Incorporation of an Aldehyde Function in Oligonucleotides

A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthe...
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Bioconjugate Chem. 2001, 12, 451−457

451

Incorporation of an Aldehyde Function in Oligonucleotides Jean-Marc Tilquin, Michel Dechamps, and Etienne Sonveaux* Laboratoire de Chimie The´rapeutique et de Radiopharmacie, Universite´ Catholique de Louvain, 73, Avenue Emmanuel Mounier, p. b. CMFA 7340, B-1200 Bruxelles, Belgium. Received June 1, 2000; Revised Manuscript Received November 15, 2000

A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CHdO chain was synthesized and conjugated with biotin derivatives.

INTRODUCTION

The aldehyde function is often used to couple biopolymers to other molecules by the process of reductive amination. In a typical reaction, this function is condensed with an amino group to form an imine bond, reduced in situ by sodium cyanoborohydride, under slightly acidic conditions (Lane, 1975 and recent examples: Westfall et al., 1998; Maruyama et al., 1998, Bentley et al., 1998; Zhao et al., 1999). A fully irreversible linkage is so obtained. The imine bond itself is quite labile. More stable links are obtained using hydroxylamines, hydrazines, and semicarbazides as nucleophiles. Stable oximes and semicarbazones functions are indeed present in well-established drugs such as cefuroxime and nitrofurantoin. The chemical properties of the aldehyde adducts are such that a tuning of the reversibility of the cross-link is thus possible. This is of prime importance if the in vivo targeting and delivery of a molecule of interest is envisioned. Modification of oligonucleotides (ODNs) through incorporation of an appropriate molecule such as intercalating agent, nonnatural analogue of nucleic bases, metal chelating agent, fluorescent dye, etc., has been widely used to obtain functionalized ODNs as tools for biological and biophysical studies (Shinozuka et al., 2000 and references therein). The coupling of ODN’s to cellular penetration enhancing polymers or peptides is also an important topic in the antisense ODN field (Lemaitre et al., 1987; Bongartz et al., 1994; Soukchareun et al., 1998; Peyrottes et al., 1998; Aubert et al., 2000). Aldehyde functions were rarely introduced in ODNs. It is surprising, as reductive amination is largely used in protein chemistry and does not interfere with the functions present in DNA. The reason is probably that suitable aldehyde building blocks for automatic ODN synthesis are not currently available (Dechamps and Sonveaux, 1998). Aldehyde functions are randomly generated on DNA by depurination: these are the apurinic sites or, more broadly, the abasic sites. Abasic sites condense with hydroxylamine and methoxyamine to form rather stable adducts (Coombs and Livingston, 1969; Talpaert-Borle´ and Liuzzi, 1983; Liuzzi and Talpaert* To whom correspondence should be addressed. Phone 2 7647349. Fax 2 7647363. e-mail [email protected].

Borle´, 1985; Liuzzi et al., 1987; Vasseur et al., 1986). When a ribose residue is introduced at the 3′ or 5′-end of an ODN (by a 3′-5′ or a 5′-5′ linkage, respectively), the sodium periodate oxidation of the glycol function gives a dialdehyde that can be coupled with, for example, polylysine (Agrawal et al., 1986; Lemaitre et al., 1987). The intermediate dialdehyde is, however, prone to β-elimination in basic media (Nadeau et al., 1984). Postsynthetic 3′, 5′, or internal functionalizations of ODNs by aldehyde functions have also been reported (Kremsky et al., 1987; Urata and Akagi, 1993; Tre´visiol et al., 1997). It should, however, be more practical to have at one’s disposal aldehydo phosphoramidite building blocks mimicking nucleotides, especially if ODNs bearing multiple aldehyde functions are required. This could allow the polylabeling of ODNs (e.g., Teigelkamp et al., 1993). More specifically, a series of aldehyde functions situated at the end of an antisense ODN could in principle allow attachment of a bouquet of fusogenic peptides to mimic the viral proteins responsible for the cellular penetration of viruses. The influenza virus, for example, needs the concerted action of numerous fusogenic peptides to induce membrane destabilization and fusion (Chernomordik et al., 1998). Devising a method to replace a nucleic acid base by a benzaldehyde motif also opens perspectives in hybridization-triggered cross-linking. Formaldehyde (Chaw and al., 1980; Huang and Hopkins, 1993), acetaldehyde (Matsuda et al., 1998), and glucose (Bucala et al., 1984) react with DNA. Inside a double helix, an aldehyde function placed in the right position on one strand could form an adduct with the exocyclic amino function of cytosine, adenine, or guanine on the other strand (Gao and Orgel, 1999) and could, if the reaction is exoergonic, enhance the stability of the duplex. The testing of these ideas required easy access to aldehydo-oligonucleotides. This paper describes the synthesis of two phosphoramidite building blocks tailored for the construction of such ODNs. EXPERIMENTAL PROCEDURES

The proportions in solvent and eluent mixtures are expressed as volumic fractions. The NMR spectra were recorded on a 200 MHz instrument, except when otherwise stated. Chemical shifts are given in ppm relative to TMS (1H, 13C) or phosphoric acid (31P).

