Synthesis and Properties of an Oligonucleotide Modified with an

Bioconjugate Chem. 1996, 7, 349−355

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Synthesis and Properties of an Oligonucleotide Modified with an Acridine Derivative at the Artificial Abasic Site Keijiro Fukui, Minoru Morimoto,† Hiroshi Segawa,‡ Kazuyoshi Tanaka, and Takeo Shimidzu* Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Received September 27, 1995X

The synthesis of an oligodeoxynucleotide (ODN) modified with 2-methoxy-6-chloro-9-aminoacridine (Acr) at an abasic site is described. A stereochemically defined aminodiol, L-threoninol, was used to serve as artificial abasic nucleoside. The molecule was modified so as to be suitable for the standard phosphoramidite method and was incorporated into the interior of an ODN. In addition, Nhydroxysuccinimidyl N-[9-(6-chloro-2-methoxy)acridinyl]-6-aminocaproate has been synthesized for postsynthetic modification of the amino substrate of the L-threoninol moiety in the ODN. By using absorption spectroscopy, it is shown that oligo(dA) conjugated with acridine binds with complementary strand in a 1:1 ratio. The melting temperature showed that the nonmodified (abasic) duplex is destabilized as a result of lacking in base at the abasic site, but the covalently linked acridine ring compensates for the destabilization effect. The fluorescence quantum yield of the acridine ring was enhanced by connection to oligo(dA) and, further, by formation of a double-strand with the complementary ODN. The quantum yield is larger than that of intermolecular intercalation. The excitation spectra of Acr-ODN in the duplex is quite similar to the absorption spectra. The results indicate that the covalently linked acridine ring is selectively intercalated into the adjacent abasic site.

INTRODUCTION

Oligonucleotides covalently linked with functional molecules have been attracting current interest because of their widespread biological usage as antisense (1) and DNA probes (2) and so on. For example, He´le`ne et al. (3) have reported that oligodeoxynucleotide (ODN) attached to a phenanthroline-copper chelate at the 5′-termini cleaves DNA by triple-stranded formation showing high specificity. Miller et al. (4) have shown that ODN methylphosphonates covalently linked to psoralen and targeted single-stranded DNA give cross-linked duplexes by cycloaddition reaction under photoirradiation. In these investigations, functional molecules are connected to the oligonucleotide or the derivative so that its functions are selectively directed toward the target position of DNA or RNA. Therefore, it is necessary for improving efficiency and specificity that the molecule should be correctly anchored to the target position. There are important factors for such molecular design: (i) the number and species of linker atoms connecting the functional molecule and ODN; (ii) the linking position of the ODN. It has been previously reported that ODN can covalently link to functional molecules through linker atoms at the 5′or 3′ terminus (3-10), base (11-13), internal phosphate (14, 15), sugar 2′ (16-20), and nonnucleotide (21-29). The 5′ or 3′ terminus brings about difficulties for anchoring the molecule at the desired position; the base may interfere with properties such as base pairing and generate at least two interaction sites neighboring the base; the internal phosphate loses the * Author to whom correspondence should be addressed. † Present address: Department of Material Science, Faculty of Engineering, Tottori University, 4-101 Koyamacho minami, Tottori 680, Japan. ‡ A member of “Field and Reaction”, PRESTO, JRDC. Present address: Department of Chemistry, College of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153, Japan. X Abstract published in Advance ACS Abstracts, April 1, 1996.

