Electrochemical oxidation of guanosine formation of some novel guanine oligonucleosides

137

J. Electroanal. Chem, 224 (1987) 137-162 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

ELECTROCHEMICAL FORMATION

P. SUBRAMANIAN Department

OXIDATION

OF GUANOSINE

OF SOME NOVEL GUANINE

and GLENN DRYHURST

of Chemistry,

OLIGONUCLEOSIDES

*

Universrty of Oklahoma, Noman,

OK 73019 (U.S.A.)

(Received 25th July 1986; in revised form 25th November 1986)

ABSTRACT The electrochemical oxidation of guanosine has been studied in aqueous phosphate buffer solutions at a pyrolytic graphite electrode. The primary electrooxidation step appears to involve a 1 e-, 1 Hf reaction leading to a free radical with the unpaired electron being located at the Cs-position. This primary radical reacts with guanosine and water to yield several other radicals which then undergo a series of chemical and electrochemical reactions yielding some novel guanine di- and tri-nucleosides.

INTRODUCTION

There have been few studies of the electrochemical oxidation of purine nucleosides and nucleotides. Recent reports from this laboratory describe some preliminary results concerning the electrochemical oxidation of 9-/3-D-ribofuranosyluric acid [l] and xanthosine [2]. The electrochemistry of the latter nucleoside was of particular interest because the initial oxidation reaction involved transfer of 1 eand 1 H+ to give a free radical which, when high concentrations (ca. 2 mM) of xanthosine were oxidized, subsequently reacted to give several new dimeric xanthine nucleosides as well as a number of monomeric products. Our investigations have now been extended to the more biologically significant nucleoside guanosine (1). Yao and co-workers [3] have shown that 1 can be electrochemically oxidized at a glassy carbon electrode over a wide pH range (2-10) although no product or mechanistic information was obtained. In this paper the electrochemical oxidation of 1 at a pyrolytic graphite electrode will be described. Attention has been focused on the oxidation of 1 at potentials corresponding to its first voltammetric oxidation peaks in near-saturated solutions

l

To whom correspondence should be addressed.

0022-0728/87/%03.50

0 1987 Elsevier Sequoia S.A.

138

at pH 7. Under such conditions a number of new oligomeric guanine nucleosides are formed, the structures of which provide a guide to understanding the very complex oxidation chemistry of 1.

OHOH

1 EXPERIMENTAL

Chemicals Guanosine was obtained from Sigma (St. Louis, MO) and was used without further purification. 8-Hydroxyguanosine was synthesized by the method of Holmes and Robins [4]. Dioxane and formic acid were obtained from Aldrich (Milwaukee, WI) and triethylamine from Pierce (Rockford, IL). Phosphate buffers of known ionic strength were prepared according to Christian and Purdy [5]. Apparatus Linear sweep and cyclic voltammetry, controlled potential electrolysis and coulometry were carried out with equipment which has been described elsewhere [6]. Voltammetry was performed at either a pyrolytic graphite electrode (PGE, Pfizer Minerals, Pigments and Metals Division, Easton, PA) having an approximate surface area of 4 mm2 or at a glassy carbon electrode (GCE, Princeton Applied Research Corporation, Princeton, NJ) having an electrochemical surface area (determined potentiostatically with 1 mM K,Fe(CN), in 0.5 A4 KC1 [7]) of 0.33 cm2. The PGE was resurfaced on a sheet of 600-g& silicon carbide paper (Buehler, Inc. Evanston, IL) mounted on a metallographic polishing wheel. The GCE was polished with 0.05 pm alumina (Buehler) impregnated into a piece of soft felt. Large-scale electrolyses and conventional coulometry were carried out using several plates of pyrolytic graphite having a total surface area of about 96 cm2. These electrodes were immersed in 30 ml of buffer solution containing guanosine. Voltammetry and controlled potential electrolysis were carried out in conventional threeelectrode cells containing a platinum counter electrode and a saturated calomel reference electrode (SCE). All voltammetric measurements were made in solutions which had been thoroughly deaerated with nitrogen. Controlled potential electrolyses were carried out in solutions which were stirred by a Teflon-coated magnetic stirrer with nitrogen gas bubbling vigorously through the solution. All potentials are referred to the SCE at 25 f 3OC. Spectroelectrochemical studies used thin-layer cells containing an opticaly-transparent reticulated vitreous carbon (RVC) electrode (Normar Industries, CA, 100 ppi grade) similar in design to that described by Norvell and Mamantov [8] except that optical quality quartz microscope slides were used (Esco, Optical Products, Oak

139

Ridge, NJ). Thin-layer spectroelectrochemistry used a rapid scanning spectrometer designed and built in-house which was controlled by a CompuPro System 8/16 computer. High performance liquid chromatography (HPLC) used a reversed phase column (Brownlee Laboratories RP-18, Santa Clara, CA, 25 x 0.4 cm) a Bio-Rad (Richmond, CA) Model 1300 dual piston pump and a Waters (Milford, MA) Model 440 UV detector (254 nm). The column was always equilibrated for at least 1 h with 0.6% dioxane in buffer A, which consisted of 10 mM formic acid in water adjusted to pH 5.0 with triethylamine, at a flow rate of 1 ml mm-‘. For separation of the electrooxidation products of 1, 2 ml of the oxidized solution was injected onto the column and eluted with the latter mobile phase. After 40 min the mobile phase was changed to 3% dioxane in buffer A. Under these conditions a number of components were eluted up to liquid chromatographic (LC) component 10 (see later discussion). After several injections of the crude electrolysis product of 1 the solvent was changed to 30% methanol in water (v/v). Several small chromatograhic peaks then were observed and one major peak, LC component 11. Most of the products separated under the latter conditions were quite stable; exceptions to this are described elsewhere in this report. However, if the pH of the initial mobile phase was < 4 most products showed evidence for some decomposition. The use of an initial mobile phase at pH 5 was selected because it gave the best overall separation of components with minimal product decomposition. Low and high resolution fast atom bombardment mass spectrometry (FAB-MS) was performed at the Midwest Center for Mass Spectrometry at the University of Nebraska-Lincoln. All FAB-MS were obtained using a matrix of dithioerythritol plus dithiothreitol. ‘H-NMR spectra were obtained with a Varian XL-300 spectrometer with Me$i as the internal standard. UV-visible spectra were recorded on a Hitachi 100-80 spectrophotometer. REmJLTs

Cyclic voltammetry Cyclic voltammetry (CV) of 1 was carried out at the PGE in phosphate buffers pH 2-11 having an ionic strength (p) of 0.5 M. Over this pH range 1 shows up to three oxidation peaks (III,, IV, and V,). Representative CVs of 1 at pH 7.0 are shown in Fig. 1. At very slow sweep rates (5 mV s- ‘) oxidation peak IV, is much smaller than peak III,. With increasing sweep rate the height of peak IV, grows relative to that of peak III,. Thus, at a sweep rate of 200 mV s-l peaks III, and IV, are of approximately equal height (Fig. IA-C). At higher sweep rates peak III, and IV, merge together. These behaviors suggest that 1 is adsorbed at the PGE and that peak IV, is an adsorption post-peak. Peak potentials (E,) for peaks III,, IV, and V, were pH-dependent according to the following equations. The data for these equations were obtained with 0.1 mM 1 at the sweep rate indicated. The fact that Ep values for all peaks are dependent on pH indicates that protons and electrons are involved in the rate-controlling steps for

Potential/ Fig. 1. Cyclic voltammograms Sweep rate: 200 mV s-‘.

