Characterization of a 5 '-Aldehyde Terminus Resulting from the

Chem. Res. Toxicol. 2001, 14, 1413-1420

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Characterization of a 5′-Aldehyde Terminus Resulting from the Oxidative Attack at C5′ of a 2-Deoxyribose on DNA Ariane Angeloff,† Igor Dubey,†,‡ Genevie`ve Pratviel,† Jean Bernadou,*,† and Bernard Meunier*,† Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, F-31077 Toulouse Cedex 4, France and Institute of Molecular Biology and Genetics, National Academy of Sciences, Kiev, Ukraine Received April 19, 2001

The 5′-aldehyde terminus is a DNA oxidative damage resulting from attack at C5′ of 2-deoxyriboses by some potent natural or chemical DNA cleavers. To offer a fast and specific method for characterization of this type of damage, we used on-line electrospray ionization mass spectrometry (ESI-MS) detection during liquid chromatography analyses. The intrinsic reactivity of 5′-aldehyde terminus with nucleophiles (formation of hydrate with water, of a Tris adduct with Tris buffer) or through β-elimination reaction resulted in complex LC profiles and MS data. We showed that derivatization of the aldehyde function as an oxime ether gives a stable derivative easy to characterize during on-line ESI-MS analyses. Complete structural characterization of the Tris adduct and the oxime ether derivative were obtained from MS and detailed NMR studies performed on derivatized 5′-aldehyde thymidine models.

Introduction Electrospray ionization mass spectrometry (ESI-MS) has proved to be a powerful technique for the analysis of modified oligonucleotides (1-5). As a promising development, liquid chromatography coupled to electrospray ionization mass spectroscopy (LC/ESI-MS) is a further emerging convenient and versatile method, especially for the analysis of chemical damage of DNA (6-9). The online ESI-MS analysis allows the determination of the molecular mass of the separated peaks of the different products formed by chemical modification of DNA or DNA models. However, some DNA products may be degradated within the electrospray source leading to a misinterpretation of the data if the transformation is complete or to a decrease of sensitivity if the resulting mass spectrum consists of a complex mixture of ionizable species. In the present report, the potentiality of the LC/ESI-MS method has been applied to the case of 5′-aldehyde ending oligonucleotides. Such type of damaged ending DNA fragments has been previously observed during the analysis of DNA oxidized by chemical nucleases, like metalloporphyrins, or natural products, such as enediyne compounds (10). We report the LC/ESI-MS analysis of the complex reaction mixture resulting from the oxidative damage of a 7-mer double-stranded duplex by an oxo-metalloporphyrin. The DNA oxidation was performed with the chemical nuclease mesotetrakis(4-N-methylpyridiniumyl)porphyrinatomanganeseIII (Mn-TMPyP) activated by sodium sulfite/dioxygen (11, 12). This system can oxidize * To whom correspondence should be addressed. Fax: + 33-561553003. E-mail: (J.B.) [email protected] and (B.M.) [email protected]. † Laboratoire de Chimie de Coordination du CNRS. ‡ Institute of Molecular Biology and Genetics.

2-deoxyribose units, being responsible for DNA strand breaks. From previous studies, we expected one of the cleaved fragment to be a 5′-aldehyde ended oligonucleotide, arising from hydroxylation at C5′ of a 2-deoxyribose. This hydroxylation promotes the spontaneous elimination of the attached phosphate group and the cleavage of DNA accompagnied by the formation of two DNA products: a 5′-aldehyde end and a 3′-phosphate end (Schemes 1 and 2) (11-13). Three classic reactions can be applied to LC/ESI-MS analyses for the transformation of the 5′-aldehyde end of an oligonucleotide into a stable derivative (14-18). They consist of adding a reduction, an oxidation, an alkaline treatment or a “heating” step (Scheme 2). A DNA fragment ending with a 5′-aldehyde nucleotide subjected to NaBH4 reduction transforms into a stable 5′-alcohol ending DNA fragment which can then be compared with a synthetic standard (11, 14-17). Oxidation of the 5′OH with alkaline sodium hypoiodite into a stable 5′-acid ending fragment is also possible (15, 17, 18). Upon heating at 90 °C for 30 min or upon alkaline treatment, two β-eliminations at the 5′-modified 2-deoxyribose unit induce the release of furfural and of the corresponding nucleobase, resulting in the formation of a one-nucleotide shorter 5′-phosphate DNA fragment (11, 13, 15-17). The 5′-phosphate fragment of DNA can be synthesized separately for identification. These three classic reactions represent a set of experiments that have been used previously for the identification of 5′-aldehyde ending fragments of 3′-32P-labeled DNA by polyacrylamide gel electrophoresis (15-18). These characterizations are limited by the need of noneasily available reference compounds (5′-acid ending oligonucleotides) or by the use of 5′-OH or 5′-phosphate oligonucleotidic references that are not really characteristic of the reduction of 5′aldehyde (except in the case of reducing the aldehyde

10.1021/tx0100800 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/13/2001

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Chem. Res. Toxicol., Vol. 14, No. 10, 2001

Scheme 1. Selective Cleavage of Strand I in Oligonucleotide Duplex I/II Depends on the Presence of an A‚T Triplet (dashed box)a

a

The arrow indicates the site of selective cleavage.

