.1
/
>
1.8
/
.I
0.3
" 1\
L-.-J
I
dCV) I
-08
I
-0.4
a.c.
06
-0.5
pH I
4
I
I
6
I
I
8
I
I
10
Fig. 2. (A) polarograms of 2-thiouracil at various concentrations in p H 7 Britton-Robinson buffer (0.5 M). ( - - - - I ) Background; ( X - X - X ) 0.1 m M ; ( . . . . . ) 1 mM: ( ) 4 m M . (B) Potential range of the sharp depression (pit) observed in a.c. polarography of 0.5 m M 2-thiouracil as a function of pH.
173
p H 8 a new depression appears at about - 0 . 1 V. This depression is located at a positively charged electrode and its occurrence at a p H above p K a of 2-thiouracil suggests that it corresponds to the adsorption of the anionic species. Depression of the base current at potentials close to - 0 . 5 V and at low concentrations, followed by formation of a sharp pit in a.c. polarograms at higher concentrations, has been observed for other biological pyrimidines and purines [35]. The pits have been attributed to intermolecular association of the adsorbed species which seem to form a compact film on the electrode surface [36,37]. The model of such adsorption behaviour for pyrimidine and purine derivatives assumes the existence of two adsorption regions with respect to concentration [38,39]. In the so-called dilute adsorption region the molecules exhibit a flat orientation, with the plane of the base ring parallel to the surface of the electrode. At higher concentrations the molecules undergo a surface reorientation so that the planes of the rings are nearly perpendicular to the electrode surface. Such effects are thought to be controlled by interaction of the dipole moments of adsorbate molecules with the charged surface of the electrode. In the perpendicular orientation a strong stacking interaction between the adsorbed molecules occurs and association takes place, causing formation of the pits in a.c polarograms. Interestingly, the association of 2-thiouracil in the adsorbed state occurs at a relatively low concentration of 0.5 m M (e.g. for uracil it was reported [38] to occur at 24.1 r a M ) and it is significantly more pronounced at neutral p H than in acid or alkaline medium.
2- Thio-4-ethoxypyrimidine d.c. and a.c. polarography Over the p H range 2.7-12, 2-thio-4-ethoxypyrimidine was found to give two d.c. polarographic waves, as illustrated in Fig. 3A. Wave I shows up at p H < 6 with a limiting current considerably in excess of the anticipated for a diffusion-control process. The temperature coefficient of wave I was about 4 . 0 % / ° C . The height of the polarographic wave increased with drop time much more slowly than expected for a diffusion-controlled wave. The dependence of the wave height on depolarizer concentraction in the range 0.1-1.0 m M was linear. However, for concentrations above 1 m M a saturation effect was exhibited. Above p H 7 wave I gradually decreased in height and completely disappeared at p H 8. The variation of the half-wave potential, UI/2 with p H for wave I is given by the equation U1/2 = [ - 1.140 - 0.060 pH] V over the p H range 4.0-7.8. The properties of wave I point to its partial catalytic nature at p H < 6 and to kinetic control at higher pH. As wave I disappears at about p H 8, simultaneously wave II appears at more negative potentials. Its height is p H independent and U1/2 shifts linearly more negative with increasing p H according to the equation U1/2 = [ - 1.740 - 0.025 pH] V over the p H range 7-11. The limiting current of wave II is proportional to the concentration of the depolarizer over the range 0.1-1.0 m M . The drop time and temperature
174
6~
6 \
q
4
X 4 ;~
:<
;4
X
2 pH I
6
I
8
I
~
1
I
10
I
2
I
4
8
5
Fig. 3. Dependence of diffusion current constant (la) on pH for: (A) 2-thio-4-ethoxypyrimidine: (B) 2-thiomethyluracil at the d.m.e. (O) Wave I; ( x ) wave II.
d e p e n d e n c e of the wave II limiting current are indicative of diffusion control at all p H values. Over the p H range 2.7-6.5, 2 - t h i o - 4 - e t h o x y p y r i m i d i n e causes a base current d e p r e s s i o n in a.c. p o l a r o g r a p h y , indicative of a d s o r p t i o n p h e n o m e n a at the electrode surface (Fig. 4). Below p H 7 this depression of the base current occurs n e a r the p o t e n t i a l of zero charge, suggesting that a n e u t r a l f o r m of the d e p o l a r i z e r is a d s o r b e d . This suggestion is s u p p o r t e d b y the fact that the m a g n i t u d e of the d e p r e s s i o n decreases with increase in p H over the p H 7 e m b r a c i n g the p K , of dissociation of 2 - t h i o - 4 - e t h o x y p y r i m i d i n e . A t a p o t e n t i a l of - 1.0 V a b r o a d desorption p e a k is observed, a n d at m o r e negative p o t e n t i a l s a r e d u c t i o n p e a k appears, whose s u m m i t p o t e n t i a l ~ c o r r e s p o n d s to Ul/2 of wave I (Fig. 4). This p e a k
/
pH 4.3
/A L -t6
[
;'
/J 1/
5.4-
pHz8 //
,
phl14
r~
]
"~
il
/.,/1] :.----)!v,, u
u(v) -12 -o.8 -0.4
~
--1.6 -1.2 -0.8 -0.4
4.2 3.0 1.8
-1.6 -1.2 - Q 8 -0.4
Fig. 4. Dependence on pH of alternating current polarographic behavior of 0.5 mM 2-thio-4-ethoxypyrimidine in Britton-Robinson buffer (0.5 M) at 25°C. (-- - - --) Background.
175 decreases in height with increasing p H and completely disappears at p H 6, i.e. two p H units less than in the case of the d.c. polarographic wave. There is no observed a.c. peak corresponding to wave II, indicating the irreversibility of the wave II reduction process. Above p H 7 a.c. polarograms exhibit an additional depression at around - 0 . 2 V, which is undoubtedly associated with the adsorption of the anionic form of 2-thio4-ethoxypyrimidine.
Electrolysis and coulometrv During the course of electrolysis at wave I potentials of 2-thio-4-ethoxypyrimidine, a high current flow was observed. Furthermore, it was not possible completely to eliminate the polarographic wave even after m a n y hours of electrolysis. The electrolysis current remained at a high value and the solution p H increased. Such behavior indicates that a major part of the current-controlling process is the catalytic reduction of hydrogen ions. As a result coulometric measurement of faradaic n-values were unsuccessful. However, it has found that prolongated electrolysis led to a gradual decrease of the U.V. absorption band of 2-thio-4-ethoxypyrimidine ()~ ..... = 276 nm at p H 7), and to formation of a new U.V. band ()~,~,x = 250 nm p H 7). Thin-layer chromatography indicated formation of a single ultimate reduction product. The course of electrolysis at wave II potentials was more rapid. Coulometry gave average n-values of 3.9 _+ 0.1. The same product was formed during reduction at wave II as in the case of wave I. It was also found that the U.V. absorption and chromatographic properties of this product were identical to the two-electron reduction product of 2-thiopyrimidine, identified as 3,4(6)-dihydro-2-thiopyrimidine
[4]. Electroreduction mechanism of 2-thio-4-ethoxypyrimidine The observed dependence of the wave I height and U~/2 on p H suggests that protonation of the depolarizer precedes electron transfer [40]. Thus, wave I is associated with electroreduction of the protonated form of the depolarizer. However, at higher p H values and at more negative potentials there is also possibly reduction of the neutral form of the depolarizer in the wave II process. It has been found that both processes lead to the same ultimate product--3,4(6)-dihydro-2-thiopyrimidine, formation of which requires the uptake of four electrons. More detail regarding the electrode mechanism was obtained from analysis of electrode kinetic parameters (Table 1). Since the theoretical value of a should be ca. 0.5, the experimentally determined ~n~ and p values suggest that two electrons and one proton are involved in the rate-controlling process of wave I, and two electrons are transferred in the rate-controlling process of wave II. For wave II the experimental value of p --- 0.42 is difficult to explain. However, the proposed electroreduction mechanism may be represented by the initial electron attack at the N 3 z C 4 double bond (Scheme 3) as proposed for reduction of other pyrimidines having this double bond available i.e. cytosine [41], 4-thiomethyluracil [1], 4-methylpyrimidine [41]. Under polarographic
176 TABLE 1 Effect of pH on the rate-determining step in the polarographic reduction of 2-thiouracil derivatives ( c = 0.5 m M )
Compound
pH range
Wave
d Uj/2
Aa
an,, h
ph
dpH (s2EtU) c
5- 7 8-10
1 II
0.060 0.025
0.059 0.053
0.93 1.02
0.94 0.42
(Mes2U) c
3- 6 3- 6
I II
0.050 0.085
0,043 0,040
1,25 1.32
1.06 1.91
Pyrimidone-4
3- 6
I
0.085
0.042
1.26
1.83
" A, slope of logarithmic plot. b Average values for given pH range. c s2EtU; 2-thio-4-ethoxyuracil; Mes2U: 2-thiomethyluracil.