10.1021/bc000060u CCC: $20.00 © 2001 American Chemical Society Published on Web 04/11/2001

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3,5-Di-O-p-chlorobenzoyl-1,2-dideoxy-1-(4-vinylphenyl)-β (and r)-D-ribofuranose (3a). Commercial 4-bromostyrene is stabilized by sterically hindered catechols. Such contaminants have to be removed in order to allow the formation of the Grignard reagent. 4-Bromostyrene (25 mL) was thus diluted by dichloromethane (75 mL), this solution was washed with 1 M NaOH (3 ×), dried (MgSO4), filtered, and concentrated, and the residue was distilled under vacuum (bp 88 °C, 0.2 mmHg). The so-purified 4-bromostyrene was used immediately. A solution of 4-bromostyrene (2.56 g, 14 mmol) in dry ether (10 mL) was added dropwise to a suspension of magnesium (0.374 g, 15.4 mmol) in the same solvent (40 mL) (the reaction was started with iodine and a few drops of the halide solution). After 2 h stirring, cadmium chloride (1.608 g, 8.75 mmol, dried at 100 °C for 1 h) was added portionwise. The mixture was further stirred for 1 h at RT. Solid 3,5-di-O-p-chlorobenzoyl-1,2-dideoxy-1chloro-D-ribofuranose 1 was then added (1.5 g, 3.5 mmol, synthesized according to Fox et al., 1961. See also Ness et al., 1961 for the first step of this synthesis). After 40 h, cold water was added, the organic phase was collected, and the aqueous phase was washed with toluene (2 × 100 mL). The gathered organic extracts were washed with NaHCO3 (5%) and water (2 x), dried on MgSO4, filtered, and evaporated under vacuum. The isomers were isolated by flash chromatography on silica (eluent: hexane/ethyl acetate 19:1), giving an amount of pure β epimer (10%), a mixture of β and R (18%) epimers, and an amount of pure, slower eluting R epimer (22%). A major side-product was p-chlorobenzoyl furfuryl alcohol, obtained from 1 by two successive β-eliminations. Anal. β epimer (C27H22Cl2O5) C, H. 1 H β epimer (ppm, CDCl3): 2.2 (1H, H2′, ddd, Jgem ) 13.5 Hz, J2′-1 ) 10.8 Hz, J2′-3 ) 6.3 Hz), 2.55 (1H, H2′′, m, Jgem ) 13.5 Hz, J2′′-1 ) 5.1 Hz), 4.5 (1H, H4, m), 4.65 (2H, H5, m), 5.2 (2H, H1 and one of the olefinic CH2’s, m), 5.6 (1H, H3, m, J3-2′ ) 6.3 Hz), 5.75 (1H, the second olefinic CH2, d, Jvic ) 17.7 Hz), 6.7 (1H, isolated olefinic CH, dd, Jvic1 ) 17.7 Hz, Jvic2 ) 10.8 Hz), 7.2-7.5 (8H, phenyl and p-chlorobenzoyl, m), 7.95 and 8.0 (4H, pchlorobenzoyl, 2 × d, J ) 8.5 Hz). 13C β epimer (ppm, CDCl ): 41.90 (C ), 65.54 (C ), 78.12 3 2 5 (C3), 81.06 (C1), 83.29 (C4), 114.54 (olefinic CH2), 126.50 to 131.56 and 136.89 to 140.59 (11 resolved signals, aromatics and olefinic CH), 165.63 and 165.84 (2 × CdO). 1 H R epimer (ppm, CDCl3, 300 MHz NMR): 2.34 (1H, H2′′, ddd, Jgem ) 14.0 Hz, J2′′-1 ) 5.5 Hz, J2′′-3 ) 3.5 Hz), 2.89 (1H, H2′, m, Jgem ) 14.0 Hz, J2′-1 ) 7.2 Hz, J2′-3 ) 6.5 Hz), 4.58, (2H, H5, m), 4.65 (1H, H4, m), 5.27 (1H, one of the olefinic CH2’s, d, Jvic ) 10.7 Hz), 5.38 (1H, H1, pseudo-t, J1-2′ ) 7.2 Hz, J1-2′′ ) 5.5 Hz), 5.58 (1H, H3, m, J3-4 ) 3.1 Hz, J3-2′ ) 6.5 Hz, J3-2′′ ) 3.5 Hz), 5.76 (1H, the second olefinic CH2’s, d, J ) 17.6 Hz), 6.73 (1H, isolated olefinic CH, q, Jvic1 ) 17.6 Hz, Jvic2 ) 10.7 Hz), 7.30-7.45 (8H, phenyl and p-chlorobenzoyl, m), 7.64 and 8.01 (4H, p-chlorobenzoyl, 2 × d, J ) 8.5 Hz). 13C R epimer (ppm, CDCl ): 40.11 (C ), 64.75 (C ), 76.69 3 2 5 (C3), 79.94 (C1), 81.99 (C4), 113.99 (olefinic CH2), 125.66 to 131.11 and 136.40 to 141.85 (13 signals, aromatics and olefinic CH), 165.13 and 165.45 (2 × CdO). (2R,3S,5S)-1,3-O-Bis(p-chlorobenzoyl)-1,2,3,5-tetrahydroxy-2,5-anhydropentadec-14-ene (3b). This stereoisomer (β epimer) was obtained along the same lines. Yield, 30%. Anal. (C29H34Cl2O5) C, H. Reference Compounds: 3,5-O-Bis(p-chlorobenzoyl)-1,2-dideoxy-1-(phenyl)-β (and r)-D-ribofuranose (3c). These compounds were also obtained by the

