Electrochemical oxidation of xanthosine

137

J. Electroanal. Chem., 216 (1987) 137-156 Plscvier Sequoia S.A., Lausamre - Printed in The Netherlands

S.K. TYAGI and GLENN Department

of Chemistry,

DRYHURST

*

Unmersiry of Oklahoma, Norman, OK 73019 (U.S.A.)

(Received 20th February 1986; in revised form 25th June 1986)

ABSTRACT The electrochemical oxidation of xanthosine in aqueous solution at pH 2.0 at the pyrolytic graphite electrode has been studied. The primary electrochemical oxidation reaction is an irreversible 1 e-, 1 H+ reaction giving the C(8)’ free radical. In order to account for the ultimate products formed, the latter primary radical reacts with xanthosine to give at least one N’ free radical and with water to give an I-hydroxylated free radical. The C(8)’ and N’ radicals couple to give a xanthosylxanthosine dimer which rapidly loses one ribosyl residue to give a xanthosylxanthine dimer. The I-hydroxylated radical reacts with the C(8)’ and N’ radicals to give two isomeric hydroxylated xanthosylxanthosines. The I-hydroxylated radical can also undergo further electrochemical oxidation (1 e-, 1 H+ ) to 9-/3-D-ribofuranosyhnic acid which is immediately oxidixed (2 e-, 2 H+ ) to a very reactive quinonoid. Attack by water on the quinonoid gives two isomeric tertiary alcohol intermediates which have been isolated and characterized by their UV and mass spectra and by their reaction with water to give a diol. The latter diol decomposes to 5-hydroxyhydantoin-5-carboxamide-3-riboside.

INTRODUCTION

There have been very few studies reported of the electrochemical oxidation mechanisms of purine nucleosides and nucleotides. Indeed, the first detailed study was recently reported from this laboratory and was concerned with the electrochemical oxidation of 9-p-D-ribofuranosyhuic acid (1) [l]. While this study was incomplete and work continues to understand more fully both the electrochemical and enzymatic oxidation of 1, it did indicate that the oxidation chemistry of purine nucleosides is likely to be significantly more complex than that of the parent bases. We are now extending this study to include many other biologicahy significant purine nucleosides, beginning with xanthosine. Xanthosine (2) is electrochemically oxidized at a pyrolytic graphite electrode in aqueous solution and preliminary studies [2,3] revealed that this oxidation reaction is appreciably more complex than

* Author to whom reprint requests and further correspondence should be addressed.

138

0

that of the parent base xanthine [4]. For example, xanthine shows a single voltammetric oxidation peak while xanthosine can show up to four peaks. Furthermore, xanthine is apparently rather simply electro-oxidized to uric acid which, being more easily oxidized than xanthine, is immediately further oxidized so that the intermediates and products formed from xanthine are the same as from uric acid. It will be shown that such a simple reaction scheme is not observed in the electrochemical oxidation of 2. In this report the electrochemical oxidation of xanthosine in acidic solution (pH 2) will be described. We have focused our attention on the oxidation chemistry of 2 at low pH because under these conditions it has been possible to isolate and characterize some intermediates and several new compounds as products. This has permitted the first real insights into the underlying mechanisms to be obtained. At higher pH values very much more complex chemistry is observed which it is hoped to report on at some later time.

EXPERIMENTAL

Conventional equipment was employed for linear sweep and cyclic voltammetry, controlled potential electrolysis and coulometry [4]. The pyrolytic graphite electrode (PGE, Pfizer Minerals, Pigments and Metals Division, Easton, PA) used for voltammetry had a surface area of 5.4 mm2 (determined electrochemically using K,Fe(CN),). The PGE was resurfaced, before running each voltammogram, on a sheet of 600 grit silicon carbide paper (Buehler Inc., Evanston, IL) mounted on a metallographic polishing wheel. The electrode was then further polished on 0.05 pm alumina (Buehler, Inc., Evanston, IL) impregnated into a piece of soft felt. Controlled potential electrolyses were carried out using a cylindrical piece of reticulated vitreous carbon (RVC, 100 ppi grade, Normar Industries, CA) as the working electrode (2.0 cm diameter X 7.0 cm). This electrode was dipped into 30 ml of the solution containing the nucleoside of interest. The RVC electrode was cleaned before each electrolysis by immersing it in a bath of boiling water for about 5 min. All voltammetry and controlled potential electrolyses were carried out in conventional three electrode cells containing a platinum counter electrode and a saturated calomel reference electrode (SCE). Most controlled potential electrolyses were carried out with the cell maintained at ca. 4°C using an ice bath. However, all potentials reported are referred to the SCE at 23 f 3°C.