conditions the reduction process terminates after the transfer of two electrons and formation of compound III (the height of wave I at pH 6-7 and wave II correspond
H
H
(I)
(11)
2e'2H+
~
Z
I 2e, H+
;
OC2H5
H (r~)
I
-C2H5OH
H
s
H
H (v)
S
H (~)
Scheme 3.
to a 2 e - process). However, coulometry gave n-values of about 4 and prolonged electrolysis led to formation of 3,4(6)-dihydro-2-thiopyrimidine (V) indicating the
177 elimination of the ethoxy substituent. After elimination of the ethoxy group, 2-thiopyrimidine (IV) is formed which immediately undergoes a further two-electron reduction to give the ultimate product (V).
2- Thiomethyluracil d.c. and a.c. polarography 2-Thiomethyluracil yields two polarographic waves over the p H range 1.5-8.5, whose heights and U1/2 values are pH-dependent (Fig. 3B). Wave I. Here, UI/2 shifts linearly more negative with increasing pH; U~/2 = [ - 0.935 - 0.050 pHI V over the p H range 1.5-7.0. The height of wave I between p H 2 and 6 is pH-independent and its diffusion current constant is indicative of a two-electron process. Within this p H range, the observed linear dependence of the limiting current on h 1/2 and on depolarizer concentration (0.1-1.5 r a M ) , and a temperature coefficient of 2 . 1 % / ° C point to diffusion control of the wave I current. At p H > 6, wave I gradually decreases in height and completely disappears at p H 8.1. The limiting current increases with h, but less than predicted by the square-root relationship, hence it was concluded that wave I between p H 6 and 8 is partially under kinetic control. Wave IL This exhibits very similar behavior (Fig. 3B). The linear dependence of U1/2 on p H is given by the equation U1/2 = [ - 1.055 - 0.085 pH] V for the p H range 1.5-7.5. Between p H 3 and 5 the wave is constant in height and the dependence of the limiting current on drop time, temperature and depolarizer concentration indicate the diffusion-controlled nature of wave I. At p H > 5, wave I gradually decreases in height and disappears at p H 7.5. Within this p H range the wave shows increasing kinetic control of the current. At p H ~< 2.5, both polarographic waves exhibit very large current values which are strongly dependent on pH, indicating that the reduction process is accompanied by a catalytic hydrogen discharge. The neutral form of 2-thiomethyluracil is only slightly adsorbed on the mercury electrode near the potential of zero charge. A t - 1.0 V a gradual desorption of 2-thiomethyluracil occurs and at more negative potentials two faradaic peaks may be observed, corresponding to the d.c. polarographic reduction waves. The heights of the peaks and their Us values depend on p H in the same way as was observed for waves I and II; for the first peak, Us = [ - 0 . 9 0 - 0.050 pH] V and for the second peak, Us = [ - 1.080 - 0.085 pH] V over the p H range 2-5.2. Electrolysis and product(s) analysis During electrolysis at wave I potentials the liberation of methyl mercaptan and disappearance of the U.V. absorption band of 2-thiomethyluracil (2~. . . . 230 nm, ~,,,~ = 265 nm, X-,,x2 = 290 nm at p H 6.5) were observed. Chromatographic analysis of the electrolysed solution indicated formation of a single product which exhibited =
178
an U.V. absorption spectrum w i t h ~maxt = 222 nm, X,.~ = 250 nm, ~,,a~, = 255 nm at p H 6.5. After termination of the electrolysis at the potentials of wave I the second wave remained. Coulometry gave an average n value of 2.0 + 0.1. Comparison of the U.V. absorption, chromatographic and electrochemical properties of the wave I reduction product with authentic pyrimidone-4 proved the identity of both compounds. Electrolysis at wave II potentials led to the elimination of both polarographic waves. Methyl mercaptan was evolved and the U.V. absorption spectrum of 2thiomethyluracil disappareared. After termination of the electrolysis the solution showed only residual U.V. absorption. Coulometry gave an average n value of 5.6 + 0.2. Chromatographic and spectrophotometric analysis of the electrolysed solution gave evidence of the formation of a major product with residual U.V. absorption and traces of a second product exhibiting an U.V. absorption band at ~max = 305 nm. Formation of the same product has been observed upon electroreduction of pyrimidone-4 [42]. These results clearly indicate that wave II is due to reduction of pyrimidone-4 formed upon electroreduction of 2-thiomethyluracil at potentials of wave I. The major reduction product of pyrimidone-4, having residual U.V. absorption, has been identified as a 1,2,5,6-tetrahydropyrimidone-4 [42]. Formation of this product requires reduction of both double bonds of the pyrimidine ring and the uptake of four electrons. The second product, exhibiting an U.V. absorption band a t ~kmax = 305 nm (pH 7) has not been identified. However, it is probably formed by two-electron reduction of one of the two possible tautomeric forms of pyrimidone-4 [42]. The chemical nature of this product is under current investigation. Interestingly, Skari~ et al. [43] have reported formation of a compound showing very similar U.V. absorption properties during partial catalytic hydrogenation of 1-methylpyrimidone-4. Electroreduction process Spectroscopic studies [31] give evidence that in aqueous medium 2-thiomethyluracil may exist as an equilibrium mixture of two tautomeric forms II1A and III B (scheme 2). The course of electrolysis and coulometric data indicate that wave I is due to the two-electron reduction of 2-thiomethyluracil to pyrimidone-4. The properties of wave I and the formation of the single product suggest that both tautomeric forms undergo the same reduction process. Elimination of methyl mercaptan during this process indicates that the initial electron attack is directed at the N ~ C bond adjacent to the sulphur substituent or directly to the C - S R bond. On the basis of experimental data it is not yet possible to elucidate the actual mechanism. However, additional information can be obtained from the electronic structure of 2thiomethyluracil, see below. The dependence of the wave height and U~/2 on p H suggest that only the neutral and not the monoanionic form of 2-thiomethyluracil ( p g a 7.5) is polarographically reducible. The analysis of the electrode process kinetic parameters (Table 1) indicates that two electrons and one proton are involved in the rate-controlling process of wave I, and that two electrons and two protons are involved in the rate-control-
179
ling process of wave II. The kinetic parameters of wave II of 2-thiomethyluracil are identical to those determined for pyrimidone-4 (Table 1), hence supporting the proposed reaction mechanism presented in Scheme 4. The process occurring at the Wave I
O
-HSCH 3 H3S
CH3S
CH3S
H
CH3S
H
Wave TT
4e,
~
HN'~ H
Scheme 4.