Scheme 2. Formation of the 5′-Aldehyde Terminus Followed by Various Chemical Transformations

function by NaBD4) or of the loss of one nucleoside unit since 5′-OH or 5′-phosphate may also result from an hydrolytic pathway. So, the direct characterization of the 5′-aldehyde DNA fragment by on-line ESI-MS spectrometry was an attractive goal. Unfortunately, the observed spectrum of this fragment under these conditions showed a mixture of different species making the direct identification of this oxidative damage not unambiguous. The failure of this method is mainly due to the intrinsic reactivity of the aldehyde function with nucleophiles (water or nucleophiles present in the reaction medium) or/and to the unstability of the 5′-aldehyde ending oligonucleotides during ESI-MS analysis. So, we wish to point out a convenient and specific chemical derivatization process that prevents any further chemical or physicochemical transformation and will facilitate their characterization by on-line LC/ESI-MS. Formation of an oxime ether derivative by reaction between carboxymethoxylamine and the aldehyde function complies with this aim and will provide a convenient way for the characterization of 5′-aldehyde ends. In addition, we also report a side-reaction that we observed between the 5′-aldehyde oligonucleotide and Tris buffer, commonly used in many biological experiments. The formation of a Tris adduct, stable enough to be analyzed under LC/ESI-MS conditions, could be a source of misinterpretations. This adduct was generated when the DNA cleavage reaction was performed in Tris buffer at pH 9; however, an early derivatization into oxime ether precluded this side reaction. Finally, both structures of the O-carboxymethyl oxime derivative and the Tris adduct were fully established on 5′-aldehyde modified thymidine derivatives used as models of the 5′-aldehyde ending oligonucleotides.

Experimental Procedures Materials. Mn-TMPyP was prepared according to ref 21. Oligodeoxyribonucleotides I and II (ODNs I and II) were synthesized by β-cyanoethylphosphoramidite chemistry. Thymidine, tert-butyldimethylchlorosilane (TBDMSCl), dicyclohexylcarbodiimide (DCC), carboxymethoxylamine hemihydrochloride and Tris (2-amino-2-hydroxymethylpropane-1,3-diol) were purchased from Aldrich. Sodium sulfite was purchased from Fluka. Nucleoside derivatives have been purified by preparative column chromatography on Kieselgel 60 silica (Merck) and TLC were performed on Kieselgel 60F254 plates (Merck) in chloroformmethanol (9/1, v/v).