Tilquin et al.

same procedure, except that the sugar derivative was condensed to the cadmium compound in a hot (40 °C) THF/benzene solution (1:1), distilling ether out of the reaction mixture (1 h). The crude mixture was chromatographed on silica (eluent: ethyl acetate/petroleum ether 1:20), giving a crop of pure R anomer as fast-migrating product, followed by the R/β mixture. The two constituents of this last fraction were separated by thin-layer chromatography, eluting three times consecutively with ethyl acetate/petroleum ether 1:15 (Rf R ) 0.23; Rf β ) 0.20). Total yield 50% (O-p-chlorobenzoylfurfuryl alcohol was a major side-product). Yield of the R anomer (mp 232-233 °C): 37%. Yield of the β anomer (mp 227-228 °C): 13%. The β anomer was also synthesized by a stereospecific method, starting from β-1-phenyl-1-deoxy-D-ribofuranose (Klein et al., 1971), by deoxygenation at position 2 followed by acylation (Robins et al., 1983). The properties of the compound obtained by this stereospecific synthesis were identical to those of the slow-eluting isomer. 500 MHz NMR data: 1H β epimer (ppm, CDCl ): 2.23 (1H, H , ddd, J 3 2′ gem ) 13.7 Hz, J2′-1 ) 10.9 Hz, J2′-3 ) 6.1 Hz), 2.55 (1H, H2′′, m, Jgem ) 13.7 Hz, J2′′-1 ) 5.1 Hz, J2′′-3 ) 1.0 Hz) (lit., 100 MHz: Millican et al., 1984: H2′ and H2′′: 2.05-2.70), 4.53, (1H, H4, m), 4.66 (2H, H5, m) (lit., ibidem: H4 and H5: 4.50-4.80), 5.26 (1H, H1, dd, J1-2′ ) 10.9 Hz, J1-2′′ ) 5.1 Hz) (lit., ibidem: H1: 5.51-5.30, J1-2′ ) 9 Hz, J1-2′′ ) 5 Hz), 5.60 (1H, H3, m, J3-4 ) 1.5 Hz, J3-2′ ) 6.1 Hz, J3-2′′ ) 1.0 Hz) (lit., ibidem: H3: 5.5-5.7), 7.24-7.46 (9H, phenyl and p-chlorobenzoyl, m), 7.95 and 8.02 (4H, p-chlorobenzoyl, 2 × d, J ) 8.5 Hz). 