g=i&gj- Chemico-Biological Interactions 102 (1996) 93- 100

ELSEVIER

Structure and mechanism of formation of a new adduct from formaldehyde and* guanosine

Gordon Kennedya, Pritpal K. Slaich”, Bernard T. Gelding”**, William P. Watson’*b

“Department of Chemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne NEI 7RV, UK

‘Shell Research Ltd, Sittingbourne Research Centre, Sittingbourne, Kent ME9 SAG, UK

Received 9 May 1996; revised 7 August 1996; accepted 13 August 1996

Abstract

Reactions between formaldehyde and guanosine in the range pH 4-10 gave N2-hydroxy- methylguanosine 1, bis-N2-guanosinylmethane 2 and 7-[7-(/I-D-ribofuranosyl)-purin-6(7H)- on-2-yl]-3-(P-D-ribofuranosyl)-5,6,7,8-tetrahydro-sym-triazino[l,2-a]purin-l0(3H)-one 3a. The latter is a new adduct, the formation of which occurs in neutral solution, is favoured at higher pH and can be rationalised by a sequence of condensations involving two molecules each of guanosine and formaldehyde.

Keywords: Guanosine adduct; Formaldehyde; Mechanism

Abbreviations: CI, chemical ionisation; COSY, correlation spectroscopy; DEPT, distortions enhanced polarisation transfer; EI, electron impact; FAB, fast atom bombardment; ODS, octadecylsilyl.

* Corresponding author. ’ Present address: Shell International Chemicals BV, Shell Research and Technology Centre Amster-

dam, Toxicology Department, P.O. Box 38000, 1030BN Amsterdam, The Netherlands.

0009-2797/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved

PII SOOOS-2797(96)03737-4

94 G. Kennedy et al. / Chemico-Biological Interactions 102 (1996) 93-100

1. Introduction

Formaldehyde is an important industrial and laboratory chemical. It occurs as an environmental contaminant formed in the combustion of organic matter. Importantly, formaldehyde is produced by the natural metabolism in all cells and is required for the biosynthesis of serine [l]. Human exposures are therefore unavoidable. Formaldehyde is also a product of the metabolism of many xenobi- otics, e.g. hexamethylphosphoramide [2]. Studies with cultured mammalian cells and bacteria have shown that formaldehyde is genotoxic [3]. Long-term inhala- tion experiments with rats have established that it is a nasal carcinogen [4]. During our studies on the formation of glycidaldehyde-guanosine adducts [5] we discovered that formaldehyde is released from the major adduct, and this leads to the additional formation of a formaldehyde-guanosine adduct (for a prelimi- nary account see Ref. [6]). To aid the analysis of the range of adducts in the glycidaldehyde-guanosine system, we investigated the reactions of formaldehyde with guanosine at a range of pH values (previous studies have described the formation of N2-hydroxymethylguanosine 1 [7] and bis-iV*-guanosylmethane 2 [S]). We have identified a new adduct 3a, the formation of which is favoured at high pH. The present report fully details our studies on the chemistry of formaldehyde with guanosine.

2. Materials and methods

2.1. Instruments

NMR spectra were measured with Bruker, Perkin Elmer or Varian instruments operating at frequencies given below with the data for each compound. Unless stated otherwise, 13C NMR spectra were measured with broad band proton decoupling. To aid assignment of resonances, where appropriate, the techniques of spin decoupling and 2D COSY (‘H spectra) and DEPT (i3C spectra) were used. The spectra were determined for solutions in the solvents cited below. Tetramethylsilane was used as internal standard for organic solvents and 3- trimethylsilylpropionic acid for D20. Mass spectra (EI or CI or FAB mode) were recorded with a Finnigan 4500, Kratos MS50, Kratos MS80 RF or VG 7070E instrument. The pH values of solutions were measured with a Corning model PTl-5 or EEL 12 meter.

2.2. HPLC

The analyses of reaction mixtures and the isolation of products were carried out using either Waters or Altex instruments. The column and solvent conditions are described below for specific separations.