Angeloff et al. Instrumentation. HPLC analyses were done on a reversedphase Nucleosil C18 10 µm column (250 × 4.6 mm) eluted by 0.1 M triethylammonium acetate (TEAA, pH 6.5) with a linear gradient of 1 to 13% (v/v) acetonitrile for 60 min at a flow rate of 1.5 mL/min. Under these conditions, the two DNA strands were not hybridized and were separated as single-stranded ODNs. All products were detected at 260 nm. LC/ESI-MS analyses were performed as indicated above but with a quaternary pump, the molarity of TEAA was lowered to 0.01 M and the flow rate was 1 mL/min. Only 4% of the flow eluted from the column was introduced into the electrospray source. The ESI-MS spectrometer was a Perkin-Elmer SCIEX API 365 and the analyses were performed in the negative mode. Fast atom bombardment mass spectrometry (FAB-MS) analyses were performed on a Nermag R10-10 apparatus in the positive mode (dimethylformamide, m-nitrobenzyl alcohol matrix). Proton NMR spectra were recorded on Bruker AM-250 and Bruker AMX-400 spectrometers at 250 and 400 MHz, respectively, with tetramethylsilane as external standard. Analysis of the Cleavage Pattern of ODNs I/II Oxidized by Mn-TMPyP/Sulfite/O2. All reactions were performed on an ice bath (4 °C), and reaction mixtures contained 100 mM NaCl and 50 mM Tris‚HCl (pH 9). Before the reaction, the two complementary single-stranded ODNs I and II were allowed to anneal for 15 min (resulting duplex ) 5 µM) before Mn-TMPyP (5 µM) was added to the mixture. After another 15-min incubation, cleavage of the duplex was initiated by adding a freshly prepared solution of sodium sulfite (500 µM). After cleavage for 15 min at 4 °C, Hepes buffer (90 mM, pH 8) was added to stop the reaction. All indicated concentrations are final concentrations. The reaction mixture was then directly analyzed by HPLC (Figure 1A) or after incubation for 2 h (Figure 1B) or 5 h (Figure 1C). To form the oxime derivative, no stopping buffer was added but the reaction mixture was incubated with carboxymethoxylamine (40 mM) for 1 h at r.t. before injection in HPLC (Figure 1D). Analyses were also performed by LC/MS using slightly modified conditions of elution (Table 1). 3′-O-tert-Butyldimethylsilylthymidine (1). 3′-O-Silyl protected thymidine was prepared by silylation of 5′-O-dimethoxytrityl thymidine (DMTrT) with TBDMSCl followed by detritylation of the fully protected intermediate (22). Product was purified by silica gel column chromatography in the gradient 0 to 2.5% methanol in dichloromethane, and precipitated from CH2Cl2 into hexane. Yield 79% (based on DMTrT). Rf ) 0.58. FAB-MS: m/z 357 (M + H)+, 231, 188, 146, 127 (Thy + H)+. M calculated for C16H28N2O5Si: 356.18. Found: 356.18. 3′-O-tert-Butyldimethylsilylthymidine 5′-carboxaldehyde (2). 3′-Silylated thymidine 1 (231 mg, 0.65 mmol) and dicyclohexylcarbodiimide (536 mg, 2.6 mmol) were dissolved in dry DMSO (2.5 mL). Then, a solution of dichloroacetic acid (28 mL, 0.33 mmol) in 0.5 mL of DMSO was added dropwise over 1 min with stirring. The reaction mixture was stirred at ambient temperature overnight. Ethyl acetate was added (2 mL), the mixture was cooled to about 5 °C, and oxalic acid (180 mg, 2 mmol) was added by little portions with stirring. Then, additional 2 mL of EtOAc were added, and cold mixture was stirred for 1 h. Precipitate of dicyclohexylurea was filtered off and washed with EtOAc (2 × 2 mL). The organic solution was washed with aqueous sodium bicarbonate (2 × 3 mL) and NaCl (3 mL), dried over sodium sulfate and evaporated to dryness. Aldehyde product 2 was precipitated from dichloromethane by pentane addition. Yield of the white powder: 193 mg (84%). Rf ) 0.61. FAB-MS: m/z 377 (M + Na)+, 355 (M + H)+, 229, 127 (Thy + H)+, 115 (BuMe2Si)+, 101. M calculated for C16H26N2O5Si: 354.16. Found: 354.16. 1H NMR (CDCl3, 250 MHz): δ 9.75 (s, 1H, H5′), 8.27 (br.s., 1H, NH (Thy)), 7.56 (q, J ) 1.2 Hz, 1H, H6 (Thy)), 6.31 (dd, J ) 5.8, 8.2 Hz, 1H, H1′), 4.66 (dt, J ) 1.9, 5.4 Hz, 1H, H3′), 4.48 (d, J ) 1.9 Hz, 1H, H4′), 2.25-2.35 (ddd, J ) 1.9, 5.8, 13.5 Hz, 1H, H2′′), 1.95-2.10 (ddd, J ) 5.4, 8.2, 13.5 Hz, 1H, H2′), 1.95 (d, J ) 1.2 Hz, 3H, CH3 (Thy)), 0.90 (s, 9H, (CH3)3C-Si), 0.13 (s, 3H, CH3-Si), 0.12 (s, 3H, CH3-Si).

LC/ESI-MS Detection of 5′-Aldehyde End in Oxidized DNA

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Figure 1. HPLC profiles of oxidized duplex I/II by Mn-TMPyP/sulfite/O2. (A, B, C) Incubation in Tris/HCl buffer pH 9 for 0, 2, and 5 h, respectively; (D) incubation for 1 h in the same conditions but in the presence of carboxymethoxylamine added at t ) 0. Table 1. LC/ESI-MS Data Corresponding to the Cleavage of Duplex I/II and Reaction of the 5′-Aldehyde Fragment tR (min)

attribution

m/z peak intensity

[charge]

obsd mass

calcd mass

neutral mass [1-] [2-] neutral mass [1-] [2-] neutral mass [1-] neutral mass [2-] neutral mass [2-] [3-] [4-] neutral mass [2-] [3-] [4-]

883.2 882.3 440.7 901.4 900.4 449.7 636.2 635.2 1246.4 622.3 2113.6 1055.8 703.8 527.2 2086.9 1042.6 694.7 520.6

883.2 882.2 440.6 901.2 900.2 449.6 636.1 635.1 1246.8 622.4 2114.5 1056.2 703.8 527.6 2087.4 1042.7 694.8 520.8

injection at T5 h (only MS data for Tris adduct are shown) TRIS adduct on 5′OdCHGCG (ODN I)b neutral mass 31 [1-] 60 [2-] pCG (fragmentation) (ODN I) neutral mass 100 [1-]

986.5 985.1 492.4 636.1 635.2

986.2 985.2 492.1 636.1 635.1

derivatization experiment (only MS data for O-carboxymethyl oxime derivative are shown) 5′HOOC-CH2-O-NdCHGCG (ODN I) neutral mass 50 [1-] 100 [2-] pCG (fragmentation) (ODN) neutral mass 31 [1-]

956.3 955.4 477.1 636.1 635.0

956.2 955.2 477.1 636.1 635.1

injection at T0 30.0

5′OdCHGCG (ODN I) 24 34 5′(HO)2CHGCG

(ODN I) 28 100

pCG (fragmentation) (ODN I) 90 32.5

CAAAp (ODN I)

39.3

ODN I

100 100 69 24 49.3

ODN II 100 45 13

33.5a

35.9

a In addition to the signals of Tris adduct, a signal corresponding to CAAAp was also detected (see caption of Figure 2). b See the text and Scheme 3 for the exact structure.