13C β epimer (ppm, CDCl ) (DEPT): 42.22 (C , t) (lit., 3 2 ibidem: C2, 41.6), 65.80 (C5, t) (lit., ibidem: C5, 64.9), 78.32 (C3, d) (lit., ibidem: C3, 82.8), 81.55 (C1, d) (lit., ibidem: C1, 80.8), 83.51 (C4, d) (lit., ibidem: C4, 77.4), 126.53 to 131.84 and 140.41 to 141.13 (12 signals, aromatics), 166.01 and 166.26 (CdO, s). 1H R epimer (ppm, CDCl ): 2.35 (1H, H , ddd, J 3 2′′ gem ) 13.7 Hz, J2′′-1 ) 5.5 Hz, J2′′-3 ) 3.7 Hz), 2.90 (1H, H2′, m, Jgem ) 13.7 Hz, J2′-1 ) 7.0 Hz, J2′-3 ) 6.9 Hz), 4.58, (2H, H5, m), 4.67 (1H, H4, m), 5.38 (1H, H1, pseudo-t, J1-2′ ) 7.0 Hz, J1-2′′ ) 5.5 Hz), 5.58 (1H, H3, m, J3-4 ) 3.2 Hz, J3-2′ ) 6.9 Hz, J3-2′′ ) 3.7 Hz), 7.25-7.41 (9H, phenyl and p-chlorobenzoyl, m), 7.64 and 8.01 (4H, p-chlorobenzoyl, 2 × d, J ) 8.5 Hz). 13C R epimer (ppm, CDCl ) (DEPT): 40.68 (C , t), 65.49 3 2 (C5, t), 78.05 (C3, d), 80.83 (C1, d), 82.70 (C4, d), 126.17 to 131.72 and 140.40 to 142.95 (12 signals, aromatics), 165.85 and 166.18 (CdO, s). 3,5-O-Bis(p-chlorobenzoyl)-1,2-dideoxy-1-[4-(1,2dihydroxyethyl)phenyl]-β-D-ribofuranose (4a). Compound 3aβ (1.8 mmol) was dissolved in an dichloromethane/acetone/water mixture (2:9:1, 35 mL) kept at 0 °C. N-methylmorpholin N-oxide (3 mmol) was added, followed by osmium tetraoxide (0.16 mmol, i.e., 2 mL of a 2.5 (weight %) solution in tert-butyl alcohol). The mixture was magnetically stirred and the progress of the reaction was followed by TLC. (CH2Cl2/CH3OH, 95:5; 3a, Rf ) 0.8, 4, Rf ) 0.1). The mixture was evaporated after 8 h and partitioned between water and CH2Cl2. The organic phase was dried (MgSO4), filtered, and evaporated. The compound was purified by column chromatography (eluent, CH2Cl2). Yield: 94%. Anal. (C27H24Cl2O7) C, H. 1H NMR (ppm, CDCl ): 2.2 (1H, H , m), 2.5 (1H, H , 3 2′ 2′′ dd, Jgem ) 13.5 Hz, J2′′-1 ) 5 Hz), 3.25 (2H, broad band, 2 × OH), 3.55 (1H, one of the two diastereotopic CH2OH’s, pseudo-t, Jgem ) 10.5 Hz, Jvic ) 7.5 Hz), 3.7 (1H,

