281 - Electroreduction mechanism of 2-thiopyrimidine derivatives

BioeZectrochetnistry j.

@

EZecfvoanaL

Elsevier

and

Bioenergetics

Chewa_ 104

Sequoia

S.A.,

(1979)

6 (x979)

__;::

zq3--26r ._

243-261

Lausanne

-

Printed

in Italy

.

’ -..

: :_--. .~s.~:.-~.~~-_-~~~:: __/ _ z_-_::.__.L -_._-I -. :_. ._. __ - i.~_..

282 - Electroreduction Mechanism. of: &hiop&d& Derivatives Part I. Polarographic Study by MONIKA

WRONA

University of Warsaw, Institute of E~eri~eatal physics, 93 zwirki C%Wigury, 02-089 Warszawa, Revised

manuscript

--r-i

received

J&&y

10th

Physics, Poland

Department

of Bio-

1979 -.

The mechanism of the polarographic reduction -of zz-thiopyrimiditie and its N- and S-methyl derivatives, with stabilized tautomerie- thieneor thiol structure, has been studied over. the~normal $i grange; -_Z+Thic+ pyrimidine undergoes electroreduction via -the same -pathway as :-r&6+ The C+$ -atom:-is the trimethyl-z-thiopyrimidine in the thione form: electroactive center and the sulfur substituent -remains intact;: :: In: a I e-, IH+ process a free radical is formed- which dimerizes to-p&hi& identified as 4,4’-bis-(3,+diydro-z-thiopyrimidirie) ~ +nd :the correspondiug C,-, C6 and/or N,-methyl derivatives. For. z-thiopyGrnid@e the free radical can be further reduced (I e- process) to 3,4-dihydro-zz~ thiopyrimidine_ In the use of 4,6-dimethyl-2-thiomethylpyrimidine, a model compound of the thiol form, the electroactive ~centeris:shifted to the carbon C-2 and the sulphur substituent undergoes elimination.~ --

:_ i Introduction

..

Recent electrochemical studies of nucleic &ids ~&ovid& int&&tiri~ information about the structure of these biopolymers and- tlieir-iritemc-~ tions with a charged electrode interface.1-4 Since. poltiror+hic~actiGit~ of nucleic acids is essentially determined by their constituents, systematic studies of the electrochemical -properties of the nuelei_c bas& ha_ve,heen undertaken.5~7 In our laboratory, interest has been ‘. directed : toyi?d thiopyrimidines which are the minor components of _..certain.trarisfer RNAs. +Thiouracil has been studied- by-- US previously?~~=_.~ Currently our attention has been turned to the derivatives of z&riouracil.~~~.Initially a simpler compound, z-thiopyrimidine (I), was-. examined_ _Becar_ise:.of .

Wrona

244

thiol-thione tautomerism our study was extended to NL or S-methyl derivatives with stabilized tautomeric structure thione (III) or thiol (IV), respectively. Polarographic properties of forms II and III were investigated by us earlier. 10 In this paper, the results of more advanced methods are reported, in&ding compounds I and IV. It affords a deeper insight into the reduction mechanism, the reactivity and the tautomerism of z-thiopyrimidine.

RZ 1. R,=R*=H II.

R,=CH,,

IV. U, =R2=CH3 R,=H

111. R, = Rr =CH

Only a few othe r reports on electrochemical studies of thiopyrimidines have appeared. ZVWAW and I
Experimental

a-Thiopyrimidine (I) was prepared according to the procedure of BOARLAXD et nZ.fS ~,64imethyl -z-thiop_ykn.idine (II) and r,q,6-trimethyl-z-thiopyrimidine (III) were synthesized by the procedure described earlier, lo 4,6-Dimethyl -z-thiomethylpyrimidine (IV) was prepared by standard methodszo All compounds were chromatographically homogenous and e_xhibited correct melting points and UV spectra.

&c. poiarograms were recorded on a RADIOMETERpolarograph Polariter PO4, using.a dropping mercury electrode (DALE.) with the following characteristics : the mercury flow rate at open circuit HL = 2.85 mg s-~, drop time t = 4.10 s in distilled water, at a mercury pressure

Wrona

246 &sults

and discussion I-rrr

The &-pounds I and II may be in thione or thiol form, on the contrary compound III has a fixed thione structure. However, all three compounds show an identical electroreduction mechanism which supports the argument that all of them have the same thione structure.

In the pH range 5-1~3 derivatives I-III exhibit a reduction wave (wave I) whose height corresponds to the transfer of one electron (Fig. I, 2).

Fig_ I_ DependenceofdiffusioncurrentconstantI~andpotential at the DX_E_

C,.on

pH

fora-thiopyrimidine

For compounds- II-III this wave is masked by an overlapping catalytic wave (wave II) ; in the case of derivative I both waves are well separated_ Up to pH 7 wave I is preceded by a well-defined adsorp‘CVithin the pH range 7.7-10.7 an additional reduction tion pre-wave. wave (wave III) occurs for compound I. Its height corresponds to a one electron process.