3′-O-tert-Butyldimethylsilylthymidine 5′-carboxald(Ocarboxymethyl)oxime (3). Carboxy-methoxylamine hemihydrochloride (52 mg, 0.48 mmol of the base) was dissolved in 3 mL of 0.25 M triethylammonium bicarbonate buffer (TEAB, pH 8). After the CO2 evolution was over, the aqueous solution was added dropwise with stirring to a solution of 2 (84 mg, 0.24 mmol) in acetonitrile (6 mL). The reaction mixture was kept at room temperature for 3 h. The mixture was diluted with 20 mL of 0.25 M TEAB and extracted with dichloromethane (3 × 20 mL). The organic layer was washed with 0.25 M TEAB and water (20 mL each), dried over sodium sulfate and evaporated. The product was purified by silica column chromatography. Impurities were washed away with 4% MeOH in dichloromethane, and product was eluted with 6% methanol in CH2Cl2

containing 0.5% triethylamine. Fractions containing product were evaporated, and 3 was precipitated from dichloromethane into pentane and dried in vacuo. White hygroscopic powder was obtained, yield 68 mg (54%, as TEA salt). Rf ) 0.08. FAB-MS: m/z 450 (M + Na+), 428 (M + H)+, 295, 226, 199, 183, 170, 146, 136, 127 (Thy + H)+, 115 (BuMe2Si)+, 102; negative mode, m/z 426 (M - H)-, 305, 199, 168, 151, 125 (Thy - H)-. M calcd for C18H29N3O7Si (acid): 427.18. Found: 427.18. 1H NMR (DMSOd6, 250 MHz): δ 11.47 (br.s., 1H, NH (Thy)), 7.82 (d, J ) 6.7 Hz, 0.75H, NdCH major isomer), 7.79 (s, 0.25H, H6 (Thy) minor), 7.73 (s, 0.75H, H6 (Thy) major), 7.22 (d, J ) 4.9 Hz, 0.25H, NdCH minor), 6.34 (t, J ) 6.0 Hz, 1H, H1′), 4.90 (d, J ) 4.9 Hz, 0.25H, H4′ minor), 4.83 (m, 0.25H, H3′ minor), 4.66 (pseudo-quintet, J ) 2.9 Hz, 0.75H, H3′ major), 4.60 (s, 0.5H,

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Figure 2. Mass spectra of 5′OdCHGCG (A), Tris adduct on 5′OdCHGCG (B) and 5′HOOC-CH2-O-NdCHGCG (C) from LC/ESI-MS analyses. (*) In addition to the signals of Tris adduct, the presence of a signal from CAAAp was due to the overlapping peak of this fragment in the eluting conditions of LC/MS analysis. CH2O minor), 4.57 (s, 1.5H, CH2O major), 4.35 (dd, J ) 2.9 Hz, 6.7 Hz, 0.75H, H4′ major), 2.91 (quart, J ) 7.5 Hz, 3H, 1.5 CH2 (TEA)), 2.05-2.55 (m, 2H, H2′,2"), 1.92 (s, 3H, CH3 (Thy)), 1.18 (t, J ) 7.5 Hz, 4.5H, 1.5 CH3 (TEA)), 0.98 (m, 9H, (CH3)3C-Si), 0.20 (s, 6H, CH3-Si). Tris Adduct (4). Tris (60 mg, 0.5 mmol) was dissolved in 4 mL of 0.25 M TEAB and added dropwise with stirring to a solution of 90 mg of 2 (0.25 mmol) in acetonitrile (8 mL). The reaction mixture was kept overnight at room temperature and then was diluted with 50 mL of 0.25 M TEAB and extracted with dichloromethane (3 × 30 mL). Organic extract was washed with water (20 mL), dried over sodium sulfate and evaporated to a volume of about 20 mL. This solution was applied to a silica column, and crude 4 was isolated by elution with a gradient 0 to 6% methanol in dichloromethane. Product 4 was then precipitated from methanol (5 mL) into pentane (100 mL). The resulting gellike mixture was kept at 4 °C overnight, and 4 was filtered and dried in vacuo. Yield of the white powder was 77 mg (67%). Rf ) 0.45. FAB-MS: m/z 480 (M + Na)+, 458 (M + H)+, 200, 174, 132, 115 (BuMe2Si)+. M calcd for C20H35N3O7Si: 457.22. Found: 457.22. 1H NMR (acetone-d6, 400 MHz): δ 9.99 (br.s., 1H, NH (Thy)), 7.75 (q, JH6,Me ) 1.2 Hz, 1H, H6 (Thy)), 6.47 (dd, J ) 5.5, 9.0 Hz, 1H, H1′), 4.63 (br.m, H5′), 4.61 (m, J ) 1.6, 1.8, 5.1 Hz, H3′) (the latter two signals are partially overlapped, total 2H), 4.24 (t, J ) 4.6 Hz, 1H, CH2OH), 4.04 (t, J ) 1.6 Hz, 1H, H4′), 3.87 (d, J ) 7.9 Hz, 1H, CH2 cycle, part A of system AB), 3.75 (dd, J ) 4.4, 6.8 Hz, 1H, CH2OH, part X of system ABX), 3.71 (d, J ) 4.6 Hz, 2H, CH2OH), 3.69 (d, J ) 7.9 Hz, 1H, CH2 cycle, part B of system AB), 3.57 (dd, J ) 4.4, 11.0 Hz, 1H, CH2OH, part A of system ABX), 3.49 (dd, J ) 6.8, 11.0 Hz, 1H, CH2OH, part B of system ABX), 2.99 (br., 1H, NH), 2.28 (ddd, J ) 5.1, 9.0, 13.0 Hz, 1H, H2′), 2.16 (ddd, J ) 1.8, 5.5, 13.0 Hz, 1H, H2"), 1.85 (d, J ) 1.2 Hz, 3H, CH3 (Thy)), 0.94 (s, 9H, (CH3)3C-Si), 0.161 (s) and 0.159 (s), (total 6H, CH3Si).