V vs. SCE

at the PGE of 0.1 mM guanosine in pH 7.0 phosphate buffer (p = 0.5 M).

the electrode reactions. Having scanned through oxidation peaks III,, Peak III,:

Eti(,n 4-11j = [1.16 - 0.050 pH] V

Peak IV,: EtipH 2-llj = [1.24 - 0.035 pH] V Peak V, : QpH z_8J= [1.28 - 0.011 pH] V

IV, and V,

at 5 mV s-l at 200 mV s-i at 200 mV s-l

several reduction peaks are observed on the reverse sweep (Fig. 1A) and, on the second anodic sweep, two new oxidation peaks (Ii and II:) appear at less positive potentials than peak III,. Reduction peak I, appears to form a quasi-reversible couple with oxidation peak I:. In order to observe peaks 1: and II: it is necessary only to sweep through reduction peak I, (Fig. 1B). If the initial positive sweep is reversed after scanning through peak III, or peaks III, + IV, reduction peak I, still appears on the reverse sweep although an additional reduction peak II, also appears (Fig. 1C). It should be noted that 1 is not electrochemically reducible (Fig. 1A). At concentrations of 1 above 0.1 mM, peaks IIIa and IV, merge together to give what will be referred to as peaks IIIJIV,. In addition, with increasing concentrations of 1, peaks I,, II,, 11 and II: become smaller relative to peaks IIIJIV,. However, at a given concentration of 1, increasing sweep rate causes reduction peak I, and oxidation peaks 1: and II: to grow relative to peaks IIIJIV,. It was also noted, at pH 7.0 for example, that at a given sweep rate the peak current (i,) for the peak IIIJIV, process increases with increasing concentration of 1 up to about 0.5 mM and then reaches an almost constant value. Similarly, at a given concentration

141

of 1 (e.g., 0.2 mM) the experimental peak current function (~,/Acv’/~) for peaks IIIJIV, increases with increasing sweep rates. Both of these observations indicate that 1 is adsorbed at the electrode surface [9]. As the pH is increased above 7 reduction peaks I, and II, and oxidation peaks 1: and II: decrease in height relative to peaks IIIJIV,. At pH d 4.3 peaks 1: and II: merge to give a single peak. Furthermore, at pH < 4 peaks III, and IV, are always merged together. Under pH and concentration conditions where peaks III, and IV, appear as a single peak (III.JIV,), the Er shifts 50 f 20 mV more positive for every decade increment in sweep rate (0.005-0.05 V s-l, 0.02-0.2 V s-l, 0.05-0.5 V s-l). For an irreversible electrode process there should be a positive shift of EP of 30/cwn mV for each ten-fold increase in sweep rate [lo]. An approximate value of LYE,0.83, was obtained by using eqn. (1) [lO,ll]: (in = 0.048/( EP - Ep,2)

(I)

Thus, theoretically [lo] E, for 1 should shift 36 mV more positive for each ten-fold increase in sweep rate. This theoretical shift agrees reasonably well with the experimental value. The results outlined above suggest that 1 is quite strongly adsorbed at the PGE. The lack of a reduction peak coupled to peaks IIIJIV, and the positive shift of EP for peaks IIIJIV, with increasing sweep rate indicates that the peak IIIJIV, process is, under the conditions investigated, electrochemically irreversible. It was not possible to measure the number of electrons transferred in the initial voltammetric oxidation step of 1 using the PGE owing to the irreproducibility of the surface area of this electrode. However, a GCE could be used at which peak current (i,) measurements were far more reproducible ( f 5-10%). At the GCE the peaks III, and IV, processes were always merged together. Estimations of voltammetric n values for the peaks IIIJIV, reaction were determined at pH 2.0 and 7.0 using sweep rates ranging from 0.005 to 20 V s-l and concentrations of 1 ranging from 0.7 to 1.6 mM using the equation [lo] n = iJ2.99

x 105(~n)“2AcD1’2y1’2

(2)

where all terms have their usual significance. The diffusion coeffient (D) for 1 was assumed to be 9.5 X lop6 cm2 s- ‘. This is the value of D for adenosine [12], a molecule of similar size and shape to 1. At pH 2.0 and pH 7.0 the calculated voltammetric n values were 1.5 f 0.5 and 1.4 f 0.3, respectively. However, owing to the adsorption of 1 it must be noted that these n values are probably of only limited significance. Controlled potential coulomety Controlled potential coulometry of 1 (0.1-1.5 mM) was carried out in phosphate buffers (p=O.l and 0.5 M) at pH 2.0, 4.45 and 7.0 at 1.2 V, 1.1 V and 1.0 V, respectively (i.e. at a potential corresponding to the peaks IIIJIV, process). Initially, electrolyses were allowed to proceed until CV showed that peaks IIIJIV,