Reduction

Mechanism

of n-Thiopyrimidiae

Derivatives.

6

8

‘\

Linear

Us

z’s_ pH

10

297

12

PH

Fig. 2. Dependence of diffusion current constant 1, and potential yl-n-thiopyrimidine at the D.M.E.

Table I. rivatives.

I.

uyt

relationship

for

0.5

mM

P&Z*

\ave

pH

range

7-14

I

on

pH for I&6-trim&h-

n-ghiopyrimidine

de

Compound -f

I

i

pKI*

_-

I r-35

i

III

i

Ir

;

s-50

2.80

I

+ 0.036 pH r.oz$o-+ O-140 pH

o 7-6 7-6 - 12.2 7-7 - IIt0

0.770

o 7-5 12.2 7-5

1.070 +

0.020

1.220

0.140 pH

I-590

+

pH

!

III

1

3-15

IV

!

2.50

I !

“.

I

-

i

I I II III

o-

12.2

0 -

5.7

4 .2 6.0 -

5-7 8.7

o-740 + 0.075 PH o.go5 1.36 I-545

+ 0.05s

pH

+ 0.034 pti

i

* pK

values

taken

from Ref. 33. 34.

Wave I_ - For derivatives I-II the wave height and potential Uy=are pH-dependent (Fig. I, Table I). The changes in slope of the 77% vs. pH curves can be correlated with pK, values (Table I). In the case of the non-dissociable derivative III the wave is constant in height over the normal pH range (Fig. 2). Moreover, there is no- break in the U%

245

Wrona

vs. pH curve. with associated

Over thk pH range 5--x0 wave f is diffusion-like in nature adsorption

effects.

At pH < 6 the limiting

current 1l is

propo~ional to the mercury pressure fz (corrected on the back pressure). With increasing pH this dependence becomes more diffusion-like, i.e. the proportionality of 11 0s. X83is observed_ The average temperature coefAt pH < 6.5 X2 is proportional to ficient is of the order of 2.1 %/C. the concentration of depolarizers I-III over the range o.z~-2 m&1, at concentration less than 0.25 m&1 only an adsorption pre-wave is observed. A well-defmed pre-waves for all three compounds po:nts to strong adsorption of their reduction products. At pH > 7 the adsorption of the reduction products decreases, leading to the disappearance of the pre-wave. In the pH range 7.5-10 for derivatives II-III, at concentration higher than 0.5 mfif a typical post-wave appears, due to the adsorption of their osidized forms. Bekeen pH g-5-10.5 the adsorption effects disappear and wave f is actually under di&sion control. At pH > 10 wave I

for compounds I-II begins to be under kinetic control. Wave II - This weve appears only in an acid medium with ex(Fig. r, 2). traordinary current values, strongly pH-dependent Ifs height does not depend on 1awhile the dependence of Ir on the depolarizer concentration exhibits a saturation effect for concentration higher than These properties, in conjuction with the disappearance of 0.7 m&1. wave II in the presence of surfactants and its behaviour during electrolysis (v&Yez?zfi-n),indicate that this is a surface, catalytic wave. Its height depends on pH in the same way as wave I, Wave whereas-the -potential U s is pH.-independent (Fig. I, Table I). The prop-

rrr. -

erties of wave

III

indicate

its pure diffusion

character_

a-c. $oZnro,ova$lt_v a-c. polarographic studies confirm the high surface activity of the examined compounds and their reduction products (Fig. 3 I-i-C).

The base current for the solutions of derivatives I-11 forms deep Their location at the potential depressions at the potential of --O-I V. where the electrode is positively charged suggests that a charged species is predominantly adsorbed_ These depressions appear for the first time

at a pH close to pk”, for the dissociation of compounds I-II

(in the case

of compound I at pH 5-1 and for II at pH 56), gradually becoming deeper with increasing pH, and at pH > pK2 their magnitude is almost pH-independent _ This pH behaviour and also the lack of such a depression for the non-dissociable derivatives III indicate that anionic forms of compounds f-II are involved in the adsorption at ---O-I V. Near the potential of zero charge of DX.E. (Le. -o.6 V) an additional small depression appears for compounds III-III. Its association with adsorption of the neutral molecules is supported by the increase of the depression magnitude with an increase in pH over the pH embracing of molecules II-III. the pK, of the protonation The deep depressions formed sharp pits which appear at more negative potentials, sufkient for the reduction of molecules I-III (Fig.

Reduction Mechanism of I-Thiopyrimidine Derivatives. I.