G. Kennedy et al. / Chemico-Biological Interactions 102 (1996) 93-100 95

2.3. Chemicals

Chemicals were either AnalaR grade, which were used directly, or laboratory reagent grade purified further when appropriate. Solvents for HPLC were commer- cially available HPLC-grade.

2.4. Isolation of adducts from the reactions of guanosine with formaldehyde

To a solution of guanosine (146 mg, 0.5 mmol) in aq. sodium hydroxide (pH 10.1) at 75°C was added formaldehyde (37% w/v aq. soln, 200 ~1, 2.4 mmol). The reaction was monitored by HPLC (Ultrasphere ODS 25 x 1 cm, eluted with an aq. methanol gradient, 2.5 ml min ‘). After 48 h, three adducts 1, 2 and 3 were separated by semi-preparative HPLC (retention times relative to guanosine: 1.1, 2.1 and 1.75, respectively) and isolated by evaporation of column eluates:

Compounds 1 (N2-hydroxymethylguanosine) and 2 (bis-N2-guanosylmethane) exhibited spectroscopic properties in agreement with data reported [7,8]

7-[7-(B-D-ribofuranosyl)-purin-6(7H)-on-2-y1]-3-(P-~-ribofuranosyl)-5,6,7,8- tetrahydro-sym -triazino[ 1 ,Za]purin- 10(3H)-one, 3a, white hygroscopic solid, ‘H NMR [360 MHz, (CD,), SO] 6 3.35 (4 H, m, 2 x 5’-CH,OH), 3.85 (2 H, m, 2 x H-4’) 4.06 (1 H, m, H-3’), 4.12 (1 H, m, H-3’), 4.37 (1 H, m, H-2’), 4.52 (1 H, m, H-2’), 4.99 (2 H, m, 2 x 5’-OH), 5.06 (2 H, m, 2 x 3’-OH), 5.13 (2 H, AB q, J 11 HZ, 6-CHJ, 5.27 (1 H, d, J 6 HZ, 2’-OH), 5.37 (1 H, d, J 6 HZ, 2’-OH), 5.63 (1 H, d, J 6 Hz, H-l’), 5.67 (1 H, d, J 6 Hz, H-l’), 5.75 (2 H, AB q, J 12 Hz, 8-CH,), 7.73 (1 H, br s, H-2 or H-12), 7.88 (1 H, s, H-2 or H-12), 8.11 (1 H, br s, 5-NH) (n.b. 15-NH not observed) (n.b. addition of D,O caused the disappearance of the resonances at 4.99, 5.06, 5.27, 5.37, 8.11); MS (positive ion FAB) m/z 591 (MH+); UV similar to that of guanosine.

Adducts 3b-3d were prepared analogously from [13C]formaldehyde ( > 99% ‘C) and either guanosine (for 3b) or a [“Nlguanosine (for 3c and 3d)(see Refs. [9] and [lo] for the preparation of the [‘5N]guanosines):

3b ‘H NMR identical to 3a except for further coupling in the resonances at 6 5.12 [J (‘H-13C) 155 Hz] and 5.75 [.I (‘H-13C) 163 Hz]; MS (positive ion FAB) m/z 593 (MH+).

3c and 3d MS (positive ion FAB) m/z 595 (MH+); ‘H NMR similar to 3b with ‘J coupling of 93 Hz to “N evident with &I;

Both 3c and 3d theoretically contain 9 species with calculated isotopic distribu- tions exemplified for 3c: ‘5N-9/15N-15 56%, 15N-9/15N-7 15%, ‘5N-9/14N 4%, 14N/15N-15 4%, 14N/15N-7 l%, 14N/14N 0.25%, 15N-5/15N-15 15%, ‘5N-5/‘5N-7 4%, ‘5N-5/‘4N 1%.