139

In a typical controlled potential electro-oxidation 15-20 mg of 2 was dissolved in 30 ml of water adjusted to pH 2.0 with trifluoracetic acid (TFA) and electrolyzed at the RVC working electrode. The solution in the working electrode compartment was agitated with a stream of nitrogen gas. Complete oxidation of 2 typically took between 15-20 min under these conditions. The electrolyzed solution was then immediately filtered through a 0.2 pm filter (Durapore, 13 mm diam., Millipore, Bedford, MA). In order to prevent product decomposition the filtered solution was divided into aliquots of about 2.5 ml and transferred to 3 ml vials maintained at ca. - 70°C. The frozen solutions were then stored in dry ice until needed. The solution in each vial was melted just before separation by high performance liquid chromatography (HPLC). For HPLC a reversed phase semi-preparative column was used (Brownlee Laboratories, CA, RP-18, 25 x 0.7 cm). A Bio-Rad dual pump HPLC system was used which was controlled by an Apple IIe computer. The UV detector was a Waters Model 440 set at 254 nm. The mobile phase was Hz0 : CH,OH : CH,CN (98 : 1: 1, v/v) adjusted to pH 3.0 with TFA. Typically the flow rate employed was 2 ml min-’ for 20 min then 4 ml min-l. Fractions were collected in freeze drying flasks maintained at ca. - 70° C in dry ice. After multiple collections of each peak the samples were freeze dried. UV spectra were recorded on a Hitachi 100-80 spectrophotometer. IR spectra were recorded on a Perk&Elmer Model 1420 spectrophotometer. Gas chromatography-mass spectrometry (GC-MS) used a Hewlett-Packard Model 5985B instrument equipped with a J k W (Ranch0 Cordova, CA) DB-1 capillary column (15 m x 0.319 pm i.d.) having a film thickness of 1.0 pm. Helium was used as the carrier gas at a flow rate of 2.6 ml mir-‘. The GC temperature program was as follows: 100°C for 5 min, then 6°C rnin-’ to 280°C then 10 min at 280°C. Electron impact (EI)-MS used an electron beam energy of 70 eV. Chemical ionization (CI)-MS used methane as the reactant gas at a pressure of approximately 2 x 10e4 Torr in the source chamber. High resolution fast atom bombardment (FAB)-MS was carried out at the Midwest Center for Mass Spectrometry at the University of Nebraska-Lincoln. Spectroelectrochemical studies used thin-layer cells containing an optically-transparent RVC electrode (100 ppi grade, ca. 0.5 mm thick) similar in design to that described by Norvell and Mamantov [5] except that optical quality quartz microscope slides were used. Thin-layer spectroelectrochemical studies and rapid scanning spectrophotometric measurements used a rapid scanning spectrometer designed and built in-house. This spectrometer is controlled by and data is acquired and analyzed using a CompuPro System 8/16 computer. Xanthosine was obtained from Vega-Fox Biochemicals (Tucson, AZ) and was shown by HPLC to be chromatographically pure. 9-/3-D-Ribofuranosyhtric acid was synthesized by the method of Holmes and Robins [6]. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and silylation grade pyridine were obtained from Supelco (Bellefonte, PA).

RESULTS

Electrochemical studies Cyclic voltammograms of 2 in phosphate buffer at pH 2.0 and in TFA at pH 2.0 are shown in Fig. lA, B. One well-defined voltammetric peak (I,) appears at this pH followed by a second, rather indistinct peak (II,) at slightly more positive potentials. Between pH 2 and 5 (phosphate buffers, p = 0.5 M) the peak potential (E,) for peak I, for 2 (0.5 mM) is pH-dependent according to the following equations:

E P(PH

2 - 5)

E P(PH

2 - 5)

=[1.27-0.086p~lV =

[1.35 - 0.090 pH] V

at5mVs-’ at 200 mV s-l

After oxidation peak I, has been scanned several poorly resolved bumps appear on the reverse sweep indicating formation of reducible species. On the second positive sweep a new oxidation peak (I’,) appears before peak I,. Ep for oxidation peak I; is the same as that for the first oxidation peak of 1. The cychc voltammograms shown in Figs. 1A and 1B indicate that the electrochemical behavior of 2 is virtually the same in phosphate buffer and TFA supporting electrolyte at pH 2.0. Cyclic voltammograms of 2 at sweep rates up to 20 V s- ’ show no evidence for a reversible reduction peak coupled to oxidation peak I,. Furthermore, Ep for peak I,

Fig. 1. Cyclic voltammograms at the PGE of 2.0 mM xantbosine in (A) pH 2.0 phosphate buffer, ionic strength 0.1 and (B) pH 2.0 trifluoracetic acid. Sweep rate: 200 mV s-l.

