Indian 10umal of Chemistry VoI.38A, October 1999, pp. 1015-1023

Electrochemical oxidation of 6-hydroxy-2,4,5- triaminopyrimidine at pyrolytic graphite electrode

Rajendra N Goyal*, Anil Kumar, Neena Jain & Priyanka Gupta

Department of Chemistry, Uni versity of Roorkee, Roorkee 247667, Indi a

Received 15 July 1999

The electrochemical ox idation of 6-hydroxy-2,4,5-triaminopyrimidine (I) at pyrolytic graphite electrode has been studied in phosphate buffers of pH range 2.5-10.8. Under cyclic voltammetric conditions, the 2e, 2H+ ox idation of this compound is fo und to give alloxan, which has been characterized on the basis of IR, mass and NMR spectral data. The kinetics of the decay of the UV -absorbing intermediate generated during electrooxidation of compound (I) has been studied and fi rst order rate constants for the di sappearance of UV -absorbing intermediate have been calculated. A tentative mechani sm in which electrode reaction is followed by chemical steps (EC) for the oJ\idation ofI has also been suggested.

Pyrimidine in various substituted forms occurs in every living cell , usually as a constituent of. larger molecules. In the form of cytosine, uracil and thymine it occurs in three of the five common bases found in nuc leic acids. The human system does not require pyrimidine derivatives in diet but can synthesize them de novo from the products of protein and carbohydrate metabo lism 1,2 . Besides that, the pharmaceuti cal potentia l of pyrimidine derivatives has also been recognized. The antineuritic action of Vitamin B" a derivative of 2-methyl-4-aminopyrimidine has been well establi shed3

,4. A number of other pyrimidine derivatives have a lso fo und applications as antibiotics5 and anti vira l6,7

agents. Work concerning preparation and application of pyrimidine derivatives as antimicrobia l8, anti­inflammatory and antia llerg ic agents has also appeared in literature9

. Barbituric acid and its a lky l and thio derivatives have a lso been used as anaesthetic agents 10,11. In view of the importance of pyrimidine nucleus in nature, many attempts have been made to determine pyrimidines in biological sampl es. Recently, S inghal and Kuhr l2 have presented a method for the electrochemical detection of purine and pyrimidine ' based nuc le ic acids by us ing s inuso idal vo ltammetry at a copper e lectrode . HPLC has also been w idely used for the analysis of pyrim id in e derivatives in various bio logica l fluid s13

-18.

T he im portance of electrochemical techniques in probing in formation related to oxidation-reduction reactions in biosystems was rea li zed long ago; the polarographic behaviour of pyrimidines has been studied by various workers l9,20 Smith and Elving21

have investigated the electroreduction of unsubstituted pyrimidine at DME and five reduction waves between pH 0.5 and 13 .0 were observed. T he involvement of s ix e lectrons leading to the formation of tetrahydropyrimidine has been suggested. Zeng et al22

. have also reported the reduction of mercaptopyrimidine at si lver electrode. Thus, most of the studies reported in literature deal w ith the reduction of the C=N bond of pyrimidines. Thi s paper deals with the oxidation chemistry of 6-hydroxy-2,4,5-triaminopyrimidine (I), a well known precursor of guanine. The amino groups at positions 4 and 5 in the pyrimidine were selected because they are used in the synthesis of purines .

T he product of oxidation has been isolated, characterized and a complete interpretation of the redox chemistry of compound (I) is presented .

Materials and Methods 6-Hydroxy-2,4,5 -triaminopyrimidine sulphate,

obtained from Aldrich Chemical Co., USA, was used as received. Phosphate buffers23 of ionic strength 1.0 M were prepared from reagent grade chemicals.

Pyro lytic graphite electrode (PGE) used as a working e lectrode for the e lectrochemical studies was fabricated in the laboratory by the reported method24

I016 INDIAN J CHEM, SEC. A, OCTOlBER 1999

and had a surface area of ca. 0.01 cm2. The pyrolytic

graphite surface was renewed each time by polishing on a 600 grit metallographic disc, washing with a jet of distilled water and drying by touching onto soft tissue paper. An average of at least three runs was taken for determining peak current values. The reference electrode used was a saturated calomel electrode (SCE) and the auxiliary electrode was a platinum wire.