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negative charge of the phosphodiester and forms diastereoisomers, which will give at least two possible structures as a result of duplex formation; and although the sugar 2′ may have some advantages for the purpose, derivatives of four nucleosides must be synthesized to accommodate an automated DNA synthesizer. Therefore, one ought to select the non-nucleoside that has no base. It has been indicated that some aromatic rings strongly interact with an abasic site in DNA (30). In this paper, we describe the synthesis of a stereochemically defined non-nucleoside suitable for the incorporation of a reactive alkyl amine into an oligonucleotide as illustrated in Figure 1. We also report the synthesis of a 9-amino-6-chloro-2-methoxyacridine reagent activated as the succinimidyl ester for postsynthetic modification of the amino substrate. The acridine derivative covalently linked to ODN (Acr-ODN) through a linker was prepared with these reagents. We will also describe the interaction between the covalently linked intercalating agent and the adjacent artificial abasic site in doublestranded DNA using spectroscopic and thermodynamic measurements. EXPERIMENTAL PROCEDURES 1H NMR and 31P NMR spectra were obtained on a JEOL EX-90 spectrometer, using tetramethylsilane and 85% H3PO4 as internal and external standards, respectively. High-performance liquid chromatography (HPLC) was performed on a Shimadzu Model LC/CTO-6A equipped with a Waters 991J 3D-UV detector, using a reversedphase COSMOSIL AR-300 column (4.6 × 150 mm). Absorption spectra were recorded on a Shimadzu UV2200 spectrophotometer with a thermoelectrical cell holder. Fluorescence spectra were recorded on a Shimadzu RF-503A spectrofluorometer. Fluorescence lifetimes were determined by a single-photon-counting method on a Horiba NAES-550 with a flash lamp filled with hydrogen. The fluorescence decay curves were analyzed by a deconvolution program. Synthesis of O1-(4′-Monomethoxytrityl)-N-[(fluoren-9-ylmethoxy)carbonyl]-L-threoninol (4). Prepa-

© 1996 American Chemical Society

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Figure 1. Oligonucleotide containing L-threoninol backbone modified with acridine derivative (left) and schematic representation of selective intercalation into the abasic site (right).

ration of N-[(fluoren-9-ylmethoxy)carbonyl]-L-threoninol (3) was described elsewhere (28). N-[(Fluoren-9-ylmethoxy)carbonyl]-L-threoninol (1.5 g, 4.58 mmol) was coevaporated with dry pyridine (3 × 15 mL) and then dissolved into dry pyridine (10 mL). Monomethoxytrityl chloride (1.49 g, 4.81 mmol) was added with stirring, and the reaction was allowed to proceed at room temperature for 4 h. Then the reaction was quenched with methanol (1 mL), and stirring was continued for 10 min. The solvent was removed under reduced pressure, and the residue was dissolved into chloroform (50 mL) and washed with 5% sodium hydrogen carbonate solution (3 × 30 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. After filtration and removal of the solvent, the product was purified with silica gel column chromatography (25 × 3.6 cm) eluting with chloroform/hexane (6:4) (2.63 g, 96% yield): 1H NMR (CDCl3) δ 1.18 (m, 3H, CH3), 2.72 (s, 1H, OH), 3.48-3.47 (m, 7H, OCH2, NCH, OCH, OCH3 singlet at 3.76), 4.114.46 (m, 3H, CH, CH2), 5.53 (d, 1H, NH), 6.57-7.79 (m, 22H, Ar). Synthesis of O1-(4′-Monomethoxytrityl)-N-[(fluoren-9-ylmethoxy)carbonyl]-O3-[2-cyanoethyl-N,Ndiisopropylaminophosphinyl]-L-threoninol (5). O1(4′-Monomethoxytrityl)-N-[(fluoren-9-ylmethoxy)carbonyl]L-threoninol (4) (1.99 g, 3.32 mmol) was coevaporated with dry acetonitrile (10 mL) and dissolved into dry acetonitrile (10 mL), and then tetrazole (232 mg, 3.32 mmol) and 2-cyanoethylbis(diisopropylamino) phosphite (1.00 g, 3.32 mmol) were added; the mixture was reacted for an hour at room temperature. The reaction mixture was extracted with ethyl acetate (150 mL). The organic layer was washed with saturated sodium hydrogen carbonate solution (150 mL) and dried over anhydrous sodium sulfate. The solution was filtered. The solvent was evaporated, and the product was coevaporated with dry acetonitrile (3 × 10 mL) for removing a small amount of water. The product was used in the DNA synthesizer without further purification (2.41 g, 91% yield): 1H NMR (CDCl3) δ 0.97-1.30 (m, 15H, CH3), 2.27-2.80 (m, 2H, CH2CN), 3.16-3.76 (m, 11H, POCH2, PNCH, OCH2, OCH, OCH3 singlet at 3.76), 4.04-4.47 (m, 3H, CH, CH2 of Fmoc), 5.53 (d, 1H, NH), 6.75-7.79 (m, 22H, Ar); 31P NMR (CDCl3) 147.9, 148.2. The only impurities observed were ca. 1% of dimer [(MMTr-Fmoc-L-threoninol)2POCH2CH2CN, δp 140.4] and two hydrolysis products [ca. 1% of MMTr-Fmoc-L-threoninol-P(OH)OCH2CH2CN, δp 7.5 and ca. 5% of NCCH2CH2OP(OH)NiPr2, δp 14.0] assigned from the literature (31).