142

had been eliminated. Under these conditions the average experimental n value was 4.2 k 0.6. However, at the point in such electrolyses where the characteristic UV spectrum of 1 (A max= 274 (sh), 245 nm at pH 7.0) disappeared (leaving a new band 232 nm) CVs showed that voltammetric oxidation peaks remained in the at A,,,= vicinity of peaks IIIJIV,. This behavior suggested that electrooxidation of 1 leads to formation of products which are oxidized at similar potentials. It will be shown subsequently that several oligomeric products are in fact formed which give voltammetric oxidation peaks in the vicinity of peaks 111,/W,. Thus, the coulometric n value reported above represents the electrochemical oxidation of 1 and of several oxidizable products. In order to obtain more meaningful coulometric n values for the peaks IIIJIV, process, short (15 min) electrolyses were performed which purposely did not remove all of 1. The amount of unoxidized 1 was determined by HPLC analysis of the product solution using conditions described in the Experimental section and a calibration curve prepared with pure 1. Background electrolyses were carried out under identical conditions and the charge required to oxidize supporting electrolyte solutions were subtracted from the charge measured when solutions of 1 were oxidized. Such coulometric experiments were carried out in phosphate buffer pH 7.0 (p = 0.5 M) at 1.2 V using initial concentrations of 1 of 0.1, 0.2, 0.5, 1.0 and 1.5 mM and experimental n values (average of at least two replicate determinations) of 4.5 ( +0.2), 3.2 ( + 0.1) 3.0 (k 0.1). 2.6 ( +O.OS), and 2.45 (+0.03), respectively, were obtained. Clearly, with increasing bulk solution concentrations of 1, experimental n values show a systematic decrease. Similar results were obtained at pH 4.4. Thin-layer spectroelectrochemistry The spectrum of 0.1 mM 7 in pH 7 phosphate buffer (cl = 0.5 M) in a thin-layer cell containing an optically-transparent RVC electrode is shown in curve 1 of Fig. = 274(sh), 245,200 nm). Initiation of the peak 111,/W, electrooxidation 2A (A,,, (1.20 V) caused the bands at 245 and 200 nm to decrease and, correspondingly, the absorbance centered at about 304 nm and 219 nm increases. Curve 6 is the spectrum observed after electrooxidation of 1 for 60 s. After scanning this spectrum the RVC electrode was open-circuited and the changes shown in Fig. 2B were recorded. Thus, the absorbance centered at 219 nm increases while that centered at 267 nm and 292 nm decreases. The absorbance vs. time changes at the latter three wavelengths followed first-order kinetics. Following electrooxidation of 1 (0.1 mM) in pH 7 phosphate (p = 0.5 M) the rate constants measured at 219, 267 and 292 nm were (9.9 + 0.32) X 10p3, (9.3 + 0.22) X 10e3 and (6.0 + 0.42) X lop3 s-l, respectively. Electrolyses of 1 at concentrations > 0.1 mM in a thin-layer cell proceeded so slowly that it was not possible to observe the subsequent decay of any intermediate species easily. Electrochemical oxidation of 8-hydroxyguanosine (7, 0.1 mM) in a thin-layer cell under identical conditions to those described above for 1 also generated a UV-absorbing intermediate. The first-order rate constants measured following oxidation (1.2 V) in pH 7.0 phosphate buffer (p = 0.5 M) at 219, 267 and 292 nm were (9.5 f 0.23) X 10e3. (6.3 + 0.30) X lop3 and (9.9 + 0.29) X 10e3 s-t,

Fig. 2. Spectra of 0.1 mM guanosine in pH 7.0 phosphate buffer (p = 0.5 M) during and after electrooxidation at 1.2 V in a thin-layer cell containing an RVC electrode. (A) Changes which occur during the electrooxidation, curve 1 being the initial spectrum of guanosine. Subsequent traces were recorded every 10 s. (B) Changes which occur after the RVC electrode was open-circuited. Spectra were recorded at 10 s intervals.

respectively. Details of the electrooxidation chemistry of 7 will be described elsewhere. Nevertheless, it appears that electrooxidation of dilute solutions of both 1 and 7 leads to formation of the same UV-absorbing intermediate which can be observed by thin-layer spectroelectrochemistry. Further evidence for a common intermediate being formed upon electrochemical oxidation of 1 and 7 was obtained by HPLC analysis. Basically, 0.5 mA4 solutions of 1 and 7 were electrooxidized at 1.2 V in pH 7.0 phosphate buffer (p = 0.5 M) for 15 min. After this time a 20 ~1 sample of the resulting solution was analyzed by HPLC (Brownlee RP-18 column, 5 pm, 25 X 0.4 cm) using a mobile phase of water + MeOH + MeCN (98 : 1: 1, v/v) adjusted to pH 3.0 with trifluoroacetic acid at a flow rate of 2 ml mm’. The product mixture from 7 gave one large product peak having a retention time of 8.0 min. Repetitive injections showed that the species responsible for this peak completely disappeared within about 30 min. The product mixture from 1 also showed a peak at the same retention time which also

144

disappeared with time. In addition, the product from 1 also showed one stable product having the same retention time as 7 (14.0 n-tin). It might be noted that the HPLC conditions described here could not be employed for separation of reaction products because injection of larger volumes caused all products to co-elute with inorganic phosphate, i.e. the chromatogram collapsed completely. Nevertheless, the HPLC analysis revealed that electrooxidation of 1 and 7 leads to formation of a common intermediate. Isolation and identification of electrooxidation products Results reported above indicate that electrochemical oxidation of 1 at peaks IIIJIV, potentials initially gives products which are also electrooxidizable. Thus, if electrolyses were allowed to proceed until all oxidizable species were electrolyzed the resulting solution would contain products of oxidation of these initially formed oxidizable products. If was judged, therefore, that the oxidation reactions of 1 could be best understood by isolating and identifying the latter primary oxidation products. In order to accomplish this, solutions of 1 were oxidized for only a short period of time. Typically 12-13 mg of 1 dissolved in 30 ml (- 1.5 mM) of pH 7.0 phosphate buffer (p = 0.5 M) were oxidized at 1.2 V for 15-20 min at room temperature. Under these conditions about 45% of 1 initially present was oxidized. Then, 2.0 ml aliquots of the resulting solution were separated by HPLC using a reversed phase column (see Experimental section). The components eluted under each chromatographic peak were collected. A representative liquid chromatogram is shown in Fig. 3. This chromatogram shows at least 16 liquid chromatographic (LC) peaks. The chromatogram shown in Fig. 3 was obtained when 200 ~1 of a product solution was injected. When 2 ml was injected LC peaks 5 and 6 emerged as a single peak and were collected together and subsequently the individual components were separated under different chromatographic conditions (see later discussion). If electrolyses were allowed to proceed for longer periods of time the chromatographic peaks due to the various oxidizable monomers and oligomers (see later discussion) became smaller and after sufficiently long periods completely disappeared. LC peak 1 was due largely to inorganic phosphate and attempts to remove this phosphate from any co-eluted organic products were unsuccessful. The products eluted under LC peaks 2 and 3 were also heavily contaminated with inorganic phosphate which could not be removed even after repeated chromatography. LC component 2 had a characteristic UV spectrum (X,,, = 262, 210 nm at pH 5 l). LC component 3 had a very similar spectrum (X max= 260. 207 nm at pH 5 *). Because of the heavy contamination by phosphate and the small amounts of LC components 2 and 3 it was not possible to gain further information about the structure of these products. LC component 4 was also isolated in only very small amounts. It showed a broad UV band, at pH 5.0, extending from 200-360 nm with A,,, = 235 (sh) and

l

Chromatograpbic mobile phase.

145

Retention t ime/min Fig. 3. High performance liquid chromatogram of the product mixture (200 PL) formed upon electrooxidation of ca. 1 m M guanosine at 1.2 V in pH 7.0 phosphate buffer (p = 0.5 M). A reversed phase column (Brownlee Laboratories, RP-18, 5 pm, 25 X0.4 cm) was used. The mobile phase was 0.6% v/v dioxane in aqueous 10 mM formic acid adjusted to pH 5.0 with triethylamine. After 20 min the concentration of dioxane was increased to 3X v/v. Flow rate: 1 ml min-‘.