141

shifts to more positive potentials with increasing sweep rate (Er = 1.10 V at 5 mV s-l and 1.24 V at 500 mV s-‘) and concentrations of 2 (Et, = 1.10 V at 0.10 mM and 1.18 V at 2.0 mM). These results suggest voltammetric peak I, is an irreversible process [7]. At bulk solution concentrations of 2 Q 0.5 mA4, the experimental peak current function (i,/AcW, where all terms have their usual electrochemical significance) increased with increasing sweep rate (5-500 mV s-l) particularly at concentrations < 0.1 mM. However, at concentrations > 1.0 mM this effect virtually disappeared and the peak current functions remained constant with sweep rate. However, it was noted that the peak current functions became slightly smaller with increasing concentrations of 2 (l-2 mM). This behavior suggests that 2 is weakly adsorbed at the PGE but that at concentrations z 1.0 mM the electrode reaction becomes largely diffusion controlled. Apparent voltammetric n values were measured using the equation for a linear diffusion controlled irreversible peak voltammogram [7]: n = iJ2.99

X

105(cyn)“*AcDv’/*

(I) where all terms have their usual significance. Experimental values of an were determined from the slope of the peak voltammograms using the equation [7,8]: an =

0.048/( Ep - Ep,*)

(2) The diffusion coefficient, D, for 2 was taken to be 9.5 X 10e6 cm* s-l. This is the value of the diffusion coefficient for adenosine [9], a molecule of similar size and shape to 2. Using concentrations of 2 of 1.0, 1.5 and 2.0 mM in pH 2.0 TFA and sweep rates ranging from 5 to 500 mV s-l, experimental an values were constant at 0.61 k 0.10. At a concentration of 2 of 1.0 mM the experimental voltammetric n value was 1.2 f 0.2. At higher concentrations this decreased slightly to 1.0 f 0.2. Controlled-potential coulometry of 2 at peak I, potentials in phosphate buffer or TFA at pH 2.0 using plates of pyrolytic graphite as the working electrode typically took 6-8 h for the current to decay to the background level. Under such conditions experimental n values were 3.5 It 0.4. However, by monitoring UV spectra throughout such an electrolysis it became apparent that electro-oxidation reactions continued after all 2 had disappeared, i.e., reaction products were formed which were electro-oxidized. In order to obtain more reliable coulometric n values related to the primary electro-oxidation of 2, very short electrolyses (5 f 1 min) were carried out using an RVC electrode, which has a very large surface area, and a potential (1.07 V) considerably lower than Ep (1.25 V). Such electrolyses did not remove all 2. The amount of unoxidized 2 was determined by HPLC analysis of the product solution (see Experimental) using a calibration curve prepared with pure 2. Background electrolyses were carried out under identical conditions and the charge required to oxidize the supporting electrolyte solutions were subtracted from the values when solutions of 2 were oxidized. Such coulometric experiments were carried out using initial concentrations of 2 of 0.1, 0.5, 1.0, 1.5 and 2.0 mM * and

l

This corresponds to an almost saturated solution of 2 in pH 2.0 TFA.

143

gave experimental n values of 2.1, 1.7, 1.5, 1.3 and 1.2 & 0.2, respectively. Virtually identical results were obtained in phosphate buffer at pH 2.0. The cyclic voltammogram shown in Fig. 2A is for 2 in pH 2.0 TFA before electrolysis at an RVC electrode. A cyclic voltammogram of an exhaustively electrolyzed solution (Fig. 2B) shows the presence of several poorly defined reduction peaks on the first negative sweep and, on the reverse sweep, at least eight oxidation peaks. If the initial sweep on the product solution was towards positive potentials only the oxidation peak at ca. 1.2 V appears, i.e., all of the other oxidation peaks are caused by oxidation of species formed in the electrochemical reduction processes. The peak at 1.2 V is not due to unoxidized 2. Figure 2C shows the UV spectra before and after the rapid electro-oxidation of 2. Thin-layer spectroelectrochemistry The spectrum of 2 in pH 2.0 TFA in a thin-layer cell containing an opticallytransparent RVC electrode is shown in curve 1 of Fig. 3A (X,, = 260, 230, 196 nm). Upon initiation of the electrolysis, the bands at 260 and 195 nm decrease and the absorbance in the regions 280-315 nm and 200-230 nm increases. After scanning spectrum 8 (Fig. 3A) the RVC electrode was open-circuited and the subsequent spectral changes are shown in Fig. 3B. Thus, the absorbances of the bands at X,, = 258 and 226 nm decrease while the absorbance at 196 nm increases slightly. The decay of absorbance at 258 and 226 nm followed first order kinetics (k = 1.54 X lop3 f 1.5 x lo-’ s-l at room temperature 23 f 2OC) and was independent of the initial concentration of 2 oxidized (0.5-1.5 mM). In pH 2.0 phosphate buffer (CL= 0.1 M) the thin-layer spectroelectrochemical behavior was identical to that observed in pH 2.0 TFA. Isolation and characterization

of intermediates

and products

Based on thin-layer spectroelectrochemical evidence, an intermediate species having a half-life of about 8 min (at room temperature) is formed upon peak I, electro-oxidation of 2. Subsequent HPLC studies (see later discussion) indicated that at least one other product was unstable. Thus, in order to oxidize 2 rapidly but without appreciable electrolysis of one or more oxidizable products under conditions where product decomposition was not severe, the following conditions were employed. Approximately 15 mg of 2 was dissolved in 30 ml of pH 2.0 TFA (ca. 1.8 mM 2) and electrolyzed at 1.07 V for lo-15 min at about 4°C. Under these conditions 80-90% of 2 was oxidized. The product solution was then rapidly filtered, frozen and stored at -70°C. Aliquots (ca. 2.5 ml) of the product solution

