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Nucleic Acids Research Pages 5067-5072  

Effects of 2[prime]-O-(trifluoromethyl)adenosine on oligodeoxynucleotide hybridization and nuclease stability
Introduction
Results and Discussion
   Synthesis
   Thermal stability
   Circular dichroism
   Nuclease resistance
   Conclusions
Materials and Methods
   2'-O-[(Methylthio)thiocarbonyl]-3',5'-O-(TIPDS)ademoisine (4)
   2'-O-(Trifluoromethyl)adenoisine (2)',3'-O-(difluoromethylidene)adenoisine (9)
   N6,N6-(Dibenzoyl)-2'-O-[(methylthio)thiocarbonyl]-3',5'-O-(TIPDS)adenosine (5)
   N6-(Benzoyl)-2'-O-(trifluoromethyl)adenoisine (6) and N6-(benzoyl)-2',3'-O-(difluoromethylidene)adenoisine (10)
   N6-(Benzoyl)-5'-O-(dimethoxytrityl)-2'-O-(trifluoromethyl)-adenoisine (7)
   N6-(Benzoyl)-5'-O-(dimenthoxytrityl)-2'-O-(trifluoromethyl)-adenoisine 3'-O-(2-cyanoethyl N,N-diisopropyl-phosphoramidite) (8)
   Synthesis of ODNs
   Electrospray ionization mass spectrometry
   Thermal denaturation and CD spectroscopy
   Partial hydrolysis of ODNs with snake venom phosphodiesterase
Acknowledgements
References

Effects of 2[prime]-O-(trifluoromethyl)adenosine on oligodeoxynucleotide hybridization and nuclease stability

Effects of 2[prime]-O-(trifluoromethyl)adenosine on oligodeoxynucleotide hybridization and nuclease stability

Naozumi Nishizono, Yuji Sumita, Yoshihito Ueno and Akira Matsuda*

Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

Received August 21, 1998; Revised and Accepted September 29, 1998

ABSTRACT

The synthesis and properties of oligodeoxynucleotides (ODNs) containing 2[prime]-O-(trifluoromethyl)adenosine (2) are described. 2[prime]-O-(Trifluoromethyl)adenosine (2) or N6-(benzoyl)-2[prime]-O-(trifluoromethyl)adenosine (6) was obtained in 22 or 32% yield by treating 2[prime]-O-[(methylthio)thiocarbonyl]-3[prime],5[prime]-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)(TIPDS)adenosine (4) or N6,N6-(dibenzoyl)-2[prime]-O-[(methylthio)thiocarbonyl]-3[prime],5[prime]-O-(TIPDS)-adenosine (5), respectively, with pyridinium poly-(hydrogen fluoride) in the presence of 1,3-dibromo-5,5-dimethylhydantoin. Nucleoside 2 was incorporated into DNA hexadecamers. ODNs that contained 2 reduced the thermal stability of duplexes with their complementary DNAs but increased the thermal stability of duplexes with their complementary RNAs. Furthermore, ODNs containing 2 were slightly more resistant to snake venom phosphodiesterase than an unmodified ODN.

INTRODUCTION

Many types of oligodeoxynucleotides (ODNs) that have been modified at their base, sugar or phosphodiester moieties have been synthesized (1,2) and used for biological and biophysical studies, such as antisense studies (1-4). In particular, since we can easily introduce various substituents into the 2[prime] position of a nucleoside, ODN analogs containing various 2[prime]-modified nucleosides have been synthesized and their biochemical and biophysical properties have been studied (5-22). Based on these studies, it is apparent that the affinity of ODNs for their complementary DNA or RNA strands and the stability of ODNs against nucleolytic hydrolysis by nucleases, which are important factors for antisense molecules, are highly dependent on the properties of the 2[prime]-substituents. For instance, it has been reported that 2[prime]-O-alkyl modifications such as 2[prime]-O-methyl and 2[prime]-O-allyl increase the thermal stability of DNA/RNA hybrids as well as the stability of ODNs against nucleolytic degradation by nucleases (5-7,11-17). However, 2[prime]-C-alkyl substituents such as 2[prime]-[alpha]-methyl, 2[prime]-[alpha]-ethyl, 2[prime]-[alpha]-allyl and 2[prime]-[beta]-methyl groups increase the stability of ODNs against nucleolytic degradation by 3[prime]-exonuclease, but reduce the thermal stability of DNA/RNA hybrids (10,18). On the other hand, Kawasaki et al. reported that the 2[prime]-[alpha]-fluoro substituent dramatically increases the thermal stability of DNA/RNA duplexes although it does not increase stability against nucleolytic hydrolysis by nucleases (9). The stability of the duplex with a 2[prime]-[alpha]-fluoro substitution could be due to the high electronegativity of the fluorine atom, which shifts the conformational equilibrium of the sugar to 3[prime]-endo. Shifting the conformation of the DNA strand to 3[prime]-endo will put it in a more RNA-like conformation and will cause the hybridization properties to be more like those of an RNA/RNA geometry.