208 nm. Owing to the very small amounts studied further.

of LC component

4 obtained

it was not

Liquid chromatographic components 5 and 6 As was noted earlier, relatively large injections (2 ml) of the crude product mixture obtained by electrooxidation of 1 caused LC components 5 and 6 to elute together. After 50-60 repetitive injections the combined eluent containing LC components 5 and 6 was freeze dried. The resulting solid product was dissolved in water (ca. 10 ml) and chromatographed a second time. The resulting mixture was freeze dried at least 3-4 times to remove triethylammonium formate. The resulting mixture of LC components 5 and 6 was dissolved in 10 ml of water and separated by HPLC (Brownlee RP-18, 5 pm, 25 X 0.7 cm) using aqueous 0.1 M HCOOH adjusted to pH 4.3 with ammonia as the mobile phase. Using a flow rate of 5 ml min-’ and 2 ml injections LC component 5 emerged with a retention time of 16.5 min and LC component 6 emerged with a retention time of 20.5 min. A baseline separation of the two components was obtained. The individual components were collected and freeze dried 2-3 times to remove ammonium formate.

146

Liquid chromatographic component 5 LC component 5 was a white powder.

The UV spectrum of this product (%ll, = 295, 262 (sh), 203 nm at pH 5.0) and a CV at pH 7 are shown in Figs. 4A and 4B, respectively. The first negative scan, starting at 0.0 V, showed no reduction peaks, i.e., the compound is not electrochemically reducible. However, the first positive scan shows that LC component 5 gives oxidation peaks at 0.86 V and 1.17 V (Fig. 4B). On the reverse sweep several small reduction peaks appear at 0.45 V, - 0.22 V, - 0.45 V, -0.52 V and - 0.86 V. On the second positive sweep a new oxidation peak appears at 0.48 V. FAB-MS of LC component 5 gave an intense pseudomolecular ion (MH+) at m/e = 581.1688 (C,,H,,N,,O,,, calculated m/e = 581.1704). Before discussing the ‘H-NMR spectrum of LC component 5 and other products it is of value to summarize that of 1 (300 MHz). In Me,SO-d, the spectrum of 1 shows S = 10.64 ppm (broad s, 1 H, Ni-H), 7.92 (s, 1 H, Cs-H), 6.48 (s, 2 H, C,-NH,). At higher field regions the spectrum of 1 becomes extremely complex

1

L 2c10

300

/

1

C

\ 300

J ‘0 0

400 Wavekwth/nm

I’>.1

Potential/

V vs SCE-

Fig. 4. UV spectra of (A) LC component 5 and (C) LC component 6 at pH 5.0. Cyclic voltammograms at the PGE of (B) LC component 5 and (D) LC component 6 in pH 7.0 phosphate buffer (p = 0.5 M) at a sweep rate of 200 mV s- ‘.

147

owing to the various sugar residue C-H and O-H resonances. In Me,SO-d, exchanged with D,O, a much simpler spectrum was obtained. At room temperature: S = 7.92 ppm (s, 1 H, Cs-H), 5.71 (d, 1 H, C&H), 4.41 (t, 1 H, C&H), 4.10 (t, 1 H, C&-H), 3.88 (q, 1 H, C&H), 3.60 (m, 2 H, C&H,). LC component 5 (Me,SO-d,): S = 8.31 ppm (broad s, 2 H, C,-NH,, A), 7.17 (broad s, 2 H, &--NH,, B), 8.14 (s, 1 H, C,-H, B). In Me,SO-d, exchanged with D,O at room temperature (ca. 22°C): S = 8.11 ppm (s, 1 H, C,-H, B), 5.90 (broad d, 1 H, C;-H, A), 5.24 (broad s, 1 H, Ci-H, B), 4.36 (broad m, 2 H, C;-H, A) and C&H, B), 4.10 (t, 1 H, C&H, A), 3.76 (1 H, C&H, B), 3.95 (q, 1 H, C,‘-H, A), 3.63 (m, 1 H, C;-H, B); between 3.6-3.2 ppm complex overlapping multiplets were observed (4 H, C-H, A, B). The latter spectrum became very much better defined at 60°C: S = 8.04 ppm (s, 1 H, C,-H, B), 5.86 (d, 1 H, C;-H, A), 5.28 (d, 1 H, C/;-H, B), 4.39 (t, 1 H, C&H, A), 4.29 (t, 1 H, C&H, B), 4.13 (t, 1 H, C&H, A), 3.83 (t, 1 H, C&H, B), 3.97 (q, 1 H, C&H, A); between 3.7-3.4 ppm complex, overlapping multiplets (4 H, C&H,, A, B) were observed. The interpretation of these NMR spectra, particularly assignments for individual ribose residue protons, was based on decoupling experiments. The NMR spectra indicate that only one Ca-H group remains in the dimer (MW = 580, C,,H,,N,,O,,) and that both C,-NH, groups are intact although in different environments. LC component 5 did not show any N,-H resonances up to 11.5 ppm. In view of the fact that the C,-NH, resonances of LC component 5, compared to that of 1, show a pronounced downfield shift, it has been concluded that any N,-H resonance of the former compound is shifted beyond 11.5 ppm. Clearly, only a single C-H group remains in LC component 5 and hence based on ‘H-NMR and FAB-MS evidence it is concluded that LC component 5 is an oxygen-bridged dimer linked between the C,-position of ring A and the N,-position of ring B (16). A I

16

0

I*

OHOH

Liquid chromatographic component 6 LC component 6 was a white solid. The UV spectrum of this product (A max = 300, 263, 232, 203 nm at pH 5.0) and its CV at pH 7.0 are shown in Figs. 4C and 4D, respectively. Clearly, the UV spectrum and cyclic voltammetric properties of LC component 6 are very similar to those of LC components 5 (compare Figs. 4C, 4D with 4A, 4B). FAB-MS of LC component 6 gave an intense pseudomolecular ion (MH+) at m/e = 581.1712 (C2,,H25N100,1, calculated m/e = 581.1704). Thus LC component 6 has the same molar mass (580 g) and molecular formula (C,H,,N,,O,,) as LC

148

component 5. The ‘H-NMR spectrum of LC component 6 (Me,SO-d,) showed 6 = 8.37 ppm (broad s, 2 H, ($--NH,, A), 8.13 (s, 1 H, Cs-H, B), 7.94 (broad s, 1 H, C,-NH, B). Decoupling experiments were used to assign the resonances observed for LC component 6 in Me,SO-d, exchanged with D,O. At room temperature (ca. 23°C) 6 = 8.14 ppm (s, 1 H, Cs-H, B), 5.87 (broad d, 1 H, C;-H, A), 5.38 (broad d, 1 H, C;-H, B), 4.36 (broad t, 1 H, C&H, A), 4.27 (t, 1 H, C;-H, B), 4.11 (t, 1 H, Ci-H, A), 3.76 (t. 1 H, C;-H, B), 3.97 (q, 1 H, C,‘-H, A), 3.64 (m, 1 H, C&-H, B), between 3.6-3.2 ppm complex overlapping multiplets due to the C,‘-H, groups of the A and B residues were observed. At 60 o C a much better resolved spectrum was obtained: 6 = 8.04 ppm (s, lH, Cs-H, B), 5.88 (d, lH, C;-H, A), 5.36 (d, lH, C;-H, B), 4.39 (t, lH, C&H, A), 4.31 (t, lH, C&H, B), 4.13 (t, lH, C&H, A), 3.84 (t, lH, C;-H, B), 3.98 (q, lH, C;-H, A); between 3.7-3.4 ppm complex overlapping multiplets due to C;-H, (A and B) and C&H (B) were observed. These spectral results indicate that only one Cs-H residue remains in the dimeric compound. Furthermore, a C,-NH, group remains intact on one guanosyl residue but in the other only a C,-NH group is present. LC component 6 did not show any N,-H resonances up to 11.5 ppm. However, as with LC component 5, the C,-NH resonances of LC component 6, compared to that of 1, show a pronounced downfield shift. It has been concluded that the N,-H resonances of LC compund 6 are shifted beyond 11.5 ppm. Thus, it is concluded that LC component 6 consists of two guanosyl residues linked by an oxygen bridge via the Cs- (A) and C,--NH (B) residues (17).