Results Cleavage of Duplex I/II and Results of ESI-MS Analysis. A typical HPLC profile observed by injection of the reaction mixture directly after incubation of duplex I‚II with the chemical nuclease Mn-TMPyP activated by sulfite/O2 is shown in Figure 1A. Due to the tight interaction between the metalloporphyrin and the A‚T

triplet present in the sequence of duplex I‚II (11), the cleavage occurs selectively on the G nucleoside located at the 3′ side of the A‚T triplet in strand I (Scheme 1; no significant degradation of strand II was detected, as previously reported). Consequently, intact ODN II, residual amount of ODN I and two main fragments from strand I, CAAAp and 5′-ald-GCG, were observed (all through the text, schemes and figures, the prefixe d for 2-deoxyribose has been omitted for clarity). A low amount of damaged oligomers containing oxidized guanine residues (noted Gox) could be seen on the HPLC profile, but their analysis was excluded from this study (see ref 8 for a detailed characterization of such species). Concerning ODN I, ODN II, and CAAAp, their on-line ESI-MS spectra clearly corresponded to the expected data and confirmed these attributions (Table 1). On another hand, the on-line ESI-MS spectrum of the 5′-aldehyde fragment (tR ) 30.0 min, Table 1; Figure 2A) showed several m/z signals, most of them were not at the expected molecular mass. In fact, the main signals corresponded to the loss of the last 5′-aldehydic nucleoside unit (m/z 635.2, fragment pCG) and to the hydrated form of the aldehyde group (m/z 900.4 and 449.7). The transformation of the 5′-aldehyde oligonucleotide into pCG occurred in the electrospray source since a reference sample of pCG migrated faster than the 5′-ald-GCG fragment. Only weak signals at 882.3 and 440.7 corresponded to the aldehyde fragment itself. When the reaction medium was incubated for several hours (Figure 1, panels B and C), a new compound slowly appeared, which was further identified as a Tris adduct on the basis of the observed mass on LC/ESI-MS analysis, m/z 985.1 and 492.4, (tR ) 33.5 min, Table 1; Figure 2B). A signal corresponding to the loss of one nucleotide unit to give pCG was also observed (m/z 635.2). According to litterature data and supported by a complete characterization performed on a thymidine-5′-carboxaldehyde model (see results below and, also, the Discussion), the exact structure of the Tris adduct results from a cyclization following the nucleophilic attack of Tris on the

LC/ESI-MS Detection of 5′-Aldehyde End in Oxidized DNA Scheme 3. Structures of Compounds 1-4

aldehydic carbonyl to form an oxazolidine ring (Scheme 3). This observed cyclized structure 4 is probably in equilibrium with the open imine form 4′, easily hydrolyzed to give back the aldehyde fragment 2. Indeed, lowering the pH from 9 to 5 for 12 h (data not shown) allowed the complete conversion of the Tris adduct 4 into the aldehyde fragment 2. To unambiguously characterize the 5′-aldehyde terminus, we were looking for a reagent which can react fastly, quantitatively, and as specifically as possible with the aldehyde group to give a stable derivative easy to characterize by on-line ESI-MS. Carboxymethoxylamine undergoes facile condensation with aldehydes and ketones to form oximes (23). In the present case, 1 h reaction at r.t. was sufficient for complete conversion of the 5′-aldehyde terminus into the O-carboxymethyl oxime derivative (Figure 1D). ESI-MS spectrum observed in online LC analysis (tR ) 35.9 min, Table 1; Figure 2C) showed main signals at m/z 955.4 and 477.1 (mono and doubly charged, respectively) corresponding to the created 5′-carboxymethoxyimino terminus on the oligonucleotide fragment. A weak signal at m/z 635.0 (pCG) corresponded to a low level of fragmentation inside of the spectrometer with loss of one nucleosidic unit. Structural Characterization of O-Carboxymethyl Oxime Derivative and Tris Adduct on a Thymidine Model. Preparation of 3′- Protected Thymidine 5′-Carboxaldehyde 2. Starting 3′-O-silyl protected thymidine 1 was prepared by standard procedure (22). It was then oxidized into 5′-aldehyde derivative by the general procedure of oxidation of primary alcohols with DMSO in the presence of dicyclohexylcarbodiimide (24). We employed a variant of this method using 0.5 equiv of dichloroacetic acid as reaction catalyst and oxalic acid to neutralize the excess DCC (25). The obtained 3′protected thymidine 5′-aldehyde 2 was sufficiently pure (purity assessed by proton NMR was about 90%) and was used in the subsequent reactions without further purification. It should be noted that the nucleoside aldehyde is quite unstable, forming hydrate and acetal with alcohol during chromatography, so it could hardly be purified by chromatography. The silylated aldehyde 2 has been previously prepared as unstable sirup by nucleoside oxidation with chromium trioxide (26). Full Characterization of O-Carboxymethyl Oxime Derivative and Tris Adduct Obtained with Thymidine 5′Carboxaldehyde Taken as a Model. Reaction of nucleoside 5′-carboxaldehyde 2 with both carboxymethoxylamine or Tris was performed in the 2/1 mixture acetonitrile-0.25 M TEAB buffer (pH 8). Under these mild basic reaction conditions, aldehyde conjugation with highly reactive O-substituted hydroxylamine with the formation of oxime 3 was particularly fast (almost 100% for 1 h incubation),