Fukui et al.

Synthesis of N-[9-(6-Chloro-2-methoxy)acridinyl]6-aminocaproic Acid Hydrochloride (7). 6,9-Dichloro2-methoxyacridine (6) (1 g, 3.60 mmol) and phenol (5 g) were stirred at 100 °C for 40 min, and then 6-aminocaproic acid (500 mg, 3.81 mmol) was added. The reaction mixture was allowed to react at 100 °C for 2 h. The reaction mixture was cooled and dropped slowly to 300 mL of diethyl ether. The precipitate was filtrated, washed with diethyl ether, and then dried in vacuo. The resulting bright yellow solid was recrystalized from diluted HCl solution to give the product as HCl salt (1.24 g, 81% yield): 1H NMR (DMSO-d6) δ 1.43-1.87 (m, 6H, CH2), 2.41-2.28 (t, 2H, CH2COO), 3.96 (m, NCH2, OCH3 singlet at 3.96), 7.41-8.51 (m, 6H, Ar). Synthesis of N-Hydroxysuccinimidyl N-[9-(6Chloro-2-methoxy)acridinyl]-6-aminocaproate Hydrochloride (8). N-[9-(6-Chloro-2-methoxy)acridinyl]-6aminocaproic acid hydrochloride (7) (500 mg, 1.17 mmol) and N-hydroxysuccinimide (183 mg, 1.59 mmol) were suspended in DMF (15 mL). The reaction mixture was cooled at 0 °C, and then 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) (304 mg, 1.59 mmol) was added. The reaction mixture was stirred for an hour at 0 °C and reacted at room temperature. After about 2 h, the suspension became a yellow solution. The solution was allowed to react overnight. Acetic acid (2 mL) was added and stirred for 10 min to quench the small excess of EDC. The solvent was removed under reduced pressure, and the reaction mixture was partitioned between chloroform (50 mL) and water (30 mL)/acetic acid (20:1). The organic layer was washed with 3 × 30 mL of water/acetic acid (20:1), dried over anhydrous sodium sulfate, and evaporated to dryness. Purification was performed on silica gel column chromatography (6 × 25 cm) eluting with chloroform/methanol/acetic acid (80:20:3). The resulting gummy residue was dissolved in a minimum amount of chloroform and then dropped in 150 mL of cold ethyl acetate/hexane (1:2). The product was precipitated as a bright yellow solid. The reagent was stored in a desiccator to prevent hydrolysis with water (373 mg, 61% yield): 1H NMR (CDCl3) δ 1.72-2.13 (m, 10H, CH2, CH3 of acetic acid), 2.58-2.96 (m, 6H, NCH2, NCOCH2), 4.45 (m, 5H, NH, OCH3), 7.05-8.09 (m, 6H, Ar). Oligonucleotide Synthesis. O1-(4′-Monomethoxytrityl)-N-[(fluoren-9-ylmethoxy)carbonyl]-O3-[2-cyanoethylN,N-diisopropylaminophosphinyl]-L-threoninol (5) was coupled using a concentration of 0.15 M on Applied Biosystems Model 391 according to the protocols supplied by the manufacturer. The oligonucleotide was synthesized on a 1.0 µmol scale. The coupling time was set for 5 min. The coupling efficiency was determined by measuring the deprotected dimethoxytrityl cation concentration. The average coupling efficiency was 80%. The 5′-DMTr protecting group of synthesized ODN was left at the end of each synthesis to facilitate purification. Cleavage from the solid support and deprotection of the base, phosphate, and amino substrate of L-threoninol was accomplished with concentrated ammonium hydroxide according to the manufacturer’s recommendations. The tritylated oligonucleotide was purified by reversed-phase HPLC on a COSMOSIL AR-300 column (4.6 × 150 mm) eluted with a linear gradient of 6-60% acetonitrile in 0.1 M triethylammonium acetate (pH 7) over 30 min (flow rate 0.8 mL/min). Appropriate fractions were combined and concentrated to dryness. The residue was detritylated in 80% acetic acid (at room temperature, 15 min). Figure 2A shows the analytical HPLC chromatogram for the purified ODN containing L-threoninol backbone with the sequence dA7[ ]dA7 ([ ] means L-threoninol). Split-