-t6

-1.2 -0.8 -0.4 -1.6 -1.2 -0.8 -0.4

-1.6 3.2

- - 249

-0.8 -0.4

~~~~~

-1.6

-I2

-0.8

-0.4

-1.6

-1.6

-12

-0.8

-0.4

-1.6

-1.2

-08

-1.2

-0.8

-0.4

-0.4

I

-1.6

-1.6

-1.2 -0.8

-1.2

*

-0.4

-0.6

I

-04

Fig- 3_ Dependence on pH of a.Remating current poIarographic behaviour of : (A} z&opyrimidiue ; (B) 4,6_dimetbyl-z-thiopyrimidine’ :- (C} r,q.6-timethyf-z-thiopyrimidine; (-----) o-5 Y&I ; ((- - -> backgrbund electrolyte base .cument. 12 mM;

3 A-B). The front of these depressions corresponds to the potential Us of the adsorption pre-waves, implying their connection with the surface Appearance of sharply defined pits activity of a reduction products. gn (ES_ polarograms is usually identified with the occurrence of associa-

tion befween Adsorbed moledules.

This phenomenon has been reported

many biological purines and pyrimidines.2%~s For the examined compounds the magnitude of these depressions decreases with increasing pH, indicating a drop of adsorbability of the reduction products a$ higher -pH. for

Wrona

250

At concentrations higher than o-g m&1 the reduction peak appears inside the depression. Its faradaic character is testified by the linear dependence of peak height on concentration over the range 0.3-3.3 m&1, and by the %greement of the summit potential U, and UY, of the wave I. The marked decrease of the peak height in relation to the theoretically eszected value indicates a low degree of reversibility of the electrode reduction process. In acid medium a second peak appears, corresponding to the catalytic wave II. However, the peak corresponding to reduction wave III is not observed, pointin g to the irreversibility of the reduction process occurring at the potential of wave TIIMacroscale

electrolysis

and cozcloinetvy

\Vave I_ - During the course of electrolysis a gradual decrease of waves I and III is observed, while wave II remains unaltered_ This is accompanied by the disappearance of the characteristic absorption band of the compounds investigated, Amax275 nm, and the appearance of a new band at 255 nm. With each of the three starting compounds electrolysis led to formation of a white precipitate, chromatographically homoThe coulometric measurements were consistent with a onegenous. electron process. Wave l-1. - The course of electrolysis at the potential of wave II is very similar to that of wave I_ However, even prolongated electrolysis can not eliminate wave II, in accord with its origin as the catal_ytic wave of the reduction products_ Wave III- - The electrolysis of wave III occurs simultaneously with wave I and leads to the formation of two products having very similar UV absorption spectra but differing in chromatographic mobility and solubility_ One of these shows an absorption band ‘h,, 255 nm at pH 7-o and is identical with the reduction product of wave I. The second product, more soluble in water, shows an absorption band A- 250 nm at pH 7-o. Coulometric measurements led to a-values between 1.3-1.6, dependent on the concentration of the electrolyzed solution, which, in conjuciion with the formation of two products, suggests a mixed mechanism operative in a-thiopyrimidine electroreduction. The variation in the determined electron number results from a tendency to shift from

the I c- to the 2 e- mechanism (virle i@a).

The one-electron character of wave I suggests the formation of dimeric products_ Because of their insolubility in water they were easily isolated and analysed by elementary analysis and mass spectrometry. For the reduction product of z-thiopyrimidine elementary analvsis gave : C, 43-30 y0 ; H, +28 y. ; N, 24-95 o/o ; this corresponds to the dimer C,H,,,X &, denoted D, below_ The mass spectrum of D, showed the following peaks at e/rrt : 226 (20 %) L corresponding to the dimer C,H,,,N,S,;

Reduction Xechatism of z-Thiopyrimidine Derivatives. I.

z5r

corresponding to the protonated and the 1x3 (+oo %)t II2 (5I %) neutral forms of the parent a-thiopyrimidine, Additional intense peaks at e/wz : qo (1.6 %), 86 (IO %), 85 (IO %), 76 (21 %). 69 .@I %) and 59 (27 %) represent fragmentation products of dimer molecule. Similar results were obtained earlier lo for. dimer reduction products of 4,f5dimethyl-2-t~op~~~ne (D,) and for- 1,4,~t~ethyl-~-t~op~~dine (DJ. The proposed structures for the dimers D, are shown in Scheme II.