potentials of wave II of 2-thiomethyluracil corresponds to the reduction of pyrimidone-4 and is shown in this scheme 4 in a somewhat simplified form. The major route leading to formation of 1,2,5,6-tetrahydro-pyrimidone-4 is presented. However, more detailed studies [42] with 1-methyl- and 3-methylderivatives of pyrimidone-4, which have stable tautomeric forms A and B respectively, have revealed that such a route is given only by tautomer A. Tautomer B undergoes a two-electron reduction tO an ultimate product exhibiting U.V. absorption at longer wavelengths ( - 305 nm) than the starting compound. Because the contribution of the B-form to the tautomeric equilibrium of pyrimidone-4 is around 40% [34] and its reduction potential is slightly more negative than that for the A-form, the two-electron reduction of B is a minor reaction, leading to only trace amounts of the second reduction product. The nature of this product, as well as the electrochemical mechanisms leading to its formation, are under current study [42]. CORRELATION OF ELECTROCHEMICAL BEHAVIOR A N D Q U A N T U M CHEMICAL PARAMETERS
The polarographic potential, UI~ a, for a reversible process, diminished by the difference of the solvation energy of reactants, is an experimental measure of the electron affinity of molecules [44]. In fact, account should also be taken of the contribution of the adsorption energy of reactants to U~f2a and to proton participation in the rate-determining step. The energy of the lowest unoccupied molecular orbital (LUMO) is a theoretical measure of the electron acceptor affinity [44]. Correlations of U~/~ and L U M O energy are valuable because they offer a simple experimental method for verification of the theoretical value of LUMO, which in turn has been further correlated with the chemical, biochemical and pharmacological
180 activity of molecules [45]. The reliability of the correlation of a q u a n t u m - m e c h a n i c a l p r e d i c t i o n a n d p o l a r o g r a p h i c U1/2 values d e p e n d s on the calculation a n d m e a s u r e m e n t s having been m a d e on identical m o l e c u l a r species. U n f o r t u n a t e l y , theoretical calculations are b a s e d on an idealized, n e u t r a l gas-phase molecule a n d a reversible electron-transfer process. In the case of the p o l a r o g r a p h i c r e d u c t i o n of organic molecules m o s t electrode reactions are n o t perfectly reversible. In addition, it is a l m o s t i m p o s s i b l e to evaluate q u a n t i t a t i v e l y the c o n t r i b u t i o n s of the such factors as r e a c t a n t solvation, a d s o r p t i o n energy a n d p r o t o n p a r t i c i p a t i o n to [[Red ~1/2. Neverthel[Red less, Vl/2 values, even for an irreversible r e d u c t i o n process, can be used to estimate qualitatively the electron a c c e p t o r p r o p e r t i e s of molecules a n d can be c o r r e l a t e d with L U M O energies for chemically related c o m p o u n d s . Values of ~l'rRed a n d the L U M O energy a n d its s y m m e t r y for the 2-thiouracil l/2 derivatives e x a m i n e d a n d for the p a r e n t uracil are given in T a b l e 2. U n f o r t u n a t e l y , in the case of 2 - t h i o m e t h y l u r a c i l a n d 2 - t h i o - 4 - e t h o x y p y r i m i d i n e the calculations were m a d e for the i m p r o p e r structures of 2-thiouracil, 2-thiol-4-keto a n d 2-thione-4enol respectively. Nevertheless, in our previous studies of t h i o p y r i m i d i n e derivatives [4,5] calculations were m a d e taking into c o n s i d e r a t i o n the m e t h y l a t i o n of s u l p h u r s u b s t i t u e n t a n d it has been p r o v e d that the effect of m e t h y l a t i o n on L U M O energy, s y m m e t r y a n d electron d i s t r i b u t i o n is negligible. D a t a p r e s e n t e d in T a b l e 2 i n d i c a t e that the presence of a sulphur s u b s t i t u e n t at p o s i t i o n 2 of uracil decreases the L U M O energy and, c o r r e s p o n d i n g l y , increases the electron a c c e p t o r p r o p e r t i e s of the molecules. These results are in qualitative a g r e e m e n t with the changes of the o b s e r v e d U1R/~d values. The influence of thiol s u b s t i t u e n t on the increase of the electron a c c e p t o r p r o p e r t i e s of uracil is greater t h a n observed for the thione substituent. Simultaneously, in the case of thiol derivatives, the s y m m e t r y of L U M O is c h a n g e d from 7r-type to o-type, a n d the d i s t r i b u t i o n of the d e n s i t y of u n p a i r e d electron strongly suggests the C 2 - S R b o n d as TABLE 2 Comparison of U~/edpotentials with LUMO values for uracil derivatives U~fd iV]" pH 5.7
pH 7
pH 8
Uracil S2U c Mes2U c
-__ 1.215
-__ - 1.280
--1.330
S2EtU e
1.480
- 1.560
1.620
LUMO energy b [eV]
Symmetry of LUMO orbital b
0.099 0.065 0.038 d 0.070 0.051 e
~r ~r o 'z" ~r
a //Red ~1/2 at 0.5 m M concn. b Calculated by the C N D O / 2 method, [47, 46]
c s2U, 2-thiouracil; Mes2U, thiomethyluracil; s2EtU, 2-thio-4-ethoxyuracil. d Calculation for 2-thiol-4-keto form of uracil [47,46]. e Calculation for 2-thione-4-enol form of uracil [47,46].
181 the electroactive centre of the molecule. These theoretical results support the conclusion that elimination of mercaptan, observed upon electroreduction of 2thiomethyluracil, is due to the direct reduction of the C2-SR bond rather than to reduction of the C2~N bond followed by former reaction. A similar electroreduction mechanism was observed for 2-methylthiopyrimidine [4,5]. CONCLUSIONS AND BIOLOGICALIMPLICATIONS In aqueous medium 2-thiouracil exists in the 2-thione-4-keto form. This form is not polarographically reducible, whereas it exhibits a strong tendency to adsorb on the surface of the mercury electrode near the potential of the e.c.m. In the adsorbed state the intermolecular association of 2-thiouracil takes place at the relatively low concentration of 0.5 mM. Uracil, by contrast, exhibits this effect at a concentration of about 24 m M [38]. If the critical concentration value where a capacitance pit appears is taken as a measure of the magnitude of intermolecular association, then the results presented in this paper indicate that stacking interactions of electrosorbed 2-thiouracil are significantly stronger than those for uracil. Incidentally, it was reported that the synthetic polymer, poly s2U, is also able to form an extraordinarily stable double-stranded helical structure having a m . p . of 68°C [48]. Under the same experimental conditions the m . p . of polyU is only 8°C [48]. The high adsorption activity of 2-thiouracil and its ability to undergo strong intermolecular association in the adsorbed state might provide a basis to speculate about the possible role of 2-thiouracil in tRNA. Bearing in mind that 2-thiouracil is usually located in the anticodon of tRNA in the so-called wobble position it is possible that 2-thiouracil is involved in the non-specific interaction of tRNA with the ribosome. Indeed, it has been reported that selective modification of 2-thiouracil to uracil markedly decreases the binding of tRNA to the ribosome [12]. However, it is difficult to exclude the possibility that such modification changes the conformation of tRNA in such a way that its binding to the proper site of the ribosome is affected. 