Nucleosides and Nucleotides. 170. Synthesis and Properties of

deoxynucleic guanidine (DNG) modified oligonucleotides containing neutral urea linkages: effect of charge deletions on binding and fidelity. Barry...
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Bioconjugate Chem. 1998, 9, 33−39

33

Nucleosides and Nucleotides. 170. Synthesis and Properties of Oligodeoxynucleotides Containing 5-[N-[2-[N,N-Bis(2-aminoethyl)amino]ethyl]carbamoyl]-2′-deoxyuridine and 5-[N-[3-[N,NBis(3-aminopropyl)amino]propyl]carbamoyl]-2′-deoxyuridine† Yoshihito Ueno, Mai Mikawa, and Akira Matsuda* Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060, Japan. Received August 11, 1997; Revised Manuscript Received October 7, 1997X

The synthesis of oligodeoxynucleotides (ODNs) containing 5-[N-[2-[N,N-bis(2-aminoethyl)amino]ethyl]carbamoyl]-2′-deoxyuridine (BAE) and 5-[N-[3-[N,N-bis(3-aminopropyl)amino]propyl]carbamoyl]-2′deoxyuridine (BAP) is described. The thermal stabilities of duplexes containing these ODNs and either the complementary DNA or RNA strand and of triplexes consisting of these ODNs and the target duplex were studied by thermal denaturation. ODNs containing BAE or BAP stabilize duplex formation with either the complementary DNA or RNA strands but destabilize triplex formation with the target duplex. Furthermore, the resistance of these ODNs to nuclease hydrolysis was studied by using snake venom phosphodiesterase (a 3′-exonuclease) and nuclease S1 (an endonuclease). It was found that ODNs containing either BAE or BAP were more resistant to nucleolytic hydrolysis by either of the nucleases than the unmodified ODN.

INTRODUCTION

Oligodeoxynucleotide (ODN)1 analogues with a variety of functional groups (i.e. intercalating, alkylating, DNAdegrading, and fluorescent groups) have been synthesized and used for biological and biochemical studies, including antisense and antigene studies (1-6). To be able to apply these ODN analogues to antisense and antigene studies, the ODNs must form stable Watson-Crick hybrids with the complementary target RNAs and form stable triplestranded DNA with target DNA duplexes (1-4). It is well-known that naturally occurring polyamines, such as spermidine and spermine, bind strongly to DNA (7-9) and stabilize duplex (10, 11) and triplex formations (12-14), although the precise mode of binding is not clear. Their enhanced thermal stability is explained by the reduction of the anionic electrostatic repulsion between the phosphate moieties by the cationic polyamines. Therefore, ODN analogues carrying various polyamines have been synthesized to improve the thermal stability of duplexes and triplexes (15-24). We recently developed a new and convenient postsynthetic modification method for the synthesis of ODNs containing various amino linkers using either 5-(methoxycarbonyl)-2′-deoxyuridine (1) or 5-(trifluoroethoxycarbonyl)-2′-deoxyuridine (2) as a convertible nucleoside † For part 169 in this series, see the following: Shuto, S., Niizuma, S., and Matsuda, A. (1997) J. Org. Chem. (submitted for publication). * To whom reprint requests should be addressed. Phone: +81-11-706-3228. Fax: +81-11-706-4980. E-mail: matuda@ pharm.hokudai.ac.jp. X Abstract published in Advance ACS Abstracts, November 15, 1997. 1 Abbreviations: ODN, oligodeoxynucleotide; CPG, controlled pore glass; HPLC, high-performance liquid chromatography; Tm, melting temperature; WSCI, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; DMAP, 4-(dimethylamino)pyridine; TEAA, triethylammonium acetate.

(25-30). If ODNs containing 1 or 2 at specific positions are treated with one of several diaminoalkanes, the desired ODNs containing 5-(N-aminoalkyl)carbamoyl-2′deoxyuridines are readily obtained. Using this method, we found that ODNs containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridines (Hs) efficiently stabilize duplex formation with either the complementary DNA or RNA strands (25, 26, 30). The terminal amino groups of the amino linkers can be further modified by the addition of a variety of functional groups (27). By converting the amino linker to a mercapto linker, we successfully synthesized 5′-5′-linked ODNs with the potential for triplex formation (28) and succeeded in the chemical ligation of ODNs via a disulfide bond in the presence of a template (29). The enhanced thermal stability of duplexes containing Hs has been explained in the following manner. Reduction of the electrostatic repulsion between the phosphate moieties by the ammonium ion of the aminohexylcarbamoyl linker and the enhanced hydrogen-donor ability at N3-H combine to give duplex stabilization, due to incorporation of an electron-withdrawing carbamoyl group at the 5-position of the uracil (26). Tris(2-aminoethyl)amine and tris(3-aminopropyl)amine have a greater positive net charge than diaminoalkanes such as diaminoethane and 1,6-diaminohexane. Therefore, we envisioned that ODNs containing either tris(2-aminoethyl)amine or tris(3-aminopropyl)amine would stabilize duplex formation with the complementary DNA or RNA strand more efficiently than the ODNs carrying the diaminoalkanes. In this paper, we report the synthesis of ODNs containing 5-[N-[2-[N,N-bis(2-aminoethyl)amino]ethyl]carbamoyl]-2′-deoxyuridine (BAE) and 5-[N-[3-[N,N-bis(3-aminopropyl)amino]propyl]carbamoyl]-2′-deoxyuridine (BAP). The thermal stability of duplexes containing these ODNs and either the complementary DNA or RNA strand, the thermal stability of triplexes consisting of these ODNs and the target duplex, and the resistance of

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Ueno et al.