Technical Notes

the second of the two diastereotopic CH2OH’s, dd, Jgem) 10.5 Hz, Jvic ) 3 Hz), 4.5 (1H, H4, m), 4.6 (2H, H5, m), 4.75 (1H, CHOH, dd, Jvic1 ) 7.5 Hz, Jvic2 ) 3 Hz), 5.2 (1H, H1, dd, J1-2′ ) 11 Hz, J1-2′′ ) 5 Hz), 5.6 (1H, H3, m, J3-2′ ) 5.5 Hz), 7.25-7.5 (8H, phenyl and p-chlorobenzoyl, m), 7.95 and 8.0 (4H, p-chlorobenzoyl, 2 × d, J ) 9 Hz). As a further proof of structure, 4a (0.18 g, 0.34 mmol) was oxydized by NaIO4 (2.13 g, 10 mmol, 1.5 h, RT) in THF/phosphate buffer pH 7 (125 mL, 1:4) to give the corresponding aldehyde in quantitative yield. The 1H NMR of the aldehyde (CDCl3) featured the lack of the CH(OH)CH2(OH) signals, the appearance of the CHdO signal at 10 ppm integrating for 1 proton, and the modification of the aromatic CH absorptions, appearing now as two sets of three doublets (12H, J ) ca. 9 Hz) in the 7.3-7.7 and the 7.8-8.1 ppm ranges, respectively. (2R,3S,5S)-1,3-O-Bis(p-chlorobenzoyl)-1,2,3,5,14,15-hexahydroxy-2,5-anhydropentadecane (4b). The oxidation was performed as for 4a. Yield, 90%. Anal. (C29H36Cl2O7) C, H. 5-O-[(4,4′-Dimethoxy)triphenylmethyl]-1,2-dideoxy1-{4-[1,2-bis(tert-butyldimethylsilyloxy)ethyl)phenyl]-β-D-ribofuranose (7a). The reaction was performed under argon under magnetic stirring. The diol 4a (0.313 g, 0.6 mmol) was dissolved in dry DMF (5 mL), and dried imidazole (0.401 g, 6 mmol) was added, followed by tertbutyldimethylsilyl chloride (0.355 g, 2.4 mmol). The progress of the reaction was followed by TLC (eluent CHCl3, Rf 4a ) 0.1, Rf 5a ) 0.6). After 48 h, the mixture was partitioned between water (15 mL) and CH2Cl2 (15 mL). The organic phase was washed (3 ×) with water, dried (MgSO4), filtered, and evaporated to give the silylated derivative 5a. 1H NMR (ppm, CDCl ): 0.0-0.2 (12H, Si(CH ) ), 0.93 3 2 0.95 (18H, SiC(CH3)3), 2.3 (1H, H2′, ddd, Jgem ) 14 Hz, J2′-1 ) 11 Hz, J2′-3 ) 6 Hz), 2.6 (1H, H2′′, dd, Jgem ) 14 Hz, J2′′-1 ) 5 Hz), 3.55 (1H, one of the two diastereotopic CH2OSi’s, dd, Jgem ) 10 Hz, Jvic ) 5 Hz), 3.7 (1H, the second of the two diastereotopic CH2OSi’s, dd, Jgem) 10 Hz, Jvic ) 7 Hz), 4.6 (1H, H4, m), 4.7 (3H, H5 and CHOSi, m), 5.3 (1H, H1, dd, J1-2′ ) 11 Hz, J1-2′′ ) 5 Hz), 5.6 (1H, H3, m, J3-2′ ) 6 Hz), 7.3-7.6 (8H, phenyl and pchlorobenzoyl, m), 8.0 and 8.1 (4H, p-chlorobenzoyl, 2 × d, J ) 9 Hz). Compound 5a (0.405 g) was deacylated by stirring 10 h in a mixture of dichloromethane (5 mL) and hydrazine hydrate (1 mL). The reaction mixture was evaporated, the residue dissolved in ether, and the solution washed with water (3 × 20 mL). The organic phase was dried (MgSO4), filtered, and evaporated to leave 6a. 1H NMR (ppm, CDCl ): -0.06, -0.05, -0.04, and 0.05 3 (12H, diastereotopic Si(CH3)2), 0.85 and 0.87 (18H, SiC(CH3)3), 2.0 (1H, H2′, ddd, Jgem ) 13 Hz, J2′-1 ) 10 Hz, J2′-3 ) 6 Hz), 2.2 (1H, H2′′, ddd, Jgem ) 13 Hz, J2′′-1 ) 6 Hz, J2′′-3 ) ca. 2 Hz), 2,5 (2H, OH, broad signal), 3.43.9 (4H, diastereotopic CH2OSi’s and H5, m), 4.0 (1H, H4, m), 4.4 (1H, H3, m, J3-2′ ) 6 Hz, J3-2′′) J3-4 ) ca. 2 Hz), 4.7 (1H, CHOSi, dd, Jvic1 ) 5 Hz, Jvic2 ) 7 Hz), 5.15 (1H, H1, dd, J1-2′ ) 10 Hz, J1-2′′ ) 6 Hz),, 7.15-7.35 (4H, aromatics, 2 × d, Jvic ) 8.5 Hz). Compound 6a (0.204 g, 0.43 mmol) was coevaporated three times with dry pyridine and then stirred for 3 h with dimethoxytrityl chloride (0.194 g, 0.57 mmol) in pyridine (15 mL). Methanol (15 mL) was added to quench the residual chloride (during 10 min), followed by dichloromethane (15 mL), and the mixture was extracted three times with NaHCO3 (5%), to eliminate the pyridinium hydrochloride. The organic phase was dried (MgSO4),