2-Thiomethyluracil and 2-thio-4-ethoxypyrimidine, which are the models of the 2-thiol-4-keto- and 2-thione-4-enol forms of 2-thiouracil, are readily electroreducible, but by different mechanisms. For 2-thio-4-ethoxypyrimidine the N 3 ~ C 4 double bond is the electroactive centre of the molecule and the sulphur substituent remains intact upon electroreduction. In the case of 2-thiomethyluracil, the electroactive site of the molecule is shifted to C 2 and the sulphur substituent readily undergoes elimination when the reduction process occurs. Identical electrochemical behavior was observed for the 2-thiopyrimidine series [4,5]. These results might be considered in relation to the metabolism of 2-thiouracil. The mechanism of metabolic breakdown of 2-thiouracil derivatives is not known. In general, regular nucleic acid pyrimidines such as cytosine, uracil and thymine are reduced in organisms to their 5,6-dihydro derivatives and ultimately hydrolysed to derivatives of fl-ureido-acids [49]. Similar to uracil, 2-thiouracil is not electroreducible in aqueous solution. However, its L U M O energy is lower (see Table 2), indicating that introduction of sulphur at the C-2 position of uracil improves the electron acceptor properties of the
182 molecule. Thus, it is p r o b a b l e that 2-thiouracil can be m e t a b o l i z e d in a way similar to uracil. In fact, it has been r e p o r t e d [50] that m e t a b o l i s m of 6-methyl-2-thiouracil in rats leads to f o r m a t i o n of several u r in a r y m et ab o l i t es including 6-methyluracil, 5,6-dihydro-6-methyluracil, fl-ureidobutyric acid and, m o r e interestingly, 2-methylthio-6-methyluracil. T h e latter c o m p o u n d is further t r a n s f o r m e d to the same p r o d u c t wh i ch is observed to f o r m u p o n e l e c t r o r e d u c t i o n of 2-methylthiouracil, i.e. pyrimid o n e - 4 (see scheme 4). This result suggests that the t r a n s f o r m a t i o n of 2-methylthio6-methyluracil in vivo is a r e d u c t io n process. ACKNOWLEDGEMENTS T h e a u t h o r is greatly i n d e b t e d to Prof. G. D r y h u r s t (The U n i v e r s i t y of O k l a h o m a , Dep t . Chemistry, N o r m a n , O K , U.S.A.) for valuable discussions and for criticism of the manuscript, to Dr. E. Pale~ek (Institute of Biophysics, Brno, Czechoslovakia) for the use of e q u i p m e n t in his l a b o r a t o r y and for his assistance, and to Dr. M. G e l l e r fo r m a k i n g available the results of his theoretical calculations and for useful discussions. This study was s u p p o r t e d by the Polish A c a d e m y of Science (Project 09.7.1.) and by project M R - l - 5 of the Ministry of Science, T e c h n o l o g y and H i g h e r Education. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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, J. Giziewicz and D. Shugar, Nucl. Acid. Res., 2 (1975) 2209. M. Wrona, Bioelectrochem. Bioenerg., 6 (1979) 243. M. Wrona and M, Geller, Bioelectrochem. Bioenerg., 6 (1979) 263. F.G. Zachau, Angew. Chem. Int. Edn. Engl., 8 (1969) 711. Z. Ohaski, F. Harada and S. Nishimura, FEBS Lett., 20 (1972) 239. W.R. Folk and M. Yaniv, Nature (London), New Biol., 237 (1972) 165. M. Yoshida, K. Takeishi and T. Ukita, Biochim. Biophys. Acta, 228 (1971) 153. F. Kimura-Harada, M. Seneyoshi and S. Nishimura, FEBS Lett., 13 (1971) 335. P.F. Agris, D. $611 and T. Seno, Biochemistry, 12 (1973) 4331. G.C. Sen and M.P. Ghosh, Nucl. Acid. Res., 3 (1976) 523. K. Watanabe, T. Oshima, M. Saneyoshi and S. Nishimura, FEBS Lett., 43 (1974) 59. V.A. Erdman, M. Sprinzl and O. Ponghs, Biochem. Biophys. Res. Commun., 54 (1973) 942. D. Richter, D. Erdman and M. Sprinzl, Nature (London) New Biol., 242 (1973) 132. E.B. Astwood in The Pharmacological Bases of Therapeutics, L.S. Goodman and A. Gilman (Editors), MacMillan, New York, 1970, 4th edn., p. 1466. 17 L. Slechter and J.H. Hunter, Ann. N.Y. Acad. Sci., 173 (1980) 708. 18 R.K. Ralph and S.J. W6jcik, Biochim. Biophys. Acta, 444 (1976) 261. 19 M.W. Yu, J. Sedlak and R.H. Lindsay, Arch. Biochem. Biophys., 155 (1973) 111. 20 R.H. Lindsay and M.W. Yu, Biochem. Pharmacol., 23 (1974) 2273. 21 C.F. Beck and G.J. Howlett, J. Mol, Biol., 111 (1977) 1. 22 K. Longberg-Hohn, L.B. Gosser and J.C. Kauer, J. Gen. Virol., 27 (1975) 329. 23 P. Zuman and M. Kuik, Collect. Czech. Chem. Commun., 24 (1959) 3861. 24 O. Manousek and P. Zuman, Collect. Czech. Chem. Commun., 20 (1955) 1340. 25 G. Horn and P. Zuman, Collect. Czech. Chem. Commun., 25 (1960) 3401.
183 26 D. Shugar and J.J. Fox, Bull. Soc. Chim. Belg., 61 (1952) 293. 27 B. Stanownik and M. Tisler, Arzneim. Forsch., 14 (1964) 1004. 28 N. Igarashi-Yamamoto, A. Tajiri, M. Hatano, S. Shibuya and T. Ueda, Biochim. Biophys. Acta, 656 (1981) 1. 29 W.C. Schneider and I.F. Halverstadt, J. Am. Chem. Soc., 70 (1948) 2626. 30 L.N. Short and H.W. Thompson, J. Chem. Soe., 168 (1952) 187. 31 A. Psoda and D. Shugar, Acta Biochim. Pol., 26 (1979) 55. 32 T. Matsukowa and B. Ochta, J. Pharm. Soc. Jpn., 69 (1949) 489. 33 D.J. Brown, E. Hoerger and S.F. Mason, J. Chem. Soc., (1955) 211. 34 D.J. Brown. The Pyrimidines, Interscience, New York, 1962. 35 G. Dryhurst, Electrochemistry of Biological Molecules, Academic Press, 1977, pp. 95, 209, 283. 36 V. Vetterl, Abh. Dtsch. Akad. Wiss. Berlin, K1. Med., 4 (1966) 493. 37 V. Vetterl, Collect. Czech. Chem. Commun., 31 (1966) 2105. 38 V. Brabec, S.D. Christian and G. Dryhurst, Biophys. Chem., 7 (1978) 253. 39 V. Brabec, S.D. Christian and G. Dryhurst, Biophys. Chem., 8 (1978) 151. 40 P. Zuman, The Elucidation of Organic Electrode Processes, Academic Press, New York, 1969. 41 D.L. Smith and P.J. Elving, J. Am. Chem. Soc., 84 (1962) 2741. 42 M. Wrona and J. Giziewicz, unpublished results. 43 V. Skaric, B. Gaspert and I. Jerkunica, Croat. Chem. Acta, 38 (1966) 1. 44 G. Briegleb, Angew. Chem., 76 (1964) 326. 45 B. Pullman and A. Pullman, Quantum Biochemistry, Wiley, New York, 1963. 46 M. Wrona, Ph.D. Thesis, University of Warsaw, 1977. 47 M. Geller, Ph.D. Thesis, University of Warsaw, 1977. 48 W. Bahr, P. Faerber and K. Scheit, Eur. J. Biochem., 33 (1973) 535. 49 J.S. Frutton and S. Simmonds in General Biochemistry, P.Z.W.L.. Warsaw, 1966, p. 929. 50 K. Kano, A. Uetake, S. Shimizn, K. Nitta and Y. Yamamoto, Yakugaku Zasshi, 94 (1974) 332.
550-ELECTROCHEMICAL DERIVATIVES
REDUCTION
MECHANISM
169
OF 2-THIOURACIL
MONIKA Z. WRONA University of Warsaw, Institute of Experimental Physics, Department of Biophysics, 93 Zwirki & Wigury, 02-089 Warszawa (Poland) (Manuscript received October 2nd 1982)
SUMMARY * The mechanism of the polarographic reduction of 2-thiouracil and its S-methyl and ethoxy derivatives with stabilized 2-thiol-4-keto or 2-thione-4-enol structure has been studied. 2-Thiouracil is not electroreducible in aqueous solutions. However, it exhibits a strong tendency to adsorption and association on the mercury electrode. 2-Thio-4-ethoxypyrimidine undergoes a 4 e-, 4 H + reduction which involves 2 ereduction of the 3,4 N = C bond, elimination of the ethoxy substituent to form 2-thiopyrimidine and 2 e-, 2 H + reduction of the latter to 3,4-dihydroderivative. For 2-thiomethyluracil, a model compound of the 2-thiol-4-keto form, the electroactive centre is shifted to C-2 and in a 2 e-, 2 H + reduction process the sulphur substituent undergoes elimination, to give pyrimidone-4; the latter can be further reduced at more negative potentials to tetrahydroderivatives (4 e -process). Biological implications of these results have been discussed.