OHOH

17

OHOH

Liquid chromatographic component 7 The component eluted under LC peak 7, after removal of triethylammonium formate by repeated freeze drying, was a white solid. This compound showed a characteristic UV spectrum (X,, = 290, 245, 206 nm at pH 5.0). A CV of LC component 7 at pH 7.0 (Fig. 5A) showed three oxidation peaks at 0.35 V, 0.40 V and 1.03 V. On the reverse sweep several reduction peaks appear, one of which, at 0.325 V, forms a quasi-reversible couple with the oxidation peaks at 0.35 V/O.40 V. FAB-MS showed a strong pseudomolecular ion (MH+) at m/e = 300.0932 (C,,Ht,N,O,, calculated m/e = 300.0944). In Me,SO-d, exchanged with D,O at 22“C the ‘H-NMR spectrum showed S = 5.56 ppm (d, 1 H, Ci-H), 4.80 (t, 1 H, C&H), 4.08 (t, 1 H, C&H), 3.79 (q, 1 H, C&H), 3.56 (q, 1 H, C&H), 3.46 (q, 1 H, C;-H). Thus LC component lacks Cs-H and has a molar mass of 299 g. Chemically

149

r

I

I

I

+ov

)

I

I

I

Potential/

I

,

I

1

I

1

V vs. SCE

Fig. 5. Cyclic voltammograms at the PGE in phosphate buffer pH 7.0 (a = 0.5 M) of (A) LC component 7, (B) LC component 8, (C) LC component 9, (D) LC component 10, (E) 8-(8-guanosyl)guanosine (29), (F) LC component 11. Sweep rate: 200 mV s-l.

150

synthesized [4] 8-hydroxyguanosine (7) exhibited identical UV and ‘H-NMR tra, FAB-MS and cyclic voltammetric behaviors to LC component 7.

spec-

OHOH

7

Liquid chromatographic component 8 It is obvious from the liquid chromatogram shown in Fig. 3 that LC component 8 is a minor electrooxidation product of 1. A very small amount of this product was obtained and purified (100-200 pg). The white solid exhibited a characteristic UV spectrum (X max= 280, 252 nm at pH 5.0). A CV of LC component 8 (Fig. 5B) showed an oxidation peak at 0.95 V. On the reverse sweep several small reduction peaks appear and, on the second positive sweep, a new oxidation peak appears at 0.38 V. FAB-MS on LC component 8 showed intense pseudomolecular ions at m/e = 863 (MH+) and, particularly, at m/e = 885 (MNa+). High resolution FABMS on the latter ion gave an exact m/e = 885.2330 (Cj0Ha4Ni40i7Na, calculated m/e = 885.2124). Thus, LC component 8 has a molar mass of 862 g and hence is a trimeric species. This compound consists of two guanosyl residues linked to a third residue which must lack a nitrogen atom, and it contains two additional oxygen atoms. The only reasonable way to lose one nitrogen atom from a trimer of 1 is by loss of an amino group from one guanosyl residue. We have been unable to collect and purify sufficient amounts of this product to carry out ‘H-NMR studies. Nevertheless, the elemental composition obtained from FAB-MS indicates that the individual purine residues must be linked together by oxygen bridges. Furthermore, the fact that LC component 8 is electrochemically oxidized at much more positive potentials than is 8-hydroxyguanosine (compare Figs. 5B and 5A) suggests that each purine residue retains its N,=C, double bond. Although it is not possible to provide compelling evidence, it has been concluded that LC component 8 has a structure similar to 23.

OHOH

151

Liquid chromatographic

component 9

Liquid chromatographic analysis of samples of LC component 9 gave several peaks indicating that this product is not very stable. The UV spectrum of LC component 9, which was pale yellow, obtained immediately after it was collected from the column showed a broad shoulder extending from 310 to 430 nm along with peaks at A,, = 270 (sh), 251,207 nm (at pH 5.0). CV of LC component 9 (Fig. SC) showed two reduction peaks at -0.48 V and -0.86 V. On the reverse sweep five oxidation peaks appear at 0.325 V, 0.40 V, 0.925 V, 1.05 V and 1.15 V which can all be observed without scarming initially through the reduction peaks noted earlier. FAB-MS on LC component 9 showed an intense pseudomolecular ion (MH+) at m/e = 555.1917 (C,,H,,N,,O,,, calculated m/e = 555.1911). Thus, this component has a molar mass of 554 g and an elemental formula of C,,H,,N,,OlO. This product must be a dimer in which one guanosyl residue has lost one carbon atom. Unfortunately, the instability of LC component 9 in solution and the very small amount of pure compound isolated (ca. 100 pg) precluded detailed ‘H-NMR studies. Nevertheless, considerable information about the structure was obtained by collisional activation MS/MS. The pseudomolecular ion (MH+) was selected in MS1 and was activated with He gas to reduce its intensity by 50%. The pseudomolecular ion gave prominent daughter ions at m/e = 457, 423 and 325. Thus, it is

I

(m/e = 423)

(m/e = 555)

(m/e = 457) k

(m/e = 325) Scheme 1.

152

proposed that LC component 9 has structure 28a or 28b (Scheme 1). The important daughter ions are rationalized by the pathways shown in Scheme 1. Further support for the structure 28a/28b is provided by the ease of electrochemical oxidation of this product (see later discussion).