Cyclic sweep voltammetric studies were performed on a Micronics cyclic voltammeter coupled with an Omniscribe x, y-t series 2000 recorder. A Cypress Model CS-1090 microprocessor controlled electro­chemical system was used for the double potential step chronoamperometric studies. The pH of the buffer 'solutions were measured using a Century digital pH-meter model CP-90 l -P after due standardization. Coulometric studies were carried out using a potentiostat fabricated in the laboratory, having the facility for three-electrode system.

Controlled potential electrolysis was carried out in a H-type cell using a pyrolytic graphite electrode (6x 1 cm2

) as the working electrode and cylindrical platinum gauze as the auxiliary electrode. The number of electrons involved in electrooxidation was determined by connecting a coulometer in series. UV spectra were recorded using either a Beckmann DU-6 or a Shimadzu UV -21 OOls spectrophotometer. IR spectrum of the product was recorded using a Perkin Elmer 1600 FTIR spectrophotometer. 'H NMR spectrum of the product was recorded on a Varian XL 300 spectrometer and mass spectrum was recorded using a Joel, JMS 0300 instrument.

Procedure For recording voltammograms, stock solution

(0.6 mM) ofthe compound (I) was prepared in doubly distilled water. The stock solution (2 ml) of compound (I) was then mixed with 2 ml of the phosphate buffer of desired pH. The solution was degassed by passing nitrogen for about 10-15 min and the voltammograms were then recorded .

The progress of electrolysis was monitored by recording spectral changes at different time intervals. For this purpose, electrolysis was carried out at potential 100 m V more positive than peak potential of oxidation peak. For recording UV /Vis spectrum, about 2-3 ml of the so lution from the electro lysis cell was transferred each time to I cm quartz cell and spectrum was recorded .

The double potential step chronoamperometric

studies were performed for a 0.3 mM solution of compound (I) at pH 3.5 by varying the step width values in the range 50 ms to 500 ms. The other parameters selected were (a) initial £=0 mY; (b) forward step £=600 m V; (c) reverse step £=0 m V; and (d) noise filter = 5 fs.

Products of electrooxidation of 6-hydroxy-2,4,5-triaminopyrimidine were characterized at pH 3.5 and 6.9. For this purpose, about 10 mg of the compound (I) was exhaustively electrolyzed in an H-cell at potential 100 m V more positive than the oxidation peak potential. Nitrogen was bubbled throughout the course of electrolysis. The progress of electrolysis was monitored by recording cyclic voltammograms at different time intervals. When the oxidation peak completely disappeared, the electrolyzed solution was removed from the cell and lyophilized. The separation of products was achieved by using gel permeation chromatography in which a glass column (75 x 15 cm2

)

packed with Sephadex G-IO (bead size 40-120 ~l!1) was used. The dried material obtained was dissolved in distilled water and passed through the column using doubly distilled water as an eluent at a flow rate of 10-12 ml Ih. Fractions of 5 ml each were collected. The absorbance of different fractions was measured at 210 nm and plotted against the volume. The first peak (150-190 ml) emerged from the column gave positive test for phosphate and hence was discarded. The second peak (200-270 ml) was collected separately, lyophilized and was analyzed by mp, JR, 'H NMR and mass spectral measurements.

Results aUld Discussion In cydic sweep voltammetry, compound (I) at a

sweep rate of 100 mVs-' exhibited one well-defined oxidation peak Ja in the entire pH range studied, when the 'sweep was initiated in the positive direction. In the reverse sweep two reduction peaks Ie and lIe were observed below pH 4.3. In the pH range 4.3-7.9, only peak Ie was noticed . Peak Ie formed a quasi-reversible couple with peak Ia in the entire pH range . Below pH 4.3 peak lIe formed a quasi-reversible couple with peak lIa observed in a subsequent sweep towards positive potentials. The quasi-reversi ble nature of the redox couples Ia l Ie and IIa lIle was established by the small difference (25 m V) between cathodic and anodic peak potential s. Some of the typ ical cyc lic vo ltammograms of compound (I) are presented in Fig. I.