INTRODUCTION F o r several y e a r s this l a b o r a t o r y has b e e n i n v e s t i g a t i n g the e l e c t r o c h e m i c a l p r o p e r t i e s of t h i o p y r i m i d i n e d e r i v a t i v e s [1-5] in the e x p e c t a t i o n t h a t k n o w l e d g e of t h e r e d o x c h e m i s t r y of t h e s e c o m p o u n d s m i g h t l e a d to an u n d e r s t a n d i n g of their biological significance. T h i o p y r i m i d i n e s are m i n o r c o n s t i t u e n t s of v a r i o u s t R N A species [6]. H o w e v e r , t h e i r f u n c t i o n still r e m a i n s a l a r g e l y u n k n o w n a n d i n t r i g u i n g p r o b l e m . T h e e l e c t r o c h e m i c a l r e d u c t i o n m e c h a n i s m of 2 - t h i o p y r i m i d i n e [ 3 - 5 ] a n d 4 - t h i o u r a c i l d e r i v a t i v e s [1,2] has b e e n i n v e s t i g a t e d b y us p r e v i o u s l y . I n this p a p e r the e l e c t r o r e d u c t i o n m e c h a n i s m of 2 - t h i o u r a c i l d e r i v a t i v e s is p r e s e n t e d . If 2 - t h i o u r a c i l o r its d e r i v a t i v e s are p r e s e n t in t R N A t h e y are u s u a l l y l o c a t e d in t h e first p o s i t i o n (5' e n d ) of the a n t i c o d o n o f t h e t R N A m o l e c u l e [7 10]. T h e r e is a h y p o t h e s i s t h a t 2 - t h i o u r a c i l d e r i v a t i v e s p l a y the c o n t r o l l i n g r o l e in the b i n d i n g of
* Abbreviations: ~p, pseudouridine; polyU, poly(uridylic acid); poly s2U, poly(2-thiouridyfic acid). 0302-4598/83/$03.00
© 1983 Elsevier Sequoia S.A.
170
tRNA to ribosomes [11,12]. It has been demonstrated that selective chemical modification of 2-thiouracil in tRNAL2yS by removing sulphur and the resulting transition of 2-thiouracil into uracil does not affect the extent of aminoacylation of this tRNA [12]. However, the efficiency of ribosomal binding and protein synthesis is markedly reduced. 2-Thiouracil derivatives have also been discovered in the T - ~ - C - G sequences of tRNA of Flavobacterium thermophilium [13] where it has been suggested that it is important for the capacity of tRNA to synthesize protein at high temperature. Further studies have discovered the specific interaction of the T - + - C - - G sequence in tRNA of thermophilic bacteria with ribosomes [13-15]. Thus, it is very likely that 2-thiouracil derivatives are necessary instead of thymine in the majority of thermophilic tRNA for the interaction of the T - + - C - G region with ribosomes at high temperature. Numerous studies have also demonstrated that 2-thiouracil exhibits pharmacological [16], anti-viral [17,18] and anti-tumor activity [19], and is incorporated into bacterial, viral, plant and mammalian RNA replacing uracil [20]. Furthermore, 2-thiouracil has been reported to cause some mutagenic effects [21]. The present communication also describes the electrochemical properties of 2-alkylthiouracil, some derivatives of which specifically inhibit polio-virus multiplication [22]. Contrary to the widely recognized biological activity of 2-thiouracil, relatively little is known about its metabolic activity. The biological properties of 2-thiouracil derivatives mentioned above, as well as the fact that catabolic processes of the normal nucleic acid bases involve redox reaction have stimulated out interest in the redox chemistry of 2-thiouracil derivatives. Only a very few other reports on electrochemical studies of 2-thiouracil have appeared. Zuman and Kuik [23] studied the catalytic activity of 2-thiouracil derivatives in buffer solutions containing cobalt ions. Preliminary studies of polarographic oxidation of 2-thiouracil on the d.m.e, were made by Zuman et al. [24,25]. Because 2-thiouracil (I) is able to exhibit keto-enol and thione-thiol tautomerism this study has been extended to include 2-thio-4-ethoxypyrimidine (II) and 2thiomethyluracil (III), which are the models of 2-thione-4-enol and 2-thiol-4-keto structures respectively (Scheme 1). o
S
S (I)
H (IT)
CH3S (1]I)
S c h e m e 1.
Ultraviolet spectroscopic studies of the 2-thiouracil tautomeric equilibria in aqueous medium suggest that a neutral molecule exists in the 2-thiol-4-keto form I [26,27]. However, recently Ueda et al. [28], on the basis of ultraviolet absorption and magnetic circular dichroism studies have been suggested that 2-thiouracil exists as an
171
equilibrium mixture of thione and thiol forms in solution. 2-Thiomethyluracil(III) can exist in aqueous medium in three tautomeric forms (IIIA-C) (Scheme 2). 0
0
OH
"itrA
~B
11TC
Scheme 2.
Ultraviolet, infrared and dipole moment data [29,30] unequivocally exclude the existence of form IIIC, and suggest that forms IIIA and IIIB are present in a 55 : 45 ratio [31]. EXPERIMENTAL
Chemicals
2-Thiouracil was a product of Fluka A.G. 2-Methylthiouracil, m.p. 200°C, was prepared according to the procedure of Matsukowa and Ochta [32]. 2-Thio-4-ethoxypyrimidine was synthesized by the procedure described by Psoda and Shugar [31]. Pyrimidone-4 was prepared from 2-thiouracil according to the procedure of Brown [33]. All coumpounds were checked for chromatographic homogenity and by comparison of melting temperatures and U.V. absorption spectra with appropriate compounds [34]. Apparatus and procedure
Electrochemical methodology, instruments and methods of calculation of the electrode kinetics parameters have been adequately described elsewhere [4,5]. RESULTS A N D DISCUSSION
2- Thiouracil d.c. and a.c. polarography It has been found that 2-thiouracil is not polarographically reducible in aqueous medium. However, it is strongly adsorbed on the surface of the d.m.e.a.c, polarograms of 2-thiouracil exhibit a marked depression of the base current at potentials where the electrode is positively charged (Fig. 1). Over the pH range 4.3-8.8 and at about the 0.3-0.5 m M concentration level, a very well-defined pit is observed. Further increase of 2-thiouracil concentration causes no further increase in the depth
172
<
~.
4.15
pH4.3
/ 3.6
pH 7.1
3.6 pHlO/ f,,), /- 3.6
/
f~
/ /
I
- 1.O
I
I
-O.6
- 0.2
2.4
//
1.2
"~ i
4.8
48
2.4
/ //iI
j~/ll/i I
k
1.2
1.2 ..~.~-" U(V)
I
!
I
-1.0 - 0.6 -0. 2
i
-- 1.0
-
0.6
2.4
I
- 0.2
Fig. 1. Dependence on p H of the alternating current polarographic behavior of 0.5 m M 2-thiouracil in Britton-Robinson buffer (0.5 M ) at 25°C. ( - - I I ) Background.
of the pit, but the potential range over which the sharp depression occurs increases (Fig. 2A). This behavior is most marked at pH 7, i.e. for a relatively low concentration of 2-thiouracil the pit extends over the largest potential range (Fig. 2B). As the p H is changed the potential range of the pit decreases. Above pH 8 the extent of the base current depression decreases and at pH > 10.5 completely disappears (Fig. 1). Bearing in mind that p K a of 2-thiouracil is 7.74 [26], this behaviour indicates that the depression is caused by adsorption of the neutral form of the molecule. Above
A
/"
3.0
/I
/
- 0.1
/
>
1.8
/
.I
0.3
" 1\
L-.-J
I
dCV) I
-08
I
-0.4
a.c.
06
-0.5
pH I
4
I
I
6
I
I
8
I
I
10
Fig. 2. (A) polarograms of 2-thiouracil at various concentrations in p H 7 Britton-Robinson buffer (0.5 M). ( - - - - I ) Background; ( X - X - X ) 0.1 m M ; ( . . . . . ) 1 mM: ( ) 4 m M . (B) Potential range of the sharp depression (pit) observed in a.c. polarography of 0.5 m M 2-thiouracil as a function of pH.