Scheme 1a

a (a) (1) (NH CH CH ) N or (NH CH CH CH ) N, pyridine, room temperature; (2) CF COOEt, Et N, pyridine, room temperature; 2 2 2 3 2 2 2 2 3 3 3 (b) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine, CH2Cl2, room temperature; and (c) succinic anhydride, DMAP, pyridine, room temperature.

the ODNs to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3′-exonuclease) and nuclease S1 (an endonuclease) were also studied. RESULTS AND DISCUSSION

Synthesis. The synthesis of ODNs containing BAE or was accomplished in a DNA synthesizer by using a suitably protected 5-[N-[2-[N,N-bis(2-aminoethyl)amino]ethyl]carbamoyl]-2′-deoxyuridine (5) or 5-[N-[3-[N,Nbis(3-aminopropyl)amino]propyl]carbamoyl]-2′-deoxyuridine phosphoramidite (8). The synthesis of the 3′phosphoramidite derivatives 5 and 8 is shown in Scheme 1. 5′-O-(Dimethoxytrityl)-5-(trifluoroethoxycarbonyl)-2′deoxyuridine (3), prepared by a previously described method (27), reacted readily with either tris(2-aminoethyl)amine or tris(3-aminopropyl)amine. Without purifying the product, we protected the amino group with a trifluoroacetyl group to give 4 or 7. The modified nucleoside (4 or 7) was converted to the protected nucleoside phosphoramidite, 5 or 8, by the usual method of ODN synthesis (31). To incorporate BAE or BAP into the 3′end of the ODNs, 4 or 7 was further modified to produce the corresponding 3′-succinate, 6 or 9, which was then reacted with controlled pore glass (CPG) to give a solid support containing 4 (35.0 µmol/g) or 7 (34.7 µmol/g). An unmodified DNA heptadecamer (control) and heptadecamers containing BAEs or BAPs were synthesized in a DNA synthesizer (31). The average coupling yields of 5 and 8 were 96 and 95%, respectively, using a 0.12 M solution of the amidite derivatives in CH3CN and 360 s for the coupling time. The fully protected ODNs (each 1 µmol scale) linked to the solid supports were treated with concentrated NH4OH at 55 °C for 16 h. This was followed by C-18 column chromatography. Detritylation gave BAP

ODNs 11-20. Each ODN in this preparation showed a single peak by reversed phase HPLC analysis. ODNs 13 and 17 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF/MS), and the observed molecular weight supported its structure. UV Melting Studies of Duplex and Triplex Formation. Duplex formations of the heptadecamers 1020 with the complementary DNA, d[TGG(AG)9GT]-3′ (21), was studied by thermal denaturation in a buffer of 0.01 M sodium cacodylate (pH 7) containing 0.01 M NaCl. One transition was observed in a melting profile of each duplex. Melting temperatures (Tms) are listed in Table 1. All of the Tm values for the modified ODNs containing BAEs or BAPs were greater than that of the corresponding unmodified ODN 10. Therefore, the modified nucleosides, BAE and BAP, were found to thermally stabilize duplex formation. The stability of the duplexes was dependent on the position and number of the modified nucleosides. Duplexes with ODN 17 or 18 containing one molecule of BAP at either the center or 5′-end (Tm ) 56.1 and 56.2 °C, respectively) were more stable than the duplex with ODN 16 containing BAP at its 3′-end (Tm ) 54.3 °C). On the other hand, the Tm values for the duplexes containing ODNs 11-13 which had one molecule of BAE at either the 3′-end, center, or 5′-end were almost same. The duplexes became more stable as the number of BAEs and BAPs increased. ODNs containing BA Ps stabilized duplex formation more efficiently than ODNs containing BAEs. A 9.8 °C increase in Tm was observed for ODN 20. In a previous paper, we reported the thermal stability of duplexes constructed from ODNs containing Hs or 5-(N-aminoethyl)carbamoyl-2′-deoxyuridines (Es) using

Synthesis and Properties of Oligonucleotides

Bioconjugate Chem., Vol. 9, No. 1, 1998 35

Table 1. Thermal Denaturationa ODN-DNAb 0.01 M NaCl

ODN-RNAc 0.1 M NaCl

triplexd

0.1 M NaCl

ODNs

Tm (°C)

∆Tm (°C)

Tm (°C)

∆Tm (°C)

Tm (°C)

10 11 12 13 14 15 16 17 18 19 20

53.4 54.9 54.5 54.7 56.1 59.7 54.3 56.1 56.2 61.2 63.2

1.5 1.1 1.3 2.7 6.3 0.9 2.7 2.8 7.8 9.8

62.7 nde 64.1 nd 66.2 68.8 nd 65.8 nd 68.1 70.7

nd 1.4 nd 3.5 6.1 nd 3.1 nd 5.4 8.0

70.7 72.4 72.2 72.9 72.2 74.1 71.9 73.8 73.8 76.0 77.7

∆Tm (°C) 1.7 1.5 2.2 1.5 3.4 1.2 3.1 3.1 5.3 7.0

0.5 M NaCl Tm (°C)

∆Tm (°C)