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ODN Modified with an Acridine Scheme 1

both chromatograms at 2.5 and 11 min correspond to N-hydroxysuccinimide and unreacted ODN, respectively. The peak at 13.5 min corresponds to ODN modified with 8 (dA7[Acr]dA7). The peaks at 28.5 and 21.5 min were assigned to 8 and the hydrolysis product 7, respectively, which was confirmed by coinjections of the authentic samples. It is apparent that the reagent 8 was almost decomposed after an hour in pH 8 buffer solution. Absorption spectra of 21.5 and 28.5 min peaks coincide with those of 7. On the other hand, the absorption spectrum of Acr-ODN is consistent with the presence of a single acridine ring tethered to the ODN, and the absorption around 430 nm, which is attributed to the absorption of the acridine ring, showed a red shift. The result suggests that the acridine ring appreciably contacts with the neighboring adenosine base even in a single-stranded DNA. RESULTS

Figure 2. Reversed-phase HPLC chromatograms: (A) analytical HPLC for ODN containing artificial abasic site (dA7[ ]dA7); (B) analytical HPLC chromatogram for the reaction mixture containing the ODN and 8 (see text). The HPLC system used a 150 × 4.5 mm C18 column, and the flow rate was 0.8 mL/min; for both HPLC systems a gradient of 6-60% solvent B over 30 min [solvent A ) 0.1 M triethylammonium acetate (pH 7.0); solvent B ) acetonitrile] was used. Detection was by UV absorbance at 260 and 420 nm and emission (excited at 420 nm, detected at 510 nm).

ting of the peak assigned to optical isomers was not detected. This means that the stereochemistry of Lthreoninol is not rearranged during DNA synthesis cycles. The following ODNs were synthesized according to the above method: dT5[ ]dT5, dT7[ ]dT7, dA5[ ]dA5, dA7[ ]dA7. Acridine Modification of the Oligonucleotides. The ODN product was dissolved into 300 µL of 0.15 M HEPES buffer (pH 8) and 270 µL of dimethyl sulfoxide. A 30 µL sample of N-hydroxysuccinimidyl N-[9-(6-chloro2-methoxy)acridinyl]-6-aminocaproate hydrochloride (8) in dimethyl sulfoxide (ca. 0.5 mg /90 µL) was added to the ODN solution every 2 h for three times. Moreover, the mixture was vortexed at room temperature for 10 h in the dark. The crude acridine-modified ODN was then freed from 8 and a large excess of its hydrolysis product using Sep-Pak C18 cartridges. The reaction mixture was dissolved into 1 mL of water and applied to the Sep-Pak cartridge. The cartridge was washed with 4 mL of water, and the product oligomers were eluted with 1.5 mL of 20% acetonitrile in water. The products were further purified by reversed-phase HPLC in the same manner as described above. Figure 2B shows the analytical HPLC chromatogram for the reaction mixture containing the ODN (dA7[ ]dA7) and 8 after an hour. The peaks in

Synthesis of L-Threoninol Phosporamidite. The synthetic scheme for preparing O1-(4′-monomethoxytrityl)-N-[(fluoren-9-ylmethoxy)carbonyl]-O3-[2-cyanoethylN,N-diisopropylaminophosphinyl]-L-threoninol (5) is shown in Scheme 1. The starting material for the preparation of Fmoc-L-threoninol (3) has been described elsewhere (28). For the purpose of selective protection of the primary alcohol, 4,4′-dimethoxytrityl (DMTr) substrate is usually used, but this substrate was not stable for the primary alcohol even at -5 °C. Thus, in the present study, 4-monomethoxytrityl (MMTr) substrate was employed for protecting the primary alcohol. The MMTr substrate was sufficiently stable for at least 6 months at room temperature and could be completely removed by following the standard protocol in DNA synthesizer. Finally, the secondary hydroxyl group was activated with bis(diisopropylamino)-2-cyanoethylphosphine. In the purification of such a product, the solvent containing triethylamine is usually used in silica gel column chromatography to prevent decomposition of the product. The Fmoc groups, however, are slowly cleaved by triethylamine used in chromatography solvent (32). Therefore, the product was coevaporated with dry acetonitrile and used in DNA synthesis without further purification. Purity of the product was estimated with the use of 1H NMR and 31P NMR. Acridine Modification Reagent. For the acridine modification of the ODN-containing L-threoninol backbone, N-hydroxysuccinimidyl N-[9-(6-chloro-2-methoxy)acridinyl]-6-aminocaproate hydrochloride (8) was synthesized (Scheme 2). The carboxylic acid and N-hydroxysuccinimide was condensed with EDC and then purified with column chromatography on silica gel. In aqueous solvent the succinimidyl ester is hydrolyzed to the carboxylic acid according to the basicity of the solvent (33). On the other hand, in organic solvent, the succinimidyl ester is not hydrolyzed and does not react with