3. Results and discussion

3.1. Characterisation of formaldehyde-guanosine adducts

Reactions of an excess of formaldehyde with guanosine in aqueous solution at

96 G. Kennedy et al. / Chemico-Biological Interactions 102 (1996) 93-100

75°C were monitored by HPLC. At pH 4.4 adduct 1 was the major product accompanied by a small amount of 2. Similarly, at pH 6.3 the predominant adduct was 1, now accompanied by 3a as well as 2. All three adducts were observed at pH 10.1, the concentration of 3a reaching a maximum at 40 h, when it comprised 30% of the total integral.

The spectroscopic properties of adducts 1 and 2 agreed with those reported [7,8]. Adduct 3a gave ions of m/z 591 (MH+) and 613 (MNa+) in its positive ion FAB mass spectrum indicating its origin from 2 molecules each of formaldehyde and guanosine. The ‘H NMR spectrum showed inter alia two kinds of ribosyl moiety, two AB systems at 6 5.13 (J 11 Hz) and 5.75 (J 12 Hz) assigned to the methylene groups of the triaza ring, and singlets at 6 7.73 (H-2 or H-8) and 7.88 (H-2 or H-8). Although this data is consistent with structure 3a, we undertook isotopic labelling experiments to confirm the assignment.

Reaction of guanosine with [13C]formaldehyde gave 3b (13C,-labelled), the ‘H NMR spectrum of which showed doubling (J 155 and 163 Hz, respectively) of the AB systems at 6 5.13 and 5.75. Its 13C NMR spectrum was dominated by two resonances at 6 54.9 and 55.6, tentatively assigned to C-6 and C-8, respectively. The [13C,,1SN,]-labelled molecule 3c derived from [“Nlguanosine primarily labelled at N-l (75%; 15-20% at NHJ [9,10] showed two intense resonances in its 13C NMR spectrum: 6 55.0 and 55.5 (see Fig. la). The appearance of the C-8 resonance as a doublet (J (13C-15N) 7.5 Hz) with superimposed singlet (offset from the centre of the doublet because of an isotope shift) arises from the ‘J coupling between this carbon and N-9. The resonance for C-6 is principally a singlet because it is flanked by unlabelled nitrogen atoms in the dominant molecule. The [‘3C,,15N,]-labelled molecule 3d produced from [“Nlguanosine primarily labelled at NH2 (75%; 15- 20% at N-l) [9,10] also showed two intense resonances in its 13C NMR: 6 55.2 and 55.7 (see Fig. lb). The C-8 resonance exhibited a doublet because the dominant

I I I I

56.0 55.0 56.0 55.0

6, kwm) 6, Wm)

Fig. 1. ‘)C NMR spectra of ‘5N-labelled compounds: (a) 3c and (b) 3d.

G. Kennedy et al. 1 Chemico-Biological Interactions 102 (1996) 93-100 97

3a

3a r = ribose 36 r = ribose, 13C at C-6 and C-8 3c r = ribose, 13C at C-6 and C-8,

lsN at N-5 (15-200/o) and N-9 (75%); also at N-l (guanosine ring) and N-7 (15-20

3d r = ribose, *3C at C-6 and C-8, * fN at N-5 (75%) and N-9 (15-20%); also at N-l (guanosine ring) and N-7 (75%).

6

Scheme 1. Structures of adducts.

species are labelled at N-7. The resonance for C-6 is a broad multiplet because of the predominant labelling at both N-5 and N-7. Note, that the assignments for C-6 and C-S (see above) are confirmed by these results.

3.2. Mechanisms of adduct formation

Nucleophilic attack by guanosine NH, on formaldehyde leads directly to adduct 1, which gives 2 by loss of water and capture of the resulting iminium ion by guanosine. Further reaction of 2 with formaldehyde gives intermediate 4a or 4b, either of which could eliminate water to give 5a or 5b (Scheme 1). Finally, Sa or 5b cyclizes to adduct 3a. The need for deprotonation, either of 2 to give 4b, or of 4a to give 5a explains why 3a is only observed at higher pH (n.b. pK, of guanosine is

98 G. Kennedy et al. / Chemico-Biological Interactions 102 (1996) 93-100

9.4). The route to 3a is analogous to that postulated for the formation of polycyclic compounds from the reactions of formaldehyde with aromatic amines (e.g. ‘Triiger’s base’ from formaldehyde and p-toluidine [ 111). It has also been shown [ 121 that N9-substituted guanines react with formaldehyde in the presence of amines to give tricyclic adducts (e.g. 6) similar in structure to 3a (Scheme 2).