40.8 39.8 35.5 40.0 27.2 35.5 39.7 36.3 40.6 31.0 35.3

-1.0 -5.3 -0.8 -13.6 -5.3 -1.1 -4.5 -0.2 -9.8 -5.5

a Experimental conditions are described in Experimental Procedures. b The complementary DNA 5′-d[TGG(AG) GT]-3′ (21). c The 9 complementary RNA 5′-r[UGG(AG)9GU]-3′ (22). d The target sequence 5′-d[AG(TC)9C(T)5AG(GA)9CT]-3′ (23). e nd, not determined.

the same sequences and conditions described here (25, 26). The length of the amino linkers was found to affect the thermal stability of the duplexes. The ODNs containing H stabilized duplex formation with either the complementary DNA or RNA strands more efficiently than ODNs containing Es (25). If ODNs containing Hs and Es are considered, the order of ∆Tm values for ODNDNA duplexes containing three molecules of the modified nucleosides in a buffer containing 0.01 M NaCl is as follows: ODN 20 containing BAPs (∆Tm ) 9.8 °C) > ODN containing Hs (∆Tm ) 8.0 °C) > ODN 15 containing BAEs (∆Tm ) 6.3 °C) > ODN containing Es (∆Tm ) 6.0 °C). The enhanced thermal stability of duplexes containing Hs and Es is thought to be due to both a reduction of the electrostatic repulsion between phosphate moieties by the ammonium ion of the aminoalkylcarbamoyl linker and the enhanced hydrogen-donor ability at N3-H caused by incorporation of an electron-withdrawing carbamoyl group at the 5-position of the uracil (26). Therefore, to confirm the importance of the terminal ammonium ion of the N,N-bis(2-aminoethyl)aminoethyl and N,N-bis(3aminopropyl)aminopropyl linkers in stabilizing duplexes, Tms were measured under conditions with a higher ionic strength (0.1 M NaCl) and the ∆Tm values were compared with those measured under conditions with a low ionic strength (Table 1). The ∆Tm values with the high ionic strength for the duplexes containing BAE were similar to that with the low ionic strength, whereas the ∆Tm values with the high ionic strength for the duplexes containing BAP became smaller than that with the low ionic strength as the number of BAP increased. These results indicate that the terminal ammonium ion of the N,N-bis(3-aminopropyl)aminopropyl linker more effectively neutralized the phosphate anions than that of the N,N-bis(2-aminoethyl)aminoethyl linker. The N,N-bis(3-aminopropyl)aminopropyl linker is longer than the N,N-bis(2-aminoethyl)aminoethyl linker and has a more positive net charge than either the aminohexyl or aminoethyl linkers. Therefore, ODNs containing BAPs should stabilize duplex formation with the complementary DNA more efficiently than ODNs containing Hs, Es, or BAEs. Stable duplex formation with mRNA is one of the most important factors in antisense research. Therefore, duplex formation by heptadecamers with the complementary RNA strand, 5′-[UGG(AG)9GU]-3′ (22), was next studied by thermal denaturation in a buffer containing 0.1 M NaCl. As shown in Table 1, the trend shown by the Tms of the modified ODNs containing BAEs or BAPs was similar to that of the corresponding ODN-DNA duplexes. However, the ∆Tm values were evidently different. The ∆Tm values for ODNs 11-13 and 16-18,

containing one molecule of BAE or BAP, were slightly greater than those of the corresponding ODN-DNA duplexes. On the other hand, those for ODNs 14, 15, 19, and 20, containing either two or three molecules of BA E or BAP, were smaller than those of the ODN-DNA duplexes. The duplexes were more stable as the number of BAE and BAP molecules increased. A 7.0 °C increase in Tm was observed for ODN 20. If ODNs containing Hs and Es are considered, the order of ∆Tm values for ODNRNA duplexes containing three molecules of the modified nucleosides in a buffer containing 0.1 M NaCl is as follows: ODN 20 containing BAPs (∆Tm ) 7.0 °C) > ODN 15 containing BAEs (∆Tm ) 3.4 °C) > ODN containing Hs (∆Tm ) 3.0 °C) > ODN containing Es (∆Tm ) 2.0 °C). When ODN analogues containing BAEs and BAPs form duplexes with the complementary DNA or RNA strand, the amino linkers should be accommodated in the major groove, since the 5-position of 2′-deoxyuridine is in the major groove. The major groove of a DNA-RNA duplex is narrower than that of a DNA-DNA duplex (32). So bulky functional groups may not fit into the narrower major groove of DNA-RNA duplexes as well as they do in DNA-DNA duplexes. The difference in ∆Tm values between DNA-RNA and DNA-DNA duplexes may reflect this conformational difference. Triplex formation of heptadecamers with the target duplex, d[AG(TC)9C(T)5AG(GA)9CT]-3′ (23), was also studied by thermal denaturation in a buffer containing 0.5 M NaCl (pH 7.0). Two transitions were observed in the melting profile of each triplex; the transition with the higher Tm was due to melting of the target duplex 23 (88 °C), and the transition with the lower Tm corresponded to dissociation of the third strand from the triplex. The Tms are also listed in Table 1. While ODNs containing one molecule of BAE at either the 5′- or 3′-end have slightly destabilized triplex formation (Tm ) 39.8 and 40.0 °C, respectively), the ODN with one molecule of BAE in its center showed markedly destabilized triplex formation (Tm ) 35.5 °C). Furthermore, a 13.6 °C decrease in Tm was observed in ODN 14, which contains two molecules of BAE in the middle of it. ODN 15, containing three molecules of BAE, one in the middle and one at either end, had a Tm similar to that of ODN 13. Furthermore, similar results were observed in the case of ODNs containing BAPs. When the polypyrimidine ODNs form triplexes with target duplexes, the ODNs are accommodated in the major groove of the target duplexes and Hoogsteen-type base triplets (T‚T and C+‚GC) are formed (4). When ODN analogues containing BAEs and BAPs form triplexes with the target duplexes, the amino linkers should lie in the