Fig. 2. (A, B) Cyclic voltammograms at the PGE of 1.2 mM xanthosine in pH 2.0 trifluoracetic acid (A) before electrolysis and (B) after exhaustive electrolysis at 1.07 V at an RVC electrode. Sweep rate: 200 mV s-l. (C) UV spectra before electrolysis (curve 1) and after electrolysis (curve 2).

em

2.0

265

315

290

Wavelength

340

3E5

/run

Fig. 3. Spectra taken during (A) and after (B) the electro-oxidation of 1 mM xanthosine at 1.6 V at an optically transparent RVC electrode in a thin-layer cell. The supporting electrolyte is pH 2.0 trifluoracetic acid. (A) Curve 1 is the spectrum of xanthosine, curves 2-8 were recorded at 30 s intervals

145

A X

t0.01

AU

Fig. 4. Liquid chromatogram of the products formed by electro-oxidation of ca. 1.8 mM xanthosine in pH 2.0 trifluoracetic acid at an RVC electrode at 1.07 V for 15 mm at 4“C. Reversed phase column (25 x0.7 cm) with a mobile phase of Ha0 : CHsOH: CHsCN (98 : 1: 1; v/v) adjusted to pH 3.0 with trifluoracetic acid. Flow rate: 2 ml min- ’ for 20 mm then 4 ml min-‘. Volume of sample injected: 2.0 ml. Fig. 5. Liquid chromatograms of the products formed by electro-oxidation of (A) 2 mM xanthosine and (B) 0.1 mM xanthosine in pH 2.0 trifluoracetic acid at an RVC electrode at 1.07 V for (A) 15 min and (B) 5 mm at 4OC. Reversed phase column (25 X 0.7 cm) with a mobile phase of Hz0 : CHsOH : CHsCN (98 : 1: 1; v/v) adjusted to pH 3.0 with trifluoracetic acid. Flow rate: 3 ml min-t. Volume of sample injected: 20 ~1.

were melted immediately before HPLC separation. A typical liquid chromatogram is shown in Fig. 4. Thus, the major products from such an electrolysis were eluted under liquid chromatographic (LC) peaks 1, 2, 3, 4, 5, and 7 (LC peak X is due to unreacted 2). The products eluted under LC peaks 6, 8, 9,10 and 11 were formed in such minute amounts that they could not be identified and will not be discussed further. It should be noted that electrolysis of 2 in pH 2.0 phosphate buffer (CL= 0.1 M) gave identical products. However, the HPLC separation required a phosphate buffer pH 3 (CL= 0.1 M) as the mobile phase. The difficulties associated with removal of inorganic phosphate from products was, in fact, the stimulus for using volatile TFA as the supporting electrolyte and in the chromatographic mobile phase. HPLC analysis of product mixtures formed by electro-oxidation of different initial concentrations of 2 revealed that the species responsible for LC peaks 1 and 2 were formed in much larger yield, relative to those responsible for LC peaks 3, 4, 5 and 7, when low concentrations of 2 were oxidized (Fig. 5A, B). If product solutions

146

were allowed to stand at room temperature LC peaks 1 and 2 disappeared rather rapidly (ca. lo-15 min) but without any corresponding increase in the other major LC peaks. LC peak 3 also decreased in height with time but in this case a corresponding increase in the height of LC peak 4 was observed.