Liquid chromatographic component 10 This product was a white solid which showed a characteristic UV spectrum 10 (pH 7.0, Fig. 5D) (&XXX= 280, 246, 210 nm at pH 5.0). CV of LC component indicated that it was not electrochemically reducible. However, it showed voltammetric oxidation peaks at 0.82 V (sh), 0.86 V, 0.96 V and 1.15 V. On the reverse sweep reduction peaks appear at 0.35 V-O.5 V (broad), and -0.70 V and on the second positive sweep a new oxidation peak appears at 0.36 V. FAB-MS gave an intense pseudomolecular ion (MH+) at m/e = 565.1763 (C,,H2sN,,0,,, calculated m/e = 565.1755). Thus, the molar mass of this product is 564 g and it has an elemental composition C,,H,,N,,O,, indicating that it is a dimer of 1. Recent reports [ll-131 have shown that photochemical oxidation of an equimolar mixture of 8-bromoguanosine and 1 in acetone + water leads to formation of 8-(8guanosyl)guanosine (29), i.e., a dimer of 1 linked through the C, positions. However, the UV spectrum of 29 (h max = 318, 276 nm at pH 5.0) is quite different from

OHOH

29 that of LC component 10. Similarly, the CV of 29 (Fig. 5E), while similar to that of LC component 10 (Fig. 5D), shows only three oxidation peaks at 0.78 V, 0.86 V and 1.15 V. Thus, LC component 10 is different from 29 and, therefore, the guanosine residues must be linked together differently. The ‘H-NMR spectrum of LC component 10 (Me,SO-d,) shows 6 = 10.8 ppm (broad s, 1 H, N,-H, A), 8.31 (s, 1 H, Cs-H, B), 6.75 (s, 2 H, &--NH,, A), 6.50 (s, 2 H, C,-NH,, B). In the higher field region (6-3 ppm) the spectrum was extremely complex owing to the C-H and O-H resonances of the two ribose residues. In Me,SO-d, exchanged with DzO at room temperature: 6 = 8.31 ppm (s, 1 H, Cs-H, B), 5.81 (d, 1 H, Ci-H, A), 5.56(d, 1 H, C;-H, B), 4.50 (t, 1 H, C;-H, A), 4.70 (t, 1 H, C&H, B), 4.12 (t, 1 H, C&H, A), 4.05 (t, 1 H, C;-H, B), 3.94 (q, 1 H, C&H, A), 3,76 (q, 1 H, C&H, B), 3.7-3.1 (multiplets, 4 H, C;-H,, A and B). At 60°C the ‘H-NMR spectrum was virtually identical. These NMR spectra of LC component 10 indicate that in the dimer one Q-H and one Ni-H are missing but that both C-NH, groups are intact. Thus, it

153

appears that the dimer is linked through the C, position of residue A and the N, position of residue B as shown in 6. ,k+

6 component I1 This product was collected and, after removal of the eluent by freeze-drying, was a white powder. At pH 5.0 LC component 11 exhibited three UV bands at x = 283, 245, 210 run. Cyclic voltammetry (Fig. 5F) showed that LC component l?&es four oxidation peaks at pH 7.0 at 0.87 V, 0.94 V, 1.10 V and 1.13 V. The reverse sweep shows several small reduction peaks. On the second positive sweep a new oxidation peak appears at 0.35 V. FAB-MS of LC component 11 showed an intense, pseudomolecular ion (MH+) at 846.2484 (C30H36Ni5015; calculated m/e = 846.2515). Thus, LC component 11 must be a trimer of 1 having a molar mass of 845 g and a molecular formula C,,H,,N,,O,,. LC component 11 was not very soluble in MqSO-d, and hence its ‘H-NMR spectrum in D,O only was obtained. At 22°C this spectrum showed: S = 8.24 ppm (s, 1 H, Cs-H, A), 6.02 (d, 2 H, Ci-H, B), 5.74 (d, 1 H, C;-H, A,) 5.04 (t, 1 H, C&H, A), 4.70 (m, 2 H, C&-H, B). Between 4.5-3.2 ppm a complex overlapping series of multiplets were observed due to the various C;-H, C,‘--H and C;-H, resonances. These spectral results indicate that only one Cs-H is free in the trimer and that two ribose residues are very similar and are different from the third. In view of these results and the known molecular formula it is concluded that LC component 11 has the structure 14 where two residues of 1 are bound by N,-N, linkages and two are joined by C&s linkages. Liquid chromatographic

,

A

B

OHOH

Effects of concentration

I

OHOH

and pH

Controlled potential electrooxidation of 1 (l-l.5 mM) in pH 9.67 phosphate buffer (II = 0.5 M) at 1.2 V under the conditions outlined earlier followed by HPLC

154

analysis showed that LC components 3 and 4 and particularly 7 and 6 were the major products. Compounds 16 and 17 were not observed. Electrooxidations in phosphate buffer pH 4.28 (p = 0.5 M) revealed that 7 and 6 were the major products along with smaller amounts of 16, 17 and 28. Electrooxidations of 0.1 mM solution of 1 in phosphate buffer pH 7.0 (p = 0.5 M) could be completed in 15 min. Thus, such electrolyses were terminated after 5 min. HPLC analyses revealed that LC components 2 and 4 along with compounds 7 and 6 were present in the product solution. Compound 7 was the major product. Electrooxidation of 0.1 mM 1 at pH 9.67 gave 7 as the major product along with smaller amounts of LC components 3, 4 and compound 6. At pH 4.28 electrooxidation of 0.1 mM 1 gave 7 as the major product. These results indicate that 7 is a major product of incomplete electrooxidation of 1 in dilute (0.1 mM) solutions between pH 4 and 10. With increasing concentrations of 1, dimer 6 replaces 7 as the major detected product. Trimers such as 23 and 14 are observed only using almost saturated solutions of 1 (> 1 mM) at pH 7.0. REACTION

SCHEMES

The primary electrochemical oxidation reaction of 1, characterized by voltammetric peaks IIIJIV,, is pH-dependent. Cyclic voltammetry reveals that as a result of the peaks IIIJV, reaction an intermediate species is formed which can be reduced in the peak I, reaction and that the reduction product gives rise to peaks 1: and II: on the second anodic sweep (Fig. lB, C). 8-Hydroxyguanosine (7) gives the peaks Ii, II;, I, system (Fig. 5A). However, with increasing concentrations of 1 the voltammetric peaks associated with 7 become much smaller. Furthermore, coulometric results indicate that with increasing concentrations of 1 electrooxidized the apparent n value decreases. Product analyses correspondingly reveal that electrooxidations of near-saturated solutions of 1 lead to formation of a variety of dimeric and trimeric nucleosides. Electrochemical oxidations of 7 under the same conditions used for 1 do not lead to oligomeric products [14]. These behaviors suggest that the initial electrooxidation of 1 gives a primary species which, in dilute solution, can undergo further reactions leading to 7 and hence its oxidation products while in more concentrated solutions the primary species can react to give, ultimately, oligomeric products. In a recent report [2] it was demonstrated that the initial electrochemical oxidation of xanthosine (30, eqn. 3) is a 1 e-, 1 H+ reaction leading 0

0

_H+

T&j 4

-

HO

0

J&* 4 HO

0

ti

ti OHOH

30

-em

OHOH

31

to the free radical 31 (eqn. 3) which then undergoes various dimerization chemical and electrochemical reactions. In the case of 30. at concentrations

(3) or other around

155

OH OH

1

2+

0 1

-

2 +HaO -

Fig. 6. Reaction scheme proposed for the initial peaks III,/IVa the primaq radical 2 and secondary radicals 3,4 and 5.