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Figure 3. Polyacrylamide gel electrophoresis of 5′-32P-labeled ODNs hydrolyzed by snake venom phosphodiesterase: (a) 10, (b) 11, and (c) 16. ODNs were incubated with snake venom phosphodiesterase for 0 min (lane 1), 10 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 120 min (lane 5). Experimental conditions are described in Experimental Procedures.

Figure 1. Structures of the 2′-deoxyuridine analogues.

Figure 2. Sequences of ODNs synthesized. BAE ) 5-[N-[2-[N,Nbis(2-aminoethyl)amino]ethyl]carbamoyl]-2′-deoxyuridine. BAP ) 5-[N-[3-[N,N-bis(3-aminopropyl)amino]propyl]carbamoyl]-2′deoxyuridine. M ) 5-methyl-2′-deoxycytidine.

major groove of the target duplexes, since the 5-position of the 2′-deoxyuridine residue of the third strand is located in the major groove of the target duplex in the triplex. The vicinity of the 5-position of the 2′-deoxyuridine residue of the third strand in the triplex is sterically crowded. Therefore, bulky amino linkers, such as the N,N-bis(3-aminopropyl)aminopropyl linker and the N,Nbis(2-aminoethyl)aminoethyl linker, may not fit into the major groove of target duplexes, especially when they are located in the middle of the third strands. Partial Digestion of ODNs with Snake Venom Phosphodiesterase and Nuclease S1. Since resistance to nucleolytic hydrolysis by nucleases is an important factor in antisense and antigene studies, the stability of ODNs containing BAE or BAP to nucleolytic digestion was examined next (1-4). Two nucleases, snake venom phosphodiesterase (a 3′-exonuclease) and nuclease S1 (an endonuclease), were used in this study. ODNs 11, 14, 16, and 19 were labeled at the 5′-end with 32P and

Figure 4. Polyacrylamide gel electrophoresis of 5′-32P-labeled ODNs hydrolyzed by nuclease S1: (a) 10, (b) 14, and (c) 19. ODNs were incubated with nuclease S1 for 0 min (lane 1), 10 min (lane 2), 30 min (lane 3), 60 min (lane 4), and 120 min (lane 5). Experimental conditions are described in Experimental Procedures.

incubated separately with these nucleases. The reactions were then analyzed by denaturing polyacrylamide gel electrophoresis (31). While the control, ODN 10, was randomly hydrolyzed by snake venom phosphodiesterase after 120 min of incubation (Figure 3a, lane 5), ODNs containing BAP or BAE at their 3′-ends were completely resistant to hydrolysis by this nuclease (Figure 3b,c, lane 5). ODNs 14 and 19, which contained two molecules of the modified nucleoside BAE and BAP in their middles, were also more resistant to nuclease S1 than the control, ODN 10 (Figure 4). The half-lives of ODNs 10, 14, and 19 were 23, 39, and 54 min, respectively. Therefore, ODNs containing BAE or BAP were more resistant to nucleolytic hydrolysis by both snake venom phosphodiesterase and nuclease S1 than the unmodified ODN. CONCLUSION

In this paper, we report the synthesis of ODNs containing 2′-deoxyuridine analogues carrying either an N,N-bis(2-aminoethyl)aminoethylcarbamoyl or N,N-bis(3-aminopropyl)aminopropylcarbamoyl linker. The thermal stabilities of duplexes containing these ODNs and either the complementary DNA or RNA strand and of

Synthesis and Properties of Oligonucleotides

triplexes of these ODNs and the target duplex were studied by thermal denaturation. We found that ODNs containing BAE or BAP stabilize duplex formation with either the complementary DNA or RNA strand but destabilize triplex formation with the target duplex. Furthermore, resistance of these ODNs to nuclease hydrolysis was examined by using snake venom phosphodiesterase (a 3′-exonuclease) and nuclease S1 (an endonuclease). The ODNs containing BAE or BAP were more resistant to nucleolytic hydrolysis by either of these nucleases than the unmodified ODN. EXPERIMENTAL PROCEDURES