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Scheme 2

Figure 3. Change in absorption spectra of dA7[Acr]dA7 upon the addition of dT14. The numbers indicated on the figures indicate the ratio of dT14 to dA7[Acr]dA7. Measurements were carried out at 0 °C in 10 mM phosphate buffer/150 mM sodium chloride (pH 7.2). A cell of 1 cm path length was used.

alcohol (34). However, 8 was slowly hydrolyzed under the circumstances of humidity and also reacted with methanol during column chromatography, which prevented complete purification. It can be assumed that these reactions are catalyzed by the N-pyridyl of the acridine ring. It was found that the use of an eluent for column chromatography containing a few percent of acetic acid suppresses these side reactions. The modified yield estimated from the intensity ratio of the HPLC chart was about 80%. The resulting acridine-modified ODN was separated from impurities with a Sep-Pak C18 cartridge and then with reversed-phase HPLC. Absorption Spectra. The change in the absorption spectrum of dA7[Acr]dA7 upon addition of complementary ODN, dT14, is shown in Figure 3. As the concentration of the complementary ODN increased, the absorption at 425 and 450 nm decreased linearly and the new absorption peak at 475 nm appeared. This behavior is quite similar to that of the 5′-tethered acridine ODN (7). The decrease in the extinction coefficient definitely ceased at a specific concentration of the complementary ODN. The decrease in the extinction coefficient of the base moiety (260 nm) also ceased at the same concentration of the complementary ODN. The result indicates that Acr-ODN binds to the complementary ODN in the ratio 1:1 and the acridine ring is selectively intercalated into the double-stranded DNA. The nonmodified (abasic) ODN, dA7[ ]dA7, also bound to the complementary sequence in the same manner. On the other hand, acridine tethered to the oligo(dT) strand bound to the complementary ODN, oligo(dA), in the ratio at 2:1 at room temperature, suggesting the formation of triplestranded DNA (35, 36). The degree of hypochromicity at 425 nm was 21%. This value is lower than that of quinacrine and poly(dA-dT) or 5′-tethered acridine (7). However, there are adenosine bases adjacent to the acridine ring even in single-stranded DNA, and thus red shift of the absorbance was observed in the single-strand compared with free acridine ring. Therefore, stronger

Figure 4. Melting curves measured at 260 nm (A) and 425 nm (B) for complexes of dA7[ ]dA7 (+), dA7[Acr]dA7 (O), and dA14 ([) with dT14. The buffer was the same as that used in Figure 3.

hypochromicity would be obtained if compared with the free acridine ring. Thermodynamical Properties. A thermal denaturation experiment was conducted to evaluate the effect of the acridine ring incorporated into ODN on the hybridization characteristics (Figure 4). All of the oligonucleotides showed a clear sigmoid. Melting temperatures (Tm) of the native, abasic, and Acr-ODN duplex were 35.6, 26.7, and 37.9 °C, respectively. The Tm of nonmodified ODN was lower than that of native DNA. These results are considered to be attributable to the lack of π-π stacking. Acr-ODN, however, showed a higher Tm than that of abasic DNA and native DNA. Figure 4B shows melting curves detected at 425 nm characteristic of the acridine ring. Although there was some noise due to the low absorbance of the acridine ring, the thermodynamic behaviors at 425 and 260 nm were nearly the same. Therefore, it is clearly demonstrated that doublestrand formation and acridine intercalation are cooperative. The acridine ring compensates the destabilization resulting from the lack of base. Photophysical Properties. Figure 5 shows the absorption and fluorescence excitation spectra of the AcrODN duplex detected at 510 nm. These spectra were quite similar, and the shape of the fluorescence excitation is independent of the detected wavelength over 480-540 nm. The results indicate that the acridine ring experiences a single environment in double-stranded DNA. Fluorescence properties of acridine derivatives, singlestranded Acr-ODN, and double-stranded Acr-ODN were determined (Figure 6). The shapes of the spectra are similar, but the intensity strongly depends on the

ODN Modified with an Acridine

Figure 5. Absorption spectra (solid line) and fluorescence excitation spectra (broken line) of dA7[Acr]dA7 and dT14 duplex. Excitation was recorded for an emission wavelength of 510 nm. The condition was the same as that used in Figure 3.