173
p H 8 a new depression appears at about - 0 . 1 V. This depression is located at a positively charged electrode and its occurrence at a p H above p K a of 2-thiouracil suggests that it corresponds to the adsorption of the anionic species. Depression of the base current at potentials close to - 0 . 5 V and at low concentrations, followed by formation of a sharp pit in a.c. polarograms at higher concentrations, has been observed for other biological pyrimidines and purines [35]. The pits have been attributed to intermolecular association of the adsorbed species which seem to form a compact film on the electrode surface [36,37]. The model of such adsorption behaviour for pyrimidine and purine derivatives assumes the existence of two adsorption regions with respect to concentration [38,39]. In the so-called dilute adsorption region the molecules exhibit a flat orientation, with the plane of the base ring parallel to the surface of the electrode. At higher concentrations the molecules undergo a surface reorientation so that the planes of the rings are nearly perpendicular to the electrode surface. Such effects are thought to be controlled by interaction of the dipole moments of adsorbate molecules with the charged surface of the electrode. In the perpendicular orientation a strong stacking interaction between the adsorbed molecules occurs and association takes place, causing formation of the pits in a.c polarograms. Interestingly, the association of 2-thiouracil in the adsorbed state occurs at a relatively low concentration of 0.5 m M (e.g. for uracil it was reported [38] to occur at 24.1 r a M ) and it is significantly more pronounced at neutral p H than in acid or alkaline medium.
2- Thio-4-ethoxypyrimidine d.c. and a.c. polarography Over the p H range 2.7-12, 2-thio-4-ethoxypyrimidine was found to give two d.c. polarographic waves, as illustrated in Fig. 3A. Wave I shows up at p H < 6 with a limiting current considerably in excess of the anticipated for a diffusion-control process. The temperature coefficient of wave I was about 4 . 0 % / ° C . The height of the polarographic wave increased with drop time much more slowly than expected for a diffusion-controlled wave. The dependence of the wave height on depolarizer concentraction in the range 0.1-1.0 m M was linear. However, for concentrations above 1 m M a saturation effect was exhibited. Above p H 7 wave I gradually decreased in height and completely disappeared at p H 8. The variation of the half-wave potential, UI/2 with p H for wave I is given by the equation U1/2 = [ - 1.140 - 0.060 pH] V over the p H range 4.0-7.8. The properties of wave I point to its partial catalytic nature at p H < 6 and to kinetic control at higher pH. As wave I disappears at about p H 8, simultaneously wave II appears at more negative potentials. Its height is p H independent and U1/2 shifts linearly more negative with increasing p H according to the equation U1/2 = [ - 1.740 - 0.025 pH] V over the p H range 7-11. The limiting current of wave II is proportional to the concentration of the depolarizer over the range 0.1-1.0 m M . The drop time and temperature
174
6~
6 \
q
4
X 4 ;~
:<
;4
X
2 pH I
6
I
8
I
~
1
I
10
I
2
I
4
8
5
Fig. 3. Dependence of diffusion current constant (la) on pH for: (A) 2-thio-4-ethoxypyrimidine: (B) 2-thiomethyluracil at the d.m.e. (O) Wave I; ( x ) wave II.
d e p e n d e n c e of the wave II limiting current are indicative of diffusion control at all p H values. Over the p H range 2.7-6.5, 2 - t h i o - 4 - e t h o x y p y r i m i d i n e causes a base current d e p r e s s i o n in a.c. p o l a r o g r a p h y , indicative of a d s o r p t i o n p h e n o m e n a at the electrode surface (Fig. 4). Below p H 7 this depression of the base current occurs n e a r the p o t e n t i a l of zero charge, suggesting that a n e u t r a l f o r m of the d e p o l a r i z e r is a d s o r b e d . This suggestion is s u p p o r t e d b y the fact that the m a g n i t u d e of the d e p r e s s i o n decreases with increase in p H over the p H 7 e m b r a c i n g the p K , of dissociation of 2 - t h i o - 4 - e t h o x y p y r i m i d i n e . A t a p o t e n t i a l of - 1.0 V a b r o a d desorption p e a k is observed, a n d at m o r e negative p o t e n t i a l s a r e d u c t i o n p e a k appears, whose s u m m i t p o t e n t i a l ~ c o r r e s p o n d s to Ul/2 of wave I (Fig. 4). This p e a k
/
pH 4.3
/A L -t6
[
;'
/J 1/
5.4-
pHz8 //
,
phl14
r~
]
"~
il
/.,/1] :.----)!v,, u
u(v) -12 -o.8 -0.4
~
--1.6 -1.2 -0.8 -0.4
4.2 3.0 1.8
-1.6 -1.2 - Q 8 -0.4
Fig. 4. Dependence on pH of alternating current polarographic behavior of 0.5 mM 2-thio-4-ethoxypyrimidine in Britton-Robinson buffer (0.5 M) at 25°C. (-- - - --) Background.
175 decreases in height with increasing p H and completely disappears at p H 6, i.e. two p H units less than in the case of the d.c. polarographic wave. There is no observed a.c. peak corresponding to wave II, indicating the irreversibility of the wave II reduction process. Above p H 7 a.c. polarograms exhibit an additional depression at around - 0 . 2 V, which is undoubtedly associated with the adsorption of the anionic form of 2-thio4-ethoxypyrimidine.
Electrolysis and coulometrv During the course of electrolysis at wave I potentials of 2-thio-4-ethoxypyrimidine, a high current flow was observed. Furthermore, it was not possible completely to eliminate the polarographic wave even after m a n y hours of electrolysis. The electrolysis current remained at a high value and the solution p H increased. Such behavior indicates that a major part of the current-controlling process is the catalytic reduction of hydrogen ions. As a result coulometric measurement of faradaic n-values were unsuccessful. However, it has found that prolongated electrolysis led to a gradual decrease of the U.V. absorption band of 2-thio-4-ethoxypyrimidine ()~ ..... = 276 nm at p H 7), and to formation of a new U.V. band ()~,~,x = 250 nm p H 7). Thin-layer chromatography indicated formation of a single ultimate reduction product. The course of electrolysis at wave II potentials was more rapid. Coulometry gave average n-values of 3.9 _+ 0.1. The same product was formed during reduction at wave II as in the case of wave I. It was also found that the U.V. absorption and chromatographic properties of this product were identical to the two-electron reduction product of 2-thiopyrimidine, identified as 3,4(6)-dihydro-2-thiopyrimidine
[4]. Electroreduction mechanism of 2-thio-4-ethoxypyrimidine The observed dependence of the wave I height and U~/2 on p H suggests that protonation of the depolarizer precedes electron transfer [40]. Thus, wave I is associated with electroreduction of the protonated form of the depolarizer. However, at higher p H values and at more negative potentials there is also possibly reduction of the neutral form of the depolarizer in the wave II process. It has been found that both processes lead to the same ultimate product--3,4(6)-dihydro-2-thiopyrimidine, formation of which requires the uptake of four electrons. More detail regarding the electrode mechanism was obtained from analysis of electrode kinetic parameters (Table 1). Since the theoretical value of a should be ca. 0.5, the experimentally determined ~n~ and p values suggest that two electrons and one proton are involved in the rate-controlling process of wave I, and two electrons are transferred in the rate-controlling process of wave II. For wave II the experimental value of p --- 0.42 is difficult to explain. However, the proposed electroreduction mechanism may be represented by the initial electron attack at the N 3 z C 4 double bond (Scheme 3) as proposed for reduction of other pyrimidines having this double bond available i.e. cytosine [41], 4-thiomethyluracil [1], 4-methylpyrimidine [41]. Under polarographic
176 TABLE 1 Effect of pH on the rate-determining step in the polarographic reduction of 2-thiouracil derivatives ( c = 0.5 m M )
Compound
pH range
Wave
d Uj/2
Aa
an,, h
ph
dpH (s2EtU) c
5- 7 8-10
1 II
0.060 0.025
0.059 0.053
0.93 1.02
0.94 0.42
(Mes2U) c
3- 6 3- 6
I II
0.050 0.085
0,043 0,040
1,25 1.32
1.06 1.91
Pyrimidone-4
3- 6
I
0.085
0.042
1.26
1.83
" A, slope of logarithmic plot. b Average values for given pH range. c s2EtU; 2-thio-4-ethoxyuracil; Mes2U: 2-thiomethyluracil.
conditions the reduction process terminates after the transfer of two electrons and formation of compound III (the height of wave I at pH 6-7 and wave II correspond
H
H
(I)
(11)
2e'2H+
~
Z
I 2e, H+
;
OC2H5
H (r~)
I
-C2H5OH
H
s
H
H (v)
S
H (~)
Scheme 3.
to a 2 e - process). However, coulometry gave n-values of about 4 and prolonged electrolysis led to formation of 3,4(6)-dihydro-2-thiopyrimidine (V) indicating the
177 elimination of the ethoxy substituent. After elimination of the ethoxy group, 2-thiopyrimidine (IV) is formed which immediately undergoes a further two-electron reduction to give the ultimate product (V).