The peak potentials of anodic peaks Ia and 11 3 were dependent on pH and shifted to less positive

500

1 400

~ 300

a. w

200

100

0 , 0 r

1

GOYAL et al.: ELECTROCHEMICAL OX IDA TlON OF A PYRIMIDINE DERIVATIVE

A B c

~--~~~~--~~j~/~I~~~--~I~/r7~~~--~~~~--~~~ 0.0 I 1.0 0.5 0.0' 1.0

Potential (V) vs.SCE ..

Fig. I -Cyclic voltammograms of 0.3 mM solution of 6-hydroxy-2,4,5-triaminopyrimidine at PGE; pH (A) 2.5; (8) 4.9 and (C) 6.9; sweep rate =\00 mVs· l

.

1017

potentials with increase in pH. The Ep vs pH plot for the anodic peaks I. and II. is presented in Fig. 2. The dependence of Ep on pH can be expressed by the relations:

12

Fig. 2-0bserved dependance of Ep on pH for the voltammetric oxidat ion peaks of 0.3 mM 6-hydroxy-2,4,5-tri aminopyrimidinc at PGE; sweep rate = 100 mVs· I

Ep(I.) (PH 2.5-5.0] = [625-59.5 pH] mV vs SCE

Ep(I.) (PH 5.0-10.8] = [5\0-35 .7 pH] mV vs SeE

Ep(I1 .) (PH 2.5-4.3] = [3 \ 0-56.8 pH] m V vs SCE

1018 INDIAN J CHEM, SEC. A, OCTOBER 1999

5

i 4

-S III

I'C

" ~!.-.. 3 > E

<{ 2

.:Z " 'I ~1

0 0 4

Fig. 3 - Yariation of the peak current function (ip IVv) with the logari thm of the sweep rate for 0.3 mM 6-hydroxy-2,4,5-triaminopyrimidine atpH 3.5.

Thus, the Ep vs pH plot for peak la shows two linear segments having intersection at pH ca. 5.0 which represents the pKa of compound (I). This pKa va lue is similar to the value reported in the literature25

. Th us, the species predominating in the solution above pH 5.0 is a cationic one having positive charge at amino group at position 5 (Ia).

rhe peak potentials of cathodic peaks Ie and lIe were also dependent on pH and shifted to more negative potentials with increase in pH. The dependence of cathodic peaks Ie and IIe on pH can be expressed by the relations :

Ep (Ie) [PH 2.5- 7.9] = [590-68.2 pH] mY vs SCE

Ep (lIe) [PH 2.5- 4.3] = [310-68.2 pH] mY vs SCE

The ratio of peaks Ie Iia was found to increase with increase in pH. The va lue of Ie lIa in the acidic pH range was 0.5 which increased to 0.8 in alka line pH range. To determine whether reduction peaks Ie and lIe are related to the oxidation peak Ia or are due to independent reduction of compound (I), cyclic voltammograms were a lso recorded by initiating the sweep towards negative potentials . The absence of reduction peaks c learly indicated that the cathodic peaks are due to the reduction of the oxidat ion products generated in peak Ia reaction .

The peak current for peak I. increased linearly with increase in concentration of compound (I) in the concentration range 0.0 1-0.5 mM. The values of the peak current function (ipV·

1/2) increase with increase in

sweep rate (Fig. 3) suggesting strong adsorption of the

Table I - Ratio of peaks leila observed during electrochemical stud ies of 6-hydroxy-2,4,5 -triamino pyrimidine at di ffe rent

sweep rate values at PG E

Sweeep rate (mY S· I) Ie' (IlA) la' (IlA) Ie' I I: (IlA)

25 5 15 0.33 50 12 27 0.44 75 26 50 0.52 100 16 25 0.64 150 20 27 0.74 200 31 40 0.77 300 55 69 0.79 400 60 75 0.80 500 60 72 0.83 600 85 100 0.85 700 93 100 0.93 800 160 165 0.96

* Average of at least three replicate determinations.

compound (I) at the surface of pyrolyt ic graphite electrode26

. The ratio of peaks IJ lle was found to remain practically constant in the entire concentration range (0.01-0.5 mM) of the compound (J), thereby indicating that the species responsible for peak lIe is independent of the con~entration of reactants as we ll as the pririlary oxidation product.