Figure 6. Fluorescence emission spectra of quinacrine (dotted line) and dA7[Acr]dA7 free of (broken line) and bound to dT14 (solid line). Excitation wavelength was 427 nm. The condition was the same as that used in Figure 3.

chemical environments around the acridine ring. The quantum yields of acridine in single- and double-stranded DNA were 0.375 and 0.276, respectively, although that of the free acridine 7 was only 0.07 at 0 °C. Furthermore, the quantum yield of acridine in double-stranded ODN was higher than that of intermolecular intercalation [quinacrine and poly (dA-dT)] showing 0.27 (in our preliminary check). The increase in emission intensity is consistent with the fact that the acridine ring covalently linked to ODN is effectively intercalated into the double-stranded ODN and, to a significant extent, even in the single strand. In these strands, the acridine ring experiences a hydrophobic environment due to the adenine bases at both ends. The photophysical and thermodynamical behavior of another Acr-ODN, dA5[Acr]dA5, was very similar to that above. DISCUSSION

Many kinds of molecules have been used as handles of DNA at 5′, 3′, or internucleotide position at which a functional molecule such as pyrene or biotin is linked after DNA synthesis. Nelson et al. were the first to use an “aminodiol” (1-amino-2,3-propanediol) backbone for this purpose (21, 22). They also synthesized the 1,3propanediol backbone systems to avoid steric hindrance and to mimic the traditional ribose moiety (26). Reed et al. have used an optically pure L-pyrrolidinol backbone derived from the natural amino acid L-proline to prevent the appearance of several optical isomers which necessarily occur from connection of the amino substrate to the alkyldiol backbone (23). Therefore, the backbone should be optically pure if the reagent is used as therapic tool or biochemical probe.

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The L-threoninol backbone was employed to mimic the spacing of the traditional ribose moiey, which has three carbons between the primary and secondary alcohols, and to suppress the appearance of several optical isomers in the incorporated DNA (Figure 1). The backbone has already been synthesized and used by Reed et al. as nonnucleotide backbone in methylphosphonate (28). The backbone contains a secondary amine which can be modified through the amide linkage. The amino substrate of L-threoninol, (2R,3R)-2-amino-1,3-butanediol, is directed to the major groove, thereby reducing steric hindrance. Then the linker chain could lie along the major groove between the handles and the acridine ring. The backbone is also considered as an artificial abasic nucleoside. An intercalator such as acridine is inserted into DNA by spreading the stacked base (37). At the abasic site, the spreading energy is not required, although the energy would be required on the target strand. Therefore, the abasic site is considered as a “pocket” appropriate to hold the intercalator, where the spreading is not required on the abasic strand. The intercalator covalently linked to the abasic site is widely useful as a pseudobase, which can intercalate to the abasic site selectively and compensate for the lack of base stacking. 9-Amino-6-chloro-2-methoxyacridine has been extensively studied as an intercalating agent in DNA or RNA (38). The optical properties of aminoacridine derivatives are strongly perturbed upon intercalation (39, 40). These properties make the acridines effective as DNA probes and also able to construct electron-transfer (41) or energytransfer systems in DNA matrix (42). Upon intercalation, the acridine ring lies so as to maximize the stacking area with the bases above and under the acridine ring (37). He´le`ne et al. have systematically investigated the chain length of oligo(dT) covalently linked to 9-amino-6chloro-2-methoxyacridine at the 3′-phosphate group. It has been indicated that the chain length of pentamethylene gives the most stable complex (7). To satisfy this condition, the linker arm has to connect the 9-amino substrate of acridine with the amino substrate of the backbone. From the CPK model, we predict that the length of tetramethylene or pentamethylene is suitable for the linker. Acridine caproic acid was synthesized on the basis of this assumption and activated as the succinimidyl ester. Although a phosphoramidite connecting 9-amino-6-chloro-2-methoxyacridine has been reported (43), the acridine modification reagent 8 can modify not only the L-threoninol moiety but also other alkylamines tethered to oligonucleotides previously synthesized (5, 6, 11-13, 16, 17, 21-23, 26). Both Acr-ODN and nonmodified (abasic) ODN bind to the complementary oligo(dT) with the ratio 1:1. The abasic ODN was strongly destabilized compared with native duplex, which comes from the lack of π-π stacking. This destabilization brought about decrease of Tm by -8.9 °C. This large value is not consistent with the results of Reynolds et al. (28). The discrepancy is possibly due to the difference of double-stranded DNA consisting of, in their case, RNA and methylphosphonate. The results, however, show coincidence with those of Bertrand et al. (44), who used ODNs containing the true abasic site. Linking of the acridine ring at the abasic site increased Tm by 11.2 °C compared with abasic ODN. This increase is comparable with the 14 °C increase reported for a 3′-acridine tailed dT12 ODN (7). In Figure 5, the excitation spectrum of the Acr-ODN as duplex is quite similar to the absorption spectrum. This similarity indicates that the acridine ring is under the influence of