LC components I and 2 In order to maximiz e the yields of the products responsible for LC peaks 1 and 2, 0.5 mM solutions of 2 were electrolyzed for ca. 5 min at 4°C. After filtering the resulting product solution 2 ml aliquots (stored at - 70°C before injection) were separated by HPLC using a high flow rate (4 ml mini). UV spectra of LC components 1 and 2 are shown in Fig. 6A, B. Thus, both products have identical spectra (A, z 268, 220 nm at pH 3.0) and both compounds decomposed at pH 3.0 over the course of about 20 min. The decomposition reactions followed first order kinetics. At room temperature (23 f 2OC), for LC component 1, k = 1.20 x 10e3 f 8 x 10e6 s-l, and for LC component 2, k = 1.50 x 1O-3 f 2 x 1O-5 s-l in pH 3.0 TFA. Electrochemical oxidation of 1 under conditions identical to those described above for 2 gave an n value of 2.0 f 0.1. HPLC analysis of the product mixture showed two major products having identical retention times, UV spectra and decomposition reaction kinetics to those responsible for LC peaks 1 and 2 formed by electrolysis of 2. Electrolysis of 1, however, never gave products corresponding to LC components 3, 4, 5 and 7. After repetitive collection of LC components 1 and 2 (at - 70°C) followed by low temperature (ca. - 30°C) freeze drying, white, fluffy solids were obtained. A sample of each compound (0.5 mg) was then derivatized with BSTFA (50 ~1) in pyridine (20 ~1) in a sealed vial at 70°C for 15 min. The resulting silylated mixtures were then separated by capillary GC and analyzed by EI- and CI-MS. The GC and GC-MS results obtained following silylation of LC components 1 and 2 were identical and each mixture gave 4 GC peaks having retention times (tR) of 16.85, 18.27, 29.95 and 30.52 min. The GC peak at t, = 30.52 min was clearly the major component of the derivatized mixtures. EI- and CI-MS showed that the molar masses of the derivatives at t, = 16.85, 18.27, 29.95 and 30.52 min were 391, 447, 723 and 748 g, respectively. FAB-MS (thioerythritol-thiothreitol matrix) showed that the major constituents of both LC component 1 and LC component 2 had a molar mass of 316 g with a smaller amount of a second constituent having a molar mass of 334 g. Thus, the major constituent of both LC components 1 and 2 were compounds having a molar mass of 316 g which could be trimethylsilylated at 6 positions (molar mass 748 g, tR = 30.52 min). The mass spectra of the trimethylsilyl derivatives at t, = 16.85, 18.27 and 29.95 min were the same as those for the derivatives of ribosylisocyanate (3), 5-hydroxyhydantoin-5-carboxamide (4), and 5-hydroxyhydantoin-5-carboxamide-3-riboside (5) which have previously been observed as the ultimate products of electrochemical oxidiation of 1 [l]. It was noted, following derivatizations of both LC components 1 and 2, that a decrease in the

1 .?i5

2.40

zs5

315

890

Wavelength

340

365

/nm

Fig. 6. UV spectra of the electro-oxidation products of xanthosine responsible for (A) LC peak 1 and (B) LC peak 2 obtained at room temperature. Spectra were obtained in pH 3.0 trifluoracetic acid. Curve 1 in each figure is the initial spectrum and subsequent spectra were recorded at 60 s intervals.

148

relative size of the GC peak at t, = 30.52 min was accompanied by an increase in the other three GC peaks, i.e., 3, 4, and 5 are decomposition products of the species having a molar mass of 316 g. FAB-MS and GC-MS results for the species responsible for LC peaks 1 and 2 formed by rapid 2 e- electro-oxidation of 1 were identical to those reported above for the products formed by electrolysis of 2. The structure and deomposition reactions of LC components 1 and 2 (MM = 316 g) will be discussed subsequently. LC component 3

This was a white, fluffy powder. At pH 3 it was not stable and HPLC analysis showed conclusively that LC component 3 decomposed into LC component 4. At pH k 5, however, LC component 3 was stable for at least 3 h. The UV spectrum of = 262, 234 (sh), 195 nm (Fig. 7A). IR LC component 3 at pH 3.0 shows h, spectrum (KBr pellet, cm-‘): 3360 (broad, s, O-H) [ll], 3080 (m, N-H), 2940 (m, N-H), 2860 (m, N-H), 1710 (s, C-O), 1580 (w), 1540 (w), 1410 (w), 1300 (m), 1210

Fig. 7. UV spectra of LC components 3 (A),4 (B), 5 (C) and 7 (D) in pH 3.0 trifluoracetic acid.

149

(m), 1130 (m), 1085 (m), 910 (w). FAB-MS of LC component 3 (thioerythritol/ thiothreitol matrix) gave the following information, m/e (relative abundance): 567 (1.9% MH+), 435 (16.1% MH+-C,Hs04), 303 (59% MH+-2 xC,H,O,), 275 (43%, MH+-[2 X C,HsO, + CO]), 259 (lo%), 152 (27%, CSH,N,O,, i.e., xanthine). A high resolution FAB-MS of the pseudomolecular ion gave an exact mass of 567.1405. This corresponds to an elemental formula of C,H,,N,O,, (calculated mass: 567.1435). The MS results indicate that LC component 3 has a molar mass of 566 g and is a dimer of 2. LC component 4

This was a white fluffy powder having the UV spectrum at pH 3.0 shown in Fig. 7B (A,, = 262, 195 mn). This spectrum is almost identical to that of LC component 3 except that the shoulder at ca. 234 nm, clearly evident for the latter compound, is almost absent for LC component 4. IR spectrum (KBr pellet, cm-‘): 3200 (broad, s, O-H), 3070 (m, ,N-H), 2930 (m, N-H), 2810 (m, N-H), 1700 (s, C=O), 1590 (w), 1570 (w), 1540 (w), 1410 (m), 1300 (w), 1200 (m), 1130 (m), 1080 (m), 1030 (w), 905 (w). FAB-MS of LC component 4 (thioerythritol/thiothreitol matrix), m/e (relative abundance); 435 (868, MH+), 303 (44.3%, MH+-CSHs04), 275 (6.2%, MH+-[CSHsO, + CO]), 259 (2.5%), 152 (27.0%, C,H,N,O,, i.e., xanthine). High resolution FAB-MS of the pseudomolecular ion (MH+) gave a measured m/e of 435.0991 which corresponds to an elemental composition of C,,H,,N,O, (calculated m/e = 435.1015). In fact the FAB-MS of LC component 4 is virtually identical to that of LC component 3 except that the peak at m/e = 567 is absent. Thus LC components 3 and 4 differ by a single ribosyl residue. Hence, component 4 must be a dimer consisting of one xanthine and one xanthosine residue. LC components 5 and 7