General Remarks. Thin-layer chromatography was done on Merck 60F254 coated plates. The silica gel or the neutralized silica gel used for column chromatography was Merck silica gel 5715 or ICN silica 60A, respectively. Melting points were measured on a Yanagimoto MP-3 micromelting point apparatus (Yanagimoto) and are uncorrected. Fast atom bombardment mass spectrometry (FAB-MS) was measured on a JEOL JMS-HX110 instrument (JEOL) at an ionizing voltage of 70 eV. The 1H-NMR spectra were recorded with a JEOL EX-270 instrument with tetramethylsilane as an internal standard. Chemical shifts are reported in parts per million (δ), and signals are expressed as an s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad). All exchangeable protons were detected by the addition of D2O. UV absorption spectra were recorded with a Shimadzu UV-240 spectrophotometer (Shimadzu Co.). 5-[N-[2-[N,N-Bis[2-(trifluoroacetylamino)ethyl]amino]ethyl]carbamoyl]-5′-O-(dimethoxytrityl)-2′deoxyuridine (4). Tris(2-aminoethyl)amine (900 µL, 6.00 mmol) was added to a solution of 3 (27) (1.31 g, 2.00 mmol) in pyridine (20 mL), and the mixture was stirred at room temperature. After 1 h, pyridine (20 mL), triethylamine (5.60 mL, 40.0 mmol), and ethyl trifluoroacetate (4.80 mL, 40.0 mmol) were added to the solution, and the mixture was stirred at room temperature for 5 h. The solvent was concentrated, and then the residue was dissolved in CHCl3. The whole was washed with aqueous saturated NaHCO3, dried over Na2SO4, and concentrated. The residue was chromatographed over a silica gel column (3.2 × 10 cm) with 75-100% EtOAc in hexane to give 4 (1.41 g, 79% as a white foam): FAB-MS m/z 895 (M+ + 1); 1H-NMR (CDCl3) δ 8.89 (t, 1H, CONH, J ) 5.3 Hz), 8.70 (s, 1H, 3-NH), 8.64 (s, 1H, H-6), 7.52 (t, 2H, CF3CONH, J ) 4.6 Hz), 7.41-7.20 (m, 9H, DMTrphenyl), 6.84-6.81 (m, 4H, DMTr-phenyl), 6.15 (t, 1H, H-1′, J1′,2′ ) 6.4 Hz), 4.35-4.31 (m, 1H, H-3′), 3.99-3.94 (m, 1H, H-4′), 3.78 (s, 6H, DMTr-OMe), 3.52-3.34 (m, 8H, H-5′ab, CF3CONHCH2, CONHCH2), 2.69-2.61 (m, 6H, NCH2), 2.50 (ddd, 1H, H-2′a, J2′a,3′ ) 4.3 Hz, J2′a,1′ ) 6.4 Hz, J2′b,2′a ) 14.0 Hz), 2.20 (ddd, 1H, H-2′b, J2′b,3′ ) 6.4 Hz, J2′b,1′ ) 6.7 Hz, J2′b,2′a ) 14.0 Hz), 2.06 (d, 1H, 3′-OH, J ) 3.1 Hz); FAB exact MS calcd for C41H45F6N6O10 (M+ + 1) 895.3099, found 895.3077. 5-[N-[2-[N,N-Bis[2-(trifluoroacetylamino)ethyl]amino]ethyl]carbamoyl]-5′-O-(dimethoxytrityl)-3′O-[(2-cyanoethyl)(N,N-diisopropylamino)phosphinyl]-2′-deoxyuridine (5). After successive coevaporation with pyridine, 4 (537 mg, 600 µmol) was dissolved in CH2Cl2 (6 mL) containing N,N-diisopropylethylamine (310 µL, 1.80 mmol). 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (270 µL, 1.20 mmol) was added to the solution, and the reaction mixture was stirred for 20 min at room temperature. The mixture was diluted with