Based on these findings, we envisioned that ODNs containing 2[prime]-O-(trifluoromethyl)adenosine (2) instead of 2[prime]-O-(methyl)-adenosine (1) may increase the thermal stability of duplexes with their complementary DNAs and RNAs without reducing nuclease stability (Fig. 1).

In this paper, we demonstrate the synthesis of ODNs containing 2[prime]-O-(trifluoromethyl)adenosine (2). The thermal stability of the duplexes between these ODNs and their complementary DNA and RNA strands and the stability of ODNs containing 2 against nucleolytic digestion were also studied.

RESULTS AND DISCUSSION

Synthesis

To synthesize ODNs containing 2, a 2[prime]-O-(trifluoromethyl)-adenosine phosphoramidite unit 8 is needed. However, a method for preparing 2 has not been reported. Recently, Hiyama et al. reported that methyl xanthates R-OC(S)SMe are easily converted into trifluoromethyl ethers R-OCF3 by treatment with pyridinium poly(hydrogen fluoride) (HF/pyridine) in the presence of 1,3-dibromo-5,5-dimethylhydantoin (DBH) (23). Therefore, we planned to synthesize 2 using Hiyama's method.

2[prime]-O-[(Methylthio)thiocarbonyl]adenosine derivative 4, which was a precursor of 2, was obtained in 73% yield by treating 3[prime],5[prime]-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine [3[prime],5[prime]-O-(TIPDS)adenosine] (3) with n-BuLi, CS2 and MeI in THF at -78°C (Scheme 1). The methyl xanthate 4 was then treated with HF/pyridine in the presence of DBH. The desired 2[prime]-O-trifluoromethyl derivative 2 was obtained in 22% yield along with 2[prime],3[prime]-O-difluoromethylidene derivative 9, which would be derived via a 2[prime],3[prime]-cyclic thiocarbonate intermediate, in 28% yield. On the other hand, when N6,N6-dibenzoyl derivative 5, which was prepared by the reaction of 4 with BzCl in pyridine, was treated with HF/pyridine in the presence of DBH, 2[prime]-O-trifluoromethyl derivative 6 was obtained in 32% yield along with 2[prime],3[prime]-O-difluoromethylidene derivative 10 in 36% yield. N6-Benzoyl derivative 6 was used for the next reaction. To introduce 2 into ODNs, the 5[prime]-OH group of 6 was protected with a DMTr group and the resulting 7 was phosphitylated by a standard procedure (24) to give the nucleoside 3[prime]-phosphoramidite 8 in 81% yield.


Scheme 1. (a) (1) n-BuLi, CS2, THF, -78°C, (2) MeI, -78°C, then room temperature, 73%; (b) BzCl, pyridine, 0°C, then room temperature, 93%; (c) HF/pyridine, DBH, CH2Cl2, -78°C, then 0°C, 22 (2) and 28% (9) and 32 (6) and 36% (10); (d) DMTrCl, pyridine, room temperature, 83%; (e) chloro-(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, N,N-diisopropylethylamine, CH2Cl2, 81%.


Figure 1. Structures of modified nucleoside analogs.

Before 2 was introduced into ODNs, the conformation of the sugar moiety of 2 was studied by 1H NMR. The fractional population of the N-conformer of 2 was calculated by the formula, %N = J3[prime],4[prime]/(J1[prime],2[prime] + J3[prime],4[prime]) (25). The J1[prime],2[prime] and J3[prime],4[prime] values of 2 were 6.1 and 2.7 Hz, respectively. Thus, the %N value was estimated to be 31%. On the other hand, the %N values of 2[prime]-deoxyadenosine (dAdo), adenosine (Ado) and 2[prime]-O-(methyl)adenosine (1) have been reported to be 19, 36 and 42%, respectively, based on an NMR study (26). Uesugi et al. reported that there is a relatively good correlation between %N and the group electronegativity of the 2[prime]-[alpha]-substituent (26). These results imply that the group electronegativity of OCF3 is not very large. Quite recently, we determined the group electronegativity of the 3[prime]-OCF3 substituent in 3[prime]-O-(trifluoromethyl)thymidine (27) on the scales of Marriott (28), Inamoto (29-32) and Mullay (33) from calibration graphs which correlated the group electronegativity of various 3[prime]-substituents in 2[prime],3[prime]-dideoxythymidine derivatives. The group electronegativities of OCF3 were calculated to be 0.38 (Marriott's scale), 2.64 (Inamoto's scale) and 3.66 (Mullay's scale), respectively, and were smaller than those of OH [0.42 (Marriott's scale), 2.79 (Inamoto's scale) and 3.97 (Mullay's scale)] and OCH3 [0.43 (Marriott's scale), 2.82 (Inamoto's scale) and 4.03 (Mullay's scale)] (27). These results were consistent with our results regarding the relative %N values of 1, 2 and Ado.