2

CH20

H

4a

I I 4b

Q

Sa

J 5b

-H+

3b

Scheme 2. Proposed pathway from guanosine and formaldehyde leading to 7-[7-(/?-D-ribofuranosyl)- purin-6(7H)-on-2-yl]-3-(8-D-ribofuranosyl)-5,6,7,8-tetrahydro-sym-triazino[l,2-a]purin-l0(3H)-one 3a via 1 and 2.

G. Kennedy et al. 1 Chemico-Biological Interactions 102 (1996) 93SlOO 99

3.3. Mechanisms of genotoxicity of formaldehyde

In principle, the formation of adduct 3a indicates a means by which relatively stable cross links could arise from the reaction of formaldehyde with DNA, thus contributing to the genotoxic action of formaldehyde. Although the principal reaction of formaldehyde with bases in DNA has been shown to have preference for adenine residues [13], it is highly likely that reactions also occur, albeit to a lesser extent, with guanine residues. For example acetaldehyde has been shown to react at N2 of guanine residues in deoxyribonucleosides and DNA [14,15]. Previous studies of formaldehyde-nucleic acid reactions have elucidated the nature of the major products in vitro (see for example Refs. [7,8,16-181) and in vivo DNA protein-cross links formed by formaldehyde have also been identified [19,20]. The precise mechanisms of action of formaldehyde’s genotoxicity and cytotoxicity are clearly rather complex. Whilst statistically the adducts of type 3a will have a rather low probability of formation, if these are formed in the same strand of a DNA chain, they would be expected to be mutagenic. Inter-strand stable cross links of this type would be expected to lead to cytotoxicity.

Cyclic adduct formation from nucleosides and reactive bi-functional chemicals is well known (see e.g. Ref. [5] and citations therein). In this paper we have shown how a cyclic adduct can also arise by stepwise condensations of a mono functional agent. This could be a mechanism by which both inter and intra-strand cross links could occur in DNA. Although adenine residues are clearly implicated as an important site of reactivity of DNA with formaldehyde, reactions with guanine residues cannot be completely excluded. A combination of reaction pathways with DNA may contribute to carcinogenesis by formaldehyde.

Acknowledgements

We thank EPSRC and Shell Research for support of this research through CASE studentships to G.K. and P.K.S.

References

[I] S.J. Benkovic, On the mechanism of action of folate- and biopterin-requiring enzymes. Ann. Rev.

B&hem., 49 (1980) 227-251.

[2] A.R. Dahl and W.M. Hadley, Formaldehyde production promoted by rat nasal cytochrome

P-450dependent monooxygenases with nasal decongestants, essences, solvents, air pollutants.

nicotine, and cocaine as substrates, Toxicol. Appl. Pharmacol., 67 (1983) 200&205.

[3] H.d’A. Heck and M. Casanova-Schmitz, Biochemical Toxicology of Formaldehyde, in: E. Hodg-

son, J.R. Bend and R.M. Philpot (Eds.), Rev. Biochem. Toxicol., 6 (1984) 1555189.

[4] J.A. Swenberg, C.S. Barrow, C.J. Boreiko, H. d’A. Heck, R.J. Levine, K.T. Morgan and T.B. Starr,

Non-linear biological responses to formaldehyde and their implications for carcinogenic risk

assessment, Carcinogenesis, 4 (1983) 9455952.

[5] B.T. Golding, P.K. Slaich, G. Kennedy, C. Bleasdale and W.P. Watson, Mechanisms of formation

of adducts from reactions of glycidaldehyde with 2’-deoxyguanosine and/or guanosine, Chem. Res.

Toxicol., 9 (1996) 147-157.