The peak current of peaks Ia and Ie also increased with increase in sweep rate in the range 25 mYs· 1 to 800 mYs· l

. The ratio of peaks Ie lIa increased with increase in sweep rate as shown in Table I. However, the ratio did not reach 1.0 even at a sweep rate of 800 mYs· l

. This behaviour indicates that the species responsible for peak le is unstable and undergoes competitive chemica l reactions. The peak potential of peak Ja also shifted to more positive potentials with increase in sweep rate in the range 25 mYs· 1 to 800

mYs·l. The plot of Mp/2 lfi log v liS log v was S shaped

and hence the nature of the electrode reaction was

estab li shed as EC in which charge transfe r is followed by irreversible chemica l reactions27 .

In view of the fact that peak Ie was al ways smaller than peak la, it may be concluded that the primary electrode reaction product of peak Ia undergoes rapid fo llow up chemical reactions. The kinetics of this process was studied by using double potential step chronoamperometry27. The chronoam perograms of 0.3 mM of compound (I) at pH 3.5 were recorded at different times of potential reversal in the range of 50 ms to 500 ms. The resulting current was measured as a function of time. A typica l chronoamperometric curve at step width 75 ms is shown in Fig. 4A. The value of RI (i .e the normalised ratio of the backward current to the forward current) was determined and

+

-

GOY AL et al.: ELECTROCHEMICAL OXIDATION OF A PYRlMIDINE DERlV ATIVE 1019

5

t 3r-

~ 1-E

c: .. ... -1 ... r-:;J

u

-3 -

- 5 .1 I I I I I 0 30 60 90 120 150

T im~(m5) _

Fig. 4A-A typical chronoamperometric curve for 0.3 mM 6-hydroxy-2,4,5-triaminopyrimidine at PGE; pH=3.5.

plotted against time (Tr) as shown in Fig. 4B . t he tll2

was determined from R, vs Trp lot when R, is equal to O.S and the half- li fe of the intermediate generated was found to be ~60 ms. Thus, it is concluded that the intermediate formed in peak la reaction undergoes very rapid hydrolysis.

The number of e lectrons (n), involved in the oxidation was determined in a conventional H-cell using a coulometer in series and the value of n was

found as 2.0 ± 0.2 .

Spectral changes The UV spectra of compound (I) were recorded in

the entire pH range to determine its pKa value. In the pH range 2.0 to 4.9, compound (I) exhibited three absorption maxima at 200, 209, and 269 nm . At pH> 4 .9, the bands at 200 and 209 were replaced by a single absorption maximum at around 21 I nm and the maximum at 269 nm shifted to 274 nm . The absorbance at Amax was plotted against pH and an inflection at around p~ S.O clearly indicated that the pK. of 6-hydroxy-2,4,S-triaminopyrimidine is S.O. This pKa value was similar to the va lue obtained from Ep vs pH plot25

. The spectral changes during e lectrooxidation were monitored at pH 3.S and 6 .9. Studies in alkaline solutions could not be conducted because of the instab il ity of this compound in alkaline medium28 At pH 6.9, compound (I) exhibited two AlIlax at 21 I and 274 nm and a broad bump centered around 240 nm (curve I) Fig. SA). Upon application of potential 100 mY more positive than peak la, the absorbances at 211 nm and 274 nm continuously

1.00

1 ....