2- Thiomethyluracil d.c. and a.c. polarography 2-Thiomethyluracil yields two polarographic waves over the p H range 1.5-8.5, whose heights and U1/2 values are pH-dependent (Fig. 3B). Wave I. Here, UI/2 shifts linearly more negative with increasing pH; U~/2 = [ - 0.935 - 0.050 pHI V over the p H range 1.5-7.0. The height of wave I between p H 2 and 6 is pH-independent and its diffusion current constant is indicative of a two-electron process. Within this p H range, the observed linear dependence of the limiting current on h 1/2 and on depolarizer concentration (0.1-1.5 r a M ) , and a temperature coefficient of 2 . 1 % / ° C point to diffusion control of the wave I current. At p H > 6, wave I gradually decreases in height and completely disappears at p H 8.1. The limiting current increases with h, but less than predicted by the square-root relationship, hence it was concluded that wave I between p H 6 and 8 is partially under kinetic control. Wave IL This exhibits very similar behavior (Fig. 3B). The linear dependence of U1/2 on p H is given by the equation U1/2 = [ - 1.055 - 0.085 pH] V for the p H range 1.5-7.5. Between p H 3 and 5 the wave is constant in height and the dependence of the limiting current on drop time, temperature and depolarizer concentration indicate the diffusion-controlled nature of wave I. At p H > 5, wave I gradually decreases in height and disappears at p H 7.5. Within this p H range the wave shows increasing kinetic control of the current. At p H ~< 2.5, both polarographic waves exhibit very large current values which are strongly dependent on pH, indicating that the reduction process is accompanied by a catalytic hydrogen discharge. The neutral form of 2-thiomethyluracil is only slightly adsorbed on the mercury electrode near the potential of zero charge. A t - 1.0 V a gradual desorption of 2-thiomethyluracil occurs and at more negative potentials two faradaic peaks may be observed, corresponding to the d.c. polarographic reduction waves. The heights of the peaks and their Us values depend on p H in the same way as was observed for waves I and II; for the first peak, Us = [ - 0 . 9 0 - 0.050 pH] V and for the second peak, Us = [ - 1.080 - 0.085 pH] V over the p H range 2-5.2. Electrolysis and product(s) analysis During electrolysis at wave I potentials the liberation of methyl mercaptan and disappearance of the U.V. absorption band of 2-thiomethyluracil (2~. . . . 230 nm, ~,,,~ = 265 nm, X-,,x2 = 290 nm at p H 6.5) were observed. Chromatographic analysis of the electrolysed solution indicated formation of a single product which exhibited =
178
an U.V. absorption spectrum w i t h ~maxt = 222 nm, X,.~ = 250 nm, ~,,a~, = 255 nm at p H 6.5. After termination of the electrolysis at the potentials of wave I the second wave remained. Coulometry gave an average n value of 2.0 + 0.1. Comparison of the U.V. absorption, chromatographic and electrochemical properties of the wave I reduction product with authentic pyrimidone-4 proved the identity of both compounds. Electrolysis at wave II potentials led to the elimination of both polarographic waves. Methyl mercaptan was evolved and the U.V. absorption spectrum of 2thiomethyluracil disappareared. After termination of the electrolysis the solution showed only residual U.V. absorption. Coulometry gave an average n value of 5.6 + 0.2. Chromatographic and spectrophotometric analysis of the electrolysed solution gave evidence of the formation of a major product with residual U.V. absorption and traces of a second product exhibiting an U.V. absorption band at ~max = 305 nm. Formation of the same product has been observed upon electroreduction of pyrimidone-4 [42]. These results clearly indicate that wave II is due to reduction of pyrimidone-4 formed upon electroreduction of 2-thiomethyluracil at potentials of wave I. The major reduction product of pyrimidone-4, having residual U.V. absorption, has been identified as a 1,2,5,6-tetrahydropyrimidone-4 [42]. Formation of this product requires reduction of both double bonds of the pyrimidine ring and the uptake of four electrons. The second product, exhibiting an U.V. absorption band a t ~kmax = 305 nm (pH 7) has not been identified. However, it is probably formed by two-electron reduction of one of the two possible tautomeric forms of pyrimidone-4 [42]. The chemical nature of this product is under current investigation. Interestingly, Skari~ et al. [43] have reported formation of a compound showing very similar U.V. absorption properties during partial catalytic hydrogenation of 1-methylpyrimidone-4. Electroreduction process Spectroscopic studies [31] give evidence that in aqueous medium 2-thiomethyluracil may exist as an equilibrium mixture of two tautomeric forms II1A and III B (scheme 2). The course of electrolysis and coulometric data indicate that wave I is due to the two-electron reduction of 2-thiomethyluracil to pyrimidone-4. The properties of wave I and the formation of the single product suggest that both tautomeric forms undergo the same reduction process. Elimination of methyl mercaptan during this process indicates that the initial electron attack is directed at the N ~ C bond adjacent to the sulphur substituent or directly to the C - S R bond. On the basis of experimental data it is not yet possible to elucidate the actual mechanism. However, additional information can be obtained from the electronic structure of 2thiomethyluracil, see below. The dependence of the wave height and U~/2 on p H suggest that only the neutral and not the monoanionic form of 2-thiomethyluracil ( p g a 7.5) is polarographically reducible. The analysis of the electrode process kinetic parameters (Table 1) indicates that two electrons and one proton are involved in the rate-controlling process of wave I, and that two electrons and two protons are involved in the rate-control-
179
ling process of wave II. The kinetic parameters of wave II of 2-thiomethyluracil are identical to those determined for pyrimidone-4 (Table 1), hence supporting the proposed reaction mechanism presented in Scheme 4. The process occurring at the Wave I
O
-HSCH 3 H3S
CH3S
CH3S
H
CH3S
H
Wave TT
4e,
~
HN'~ H
Scheme 4.