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filtered, and evaporated. The residue was chromatographed on silica (eluent CH2Cl2/CH3OH 98:2 + a few drops of triethylamine). Overall yield: 0.255 g, 0.33 mmol, 55%). Anal. (C46H64O7Si2) C, H. 1H NMR (ppm, CDCl ): -0.1 to 0.1 (12H, diastereotopic 3 Si(CH3)2), 0.8-0.9 (18H, SiC(CH3)3), 1.9-2.1 (1H, H2′ and OH, m), 2.2 (1H, H2′′, ddd, Jgem ) 12.3 Hz, J2′′-1 ) 5.4 Hz, J2′′-3 ) ca. 2 Hz), 3.3 (1H, H5, dd, Jgem ) 10 Hz, Jvic ) 5.4 Hz), 3.4 (1H, H5′, dd, Jgem ) 10 Hz, Jvic ) 4.3 Hz), 3.5 (1H, one of the diastereotopic CH2OSi, dd, Jgem) 10 Hz, Jvic ) 4.8 Hz), 3.6 (1H, the second diastereotopic CH2OSi, dd, Jgem) 10 Hz, Jvic ) 7 Hz), 3.8 (6H, OCH3), 4.0 (1H, H4, m), 4.4 (1H, H3, m), 4.7 (1H, CHOSi, dd, Jvic1 ) 4.8 Hz, Jvic2 ) 7 Hz), 5.15 (1H, H1, dd, J1-2′ ) 10 Hz, J1-2′′ ) 5.4 Hz), 6.8 (4H, DMTr, d, J ) 8.5 Hz), 7.157.55 (13H, aromatics). (2R,3S,5S)-1-O-(4,4′-dimethoxytrityl)-14,15-O-bis(tert-butyldimethylsilyl)-1,2,3,5,14,15-hexahydroxy2,5-anhydropentadecane (7b). This compound was obtained by the same procedure. Yield, 61%. Anal. (C48H76O7Si2) C, H. 3-{5-O-[(4,4′-Dimethoxy)triphenylmethyl]-1,2-dideoxy-1-{4-[1,2-bis(tert-butyldimethylsilyloxy)ethyl)phenyl]-β-D-ribofuranosyl}(2-methoxy)(N,N′-diisopropylamino)phosphine (8a). The reaction was performed under argon. Compound 7a (0.178 g, 0.23 mmol) was coevaporated three times with acetonitrile, dried under high vacuum (2 h), and dissolved in dry CH2Cl2 (10 mL) containing diisopropylammonium tetrazolide (0.022 g, 0.13 mmol). Methyl tetraisopropylphosphorodiamidite (0.064 g, 0.24 mmol) was added. The mixture was magnetically stirred for 2 h [control by TLC, hexane/ N(CH2CH3)3, 6:1]. Sodium bicarbonate (5%, 10 mL) was added. The organic phase was washed with a phosphate buffer (pH 7, 10 mL) and then with brine (sat., 10 mL), dried (MgSO4), filtered, and evaporated. The residue was chromatographed on silica [eluent: hexane/N(CH2CH3)3, 6:1, Rf of 8a ) 0.7]. Yield, 60%. 31P NMR, δ ) 148.536 and 148.366 ppm. Anal. (C53H80NO8PSi2) C, H, N. 1H NMR (ppm, CDCl ): -0.1 to 0.1 (12H, diastereotopic 3 Si(CH3)2), 0.8-0.9 (18H, SiC(CH3)3), 1.0-1.3 (12H, NCH(CH3)2, m), 1.9-2.5 (2H, H2′ and H2′′, m), 3.2 (2H, H5, m), 3.3 and 3.4 (3H, OCH3 of the two diastereoisomers, 2 × d, JH-P ) 13 Hz), 3.6 (4H, CH2OSi and NCH(CH3)2), 3.8 (6H, OCH3), 4.2 (1H, H4, m), 4.5 (1H, H3, m), 4.7 (1H, CHOSi, m), 5.2 (1H, H1, m), 6.8 (4H, DMTr, d, J ) 8.5 Hz), 7.15-7.55 (13H, aromatics). 3-[2R,3S,5S)-1-O-(4,4′-dimethoxytrityl)-14,15-O-bis(tert-butyldimethylsilyl)-1,2,5,14,15-pentahydroxy2,5-anhydro]pentadecoxy},2(cyanoethoxy),(N,N-diisopropylamino)phosphine (8b). The same protocol was used, engaging 0.06 mmol of diisopropylammonium tetrazolide and 0.12 mmol of bis(diisopropylamino)(βcyanoethoxy)phosphine. Yield, 70%. 31P NMR, δ ) 148.811 and 148.641 ppm. Anal. (C57H93N2O8PSi2) C, H, N. Synthesis and Purification of Oligonucleotides 9a,b. Building blocks 8a,b were introduced at the X position in the sequence d(GTCGTGAC-X-GGGAAAAC) by automatic synthesis. The trityl-on oligomer was deprotected by the classical sequence of thiophenol (only for 9a), concentrated ammonia (50 °C), and tetrabutylammonium fluoride (THF, 0.1 M). The oligomer was then desalted (Pharmacia NAP-10), adsorbed on a C-18 cartridge (Pharmacia Oligo-Pak), and eluted by detritylation (2% aqueous trifluoroacetic acid). A final purification, when needed, was performed by anion-exchange chromatography (Pharmacia Mono Q column), followed by desalting (NAP-10).