electrc-oxidation of guanosine (1) to give

2 mM, voltammetric, potentiostatic and coulometric n values close to 1.0 were measured experimentally. However, it is not possible to employ such high concentrations of 1 because of solubility limitations. In addition, controlled potential electrooxidations of 1 proceeded more slowly than for 30 due to the more positive potentials required for the oxidation of 1 and, perhaps, to electrode blockage by adsorbed reactant and/or product(s). Thus, it has not been possible to find conditions under which 1 is electrochemically oxidized only to or primarily to 1 eproducts. Nevertheless, the observed electrochemistry and the nature of the isolated reaction products strongly supports the conclusion that the initial step in the peaks IIIJIV, oxidation of 1 is a 1 e-, 1 H+ process which occurs at the Cs-H position to give the free radical 2 (Fig. 6). Subsequent formation of 7 as an important product provides evidence that the initial oxidation occurs at the Cs-H position. We have been unable to detect any Cs-Cs-linked dimers as a result of the electrochemical oxidation of 1 although it has been reported [13,15,16] that photochemical oxidation of mixtures of 8-bromo-1 and 1 leads to such a dimer in very low yields. However, a major difference between the latter photochemical reaction and the electrochemical oxidation of 1 is that the radical species formed electrochemically is formed in high concentrations over a short period of time in the vicinity of the electrode surface. In the photochemical reaction very low concentrations of unknown radicals are formed over long periods of time (days) in homogeneous solution. In order to account for

156

the various monomers, dimers and trimers which are formed electrochemically it is clear that radicals in addition to 2 (Fig. 6) must be formed. While in principle a 1 e-, 1 H+ oxidation of 1 could lead directly to other free radicals there is no evidence from many studies of the electrooxidation chemistry of purines such as xanthine [6], 30 [2] and guanine 1171 that the initial oxidation reaction occurs at a site other than the C, position. Accordingly, it is suggested that radical 2, formed at the electrode surface, can react with 1 diffusing to the surface forming radicals 3 and 4 (Fig. 6) i.e., in hydrogen atom abstraction reactions. In order to account for the formation of 7 and various oxygen bridged dimers, radical 2 must also react with water giving the hydroxylated radical 5. Reaction between the primary radical 2 and radical 3 leads to S-(1guanosyl)guanosine (6, Fig. 7) which is a major peak IIIJIV, electrooxidation

OHOH

2 2

H2O

-2H+-2e- [Peak I’JI’,

]

++2H++2e- [Peak I,]

COOH

COOH

;-

Products

Fig. 7. Reaction schemes proposed for formation of 8-(l-guanosyl)guanosine (7) upon peaks III ./IV, electrochemical oxidation of guanosine.

(6) and 8-hydroxyguanosine

157

product of 1 between pH 4 and 10. Observation of the characteristic peaks of 7 (I,, I:, 111) in cyclic voltammograms of 1 is explained by further 1 e-, 1 H+ electrooxidation of hydroxylated radical 5 to 7. Since 7 is more easily electrooxidized than 1 (compare Figs. 1 for 1 and 5A for 7) it is further oxidized. The electrochemical oxidation of 7 is currently being studied and details of the reaction are not yet known [14]. Nevertheless, based upon the known electrochemical oxidation of the parent base, 8-hydroxyguanine [17], and of similar purine bases [18] it is very probable that 7 is oxidized in a 2 e-, 2 H+ reaction to the quinonoid 8 (Fig. 7). It is 8 which is responsible for reduction peak I, observed in cyclic voltammograms of 1 (Fig. 1) and 7 (Fig. 5A) which corresponds to the quasi-reversible reduction of quinonoid 8 to 7 (Fig. 7). By analogy to similar compounds formed upon electrochemical oxidation of 8-hydroxyguanine [17] and other purine bases [18] and nucleosides [l] quinonoid 8 is undoubtedly very unstable and is attacked by water to give tertiary alcohol 9 (Fig. 7) which rearranges to the bicyclic carboxylic acid 10. Based on earlier studies [1,17,18] it is probably 10 which is observed in thin-layer spectroelectrochemical oxidations of 1 and 7. Carboxylic acid 10 disappears in a (pseudo-)first-order reaction which is probably attack by water giving 11 (Fig. 7)

OHOH

3

Fig. 8. Reaction scheme proposed for formation of S-(8-guanosyl)-1-(1-guanosyllguanosine peaks 111,/W, electrochemical oxidation of guanosine.

(14) upon

R

Fig. 9. Reactlon guanosyl)guanosine

scheme proposed for formation of 8-O-(l-guanosyl)guanosine (17) upon peaks 111,/W, electrcchermcal oxidation of guanosine.

(16) and

8-0-(2-

which decomposes to products. In the case of the electrochemical oxidation of 8-hydroxyguanine an ultimate product formed is 5-guanidinohydantoin [17]; hence it is likely that 11 decomposes to the riboside of the latter compound. However, electrochemical reaction pathways for 7 will be described in a later report. Cyclic voltammograms show that with increasing concentrations of 1, peaks I,, 1: and II: become progressively smaller relative to peaks IIIJIV, (p. 140). Peak I, is due to reduction of quinonoid 8 (Fig. 7) to 8-hydroxyguanosine (7, Fig. 7) and peaks 1: and II: to oxidation of 7 on the subsequent positive sweep. The latter peaks become smaller with increasing concentrations of 1 owing to the preferential occurrence of second-order follow-up reactions of radicals 2, 3 and 4 rather than the pseudo-firstorder attack of water on radical 2 to give hydroxy radical 5 and hence 7. There are several routes which can lead to the trimer 8-(8-guanosyl)-l-(l-guanosyl)guanosine (14, Fig. 8). One route involves dimerization of radical 3 to give 1-(1-guanosyl)guanosine (12, Fig. 8). We have been unable to isolate 12 and thus it is proposed that it is further oxidized either electrochemically (- 1 H+, - 1 e-) or chemically by a hydrogen atom abstraction reaction with radicals 2, 3, or 4 to give the dimeric radical 13. This can then couple with radical 2 to give 14 (Fig. 8). Dimers 16 and 17 (Fig. 9) consist of two guanosyl residues linked by oxygen bridges. There seems to be only one reasonable route to form such dimers which involves electrochemical oxidation of the hydroxylated radical 6 to the oxyradical 15 (Fig. 9). Coupling of 15 with radical 3 leads to 8-O-(1-guanosyl)guanosine (16, Fig. 9) or with radical 4 leads to 8-O-(2-guanosyl)guanosine (17).

159

As noted earlier the structure of trimer 23 has not been fully confirmed by NMR spectra because it is obtained in only very small amounts. Nevertheless, high resolution FAB-MS indicates that 23 or a very similar structure is probable. Trimer

n

c

H+-e-

A

1

R

1%

23 Fig. 10. Reaction scheme proposed for formation of 1-O-(2-xanthosyl)-8-O-(1-guanosyl)guanosine upon peaks IIIa/IVa electrochemical oxidation of guanosine.