whereas with Tris the reaction was slower (about 50% for 2 h incubation). In the case of carboxymethoxylamine hemihydrochloride, part of TEAB neutralized the HCl. The rate dependence on the pH or the mixture composition was not specially studied. However, we observed that the reaction of the nucleoside aldehyde derivative 2 with carboxymethoxylamine in a nonpolar organic solvent (chloroform in the presence of triethylamine) was much slover than in aqueous medium. Both products 3 and 4 seem to be stable during the isolation procedure, when Tris adduct 4 appears to decompose slowly on silica during chromatographic purification. Oxime 3 is readily soluble in common organic solvents. Tris conjugate 4 contains a polar moiety and therefore is not highly soluble in nonpolar solvents such as chloroform or dichloromethane where it is swelling, but 4 is highly soluble in methanol, DMF, and DMSO, and less soluble in acetone. O-Carboxymethyl Oxime Derivative 3. Two diastereoisomers of 3 were observed in proton NMR spectra. Signals of thymine-H6, H3′, and H4′ sugar protons, and imine and methylene protons of the oxime residue are all present as pairs of resonances in the approximate ratio 3:1. Signal assignments were obtained by selective irradiations. For example, selective irradiation of the resonance at 7.82 ppm (imine proton of the major isomer) changed the dd pattern at 4.35 ppm into a doublet (J ) 2.9 Hz) at 4.35 ppm (H4′ of the major isomer), and irradiation of 7.22 ppm signal (imine proton of the minor isomer) led to the transformation of the doublet at 4.90 ppm into a singlet (H4′ of the minor isomer). About 0.5 equiv of triethylamine is present; however, the carboxylic proton signal was not observed. It is known from the literature that O-substituted hydroxylamines form quite stable Z and E diastereoisomers of O-substituted oximes, and Z/E ratio depends on the reaction conditions (27). The configuration of diastereoisomers of O-substituted oxime 3 has been determined by 1D ROESY experiments at 400 MHz. The obtained results demonstrated that the major diastereoisomer of the conjugate 3 is in E configuration. Selective irradiation of the imine proton (5′-CH; 7.22 ppm) of the minor diastereoisomer produced no NOE with the oxime residue CH2 group, whereas a NOE was observed with H3′ and H4′ sugar protons, as well as with the thymineH6. Selective irradiation of the imine proton (5′-CH; 7.82 ppm) of the major diastereoisomer was more complicated because its chemical shift was close to that of H6 of the minor diastereoisomer. However, upon its irradiation, a NOE was observed with the methylene group of the oxylamine residue of the major diastereoisomer, and with the H3′ and H4′ protons of this isomer. These data demonstrate that, in the major diastereoisomer, the imine proton is spatially close to the O-substituent of the oxime residue, whereas, in the minor diastereoisomer, NdCH and CH2 are more distant. So, the major diastereoisomer is in E configuration, and the minor diastereoisomer is Z. The fact that the E diastereoisomer of the aldehyde conjugate is the major component of the isomeric mixture is in full agreement with literature data reporting that the E diastereoisomer is thermodynamically more stable in oximes and O-substituted oximes (27). It is interesting to note that the irradiation of the thymine H6 proton of each diastereoisomer produced NOE with both imine proton and anomeric H1′. It means that the heterocyclic base is oriented around the glyco-

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Figure 3. Part of 1H 400 MHz NMR spectrum of Tris adduct 4 in acetone-d6.