454 Bioconjugate Chem., Vol. 12, No. 3, 2001

Oxidation of 9a,b to 10a,b and Titration by NaBT4. The aldehyde function was generated from the vicinal diol according to the protocol of Bayard et al. (1984). Oligonucleotide 9a (2.8 nmoles) was oxidized by NaIO4 (2.5 µL of 1.6 mM solution in 0.02 M sodium acetate buffer, pH 4.75) at 4 °C for 5 h in the dark. Ethylene glycol was added to destroy the excess NaIO4. The aldehyde function was then reduced in situ by NaBT4 (+ 2.5 µL of 10 mM solution in 0.01 M sodium borate buffer, pH 9). The excess hydride was decomposed by the addition of acetate buffer. The yield of tritium incorporation was determined by passing the sample through two successive NAP-10 cartridges, isolating the high molecular weight fraction and determining both the A260 (2.5 nmol of DNA recovered) and the immobilized tritium content (2.37 nmol, by scintillation counting). The tritium incorporation yield was thus 95%. The yield of 10b, obtained by the same procedure, was quantitative. Synthesis of 11 and 12. A solution of NaIO4 (120 mM) in phosphate buffer (20 mM, pH 7) was made. It was degassed by helium (to avoid overoxidation of the aldehyde to be obtained). This solution (1 mL) was added to the glycol-oligonucleotide 9b (100 OD in 25 µL). The reaction was stopped after 30 min in the dark at room temperature by desalting on NAP-10. The success of the reaction was checked by reversed phase HPLC [BIORAD Bio-Sil C18 HL 150 × 4.6 mm column. Linear gradient of eluent B (triethylammonium acetate 0.1 M in CH3CN: H2O, 60:40) in eluent A (triethylammonium acetate 0.1 M in CH3CN:H2O, 5:95), from 0 to 30% in 25 min. Flow, 1 mL/min]. The retention times were 15.4 and 16.3 min for 9b and 10b, respectively. The solution of the aldehyde-oligomer was not kept, but immediately used for the next step. A solution of biotin hydrazide (Sigma, 10 µL, 15 mM) and acetate buffer (5 µL, 500 mM, pH 5) and a solution of sodium cyanoborohydride (5 µL, 100 mM in acetate buffer) were added to the aldehyde-oligonucleotide 10b dissolved in water (1.5 mmol in 500 µL). The mixture was desalted on NAP-10 after 2 h at room temperature. The conjugate 11 was isolated by semipreparative HPLC, under the same conditions as indicated above. Similarly, a solution of D-biotinyl-1,8-diamino-3,6dioxaoctane (Boehringer Mannheim, 10 µL, 15 mM) and phosphate buffer (100 µL, 20 mM, pH 6.8) and a solution of sodium cyanoborohydride (10 µL, 5 mM in water) were added to the aldehyde-oligonucleotide 10b dissolved in water (1.5 nmol in 200 µL). After 4 h at room temperature, the mixture was diluted by water to 500 µL and desalted on NAP-5. The conjugate 12 was also isolated by semipreparative HPLC. A titration of conjugate 12 by avidin was performed. Aliquots of a solution of avidin (Sigma, 5.2 nmol/mL of sites of complexation) were added to solutions of the biotin-oligonucleotide conjugate (66 pmol of oligonucleotide in 200 µL). Each solution was ultrafiltrated (ULTRAFREE-MC from Millipore, membrane UFC-3LTK, cutoff 30.000), and the filtrate was checked by HPLC. Oligomer 9b was used as a not-retained internal standard. The addition of 65 pmol of avidin sites of complexation was necessary to completely retain the biotinylated oligomer on the filter. RESULTS AND DISCUSSION

Proper Protection of the Aldehyde Function. The conditions of automatic DNA synthesis should arm an

Tilquin et al.