Bioconjugate Chem., Vol. 9, No. 1, 1998 37

CHCl3 and washed with aqueous saturated NaHCO3 and brine. The separated organic phase was dried over Na2SO4 and concentrated. The residue was chromatographed over a neutral silica gel column (3.3 × 9 cm) with 50-80% EtOAc in hexane to give 5 (488 mg, 74% as a white foam): FAB-MS m/z 1095 (M+ + 1); 31P-NMR (CDCl3) δ 149.90, 149.64 (85% H3PO4 as an internal standard); FAB exact MS calcd for C50H62F6N6O11P (M+ + 1) 1095.418, found 1095.4240. 5-[N-[2-[N,N-Bis[2-(trifluoroacetylamino)ethyl]amino]ethyl]carbamoyl]-5′-O-(dimethoxytrityl)-3′O-succinyl-2′-deoxyuridine (6). After successive coevaporation with pyridine, 4 (179 mg, 200 µmol) was dissolved in pyridine (2 mL). Succinic anhydride (40 mg, 400 µmol) and DMAP (12 mg, 100 µmol) were added to the solution, and the mixture was stirred at room temperature for 3 days. The mixture was diluted with CHCl3 and washed with H2O, aqueous saturated NaHCO3, and then brine. The separated organic phase was dried over Na2SO4 and evaporated. The residue was chromatographed on a silica gel column (3.0 × 9 cm) with 80-100% EtOAc in hexane and then 0-30% MeOH in EtOAc to give 6 (117 mg, 59% as a white foam): 1H-NMR (CDCl3) δ 8.73 (s, 1H, H-6), 7.61 (s, 2H, CF3CONH), 7.417.11 (m, 9H, DMTr-phenyl), 6.88-6.78 (m, 4H, DMTrphenyl), 6.02-5.95 (m, 1H, H-1′, J1′,2′ ) 6.4 Hz), 5.125.10 (m, 1H, H-3′), 4.23-4.15 (m, 1H, H-4′), 3.73 (s, 6H, DMTr-OMe), 3.48-3.24 (m, 8H, H-5′ab, CF3CONHCH2, CONHCH2), 2.65-2.44 (m, 12H, NCH2, H-2′ab, OCOCH2CH2COOH). 5-[N-[3-[N,N-Bis[3-(trifluoroacetylamino)propyl]amino]propyl]carbamoyl]-5′-O-(dimethoxytrityl)-2′deoxyuridine (7). Tris(3-aminopropyl)amine (90 µL, 450 µmol) was added to a solution of 3 (27) (98 mg, 150 µmol) in pyridine (2 mL), and the mixture was stirred at room temperature. After 4 h, pyridine (2 mL), triethylamine (420 µL, 3.00 mmol), and ethyl trifluoroacetate (360 µL, 3.00 mmol) were added to the solution, and the mixture was stirred at room temperature for 5 h. The mixture was diluted with CHCl3, which was washed with aqueous saturated NaHCO3. The separated organic phase was dried over Na2SO4 and concentrated. The residue was chromatographed over a silica gel column (2.5 × 8 cm) with 0-20% MeOH in EtOAc to give 7 (105 mg, 75% as a white foam): FAB-MS m/z 937 (M+ + 1); 1 H-NMR (CDCl3) δ 8.66-8.59 (m, 3H, CONH, 3-NH, H-6), 7.76-7.74 (m, 2H, CF3CONH) 7.50-7.16 (m, 9H, DMTrphenyl), 6.87-6.80 (m, 4H, DMTr-phenyl), 6.15 (t, 1H, H-1′, J1′,2′ ) 6.5 Hz), 4.31 (m, 1H, H-3′), 3.97 (m, 1H, H-4′), 3.78 (s, 6H, DMTr-OMe), 3.48-3.32 (m, 8H, H-5′ab, CF3CONHCH2, CONHCH2), 2.51-2.42 (m, 7H, NCH2, H-2′a), 2.23 (m, 1H, H-2′b), 1.77-1.66 (m, 6H, NCH2CH2CH2NH); FAB exact MS calcd for C44H51F6N6O10 (M+ + 1) 937.3568, found 937.3620. 5-[N-[3-[N,N-Bis[3-(trifluoroacetylamino)propyl]amino]propyl]carbamoyl]-5′-O-(dimethoxytrityl)-3′O-[(2-cyanoethyl)(N,N-diisopropylamino)phosphinyl]-2′-deoxyuridine (8). Compound 7 (562 mg, 600 µmol) was phosphitylated as described in the preparation of 5 to give 8 (469 mg, 69% as a white foam): FAB-MS m/z 1137 (M+ + 1); 31P-NMR (CDCl3) δ 149.78, 149.57 (85% H3PO4 as an internal standard); FAB exact MS calcd for C53H68F6N8O11P (M+ + 1) 1137.464, found 1137.4710. 5-[N-[3-[N,N-Bis[3-(trifluoroacetylamino)propyl]amino]propyl]carbamoyl]-5′-O-(dimethoxytrityl)-3′O-succinyl-2′-deoxyuridine (9). Compound 7 (281 mg, 300 µmol) was succinylated as described in the preparation of 6 to give 9 (145 mg, 47% as a white foam): 1H-