sidic bond in such way that H6 can interact with H1′ as well as with 5′-CH. Perhaps the conformation of the base around the glycosidic bond is somewhere between syn and anti, to minimize the steric interaction with the 5′substituent that could rotate around the C4′-C5′ bond. Tris Adduct 4. It has been reported previously that Tris reacts with aldehydes of low molecular weight in aqueous solutions to give products with structures shown by NMR to be either the expected Schiff base (28) or, more frequently, a 2-substituted 4,4′-bis(hydroxymethyl)-1,3oxazolidine (29, 30). Reaction of nucleoside 5′-aldehyde 2 with Tris in aqueous buffer (acetonitrile-TEAB) also led to the formation of a product, 4, showing such a cyclic structure. Indeed, the 1H NMR spectra of the Tris conjugate 4 (400 MHz) confirmed the presence of the oxazolidine structure formed by cyclization of the initially formed Schiff base. Signal assignments have been made by selective irradiations. While Bubb et al. observed the formation of two diastereoisomers in the reaction of glyceraldehyde 3-phosphate with Tris (30), only one single isomer of the conjugate 4 was observed by NMR in the present case. A detailed NMR analysis supporting the chiral oxazolidine structure will be published elsewhere. Briefly, the spectrum of compound 4 recorded at 400 MHz in acetone-d6 (see Figure 3 for an expanded part of the 1H spectrum) exhibits only two OH groups from Tris at 4.24 and 3.75 ppm. The OH proton at 4.24 ppm was associated to the CH2 group at 3.71 ppm which appeared usually as a doublet. The other CH2OH hydroxylic proton showed as an ABX system. Nonequivalent CH2 protons were at 3.57 and 3.49 ppm, indicating a restricted rotation of this group whereas the rotation of the other is free. The third CH2 protons from Tris, involved in the oxazolidine cycle, appeared as an AB system (3.69 and 3.87 ppm). The three-dimensional structure of 4 was determined by 2D NOESY and 1D

TOCSY experiments. Thymine is in anti conformation around the glycoside bond, with its H6 and methyl protons interacting with the oxazolidine cycle. The latter seems to be perpendicular to the base, since only one of its methylene protons, and only one CH2OH group are interacting with thymine. The other methylene proton of oxazolidine cycle that is not interacting with the base interacts with H5′ proton. These data strongly suggest a R configuration for the CH5′ center in the conjugate 4. The “frozen” CH2OH group stabilizes this structure by hydrogen bond formation with the nitrogen atom of the Tris cycle, whereas the NH of Tris can interact with furanose oxygen thus further stabilizing the structure. In the S configuration, the oxygen atoms of Tris cycle and sugar ring would be very close, destabilizing this structure by electrostatic and steric repulsions, and perhaps explain why the S isomer was not formed. Probably, a thermodynamic control leads to the preferential formation of the more stabilized structure, via the opening-closure of the Tris cycle to shift the isomer population to the single R configuration (an initial kinetically controlled formation of the mixture of diastereoisomers can be possible).

Discussion Until now, the oxidative chemistry at C5′ of 2-deoxyribose resulting on 5′-aldehyde formation has only been demonstrated for enediynes and Mn-TMPyP (10). Characterization of such type of DNA damage is possible by conventional methods such as polyacrylamide gel electrophoresis provided that products are identified after an additional workup by comparison with reference samples. No direct and fast detection has been previously described. Since liquid chromatography coupled to electrospray mass spectroscopy is an emerging method for

LC/ESI-MS Detection of 5′-Aldehyde End in Oxidized DNA

direct analysis of damaged oligonucleotides, we investigated the possibility to directly characterize the 5′aldehyde fragment by LC/ESI-MS analysis. We used short double-stranded oligonucleotides (7-mers) as simplified models of DNA substrates that can be easily separated by LC. The sequence was chosen in order to create only one 5′-aldehyde fragment which was then directly analyzed. Unfortunately, due to the reactivity of 5′-aldehyde nucleos(t)ides with nucleophiles (water itself or buffer constituants such as the Tris base) or to the β-scission induced by the lability of H atom in R position of the aldehyde group, the signal corresponding to the molecular peak is too weak to allow direct and unambiguous identification of the 5′-aldehyde fragment. Then we looked for a derivatization of the aldehyde function as selective as possible and easy to perform just before the injection in the LC/MS apparatus. Oxylamines derivatives were used previously for the detection of abasic sites (19) and are particularly convenient for condensation with aldehydes and ketones (23) since the oxylamine function is a stronger nucleophile than a regular amine. Moreover, the covalent coupling between the two reagents is stable without the need of a reduction step, like in the case of formation of an imine between a simple amine with an aldehyde (20). This method is promising for the following reasons. The reaction media can be injected directly onto the HPLC column without any supplementary workup except the addition of carboxymethoxylamine itself. DNA lesions can thus be analyzed while they are still present on the DNA strand and can be “trapped” just after being generated. The trapping of 5′-aldehyde DNA fragments by the oxylamine reagent under the reported experimental conditions was 100% yield, and the oxime ether derivative is stable for at least several hours at pH above 4. The derivatization can be performed in any buffer since the reversed-phase column is eluted with a volatile buffer (triethylammonium acetate or ammonium acetate buffer) associated with methanol or acetonitrile. The sodium counterions of the phosphates of oligonucleotides are eliminated during the HPLC separation and thus the on-line ESIMS analyses can be operated in good conditions (without sodium adducts on the mass spectra) (8). Since the method needs a minimal amount of material (1 nmol of starting oligonucleotide), it is convenient and versatile. The on-line ESI-MS analysis allows the determination of the molecular mass of the peaks of the different reaction products with high accuracy (0.02%). The oxylamines derivatives were used previously for detection of abasic sites (19). However, no confusion could be made between a derivatized 5′-aldehyde terminus and a derivatized abasic site since only the last one is accompanied by a nucleobase loss, easily detected by the mass value. In addition, due to the intrinsic reactivity of the 5′aldehyde with nucleophiles, we have also evidenced a side reaction of this aldehyde with Tris buffer, a very commonly used buffer. When the pH value was above 7, a Tris adduct is formed and is a source of misinterpretation since it appeared as an additional well-separated peak in LC analyses with a m/z observed mass of +103 (relative to the aldehyde mass). This adduct was unstable at acidic pH and was transformed back to the starting aldehyde derivative when the pH value was below 5. Since the reaction is slow and reversible, the addition of carboxymethoxylamine in the reaction mixture com-