0,:

R,=R,

4:

R, =CH, , R,=H

43:

R,-

4-i

R2=CH3

Dimerization may occur via formation. of 4,1’, 5.5’ or 6,6’ bonds. In the case of the dimers D, and D, the 4,4’ and 6,6’ bonds are identical because of symmetry about the 2,s position. ~nfo~unately for dimers D, poor solubility in various solvents, and instability in DNSO precluded the running of NMR spectra. Attempts to obtain suitable crystals for X-ray diffraction have thus far been unsuccessful, Thus, we have no direct evidence for the proposed structure of the dimer D,. Acceptance of such a structure is based on our earlier studies of +-oxopyrimidine derivatives.24.‘5 The electroreduction mechanism of +oxopyrimidine is similar to that of the z-thiopyrimidine derivatives, and leads to the formation of a dimer, possessing analogous physico-chemical properties. Its structure was identified as +4’(6,6’)-bis-[Z;,4(6)-dihydropyrimidone-23; Dimers having on the basis of crystallographic and NNR studiesa” identical structure to D1_aare also formed during photochemical reduction of z-thio- and z-oxopyrimidine derivativesC7 Substitution of the methyl group at N---T differentiaks th& positions 4 and 6_ Thus it would be necessary to consider if dimerization occurs via the;&’ or 6,6* bond. Some evidence for 4,4’ dimerization results from studies of CPK models. The presence of the methyl groups at positions N-I and C-6 makes the formation of a 6,6’ dimer impossible because of steric hindrance whereas this is not the case for dimer 4,4’_ Additional evidence for the proposed structure of D,, is provided by their susceptibility to electrolytic and photochemical oxidation to the parent monomers. The polarographic are o.xidation potentials vs 01 and spectral properties of -dimers D, reported in Table 2_ Electrochemical oxidation of the dimers D,_3 leads to partial regeneration of the corresponding parent- monomers I-III, The reactions verified spectrophotometrically and polarographicallywere, however, not quantitative due to the fact that the monomers themselves undergo further o_xidation at potentials very similar zto di-

Table

z_

pyrimidine

Physico-chemical

Dimer

-

uyzo= P=

-

W)

of

dimer

D2

(

g-035

D,

1

--0.020

quantum

I

kur(nm) PH

7-o

-o.ozo

D,

@

properties

reduction

products

of

z-thio-

derivatives_

-_

Ema

103

Q (mol einstein-I)

7-o

255

14-6

0.24

254

20.4

o-35

22-S

0.23

258

I

yield.

of this effect has been mers D1_3_ However, no systematic investig&ion carried out_ On irradiation in aqueous medium at 254 nm, the dimers D, undergo photochemical oxidation to quantitatively regenerate the parent The course of this reaction may be followed by the monomers I-III,

CL1-

320

.

340

Fig_ + Photochemical conversion, by irradiation at 254 nm. .of dimer reduction product of 2thiopyrimidine with quantitative regeneration of z-thiopyrimidine as shown by isosbestic points at 232 and 263 nm (pH 7-o)_ 0’. absorption spectrum of dimer D, time o_ I’-5’ refer time of irradiation in minutes. After 5’ irradiation, the reaction was essentially completed.

.

Reduction

Mechanism

.

_

.

:

.

of 2--Thiopyrimid~e.:Dedrivativesi:-:~I'S:-:::-:i:-:-:=-~:3~

v e r y r a p i d d i s a p p e a r a n c e of t h e a b s o r p t i 0 n . b a n d : o f : ~ t h e : d i m e r . ~ n 0 1 e ~ e ~:-~ a n d t h e s i m u l t a n e o u s : appearance: of-the:-:characteristic~;.U3/?:~ibsi~0/i~ of t h e p a r e n t m o n o m e r (Fig.. 4).. T h e q U a n t n m ~ y i e l d s : f o r : p h 0 t ~ i d i ~ 6 ~ f f ~ i n t h e presence of t h e pure_oxygen axe-.listed in-~Table:.~:~. F;U~he/;~=-st~die~:~ prove t h a t t h e solution: c o n c e n t r a t i o n of -m o l e c u l ~ "o:-xyge~n~166h~s~ t h e rate of this reaction:- .Removal of-O~:considerably!i.intiibit~s~:tfie~p~6~dissociation process suggesting that-molei~ulaf:Oxygen~plays~:-:-i:h~i:_r~l~!~f~: a n o x i d a n t (i.e. e l e c t r o n accept0r).: F u r t h e r detailed:sft~i]i~S.:6n-:-£he~imf?~? of the .excitation states of the_dimers, and-kinetid/me-asu~eiiiefitS,~iiiia--~i~i help _to c l a r i f y t h e m e c h a n i s m of. t h e : p h o t o o x i d a t i 6 n ~ - . o f : / ~ e ~ : ~ D ~ : ~ The second r e d u c t i o n - p r o d u c t was~obtained ~dufingithd~l6Ct~-bi~s~; • of w a v e I I I of 2 - t h i o p y r i m i d i n e . - The-similarity~ of~itS-:U~.-.ab~ti614~ -: s p e c t r a to t h a t of t h e d i m e r D,, as,weii as its~better solubifify.~i~d~:hi'gfi:::: c h r o m a t o g r a p h i c m o b i l i t y , i n d i c a t e a molecule h a v i n g a S t _ r i / c t h r e ~ r : : to t h a t of d i m e r D~, b u t w i t h a s m a l l e r m o l e c u l a r W e i g h t . : I t wa~:iden "tiffed i as 3,4(6)--dilaydro--2-thiopyrlmldine. - :-. -:~.:-.-:;.:_..::.%:.~:~.~,-.:.~ ~:~-,:,-