0:

0.25

Fig. 4B - Plot of Rl vs Tr observed for 0.3 mM 6-hydroxy-2,4,5-tri aminopyrim idine in phosphate buffer of pH =3.5

Table 2-Rate constant values observed for the decomposition of UV -absorbing intermediate generated

during electrochemical oxidation of 6-hydroxy-2,4,5-triamino pyrimidine at different pH values at PGE

pH

3.5

5.0

6.9

A./nm

215 240 270

240 270

240 270 295

k' /I 0 - 3S"

0.9 1.2 1.0

0.42 0.44

0.44 0.45

* Average of at least three replicate determinations.

decreased . The absorbance in the region 2 1S-2S0 nm first increased (curves 2-S) and then decreased (curves 6-9). The absorption maximum at 274 nm completely d isappeared in the exhaustively e lectro lyzed solution

of compound (I). The absencc of isosbestic point in the region 21S-2S0 nm strongly indicated the role of fo llow-up chemical reactions involved in the e lectrode reaction. In the second set of experiments, the potentiostat was open-circuited after IS min of e lectrolysis and changes in the UV /Vis spectrum were monitored. A systematic decrease in absorbance in the region 240 nm-320 nm was observed . This behaviour indicated that a UY absorbing intermediate is generated during electrooxidation of compound (I) w hich decays in the presence of competitive chemical reactions.

1020 INDIAN J CHEM, SEC. A, OCTOBER 1999

2.0r----:---------------~

1'" Col U r::: ~ 1.0 ... 0 <II .D ~

0.5

COl 0 c: cu .0 .... 0 III .0 <

0.33

0.24

0.21

3.0

t 61 U c: «I f. 1.5 0 11" .0 ~

0.0 I 190 · 216 :~42 294 320

Wavelength (nm)-~

Fig. 5 - Spectral changes observed during the electrooxidation of 0.3 mM 6-hydroxy-2,4,5-triaminopyrimidine at POE [A] pH=6.9; potential 0.35 V vs SeE. Curves were recorded at (1) 0; (2) 5; (3) 20; (4) 55 ; (5) 120; (6) 245; (7) 490; (8) 825 and (9) 900 min of electrolysis. [B] pH=3.5; potential 0.52 V vs SCE. Curves were recorded at (1) 0; (2) 5; (3) 15; (4) 30; (5) 45; (6) 110 and (7) 150 min of electrolysis.

t -1.90

8 ~ I ~

-; -1.66 0

24 36 Time (min.)----+-

O.OoL ___ --;';;;:1 - __ -;:!-.I ----;;f;:;-----.-';;I ------....l o 12 24 36 ·48 Time (min.)

Fig. 6-0bserved variation in the absorbance vs time and log (A­Am) vs time for the decay of UV -absorbing intermediate generatcd during oxidation of compound (I) at pH=3.5, ),,=240 nm.

GOYAL el al.: ELECTROCHEMICAL OXIDATION OF A PYRIMIDINE DERIVATIVE 1021

At pH 3.0, compound (I) exhibited three absorption bands, at 200, 209 and 269 nm. When electrolysis was initiated, the bands at 209 and 211 nm first increased in intensity, merged and after 15 min of electrolysis shifted to a longer wavelength (228 nm). The absorbance in the region 260-290 nm decreased systematically (Fig. 58). The exhaustively electrolyzed solution exhibited a single absorption band around 228 nm. The kinetics of the decay of the UV-absorbing intermediate was monitored at different pH by plotting absorbance vs time curves (Fig. 6). The values of k for the decay of UV -absorbing intermediate were calculated from log (A-Ax,) vs time curves. The decay was found to follow first order kinetics and the values of k observed at different pH were in the range (0.4-1 .2)x 10-3

S-I (Table 2). Examination of Table 2 shows that the value of k at pH 3.5 is almost thrice of that observed at pH 6.9. The higher rate of decay of the intermediate at pH 3.5 indicates that the species responsible for peak Ie undergoes an acid catalyzed reaction.