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only one kind of environment, although the linker is rather long (7). The fluorescence quantum yield of the Acr-ODNforming duplex was larger than that of quinacrine plus poly(dA-dT) as seen in Figure 6. The result indicates that the acridine ring is strongly intercalated. The ratio of the absorption intensities of the peaks at 425 and 450 nm (I450/I425) increases upon the concentration of the complementary ODN. This behavior was different from that of quinacrine plus poly(dA-dT) (45). This difference is considered to be caused by the binding mode. Quinacrine is intercalated through the minor groove of the DNA. From the CPK model, the linker of the Acr-ODN is directed to the major groove. Hence, the acridine ring would be intercalated through the major groove. The acridine ring binding to the duplex with only one mode will give a single-exponential decay in emission. The acridine, however, shows biexponential decays of 26 (96%) and 8 ns (4%) (excited at 450 nm and detected at 510 nm). The lifetime of Acr-ODN was nearly identical with that of quinacrine plus poly(dA-dT), but its ratio of long lifetime components was smaller than that of AcrODN. However, the lifetime does not necessarily reflect multiplicity of the binding mode, because 9-amino-6chloro-2-methoxyacridine alone shows a complex lifetime of emission (46). Therefore, for the investigation of strict coordination of acridine ring, two-dimensional NMR or X-ray crystallography will be required. Moreover, systematic investigations about the chain length will be useful to afford stronger interaction between the acridine ring and DNA. Such studies are now in progress and will be reported in a forthcoming paper. LITERATURE CITED (1) Cohen, J. S., Ed. (1989) Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL. (2) Symon, R. H., Ed. (1989) Nucleic Acid Probes, CRC Press, Boca Raton, FL. (3) Franc¸ ois, J.-C., Saison-Behmoaras, T., Barbier, C., Chassignol, M., Thuong, N. T., and He´le`ne, C. (1989) Sequencespecific recognition and cleavage of duplex DNA via triplehelix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate. Proc. Natl. Acad. Sci. U.S.A. 86, 9702-9706. (4) Lee, B. L., Murakami, A., Blake, K. R., Lin, S.-B., and Miller, P. S. (1988) Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA. Biochemistry 27, 3197-3203. (5) Connolly, B. A. (1987) The synthesis of oligonucleotides containing a primary amino group at the 5′-terminus. Nucleic Acids Res. 15, 3131-3139. (6) Sinha, N. D., and Cook, R. M. (1988) The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol. Nucleic Acids Res. 16, 2659-2669. (7) Asseline, U., Delarue, M., Lancelot, G., Toulme´, F., Thuong, N. T., Montenay-Garestier, T., and He´le`ne, C. (1984) Nucleic acid-binding molecules with high affinity and base sequence specificity: Intercalating agents covalently linked to oligodeoxynucloetides. Proc. Natl. Acad. Sci. U.S.A. 81, 3297-3301. (8) Mann, J. S., Shibata, Y., and Meehan, T. (1992) Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at 5′-phosphate. Bioconjugate Chem. 3, 554-558. (9) Doan, T. L., Perrouault, L., Helene, C., Chassignol, M., and Thuong, N. T. (1986) Targeted cleavage of polynucleotides by complementary oligonucleotides covalently linked to ironporphyrins. Biochemistry 25, 6736-6739. (10) Chen, J.-K., Carlson, D. V., Weith, H. L., O’Brien, J. A., Goldman, M. E., and Cushman, M. (1992) Synthesis of an oligonucleotide-intercalator conjugate in which the linker

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