El¢cgroredu-ction mechanism of compounds I-III"-

:" ;:~:';:":-!:::~:S-:~:::~_:~i~-~

K i n e t i c p a r a m e t e r s - o f t h e eleetrode:process for :compo-:uhds~I~Ii! :~ are l i s t e d i n the T a b l e 3- Since theoretical-value.!0f-~ shohld~:~.a~.~0~5~ a n d for t h e : c o m p o u n d s - e x a m i n e d undoubtedly,-;n~-~m~ i,---:the:~0btained~ -

Table 3-

Effect of p H

on the rate-determ~ng step :in the:'pol~graphid~rL~luc~i':=

.tion of 2 - t h i o p y r i n m i d i n e

derivatives

(c. =

o.5m3¢):

" 3--. :

--:": -::: :-f:(~::L:- ~'~:--.

£

Compound

%Vave

range

pH

4.27.87.8-

6-4 9.6 9.6

I_ ..

v i .

III

d U~. dpH

0.036 o.I4o 0.005

•

o.049

.

II

4-77 .0 7-5 -- x o . 2

I I

.

.

.

..

o:o66 _-.

III

6 . o -- I 2 . I

I

0.075

IV

3-3 5.o 6.o

I II III

o

--

5.o 5-7

a A ~ slope of logarytbmic plot. b Average values for given pig range~

0.058 o-o34

x.xo _ 0.8 : :0.83: x-9 0,7.4 - " _ 0 : - : - : -

-

.

0.065

0 . 0 2 0

0.I4 0

-

• 0.065 i~ 6 " 0 7 4 :

,=

~ _-:.

?~

-

.

_

o.s3_ " -0:8~'~

.

o.3: -

-. L9:-:~i. "~i _.

\Vrona

254

values of a are probably heightened by the adsorption effects. For the same reason the estimated values of ~5 are also disturbed -and must be interpretated rathar cautiously_ Nevertheless they reflect some tendency in the electrode mechanism. The data suggest that for wave I one proton is involved kr the rate-determining step at a pH less than pK, of compounds I-II, and two protons at pH > pK,. For the non-dissociable compound III one proton participates in the rate determining step over A gradual decrease of wave current centering all esamined pH-ranges. at 2-3 pH units higher than the dissociation constant pK,, observed for derivatives I-II, indicates the polarographic inactivity of their anionic forms. This is also confirmed by the constant value of the wave height in the case of the non-dissociable compound III. Thus, at pH < pK, neutra1 species are transported to the electrode and in the rate determining step one electron and one proton are transferred, whereas at pH > p& dominating in the solution anionic forms are transported but they must be protonated before the electron transfer. Accordingly, in the pH > pK, one electron and two protons participate in the rate controlling step. In alkaline medium, where proton activity decreases, the wave disappears. ZUXAX2* proves that in the case where only one wave is observed on polarographic curves, and where its height remains pH-independent but U5gis shifted, the general sequence must be : proton, electron (H+, e-). On the basis of the obtained results it is difficult to be sure whether fast proton-transfer precedes the electron-transfer because the same shift of UY, can be formally obtained for simultaneous electron and proton transfer. However, since simultaneous electron and proton-transfer is not probable the obtained results suggest rather the following sequence : (H+, e-) at pH < pK, and (He, Hf, e-) at pH > pK,. Kinetic parameters of wave III indicate that only one electron participates in the rate determining step. On this basis the following scheme is proposed for the reduction mechanism of molecules I-III : R+H++e-+kH RH e

step determining U,,r

I/Z Dimer

(3)

RH+e--+RH-

(4

RH-

-+ H’ +

step determining Uy~m RH,

The proposed pathways for electroreduction of derivatives I-III are presented in Fi_g: 5_ Irreversibihty of both electron transfer steps are probably caused by a rapid chemical reaction involving the product of the electron transfer processes. As regard to wave II, its properties show that it is due to the catalytic hydrogen discharge process involving stable reduction products, i.e. dimers D,. It is possible ‘that the parent compounds I-III also

Reduction

Mechanism

of

+Hte- _

z-Tbiopyrimidine