38 Bioconjugate Chem., Vol. 9, No. 1, 1998

NMR (CDCl3) δ 8.85 (m, 2H, H-6, 3-NH), 8.14 (m, 2H, CF3CONH), 7.40-7.14 (m, 9H, DMTr-phenyl), 6.83-6.80 (m, 4H, DMTr-phenyl), 6.02 (t, 1H, H-1′, J1′,2′ ) 6.2 Hz), 4.99 (m, 1H, H-3′), 4.24 (m, 1H, H-4′) 3.75 (s, 6H, DMTrOMe), 3.55-3.14 (m, 8H, H-5′ab, CF3CONHCH2, CONHCH2), 2.97-2.21 (m, 12H, NCH2, H-2′ab, OCOCH2CH2COOH), 1.86-1.59 (m, 6H, NCH2CH2CH2NH). Synthesis of the Controlled Pore Glass Support Containing 4 and 7. Aminopropyl controlled pore glass (323 mg, 29.0 µmol, 89.8 µmol/g, CPG Inc.) was added to a solution of 6 (115 mg, 0.116 mmol) and 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (WSCI) (22 mg, 0.116 mmol) in anhydrous DMF (3 mL), and the mixture was kept for 4 days at room temperature. After the resin was washed with anhydrous pyridine, 3 mL of a capping solution (0.1 M DMAP in 9:1 pyridine/Ac2O) was added, and the whole was kept for 1 days at room temperature. The resin was washed with EtOH and acetone and was dried under vacuum. The amount of nucleoside 4 loaded onto the solid support is 35.0 µmol/g from the calculation of released dimethoxytrityl cation by a solution of 70% HClO4/EtOH (3:2, v:v). In a similar manner, the solid support containing 7 was obtained in 34.7 µmol/g of loading amounts. Synthesis of ODNs. ODNs were synthesized on a DNA/RNA synthesizer (Applied Biosystem model 392) by the phosphoramidite method (29). Each ODN (1 µmol) linked to the resin was treated with concentrated NH4OH (2 mL) for 16 h at 55 °C. After filtration of the resin, the filtrate was concentrated under reduced pressure. The residue obtained from the reaction with concentrated NH4OH was purified by C-18 silica gel column chromatography (1 × 10 cm) with a linear gradient of CH3CN in 0.1 M TEAA buffer (pH 7.0). Fractions were concentrated, and the residue was treated with 80% AcOH for 20 min at room temperature. The solution was concentrated, and the residue was coevaporated with H2O. The residue was dissolved in H2O, and the solution was washed with Et2O; then, the H2O layer was concentrated to give deprotected ODN 11 (14.4), 12 (11.1), 13 (27.7), 14 (18.0), 15 (19.7), 16 (16.8), 17 (24.2), 18 (17.5), 19 (10.6), and 20 (8.2). The yields are indicated in parentheses as OD units at 260 nm starting from a 1 µmol scale. Matrix-Assisted Laser Desorption/Ionization Timeof-Flight Mass Spectrometry. Spectra were obtained on a Voyager Elite reflection time-of-flight mass spectrometry system (PerSeptive Biosystems, Inc., Framingham, MA) equipped with a nitrogen laser (337 nm, 3 ns pulse) in the negative ion mode. 3-Hydroxypicolinic acid (HPA), dissolved in H2O to give a saturated solution at room temperature, was used as the matrix. Time-tomass conversion was achieved by calibration by using the peak representing the C+ cation of the charged derivative to be analyzed. The observed average molecular masses of 13 and 17 were 5258.33 and 5299.89, respectively, and fit the calculated molecular weights (theoretical average molecular masses) for these compounds, i.e. 5258.69 (for 13, C176H244N46O110P16) and 5300.77 (for 17, C179H250N46O110P16), within a commonly accepted error range of MALDI-TOF/MS. Thermal Denaturation. Each solution contains each ODN (3 µM) and the complementary DNA 21 (3 µM), RNA 22 (3 µM), or the target duplex 23 (3 µM) in an appropriate buffer. The solution containing each ODN was heated at 90 °C for 10 min, then cooled gradually to an appropriate temperature, and used for the thermal denaturation studies. Thermal-induced transitions of each mixture were monitored at 260 nm on a Perkin-

Ueno et al.

Elmer Lambda2S instrument. The sample temperature was increased 0.5 °C/min. Partial Hydrolysis of ODNs with Snake Venom Phosphodiesterase. Each ODN labeled with 32P at the 5′-end (10 pmol) was incubated with snake venom phosphodiesterase (0.4 µg) in the presence of Torula RNA (0.15 OD260 unit) in a buffer containing 37.5 mM TrisHCl (pH 8.0) and 7.5 mM MgCl2 (total of 20 µL) at 37 °C. At appropriate periods, aliquots of the reaction mixture were separated and added to a solution of EDTA (5 mM, 10 µL), and then the mixtures were heated at 100 °C for 5 min. The solutions were analyzed by electrophoresis on a 20% polyacrylamide gel containing 8 M urea (31). Partial Hydrolysis of ODNs with Nuclease S1. Each ODN labeled with 32P at the 5′-end (10 pmol) was incubated with nuclease S1 (0.2 unit) in the presence of Torula RNA (0.54 OD260 unit) in a buffer containing 30 mM sodium acetate (pH 4.6), 1 mM ZnSO4, and 280 mM NaCl (total of 20 µL) at 37 °C. At appropriate periods, aliquots of the reaction mixture were separated and added to a solution of EDTA (5 mM, 10 µL), and then the solutions were heated for 5 min at 100 °C. The solutions were analyzed by gel electrophoresis as described above. Densities of radioactivity of the gel were visualized by a Bio-imaging Analyzer (Bas 2000, Fuji, Co. Ltd.). ACKNOWLEDGMENT

This investigation was supported in part by a Grantin-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture of Japan and a Grant from the “Research for the Future” Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00301). LITERATURE CITED (1) Uhlmann, E., and Peyman, A. (1990) Antisense oligonucleotides: a new therapeutic principle. Chem. Rev. 90, 544-584. (2) Milligan, J. F., Matteucci, M. D., and Martin, J. C. (1993) Current concepts in antisense drug design. J. Med. Chem. 36, 1923-1937. (3) Crooke, S. T., and Lebleu, B., Eds. (1993) Antisense research and applications, CRC Press, Boca Raton, FL. (4) Thuong, N. T., and Helene, C. (1993) Sequence-specific recognition and modification of double-helical DNA by oligonucleotides. Angew. Chem., Int. Ed. Engl. 32, 666-690. (5) Beaucage, S. L., and Iyer, R. P. (1993) The functionalization of oligonucleotides via phosphoramidite derivatives. Tetrahedron 49, 1925-1963. (6) Beaucage, S. L., and Iyer, R. P. (1993) The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 49, 6123-6194. (7) Tabor, C. W., and Tabor, H. (1976) 1,4-Diaminobutane (putrescine), spermidine, and spermine. Annu. Rev. Biochem. 45, 285-306. (8) Tabor, C. W., and Tabor, H. (1984) Polyamines. Annu. Rev. Biochem. 53, 749-790. (9) Etter, M. C. (1990) Encoding and decoding hydrogen-bond patterns of organic compounds. Acc. Chem. Res. 23, 120-126. (10) Tabor, H. (1962) Protective effect of spermine and other polyamines against heat denaturation of deoxyribonucleic acid. Biochemistry 1, 496-501. (11) Thomas, T. J., and Bloomfield, V. A. (1984) Ionic and structural effects on the thermal helix-coil transition of DNA complexed with natural and synthetic polyamines. Biopolymers 23, 1295-1306. (12) Hampel, K. J., Crosson, P., and Lee, J. S. (1991) Polyamines favor DNA triplex formation at neutral pH. Biochemistry 30, 4455-4459.