100 G. Kennedy et al. 1 Chemico-Biological Interactions IO.? (1996) 93-100

[6] C. Bleasdale, S.B. Ellwood, B.T. Golding, G. Kennedy, P.K. Slaich, O.J. Taylor, and W.P. Watson, Studies to Probe the Mechanisms involved in the Genotoxic Action of Chemicals, in: R.C. Garner, P.B. Farmer, G.T. Steele and A.S. Wright (Eds.), Biomonitoring and Carcinogen Risk Assessment, Oxford University Press, London, 1991, pp. 2155221.

[7] F.A. Beland, N.F. Fullerton and R.H. Heflich, Rapid isolation, hydrolysis and chromatography of formaldehyde-modified DNA, J. Chromatog., 308 (1984) 12 I- 13 1.

[8] Y.F.M. Chaw, L.E. Crane, P. Lange and R. Shapiro, Isolation and identification of cross-links from formaldehyde-treated nucleic acids, Biochemistry, 19 (1980) 552555531.

[9] B.T. Golding, P.K. Slaich and W.P.Watson, Conversion of AICA-riboside into [“Nlguanosines, J. Chem. Sot., Chem. Commun., (1986) 901-902.

[lo] C. Bleasdale, S.B. Ellwood, B.T. Golding, P.K. Slaich, O.J. Taylor and W.P.Watson, Synthesis of [“Nlguanosines and deoxy[‘sN]guanosines from 5-amino-l-(j?--D-ribofuranosyl)imidazole-4-carbox- amide (‘AICA-riboside’), J. Chem. Sot. Perkin Trans., 1 (1994) 2859-2865.

[ll] M.A. Spielman, The structure of Troeger’s base, J. Am. Chem. Sot., 57 (1935) 583-585. [12] V.S. Volkov, A.M. Poverennyi and E.D. Sverdlov, Interaction of formaldehyde with nucleic acids

and their structural components in the presence of amines. III. Formation of tricyclic 1,2-N-adducts of 9-substituted guanines in the reaction with formaldehyde and primary amines, Biorg. Khim., 13 (1987) 204212.

[13] H. Huang and P.B. Hopkins, DNA interstrand cross-linking by formaldehyde: Nucleotide sequence preference and covalent structure of the predominant cross-link formed in synthetic oligonucle- otides, J. Am. Chem. Sot., 115 (1993) 9402-9408.

[I41 C.E. Vaca, J.-L. Fang and E.K.H. Schweda, Studies of the reaction of acetaldehyde with deoxynucleosides, Chem. Biol. Interact., 98 (1995) 51-67.

[IS] J-L. Fang and C.E. Vaca, Development of a “P-postlabelling method for the analysis of adducts arising through the reaction of acetaldehyde with 2’-deoxyguanosine-3’-monophosphate and DNA, Carcinogenesis, 16 (1995) 2177-2185.

[16] H. Fraenkal-Conrat, Reaction of nucleic acid with formaldehyde. Biochem. Biophys. Acta., 15 (1954) 307-309.

[17] D.J. McGhee and P.H. von Hippel, Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases, Biochemistry, 14 (1975) 1281~1303.

[18] Y-T. Chang and G.H. Loew, Reaction mechanisms of formaldehyde with endocyclic amino groups of nucleic acid bases, J. Am. Chem. Sot., 116 (1994) 354X-3555.

[19] M. Casanova, D.F. Deyo and H.d’A. Heck, Covalent binding of inhaled formaldehyde to DNA in the nasal mucosa of Fischer 344 rats: analysis of formaldehyde and DNA by high performance liquid chromatography and provisional pharmacokinetic interpretation, Fund. Appl. Toxicol., 12 (1989) 3977417.

[20] M. Casanova, K.T. Morgan, W.H. Steinhagen, J.1. Eve&t, J.A. Popp and H.d’A. Heck, Covalent binding of inhaled formaldehyde to DNA in the respiratory tract of rhesus monkeys: pharmacoki- netics, rat to monkey interspecies scaling, and extrapolation to man, Fund. Appl. Toxicol., 17 (1991) 409-428.