Derivatives.

&

Hx.

sJ+,*e:"

sJdR,

I.

-

. -

.’

R2

RI-R,=HorCH,

Fig. 5. Proposed vatives

reaction

(r-rrrj.

sequence

for the electrochemical

behaviour

of z-thiopyrixnidine

possess catalytic activity, but tl$s is masked by the much higher activity of their electroreduction productsConcentration studies of way-e II and its disappearance in the presence of surfactants (gelatin. or Triton X-100) suggest that it is a typical catalytic surface wave. Many organid compounds containing a sulfur atom possess a marked ability for producing such waves.“%30 The general mechanism for catalytic hydrogen evolutiop by sulfur-containing molecules, formulated by BRDIGKA~~ atid extended by 31_4IR4NOVSKII 29 for other organic catalysts, postulates that catalytic activity is due to the unshared pair of electrons on the sulfur, o%ygen or nitrogen atoms to which a proton may attach itself- Such -a process can be &hematically described as follo& ; R=

adsyption

S-R=&

,_.

R= R=

SH+&+~-+R=S~+I/~H~-

rr +

where R = S represents the sulfurYcontaining molecule. In case of surface catalytic Waves, the- adsorption- bf a &fur_ catalyst on the electrode is the factor limiting the rate df .the electrode process. Any change- in adsorption lead to an increase in activity- or inhibition of the electrochemical step. For tliis reason, _a&hqyg@_,PC+ oxidized and reduced forms of compounds I-III possess the structure suitable to producing catalytic waves, the catalytic activity.- of the reduction products is much lugher, because of their higher adsorbability ‘ ,; _.-CT dn the electrode in acid solutions.

Wrona

256 Sk&es

of 4,6~~~zet~y~-z-~?tiol~retltylpyrint2iEi~te

(IV)

d_c_ +olmogmfihy

4,6-Dimethyl-z-thiomethylpyrimidine exhibits three polarographic waves within pH r-g_ The pH-dependence of the wave height and Uy, are shown in Fig. 6 and in Table I_

2

2

4

6

4

6

8

8

Fig. 6. Dependence of the diffusioncurrentconstant ID and the potential methyl-z-thiomethylpyrimidine at the D_X_E.

. CT% on pH for 4,6-di-

wave I is under effective Wwe I_ - Within the pH range IS-4.0 diffusion control_ Its height is constant and corresponds to the transfer of three electrons. At pH > 4 the wave gradually decreases and disappears at pH about 6_ Over this pR range the wave shows kinetic control of the current_ Wave II. - It appears at the pH range 4-z-5-7 gradually increasing in height_ Simultaneously wave I disappears, but the sum of the heights of both waves remains constant_ The properties of wave If indicate its purely diffusion character_ Wave III. - It appears at pH 6 simultankously with ,the disappearance of wave II. At this pH it attains maximum value of current_ With increasing pH the wave height decreases sharply, disappearing at pN g. Within the pH range 67, wave III has a diffusion character, whereas at pH > 7.0 the wave shows kinetic control of the current. as. ~okzrogmphy .A deep depression of the base current near the DXE_ potential of zero charge (Fig. 7) indicates strong adsorption involving the neutral form of the molecules. This is sripported by the increase of the depression

Reduction Mechanism of n-Thiopyrimidine

Derivatives. I.

‘57

magnitude at pH higher than pK of proton&ion for 4,6-dimethyl-zthiomethylpyrimidine. At pH > 3 gradual desorption of the molecules occurs before reduction (broad desorption peak at li’ = -1.1 V) and at more negative potentials reduction peaks were observed. The potentials U, for all three observed peaks correspond to the polarographic Us values, whereas the heights of the peaks are much less than theoretically expected for even a completely reversible one-electron process. This indicates a low degree of electrode process reversibility.

-1.6

-1.2

-0.8

-0.4

-1.6

-1.2

-0.8

-0.4

Fi,s 7. Variationin ax. polarographicbehaviourof 1.0 mM 4.6-dimethyLz-thiomethyIpyrimidine with pH. (- - -) base current of backsound electrolyte.

MacroscnZe

electrolysis

am?codoinet~y

Wave I. - During electrolysis the UV absorption of 4,6-dimethylz-thiomethylpyrimidine (A,, 250 nm at pH 4.0) disappears and a new, poorly defined band appears at ZSI nm. It is accompanied by the simultaneous disappearance of wave II and the liberation of HSCH,. The UV absorption of the reduction product slowly disappears indicating its decomposition in aqueous medium. Coulometkic determination of the faradaic n-value gives an average value of 2.9, suggesting formation of a free-radical species. The properties of the product are very sin&?