Both of these compounds were white solids which had identical UV spectra (e.g., h = 255, 195 mn at pH 3.0, Fig. 7C, D). IR spectrum for component 5 (KBr pzk, cm-‘): 3400 (broad, s, O-H), 3080 (m, N-H), 2960 (m, N-H), 2860 (m, N-H), 1690 (s, C==O), 1580 (s), 1390 (m), 1280 (w), 1205 (m), 1080 (m), 1040 (m), 910 (w). For component 7: 3400 (broad, s, O-H), 3060 (m, N-H), 2940 (m, N-H), 2860 (m, N-H), 1690 (s, GO), 1575 (s), 1385 (m), 1275 (w), 1205 (m), 1080 (m), 1040 (w), 910 (w). FAB-MS (thioerythritol/thiothreitol matrix) for LC components 5 and 7 were virtually indistinguishable. Thus, for component 5, m/e (relative abundance): 585 (70%, MH+), 542 (9.88, MH+-NHCO), 453 (70%, MH*-C,H,O,), 410 (25%, MH+-[CSHsO, + NHCO]), 321 (54%, MH+-2 x C,H,O,). For component 7: 585 (42%), 542 (6%), 453 (22%) 410 (IS%), 321 (75%). High resolution FAB-MS for the pseudomolecular ions of components 5 and 7 gave m/e = 585.1556 and 585.1529, respectively. These masses correspond to an elemental formula of C,,HzSN,O,, (calculated m/e: 585.1543).

Fig. 8. Cyclic voltammograms at the PGE of ca. 0.6 mM LC components pH 2.0 trifluoracetic acid. Sweep rate: 200 mV s-l.

3 (A), 4 (B), 5 (C) and 7 (D) in

DISCUSSION

The results presented above show that the electrochemical oxidation of 2 at its first voltammetric oxidation peak is a pH-dependent, irreversible process. At concentrations of 2 > 1 mM under both voltammetric and controlled potential electrolysis conditions, the apparent n value is close to 1. However, coulometric n values close to unity are observed only when electrolyses are very rapid. Extended electrolyses cause the n values to increase to 3.5 f 0.4. This is due to the fact that many initially formed major dimeric products are electro-oxidizable. Figure 8 shows cyclic voltammograms of LC components 3, 4, 5 and 7 at pH 2.0 which indicates that all of the dimeric derivatives of 2 give oxidation peaks at potentials close to peak I,. Furthermore, having scanned through these oxidation peaks all compounds give several reduction peaks on the reverse cycle and on the second positive sweep several new oxidation peaks. It is particularly noticeable that all compounds give an oxidation peak at a potential close to that of peak I’, observed in cyclic voltammograms of 2 (Fig. 2A). Most of the peaks observed in the cyclic voltammogram of oxidized 2 (Fig. 2B) can be at least approximately matched with those observed for the various dimeric products (Figs. 8A-D). At this time the nature of none of these peaks is known.

151

>*

+

H20

H

R 8

6

Fig. 9. Reaction pathways proposed for the initial peak I, electrochemical oxidation of xanthosine at pH 2.0.

At concentrations of 2 > 1 mM only relatively small amounts of two intermediates are formed, LC components 1 and 2.‘The relative yields of these unstable products, however, increase when lower concentrations of 2 are electro-oxidized. These same compounds are also formed as major, unstable products of the 2 eelectrochemical oxidation of 1. This indicates that one oxidation route for 2 at pH 2.0 proceeds via 1 which, being more easily oxidized than 2 (E, for 1 at pH 2.0 = 0.69 V) is oxidized to the compounds responsible for LC peaks 1 and 2. In view of the preferential formation of dimeric products when increasing concentrations of 2 are electro-oxidzed it must be concluded that the initial reaction leads to a radical species. Based on the shift of Ep with pH for peak I, of 2 (- 0.086 per pH unit at 5 mV s-l) and the measured value of cm (0.61) it can be calculated from eqn. (3) that the number of protons ( p) involved in the rate determining irreversible electrode reaction is 1.0 [S]. d E,,/d(pH)

= - 0.059 p/an

V

(3)

A 1 e-, 1 H+ electrochemical oxidation of 2 must give a free radical. In view of the formation of 9-/3-D-ribofuranosyluric acid as the major intermediate in the electrooxidation of 2 at low concentrations the primary radical formed must be the C(8)’ species (6, Fig. 9). For reasons discussed below at least two more radical inter-