Product characterization The product of the electrooxidation of compound

(I) was isolated and characterized at pH 3.5 . The freeze-dried material obtained from the fraction collected for the second peak between volume 200 and 270 ml (P2) in gel-permeation chromatography (see experimental) exhibited a single spot in TLC (Rf =0.42). The melting point of this product was 53·C. The IR spectrum of the product exhibited strong bands at 1720 (N-H), 1640 (cyclic>C=O), 1410 (-C-H-), 1260, 1170, 1020, 808 and 780 (C-C and C-N) cm- I and was identical to that of authentic sample of alloxan. The 1 H NMR spectrum of this product further confirmed the structure as alloxan by exhibiting sharp peaks at 8=11.24 (s, N-H); 11.46 (s, N-H) and 8.24 (s, O-H). Further, the mass spectrum of the material also clearly exhibited a molecular ion peak at m/z= 160. Alloxan is reported to b I . . 29 Tl . e e ectroactlve In nature. lUS, cyclic voltammo-grams of alloxan were also recorded at PGE. A well­defined redox couple was obtained for alloxan at the same potentials with similar dEp /dpH value as was observed for peaks lIe lIIa in the case of compound (I) . A comparison of cyclic voltammograms of compound (I) and alloxan is presented in Fig. 7. Thus, peaks lIe and IIa observed have been assigned to the product of oxidation (alloxan) generated in the EC mechanism of compound (I). Hence, it is concluded that the product of oxidation is alloxan monohydrate.

1

A

8

0.5 0.0 -0.5 -to Potential (V ) vs. 5 C E ~

Fig. 7 - A comparison of the cyc li c voltammograms of 0.3 mM of a ll oxan (A) and 6-hydroxy-2,4,S-tri aminopyrimidine (8) in the phosphalc buffer ofpH=3.S at PGE. sweep rate = 100 mV s· l

.

Redox mechanism The experimental evidences presented above

indicate that the electrochemical oxidation of compound (I) proceeds by a 2e, 2H' reaction . Based on the results obtained, electrooxidation of 1 can be

1022 fNDIAN J CHEM, SEC. A, OCTOBER 1999

P(lak la -2e-2H+

>

" Peak Ie 2e+ 2H+

(TIl:)

1 o

HN~O HNJ..N,.lO

H

H2 0 <

H2 0

(1I)

( nn

Scheme-I : Tentative mechanism proposed for the electrooxidation of 6-hydroxy-2,4. 5-triaminopyrimidine.

rationalized by an EC mechanism in which a quasi­reversible electrochemical step is followed by irreversible chemical steps as shown in Scheme I. Thus, the primary electrode reaction involves 2e, 2W quasi-reversible oxidation of the monocation of I to give a protonated diimine II. The species II is highly unstable as can be inferred from the half-life obtained (/ 112=60 ms) by double potential step chronoampero­metric experiments and thus readily undergoes hydrolysis to give IV releasing ammonium ions in the solution. The presence of ammonium ions was confirmed by Nessler's reagent test. The hydrolysis of diimine n to give IV occurs in two steps via the formation of quinoneimine (ill). The first step of hydrolysis results in the formation of quinoneimine

III and ammonia which converts to ammonium ions in ac idic medium. Further hydration of III appears to be the rate determining step. The results of the study of kinetics of decay of the UV absorbing intermediate generated during electrooxidation indicated that thi s step follows first order kinetics. The higher values of k at pH<3.S indicate that the decay is fast in acidic medium, i.e. , it is an acid catalyzed reaction . Compound IV in further hydrolysis and deamination steps leads to the format ion of alloxan as shown by IR, I H NMR and mass spectra. Peaks lIa/ lIe obtained in the cyclic voltammetry at lower p H are due to the electroactive nature of alloxan.

The present studies clearly establish that 2e, 2H+ electrode reaction of 6-hydroxy-2,4,S-triaminopyri-

--

GOYAL et al. : ELECTROCHEMICAL OXIDATION OF A PYRIMIDINE DERIVATIV E 1023

midine gives alloxan as the major product at pyrolytic graphite electrode. Conversion of diiminopyrimidines to alloxan by electrochemical method is a common reaction very often observed at solid electrodes; however, the 2e, 2W oxidation of a triaminopyri­midine observed in the present studies is rather uncommon.

Acknowledgement One of the authors (N.J.) is thankful to the Council

of Scientific and Industrial Research, New Delhi for the award of a Research Associateship. P.G. is thankful to the University Grant Commission, New Delhi for the award of a Senior Research Fellowship.

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