potentials of wave II of 2-thiomethyluracil corresponds to the reduction of pyrimidone-4 and is shown in this scheme 4 in a somewhat simplified form. The major route leading to formation of 1,2,5,6-tetrahydro-pyrimidone-4 is presented. However, more detailed studies [42] with 1-methyl- and 3-methylderivatives of pyrimidone-4, which have stable tautomeric forms A and B respectively, have revealed that such a route is given only by tautomer A. Tautomer B undergoes a two-electron reduction tO an ultimate product exhibiting U.V. absorption at longer wavelengths ( - 305 nm) than the starting compound. Because the contribution of the B-form to the tautomeric equilibrium of pyrimidone-4 is around 40% [34] and its reduction potential is slightly more negative than that for the A-form, the two-electron reduction of B is a minor reaction, leading to only trace amounts of the second reduction product. The nature of this product, as well as the electrochemical mechanisms leading to its formation, are under current study [42]. CORRELATION OF ELECTROCHEMICAL BEHAVIOR A N D Q U A N T U M CHEMICAL PARAMETERS
The polarographic potential, UI~ a, for a reversible process, diminished by the difference of the solvation energy of reactants, is an experimental measure of the electron affinity of molecules [44]. In fact, account should also be taken of the contribution of the adsorption energy of reactants to U~f2a and to proton participation in the rate-determining step. The energy of the lowest unoccupied molecular orbital (LUMO) is a theoretical measure of the electron acceptor affinity [44]. Correlations of U~/~ and L U M O energy are valuable because they offer a simple experimental method for verification of the theoretical value of LUMO, which in turn has been further correlated with the chemical, biochemical and pharmacological
180 activity of molecules [45]. The reliability of the correlation of a q u a n t u m - m e c h a n i c a l p r e d i c t i o n a n d p o l a r o g r a p h i c U1/2 values d e p e n d s on the calculation a n d m e a s u r e m e n t s having been m a d e on identical m o l e c u l a r species. U n f o r t u n a t e l y , theoretical calculations are b a s e d on an idealized, n e u t r a l gas-phase molecule a n d a reversible electron-transfer process. In the case of the p o l a r o g r a p h i c r e d u c t i o n of organic molecules m o s t electrode reactions are n o t perfectly reversible. In addition, it is a l m o s t i m p o s s i b l e to evaluate q u a n t i t a t i v e l y the c o n t r i b u t i o n s of the such factors as r e a c t a n t solvation, a d s o r p t i o n energy a n d p r o t o n p a r t i c i p a t i o n to [[Red ~1/2. Neverthel[Red less, Vl/2 values, even for an irreversible r e d u c t i o n process, can be used to estimate qualitatively the electron a c c e p t o r p r o p e r t i e s of molecules a n d can be c o r r e l a t e d with L U M O energies for chemically related c o m p o u n d s . Values of ~l'rRed a n d the L U M O energy a n d its s y m m e t r y for the 2-thiouracil l/2 derivatives e x a m i n e d a n d for the p a r e n t uracil are given in T a b l e 2. U n f o r t u n a t e l y , in the case of 2 - t h i o m e t h y l u r a c i l a n d 2 - t h i o - 4 - e t h o x y p y r i m i d i n e the calculations were m a d e for the i m p r o p e r structures of 2-thiouracil, 2-thiol-4-keto a n d 2-thione-4enol respectively. Nevertheless, in our previous studies of t h i o p y r i m i d i n e derivatives [4,5] calculations were m a d e taking into c o n s i d e r a t i o n the m e t h y l a t i o n of s u l p h u r s u b s t i t u e n t a n d it has been p r o v e d that the effect of m e t h y l a t i o n on L U M O energy, s y m m e t r y a n d electron d i s t r i b u t i o n is negligible. D a t a p r e s e n t e d in T a b l e 2 i n d i c a t e that the presence of a sulphur s u b s t i t u e n t at p o s i t i o n 2 of uracil decreases the L U M O energy and, c o r r e s p o n d i n g l y , increases the electron a c c e p t o r p r o p e r t i e s of the molecules. These results are in qualitative a g r e e m e n t with the changes of the o b s e r v e d U1R/~d values. The influence of thiol s u b s t i t u e n t on the increase of the electron a c c e p t o r p r o p e r t i e s of uracil is greater t h a n observed for the thione substituent. Simultaneously, in the case of thiol derivatives, the s y m m e t r y of L U M O is c h a n g e d from 7r-type to o-type, a n d the d i s t r i b u t i o n of the d e n s i t y of u n p a i r e d electron strongly suggests the C 2 - S R b o n d as TABLE 2 Comparison of U~/edpotentials with LUMO values for uracil derivatives U~fd iV]" pH 5.7
pH 7
pH 8
Uracil S2U c Mes2U c
-__ 1.215
-__ - 1.280
--1.330
S2EtU e
1.480
- 1.560
1.620
LUMO energy b [eV]
Symmetry of LUMO orbital b
0.099 0.065 0.038 d 0.070 0.051 e
~r ~r o 'z" ~r
a //Red ~1/2 at 0.5 m M concn. b Calculated by the C N D O / 2 method, [47, 46]
c s2U, 2-thiouracil; Mes2U, thiomethyluracil; s2EtU, 2-thio-4-ethoxyuracil. d Calculation for 2-thiol-4-keto form of uracil [47,46]. e Calculation for 2-thione-4-enol form of uracil [47,46].
181 the electroactive centre of the molecule. These theoretical results support the conclusion that elimination of mercaptan, observed upon electroreduction of 2thiomethyluracil, is due to the direct reduction of the C2-SR bond rather than to reduction of the C2~N bond followed by former reaction. A similar electroreduction mechanism was observed for 2-methylthiopyrimidine [4,5]. CONCLUSIONS AND BIOLOGICALIMPLICATIONS In aqueous medium 2-thiouracil exists in the 2-thione-4-keto form. This form is not polarographically reducible, whereas it exhibits a strong tendency to adsorb on the surface of the mercury electrode near the potential of the e.c.m. In the adsorbed state the intermolecular association of 2-thiouracil takes place at the relatively low concentration of 0.5 mM. Uracil, by contrast, exhibits this effect at a concentration of about 24 m M [38]. If the critical concentration value where a capacitance pit appears is taken as a measure of the magnitude of intermolecular association, then the results presented in this paper indicate that stacking interactions of electrosorbed 2-thiouracil are significantly stronger than those for uracil. Incidentally, it was reported that the synthetic polymer, poly s2U, is also able to form an extraordinarily stable double-stranded helical structure having a m . p . of 68°C [48]. Under the same experimental conditions the m . p . of polyU is only 8°C [48]. The high adsorption activity of 2-thiouracil and its ability to undergo strong intermolecular association in the adsorbed state might provide a basis to speculate about the possible role of 2-thiouracil in tRNA. Bearing in mind that 2-thiouracil is usually located in the anticodon of tRNA in the so-called wobble position it is possible that 2-thiouracil is involved in the non-specific interaction of tRNA with the ribosome. Indeed, it has been reported that selective modification of 2-thiouracil to uracil markedly decreases the binding of tRNA to the ribosome [12]. However, it is difficult to exclude the possibility that such modification changes the conformation of tRNA in such a way that its binding to the proper site of the ribosome is affected. 2-Thiomethyluracil and 2-thio-4-ethoxypyrimidine, which are the models of the 2-thiol-4-keto- and 2-thione-4-enol forms of 2-thiouracil, are readily electroreducible, but by different mechanisms. For 2-thio-4-ethoxypyrimidine the N 3 ~ C 4 double bond is the electroactive centre of the molecule and the sulphur substituent remains intact upon electroreduction. In the case of 2-thiomethyluracil, the electroactive site of the molecule is shifted to C 2 and the sulphur substituent readily undergoes elimination when the reduction process occurs. Identical electrochemical behavior was observed for the 2-thiopyrimidine series [4,5]. These results might be considered in relation to the metabolism of 2-thiouracil. The mechanism of metabolic breakdown of 2-thiouracil derivatives is not known. In general, regular nucleic acid pyrimidines such as cytosine, uracil and thymine are reduced in organisms to their 5,6-dihydro derivatives and ultimately hydrolysed to derivatives of fl-ureido-acids [49]. Similar to uracil, 2-thiouracil is not electroreducible in aqueous solution. However, its L U M O energy is lower (see Table 2), indicating that introduction of sulphur at the C-2 position of uracil improves the electron acceptor properties of the
182 molecule. Thus, it is p r o b a b l e that 2-thiouracil can be m e t a b o l i z e d in a way similar to uracil. In fact, it has been r e p o r t e d [50] that m e t a b o l i s m of 6-methyl-2-thiouracil in rats leads to f o r m a t i o n of several u r in a r y m et ab o l i t es including 6-methyluracil, 5,6-dihydro-6-methyluracil, fl-ureidobutyric acid and, m o r e interestingly, 2-methylthio-6-methyluracil. T h e latter c o m p o u n d is further t r a n s f o r m e d to the same p r o d u c t wh i ch is observed to f o r m u p o n e l e c t r o r e d u c t i o n of 2-methylthiouracil, i.e. pyrimid o n e - 4 (see scheme 4). This result suggests that the t r a n s f o r m a t i o n of 2-methylthio6-methyluracil in vivo is a r e d u c t io n process. ACKNOWLEDGEMENTS T h e a u t h o r is greatly i n d e b t e d to Prof. G. D r y h u r s t (The U n i v e r s i t y of O k l a h o m a , Dep t . Chemistry, N o r m a n , O K , U.S.A.) for valuable discussions and for criticism of the manuscript, to Dr. E. Pale~ek (Institute of Biophysics, Brno, Czechoslovakia) for the use of e q u i p m e n t in his l a b o r a t o r y and for his assistance, and to Dr. M. G e l l e r fo r m a k i n g available the results of his theoretical calculations and for useful discussions. This study was s u p p o r t e d by the Polish A c a d e m y of Science (Project 09.7.1.) and by project M R - l - 5 of the Ministry of Science, T e c h n o l o g y and H i g h e r Education. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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