152

_H+_e-

11 -

12 H2O

I

Fig. 10. Secondary electrochemical oxidation of the hydroxyxanthosyl radical 8 to 9-/3-D-ribofuranosylurk acid (9) and S-hydroxyhydantoin-5-carboxamide-3-riboside (5).

mediates must be formed. It is proposed that the primary radical 6 reacts with 2 to give the N(1)’ radical 7 (Fig. 9). In addition, primary radical 6 is attacked by water giving the hydroxy radical 8 (Fig. 9). The latter is the predominant reaction of 6 at low bulk solution concentrations of 2. In order to account for the observed electrochemistry, hydroxy radical 8 must be oxidized (1 e-, 1 H+) to give 9-/3-Dribofuranosyluric acid (9, Fig. 10). The latter compound is much more easily electrochemically oxidized (2 e-, 2 H+) than 2 giving the quinonoid intermediate 10 (Fig. 10) which is rapidly attacked by water giving the two isomeric tertiary alcohols 11 and 12 (Fig. 10) [l]. Since these isomers have very similar chromophores (O=C(2)-N(3)=C(4)for 11 and O=C@)-N(7)=C(5)for 12) it is not surprising that they have very similar UV spectra (Fig. 6A, B). Both 11 and 12 have a molar mass of 316 g and can be trimethylsilylated at up to six positions (molar mass 748 g). All of these properties are in accord with the UV-absorbing intermediates formed upon electrochemical oxidations of 2 and 1 which are responsible for LC

153

components 1 and 2. Furthermore, it has already been speculated [l] that intermediates 11 and 12 should be attacked by water at low pH to give the common diol 13. Indeed, FAB-MS results on the isolated components responsible for LC peaks 1 and 2 show a significant peak due to a compound having a molar mass of 334 g. This peak must be due to the unstable diol 13 or perhaps a ring-opened form. This is the first direct evidence for the formation of such a diol upon electrochemical oxidation of any purine. Ring opening and hydrolysis of diol 13 leads to Shydroxyhydantoin-5-carboxamide-3-riboside (5, Fig. lo), a previously known product of electro-oxidation of 1 [l]. This compound is also known [l] to be hydrolyzed at low pH to 5-hydroxyhydantoin-Scarboxarnide (4) and other fragments in accord with the findings described earlier. Very strong evidence for formation of free radicals in the initial electro-oxidation step of 2 is provided by the isolation of the dimers responsible for LC peaks 3 and 4. LC component 3 (MM = 556 g) is clearly a dimer of 2 while LC component 4 (MM = 434 g) has lost one ribosyl residue. Recently, Joshi and Davies [lo] have reported that UV irradiation of an equimolar mixture of 8-bromoxanthosine and 2 in 30% aqueous acetone gives 8-(8-xanthosyl)-xanthosine, i.e. a dimer of 2 linked through the C(8)-positions. However, the UV spectra of this photochemical dimer, %KiX(%X X 10p3) = 312 nm (16.8 1 mol-’ cm-‘), 262 nm (9.8) at pH 1.0; 324 nm (13.1) 272 nm (14.6) at pH 7.0; 318 nm (11.3), 270 nm (14.7) at pH 13, are considerably different to those of the electrochemically-formed dimers. For example, LC component 3 shows the following spectra: Xmax(e,, X 10p3) = 264 nm (15.5) 235 nm (sh. 8.2) at pH 1.0; 265 nm (14.9) at pH 7.0; 272 nm (14.5), 258 nm (sh. 11.8) at pH 13. For LC component 4: 264 nm (13.3), 235 nm (sh. 6.0) at pH 1.0; 265 rmr (14.6) at pH 7.0; 276 nm (12.8), 255 nm (sh. 9.3) at pH 13.0. The UV spectra of 2 are as follows: 260 nm (9.3) 238 nm (6.7) at pH 1.0; 275 nm (8.0) 247 run (7.3) at pH 7.0; 275 nm (8.7), 247 nm (9.3) at pH 13.0. Joshi and Davies [lo] concluded that the photochemically-formed dimer must have extensive conjugation and overlap of the s-electron systems of the individual bases because of the red shift of the absorption spectrum by 40-50 nm relative to that of 2. This condition can only be met if, on average, the base residues tend toward coplanarity. The electrochemicallygenerated dimers responsible for LC peaks 3 and 4, however, absorb at very similar wavelengths to 2. Thus, the electrochemical dimers must be linked together at different sites to the photochemical (8-8) dimer and, probably, have the base residues arranged so that minimum r-electron overlap occurs. Thus, it is proposed that the N(1)’ radical (7) formed by a hydrogen atom abstraction reaction of the primary C(8)’ radical (6) with 2, dimerizes to give l-(l-xanthosyl)xanthosine (14, Fig. 11). This dimer has the same molar mass as 8-(8-xanthosyl)xanthosine but would be expected to have a different UV spectrum. Under the acidic conditions employed to electro-oxidize 2 and to carry out the HPLC separations, 14 clearly loses one of its ribose residues to form dimer 15 (Fig. 11). There are, clearly, dimeric structures other than 14 and 15 which could also be proposed. Work is currently under way to prepare crystals of compounds 14 and 15 so that their structures and stereochemistry can be elucidated by X-ray diffraction methods.