to the one-electron reduction product of pyrknidine, identified by _ ELVING et n1.32 as 4,4’-bis-(3,4-dihydropyrimidme) Wave II. - The course of electrolysis is very similar to that of: wave I. Coulometry gives the mean value IZ = 4. The product shows a weak absorption band at A,285 nm (in H,O). Its properties are similar to the_ product of two-electron reduction of pyrimidine, identified as 3,4-dihydropyrimidine. 32 the electrolysis liberation of HSCH, was Wave III. - During Coulometry gave an average value of 92 = 5.5. The product observed.

showed only residual UV absorption similar to tetrahydropyrimidine.32

~58

Wrona

Kinetic parameters of waves I-III are reported in Table III_ The slopes of the waves deviate from the theoretically expected values for the reversible waves. There appeared to be two distinct regions of constant a-ma values. One corresponds to wave I, the second to waves II-III. On the basis of a being cn. 0.5 the data suggest that wave I involves two electrons and one proton in the rate-controlling process. Disappearance of wave I at pH about 3 pH units higher than pK, for protonation, indicates that at potentiak of wave I only protonated species are reduced. Kinetic parameters for wave II indicate that only one electron participates in the rate-controlling stepFor wav-e III there 1s no very meaningful kinetic data because the wave is under effective diffusion control only at pH 6. The data show that, similarly to wave II one electron participates in the potentialdetermining step, but in this case the potential UK depends on the pH, Nevertheless on the basis of estimated value of p = 0.4 it is difficult to explain the role of proton in the electrode process. Additional information is obtained from the pH behaviour of waves II-III. Wave III appears at a pH where wave II disappears; its height decreases very sharply with increasing pH and a plot of the wave height ZJS-pH has the shape of a dissociation curve. Such behaviour can only be esplained by the assumption that the electroactive form is generated from the electroinactive one at a rate that depends on pH_ This pH-dependent-reaction can be either an acid-base reaction or a chemical reaction. Within the pH range where waves II-III occur, q,6-&methyl-a~thiomethylpyrimidine esists in neutral form, whose stability has been confirmed by spectrophotometric studies. Therefore only a fast protonation reaction, preceding an electron-transfer would esplain the obserx-ed pH-behaviour of waves n-m. The difference between the polarographic pK’ -value, determined for wave III and spectrophotometric pK, value is about 7 pH units, implyin g that protonation of the depolarizer procedcs at the surface of the electrode”* 14ceordingly, at pH < pl ph’, the transport of neutral molecules of compound IV to the electrode begins, but fast surface protonation of depolarizer precedes an electron transfer. For wave II protonation is very fast and it does not affect U,. At a higher pH (wave III) the rate of protonation decreases affecting the wave height and potential- i73~. For waves II-III in the rate-determining step one eIectron is transferred. The free radical species formed readily undergoes further reduction, according to a three- or four--electron process (Fig. S).

Reduction Mechanism of Wave

I

Wave

II:

a-Thiopyrimidine

Derivatives.

I.

-

259

p3

3

Wave

wave It I-II

III +2e: 2H*

*

or

:+TCH H

Fig.

3

fi? i-i

S_

Proposed reaction at the D.M.E.

sequence -for electroreduction

of ;2,6-dimethyl-z-thiomethylpyrimidiie

conch5ions

s-Thiopyrimidine in aqueous solutions exists in the thione form. This form is readily electro-reducible by a mechanism very similar to that observed earlier for other pyrimidines having similar structure, derivat~ves.S,1°,“4,45r33,34 The double bond such as z-osopyrimidine N, - C, is the electrozictive centre of molecules. In the first one-electron reduction step a free radical is formed which can be reduced at more negative potentials to 3,+dihydro-z-thiopyrimidine or dimerizes before further reduction, giving dimer 4,4-bis-&+dihydro-z-thiopyrimidine) . This dimer is readily oxidised electrochemically or photochemically regenerating parent molecules of a-thiopyrimidine.