unprotected aldehyde (nucleophilic attack of the P(III) derivatives on the carbonyl, oxidation of the aldehyde by iodine, and possible Cannizzaro reaction in the strongly basic conditions used for full deprotection). A classical acetal protection is not convenient, as it would be cleaved during the detritylation steps. Our strategy was to use a bis-silylated glycol as a precursor of aldehyde. ODNs can be treated with aqueous sodium periodate without degradation, as reported in the Introduction. This reagent selectively oxidizes glycols. A glycol is of course not compatible with the conditions of automatic synthesis. The OHs have to be properly protected. tert-Butyldimethylsilyl protection is used to cap the 2′-OH in solidsupported RNA synthesis (Wu and Ogilvie, 1990). A reasonable strategy leading to aldehydo-ODNs was thus to use a phosphoramidite synthon incorporating a O,O′bis(tert-butyldimethylsilyl)glycol function. Synthesis. The synthesis started with the coupling of organocadmium compounds 2a,b with the highly electrophilic anomeric carbon of the deoxyribofuranosyl chloride 1 (Scheme 1). This type of coupling was pioneered in the hexose field (Hurd and Holysz, 1950) and expanded later in the ribose series (Klein et al., 1971). The yields were, however, low, ca. 20%. We and others have successfully used this method for the synthesis of various C-nucleosides in the deoxyribofuranose series, with, in our hands, yields higher than 80% (Franc¸ ois et al., 1994; Chaudhuri and Kool, 1995; Ren et al., 1996; Dechamps and Sonveaux, 1998; Stra¨ssler et al., 1999). The coupling is not stereoselective, a mixture of R and β isomers being obtained. The formation of the p-styrenylmagnesium bromide, the obligate precursor of the corresponding organocadmium compound 2a, proved to be impossible if p-bromostyrene was not freed from its radical trap stabilizors. The determination of the configuration of 3a,b needed a detailed study, the Karplus equation being difficult to use (Chaudhury and Kool, 1995) because of the flexibility of the deoxyribofuranose ring (Franc¸ ois et al., 1990). O,O′-Dibenzoyl R and β 1-phenyl-1,2-dideoxy-D-ribofuranoses (e.g., 3c) are known compounds, but their NMR spectra were not reported in sufficient detail (Millican et al., 1984; Gunning et al., 1985). For the sake of recording reference NMR spectra, we thus also obtained by our method the mixture of R-3c and β-3c and separated them by chromatography, and we synthesized independently β-3c by a reported stereospecific procedure (the route used the stereospecific synthesis of β-1-phenyl-1-deoxy-D-ribofuranose, followed by deoxygenation at position 2 and benzoylation) (Klein et al., 1971; Robins et al., 1983). The stereoisomers R-3a,b and β-3a,b, separated by chromatography, were then easily identified by comparison. The synthesis was pursued with pure stereoisomers in order to simplify the analysis of the spectral data. On a practical point of view, if the method is used for labeling purpose only, the synthesis could be pursued on the mixture of epimers. We chose the less abundant β epimer because it corresponds to the configuration of natural nucleotides, a point of importance if hybridization-triggered cross-linking is envisioned. Osmium tetraoxide oxidation (Jacobsen et al., 1988), followed by silyl capping, gave the fully protected nucleoside analogues 5a,b. Selective aminolysis of the ester functions of 5a,b led to 6a,b, that were dimethoxytritylated on the primary alcohol function and then phosphitylated. Compounds 8a,b are building blocks tailored for the automatic phosphoramidite-type DNA synthesis.

Technical Notes

Bioconjugate Chem., Vol. 12, No. 3, 2001 455

Scheme 1

We incorporated 8a,b at the X position in the sequence GTCGTGAC-X-GGGAAAAC. The trityl-on oligomer was cleaved from the glass beads by concentrated ammonia, fully deprotected, and purified to homogeneity by anionexchange chromatography. The oxidation of the glycol by sodium periodate, followed by the titration of the generated aldehyde function by NaBT4, proved that the yields of these reactions were practically quantitative. We also synthesized the biotine conjugates 11 and 12, to demonstrate that the aldehyde function of the oligonucleotides can be coupled with amino compounds by reductive amination. The biotinylated oligomers were quantitatively captured by avidin. CONCLUSION

We thus described a practical method to introduce an aldehyde function and perform a reductive amination at any position in an ODN. Both 3′ and 5′ ends of the ODN remain free for further functionalization or 32P labeling.

ACKNOWLEDGMENT

This work was supported by the Belgian Fonds de la Recherche Scientifique Me´dicale (grant no. 3.4589.96), the Belgian Fonds National de la Recherche Scientifique (Cre´dit aux Chercheurs, grant no.1.5.113.00) and the Actions de Recherches Concerte´es 94/99-172 of the Direction Ge´ne´rale de la Recherche Scientifique - Communaute´ Franc¸ aise de Belgique, Belgium. Supporting Information Available: Elemental (C, H) analysis data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Agrawal, S., Christodoulou, C., and Gait, M. J. (1986) Efficient Methods for Attaching Nonradioactive Labels to the 5′ Ends of Synthetic Oligodeoxyribonucleotides. Nucleic Acids Res. 14, 6227-6245. (2) Aubert, Y., Bourgerie, S., Meunier, L., Mayer, R., Roche, A.-C., Monsigny, M., Thuong, N. T., and Asseline, U. (2000)

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