pletely inhibits the formation of the Tris-adduct and the only O-carboxymethyl oxime derivative could be detected. The structures of the oxime ether derivative and of the Tris adduct were unambiguously established by MS and NMR analyses on thymidine models. 3′-Silylated thymidine 5′-aldehyde reacted with carboxymethylamine gave a mixture of two diastereoisomeric oxime ethers E and Z in the ratio 3:1 (Scheme 3). The probable coexistence of these two diastereoisomers in the 5′-aldehyde ODNs are not perturbating the LC-MS characterization since they are not separated under the chromatographic conditions used and since they have the same mass. The 3′silylated thymidine 5′-aldehyde incubated at pH 9 with Tris buffer gave a Tris conjugate including in its structure an oxazolidine cycle with creation of a new chiral center at C5′. On the basis of NOE experiments, the only 5′(R) diastereoisomer was observed. In conclusion, the formation of O-carboxymethyl oxime derivative from a 5′-aldehyde terminus avoids β-scission reaction and its partial conversion to its hydrate form or to adducts with nucleophiles present in the reaction medium (such as Tris buffer). The O-carboxymethyl oxime derivative constitutes a stable derivative which allows the specific and fast characterization of 5′-aldehyde ended DNA strands by LC/ESI-MS analysis.

Acknowledgment. All LC/ESI-MS analyses have been performed in the “Service Commun de Spectrome´trie de Masse FR 1744-UPR 8241” with the fruitful collaboration of C. Claparols and S. Richelme. We are grateful to Y. Coppel for fruitful discussions on the NMR data and to University Paul Sabatier for a fellowship (I. Dubey).

References (1) Potier, N., Van Dorsselaer, A., Cordier, Y., Roch, O., and Bischoff, R. (1994) Negative electrospray ionisation mass spectrometry of synthetic and chemically modified oligonucleotides. Nucleic Acids Res. 22, 3895-3903. (2) Cook, G. P., and Greenberg, M. M. (1996) A novel mechanism for the formation of direct strand breaks upon anaerobic photolysis of duplex DNA containing 5-bromouridine. J. Am. Chem. Soc. 118, 10025-10030. (3) Kino, K., Saito, I., and Sugiyama, H. (1998) Product analysis of GG-specific photooxidation of DNA via electron transfer: 2-aminoimidazolone as a major guanine oxidation product. J. Am. Chem. Soc. 120, 7373-7374. (4) Gasparutto, D., Ravanat, J.-L., Ge´rot, O., and Cadet, J. (1998) Characterization and chemical stability of photooxidized oligonucleotides that contain 2,2-diamino-4-[(2-deoxy-β-D-erythropentofuranosyl)amino]-5(2H)-oxazolone. J. Am. Chem. Soc. 120, 10283-10286. (5) Duarte, V., Muller, J. G., and Burrows, C. (1999) Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidised form of 7,8-dihydro-8-oxoguanine. Nucleic Acids Res. 27, 496-502. (6) Apffel, A., Chakel, J. A., Fischer, S., Lichtenwalter, K., and Hancock, W. S. (1997) Analysis of oligonucleotides by HPLCelectrospray ionization mass spectrometry. Anal. Chem. 69, 13201325. (7) Tretyakova, N. Y., Niles, J. C., Burney, S., Wishnok, J. S., and Tannenbaum, S. R. (1999) Peroxynitrite-induced reaction of synthetic oligonucleotides containing 8-oxoguanine. Chem. Res. Toxicol. 12, 459-466. (8) Vialas, C., Pratviel, G., Claparols, C., and Meunier, B. (2000) Guanine oxidation in double-stranded DNA by Mn-TMPyP/ KHSO5: 5,8-dihydroxy-7,8-dihydroguanine residue as a key precursor of imidazolone and parabanic acid derivatives. J. Am. Chem. Soc. 122, 2157-2167. (9) Bourdat, A.-G., Douki, T., Frelon, S., Gasparutto, D., and Cadet, J. (2000) Tandem base lesions are generated by hydroxyl radical within isolated DNA in aerated aqueous solution. J. Am. Chem. Soc. 122, 4549-4556.

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Characterization of a 5 '-Aldehyde Terminus Resulting from the

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