Wrona

260

In the case of the ~&dimethyl-z-thiomethylpyrimidine - model compound of thiol form of +thiopyrimidine, the electroactive center is

shifted to carbon C-z and the electroreduction process leads to the elimination of the sulfur substituent. Methylation sf C,, C6 and N, positions in z-thiopyrimidine does not change the electroreduction mechanism but affects adsorbability of molecules I-III and their reduction products and shifts the potential Us Methylation at the sulfur atom completely to more negative values. changes the mechanism of electroreduction and the potentials involved the in electrode mechanism_ These phenomena are discussed on the basis of the electronic structure of compounds I-IV in subsequent paper.

Acknowledgments The author is greatly indebted to Professor DAVID SHUGAR for valuable discussion and for criticism of the manuscript, and Dr. EMIL PALECEK (Institute of Biophysics, Bmo. Czechoslovakia) for generally making possible a-c. polarographic facilities and for useful discussion_ I would also like to thank Dr. J_ GIZIEWICZ for the synthesis of some pyrimidine derivatives and to R. RIEDEL for expert technical assistance. This work was partly supported by the Polish Academy of Sciences within the project og.7.1. I should to thank Professor P. J_ ELVING for his incredibly thorough review and deep criticism of this paper.

References 1 2 3 4

6

6 7 6

9

10

11

E. PALECEK, XXI. p_ 3

Methods

iw E~zzymology.

&ademic

Press, N.Y.

(1971)

Vol.

V.

BRABEC and E_ PALECEK, Bio#hys_ Chem_ 4 (1976) 7g H. BERG. J_ FLEMLLIXG and G_ HORN. Bioelectvochem. Bioenerg. P_ V_U_EXTA. H. N~~RX-BERG and P_ KLAHRE, Bioelectrochem.

2 (1975) 287 Bioenerg. ‘1

(19741 457 P-J_ ELVING, J.E. ~'R_EILLY and C-0. SCHMAKEL. Methods of Biochemical Analysis, Interscience, New York (1973) Vol. 21, p_ 287 G_ DRYHURST, P-J_ ELVIXG. TaZanta 16 (1969) 855 P-J_ ELV'ISG. Ann_ N-Y. Acad_ Sci_ 158 (1969) 124 N_ WROXA, 33. CZOCHR_LSICA. D_ SHUG-AR, J_ EZectroamzL Chem_ Itevfacial EZectrochem_ 68 (x976) 355 K BRONX and B. CZOCHRALSKA, J_ EkctroanaZ. Chem. Interfacial Electro&em_ 48 (1973) 43.3 M. \~'ROKA, J_ GIZIEWCZ and.D_ SHUGAR, Nucleic Acid Res. 2 (1975) zzog P. Zunr..xx,31. KUIK, CoUect_Czech_ Chem. Commun. 24 (1959) 3861

Red&ion

Mechanism

of 2~Thiopyri&idi~e D&ivatives_~:~_--1 1 -:-:,. 26i ;

12

-.

._

f

26

J_'BARDES. RR HERR and T. EuR~J~. J_-'Am:.Chern.SOC,~~~-.(~~~~)~~~~:. O_ MANOUSEK and P. ZUMA~; CoZZect. Czech. C/rem_ Coin~u~~~~?O~(~~js~)_=,~~~o 1: G. HORN and P. &.%a~. CoZZect.Czech_ Cfrem. G+zmurr. "5_~(xg~,~);.34~i:~.-~ -.. M BREZINA and P. ZUMAN, Polarography in _Me$i$p; $och~mzktrp. and -: Pharmacy, J. Wiley, Interscience.New York- (r&8). -I ‘-_-’ -. .-T-_;’ -. W.F. S~~YTH, G. SVEHLA and P. Z&AN,- Ana& Chim. A&a 51 -(rg7+)-_$3-'. W.F. SMYTH, G. SVEHLA and_ P. ZUBKAN, Ana~~.C~im.--~ctd'52-.(ig‘70) :I2g.. W.F. SLEUTH. P. ZV~;~AN and G_ SVEHLA. J_ EZectrkzaZ_-.Chim_ ~I~t.&f&iaZ _. .._ EZectrochem_ 30 (1971) IOI M.P. BOARLAND and J.F. MCONIE, J. Chem; SOL 1951. 1218 ‘i. .A.G. WILLIAMS, -J_ Am. .Chem_ SOL 37 (Igrg) 594 : L. MEITES, PoZarographik Techniques. 2nd edition, J_ Wiley &-So~,New_: York (rg63) V. VETTERL, J_ EZectvoanaZ. CJzem. I~~terfac&zZ. EZe&ochem. 19 -(i&S) 169.. _ V_ VETTERL. BioeZectvochem. Bioenerg. 3 (x976) 338 B. CZOCHRALSKA and D_ SHUGAR. Biochim. Biophjs. Actu 281 (1972)-r. B_ CZOCHR_LSKA. M. WRONG and D. SHUGAR, Bioebctvpchem. Bioker&?_ -1.

26

B. CZOCHR~SK-k,

‘13 14 15

16 I7 18

16 26 21

22 23 24

T.

(1974)

27 26

29

36 31 32 33 34

40

__” -

:_.<

D. SHUGAR, S.K. AROR& R.B. BATES and-RX CUTLER, _: . -I_-: J_ Am. Chem. Sot. 99 (x977) 2583 K.H. PFOERTNER. HeZv_ Chim. Acfn 58 (1975) 865 -_ -- -: -_ ..~Ii‘- .- -:.P. ZUMAN, The Elzrcidation of Ovgarric EZectrode~Processes, Academic P&S, New York (1969) S.G. ~!L.IRANOVSKII,CotQZyticand Kinetic waves in PoZarogrcrphy; Izti. tizi&&; Moscow (1966) _: --. S-G_ MAI~~NOVSKII, J. EZectroanaZ. Chem. 6 (Ig63)_77 -.-_ R_ BRDIISKA. LVature (London) 139 (x937) 330. 1029 _D.L. S~XITH And P_J_ ELVING, J_ Am_ Chem. Sot. 84 -(Fg62) 2741 : _ . _ -.I)-Interscience,New Ycrk (1962) D. J_ BROIVN, The Pytimidilies, B. STANOVNIK ahd M_ TISLER. Arzneim. For&h. 14 (x964) 1004