(23)

160

R

24 OHOH

5

I

-2H+ -2e-

Fig. 11. Reactlon scheme proposed for formation of ribofuranosyl-7-amino-2,4,6,8-tetraaza-5,7-diene-bicyclo-(3.3.0)octane chemical oxidation of guanosine.

1,2-dihydro-3-hydroxy-3-(1-guanosyl)-4-/3-D(28) upon peaks III,/IV, electro-

23 differs from other identified compounds in that one exocyclic amino group has been lost. Thus, it is proposed that radical 4, which should exist in the tautomeric form 4a and contains an imino residue, is hydrolyzed to the xanthosyl residue 18a/18b (Fig. 10). Coupling of radicals 18b and 3 then leads to 2-0-(1guanosyl)xanthosine 19. Electrochemical (-1 H+, - 1 e-) or chemical (-H’) oxidation of 19 must then give the radical dimer 20 which, following attack by water, is further oxidized (- 2 H+, -2 e-) to the dimeric oxyradical 22 (Fig. 10). Coupling of radicals 3 and 22 then gives l-0-(2-xanthosyl)&O-(l-guanosyl)guanosine (23, Fig. 10). The low yield and instability of LC component 9 in aqueous solution and hence the absence of a useful ‘H-NMR spectrum make an exact structural assignment for this product difficult. However its elemental formula (C,,H,N,,O,,) and the collisional activation mass spectral data (Scheme 1) indicate that LC component 9 has the structure 28 (Fig. 11). Thus, it is suggested that the hydroxylated radical 5 couples with radical 3 to give the hydroxylated dimer 24 (Fig. 11). Unlike other

161

identified oligomeric products, 24 possesses an 8-hydroxyguanosyl residue and hence would be expected to be easily oxidized ( - 2 H+, - 2 e-) to the quinonoid dimer 25. The expected nucleophilic attack by water on 25 would lead to the tertiary alcohol 26 and, by analogy with the known electrochemical behavior of B-hydroxyguanine [17], this should rearrange to the carboxylic acid 27 (Fig. 11; see also Fig. 7). Decarboxylation of 27 would then give 1,2-dihydro-3-hydroxy-3-(1-guanosyl)-4P-ribofuranosyl-7-ao-2,4,6,8-tetraaza-5,7-diene-bicyclo(3.3.O)-octane (28). The reaction schemes outlined in Figs. 6-11 represent reasonable pathways to the various fully or partially identified products formed upon electrochemical oxidation of 1. Because of the formation of a variety of reactive radical species it would be expected that many more oligomeric products should be obtained and, indeed, liquid chromatography of product solutions clearly indicates that several additional products are formed. Thus, compounds 6, 14, 16, 17, 23 and 28 represent only those products which we have been able to isolate in sufficient quantity to permit structural information to be obtained. The reaction schemes outlined in Figs. 7, 9, 10 and 11 and products 7, 8, 14, 16, 17, 23 and 28 require further electrochemical and/or chemical oxidation steps. It is these reactions which no doubt account for the fact that coulometric n values are always in excess of 2. In an earlier report [18] it was shown that guanine, the parent base of 1, is oxidized first to 8-hydroxyguanine (2 e-, 2 H+) which, being more easily oxidized, is further oxidized (2 e-, 2 H+) to an unstable quinonoid intermediate. A series of hydration and other follow-up chemical and electrochemical reactions leads to the final products, 2,5-diimino-4-irnidazolone and Sguanidinohydantoin. Oligomeric products are not formed in the electrooxidation of guanine. This fact is almost certainly due to the low solubility of this compound (at least 20-25 times less than that of 1). The low solubility of guanine would yield low concentrations of the C(8)’ guanyl radical which in turn would not favor second-order follow-up reactions. Rather, a pseudo-first-order attack by water on the latter radical would give an 8-hydroxyguanyl radical similar in structure to 5 (Fig. 7) which would then be oxidized to 8-hydroxyguanine and hence to the final monomeric products. The isolated guanine oligonucleosides are all electrochemically oxidizable (Fig. 5) and cannot be isolated if the oxidation of 1 is permitted to proceed until all oxidizable species are removed. Furthermore, cyclic voltammograms of all of the oligonucleosides show that after scanning their voltammetric oxidation peaks a small reduction peak appears at ca. 0.33 V (at pH 7.0) and, on the second positive sweep, one or two oxidation peaks appear at about 0.4 V. This behavior suggests that these oligonucleosides are electrochemically oxidized to a species similar to the quinonoid intermediate formed upon oxidation of 7, i.e., 8 (Fig. 7) which can be reduced at 0.33 V to give either 7 or a compound having a very similar structure. No attempt has yet been made to probe the electrochemical oxidations of the various guanine oligonucleosides isolated but it is clear from their cyclic voltammograms that some very complex chemistry is involved. It does seem reasonable, however, that under certain conditions formation of even larger oligonucleosides of guanine might be possible.

162

ACKNOWLEDGEMENTS

This work was supported by NIH Grant No: GM-21034. The authors would also like to thank Dr. R. Jeremy H. Davies of the Biochemistry Department, Medical Biology Centre, Queen’s University, Belfast, Northern Ireland for providing a sample of 8-(8-guanosyl)guanosine. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

R.N. GoyaI, A. Brajter-Toth, J.S. Besca and G. Dryhurst, J. Electroanal. Chem., 144 (1983) 163. S.K. Tyagi and G. Dryhurst, J. Electroanal. Chem., 216 (1987) 137. T. Yao, T. Wasa and S. Musia, Bull. Chem. Sot. Jpn., 50 (1977) 2917. R.E. Holmes and R.K. Robins, J. Am. Chem. Sot. 87 (1965) 1771. G.D. Christian and W.C. Purdy, J. Electroanal. Chem., 3 (1962) 363. J.L. Owens, H.A. Marsh and G. Dryhurst, J. Electroanal. Chem., 91 (1978) 231. R.N. Adams, Electrochemistry at Solid Electrodes, Marcel Dekker, New York, 1969. V.E. Norvell and G. Mamantov, Anal. Chem., 49 (1977) 1470. R.H. Wopschall and 1. Sham, Anal Chem.. 39 (1967) 1514. R.S. Nicholson and I. Sham, Anal Chem., 36 (1964) 706. Ref. 7, 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. (M), (1981) 2701. A. Bhide, P. Subramanian and G. Dryhurst, work in progress. S.N. Bose, R.J.H. Davies, J.W. Anderson, J.C. van Niekerk, L.R. Nassimbeni and R.D. Macfarlane, Nature, 271 (1978) 783. 16 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. 17 R.N. GoyaI and G. Dryhurst, J. Electroanal. Chem., 135 (1982) 75. 18 R.N. GoyaI, A. Brajter-Toth and G. Dryhurst, J. Electroanal. Chem., 131 (1982) 181.