-

OH OH z ,

I

Fig. 11. Dimerization of the N(l)* l-(1-xanthosyl)xanthne (15).

xanthosyl radical to give 1-(1-xanthoxyl)xanthosine

(14) and

Two additional dimers are formed upon electrochemical oxidation of 2 which are responsible for LC peaks 5 and 7. These dimers have virtually identical UV, IR, and mass spectra and therefore must be isomers. The molar mass of both dimers is 584 g and both contain two ribosyl residues, two xanthine residues and the elements of one molecule of water. This indicates that there are two likely basic structures for these dimers. One could be linked through the C(8) position of one xanthosine residue with water added across the N(7)=C(8) double bond (17). The other could possess an oxygen bridge between the two xanthosyl residues (18). The great spectral similarities between LC components 5 and 7 argue against one having structure 17 and the other structure 1%. It seems unlikely that the hydroxy radical 8 could be readily transformed into an oxyradical leading to dimers having an oxygen

OH OH

18 -

155

Fig. 12. Dimerization of the hydroxyxanthosyl radical 8 with the C(8)’ xanthosyl radical and the N(1)’ xanthosyl radical to give isomeric hydroxy dimers 19 and 20.

bridge such as that shown in 18. Thus, at this time, it is concluded that the hydroxy dimer 8 reacts with either the primary C(8)’ radical (6) to give the C(8)-C(8)-linked hydroxy dimer 19 or with the N(1)’ radical to give the C(8)-N(l)‘-linked hydroxy dimer 20 as shown in Fig. 12. Again, it is possible that the linkage between the two purine residues occurs at other positions. Thus structures 19 and 20 must, at this time be regarded only as reasonable suggestions. Attempts are now under way to elucidate fully the structure and stereochemistry of these hydroxy dimers using single crystal X-ray diffraction methods. CONCLUSIONS

There have been a few reports of the formation of purine nucleoside dimers by the ultraviolet photolysis of 8-bromopurine nucleosides [10,12,13] including C(8)-C(I)-linked dimers of 2 [lo]. However, it is clear that the dimers formed upon electrochemical oxidation of 2 are structurally quite different from the photodimers. These electrochemical studies indicate that the primary oxidation step of 2 is a 1 e-, 1 H+ reaction leading to free radical 6 which can dimerize directly to 14 and hence 15 or be attacked by water to give the hydroxyradical8. The latter radical can either undergo further electrochemical oxidation or react with the primary C(8)’ radical 6 or the secondary N(1)’ radical 7 to form two new isomeric dimers probably having structures 19 and 20, respectively. None of the dimeric xanthine nucleosides described in this paper have been previously reported.

156 ACKNOWLEDGEMENTS

This work was supported by NIH Grant No: GM-21034. Additional support was provided by the Research Council of the University of Oklahoma. REFERENCES 1 2 3 4 5 6 7 8 9 10 11

R.N. Goyal, A. BraJter-Toth, J.S. Besca and G. Dryhurst, J. Electroanal. Chem., 144 (1983) 163. J.L. Owens, Ph.D. Dissertation, University of Oklahoma, 1977. M.T. Cleary, Ph.D. Dissertation, University of Oklahoma, 1980. J.L. Owens, H.A. Marsh and G. Dryhurst, J. Electroanal. Chem., 91 (1978) 231. V.E. Norvell and G. Mamantov, Anal. Chem., 49 (1977) 1470. R.E. Holmes and R.K. Robins, J. Am. Chem. Sot., 87 (1965) 1772. R.S. Nicholson and I. Sham, Anal. Chem.. 36 (1964) 706. R.N. Adams, Electrochemistry at Solid Electrodes, Marcel Dekker, New York, 1969, p. 136. B. Janik and P.J. Elving, J. Am. Chem. Sot., 92 (1970) 235. P.C. Joshi and R.J.H. Davies, J. Chem. Res., S, (1981) 227. C.J. Pouchert (Ed.), Aldrich Library of Infrared Spectra, 3rd ed., Aldrich Chemical Company, Milwaukee, WI, 1981. 12 S.N. Bose, R.J.H. Davies, D.W. Anderson, J.C. van Niekerk, L.R. Nassimbeni and R.D. MaCfarlane, Nature (London), 271 (1978) 783. 13 S.N. Bose, R.J.H. Davies, J.C. van Niekerk, D.W. Anderson and L.R. Nassimbeni, J. Chem. Sot. Perkin Trans. 2, (1979) 1194.