Synthesis and Properties of Oligonucleotides (13) Thomas, T., and Thomas, T. J. (1993) Selectivity of polyamines in triplex DNA stabilization. Biochemistry 32, 14068-14074. (14) Musso, M., and Van Dyke, M. W. (1995) Polyamine effects on purine-purine-pyrimidine triple helix formation by phosphodiester and phosphorothioate oligodeoxyribonucleotides. Nucleic Acids Res. 23, 2320-2327. (15) Tung, C.-H., Breslauer, K. J., and Stein, S. (1993) Polyaminelinked oligonucleotides for DNA triple helix formation. Nucleic Acids Res. 21, 5489-5494. (16) Prakash, T. P., Barawkar, D. A., Kumar, V. A., and Ganesh, K. N. (1994) Synthesis of site-specific oligonucleotidepolyamine conjugates. Bioorg. Med. Chem. Lett. 4, 17331738. (17) Barawkar, D. A., Kumar, V. A., and Ganesh, K. N. (1994) Triplex formation at physiological pH by oligonucleotides incorporating 5-Me-dC-(N4-spermine). Biochem. Biophys. Res. Commun. 205, 1665-1670. (18) Barawkar, D. A., Rajeev, K. G., Kumar, V. A., and Ganesh, K. N. (1996) Triplex formation at physiological pH by 5-MedC-(N4-spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities. Nucleic Acids Res. 24, 12291237. (19) Schmid, N., and Behr, J.-P. (1995) Recognition of DNA sequences by strand replacement with polyamino-oligonucleotides. Tetrahedron Lett. 36, 1447-1450. (20) Sund, C., Puri, N., and Chattopadhyaya, J. (1996) Synthesis of C-branched spermine tethered oligo-DNA and the thermal stability of the duplexes and triplexes. Tetrahedron 52, 12275-12290. (21) Hashimoto, H., Nelson, M. G., and Switzer, C. (1993) Formation of chimeric duplexes between zwitterionic and natural DNA. J. Org. Chem. 58, 4194-4195. (22) Hashimoto, H., Nelson, M. G., and Switzer, C. (1993) Zwitterionic DNA. J. Am. Chem. Soc. 115, 7128-7134. (23) Ozaki, H., Nakamura, A., Arai, M., Endo, M., and Sawai, H. (1995) Novel C5-substituted 2′-deoxyuridine derivatives bearing amino-linker arms: synthesis, incorporation into oligodeoxyribonucleotides, and their hybridization properties. Bull. Chem. Soc. Jpn. 68, 1981-1987.

Bioconjugate Chem., Vol. 9, No. 1, 1998 39 (24) Ono, A., Dan, A., and Matsuda, A. (1993) Synthesis of oligonucleotides carrying linker groups at the 1′-position of sugar residues. Bioconjugate Chem. 4, 499-508. (25) Ono, A., Haginoya, N., Kiyokawa, M., Minakawa, N., and Matsuda, A. (1994) A novel and convenient postsynthetic modification method for the synthesis of oligodeoxyribonucleotides carrying amino linkers at the 5-position of 2′-deoxyuridine. Bioorg. Med. Chem. Lett. 4, 361-366. (26) Haginoya, N., Ono, A., Nomura, Y., Ueno, Y., and Matsuda, A. (1997) Synthesis of oligodeoxyribonucleotides containing 5-(N-aminoalkyl)carbamoyl-2′-deoxyuridines by a new postsynthetic modification method and theri thermal stability and nuclease-resistance properties. Bioconjugate Chem. 8, 271280. (27) Nomura, Y., Ueno, Y., and Matsuda, A. (1997) Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nucleaseresistance properties. Nucleic Acids Res. 25, 2784-2791. (28) Ueno, Y., Ogawa, A., Nakagawa, A., and Matsuda, A. (1996) Facile synthesis of 5′-5′-linked oligodeoxyribonucleotides with the potential for triple-helix formation. Bioorg. Med. Chem. Lett. 6, 2817-2822. (29) Ueno, Y., Nakagawa, A., and Matsuda, A. (1997) Chemical ligation of oligodeoxynucleotides having a mercapto group at the 5-position of 2′-deoxyuridine via a disulfide bond. Nucleosides Nucleotides (in press). (30) Ueno, Y., Kumagai, I., Haginoya, N., and Matsuda, A. (1997) Effects of 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine on endonuclease stability and an ability of oligodeoxynucleotide to activate RNase H. Nucleic Acids Res. 25, 3777-3782. (31) Gait, M. J., Ed. (1984) Oligonucleotides synthesis: a practical approach. IRL Press, Oxford, U.K. (32) Saenger, W. (1984) Principle of nucleic acid structure, Springer-Verlag, Inc., New York. (33) Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Plainview, NY.

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