ODNs containing 2 were synthesized on a DNA synthesizer by the phosphoramidite method (34). In this study, 2 was incorporated into a heptadecamer. The sequences of the ODN analogs synthesized are shown in Table 1. The average coupling yield of 8 was 96% using a 0.12 M solution of the amidite in CH3CN with a coupling time of 600 s. The fully protected ODNs (1 µmol) linked to the solid supports were treated with concentrated NH4OH at 55°C for 16 h and then subjected to C-18 column chromatography; detritylation gave ODNs (17-21) in 20-77 OD260 units. Each of the resulting ODN analogs showed a single peak on reversed phase HPLC. Furthermore, ODNs 17 and 19 were analyzed by electrospray ionization (ESI) mass spectrometry and the observed molecular weights supported their structures (Materials and Methods).


Table 1. Synthesized ODNs and hybridization dataa
aExperimental conditions are described in Materials and Methods.
b1 and 2 (Fig. 1).
cThe complementary RNA: 5[prime]-r[UUG UAU UUG UUU UUC U]-3[prime] (22).
dThe complementary DNA: 5[prime]-d[TTG TAT TTG TTT TTC T]-3[prime](23).
e[Delta]Tm1 = Tm(each ODN) - Tm(control ODN 11).
f[Delta]Tm2 = Tm(each ODN containing 2) - Tm(each ODN containing the same number of 1).


Thermal stability

Stable duplex formation with mRNA is one of the most important factors in antisense research. Thus, duplex formation by the heptadecamers 11-21 with the complementary RNA, 5[prime]-r[UUG UAU UUG UUU UUC U]-3[prime] (22), was studied by thermal denaturation in a buffer of 0.01 M sodium cacodylate (pH 7.0) containing 0.01 M NaCl. One transition was observed in the melting profile of each duplex. Melting temperatures (Tm values) are listed in Table 1. The stability of duplexes containing 1 or 2 was dependent on the position and number of 1 or 2. Compared with the control ODN 11, ODNs 12 and 17, which contained two molecules of 1 or 2 at the 5[prime]-end and near the 3[prime]-end of the strands, slightly reduced the thermal stability of ODN/RNA duplexes. On the other hand, ODNs 13-16 and 18-21, which contained three or four molecules of 1 or 2, greatly increased the thermal stability of ODN/RNA duplexes. The duplexes became more stable as the number of 1 or 2 increased. The [Delta]Tm1 [Tm(each ODN) - Tm(control ODN 11)] values for ODNs containing three or four molecules of 1 or 2 were 3.8-7.7°C. Among the ODNs that contained 1, ODN 14, which contained four molecules of 1 at intervals of three or four natural nucleosides, increased the thermal stability of ODN/RNA duplexes the most ([Delta]Tm1 = 6.2°C). On the other hand, among the ODNs that contained 2, ODN 20, which contained four molecules of 2 near the 5[prime]-end of the strand, increased the thermal stability of ODN/RNA duplexes the most ([Delta]Tm1 = 7.7°C). The [Delta]Tm2 [Tm(each ODN containing 2) - Tm(each ODN containing the same number of 1)] values were calculated to compare the abilities of 1 and 2 to stabilize duplex formation (Table 1). Except for ODN 19, the [Delta]Tm2 values increased as the number of 2 increased. Therefore, nucleoside 2 was found to more efficiently stabilize duplex formation with the RNA than 1. Nucleoside 2 most efficiently stabilized duplex formation when four molecules of 2 were incorporated near the 5[prime]-end of the strand (ODN 20, [Delta]Tm2 = 1.7°C).

The thermal stabilities of DNA/RNA duplexes containing nucleoside analogs with electronegative substituents at the 2[prime]-[alpha] positions are often discussed on the basis of the gauche effect between the electronegative substituents and O4[prime] of the furanose ring (35-38). The electronegative substituent at the 2[prime]-[alpha] position shifts the conformational equilibrium of the sugar to 3[prime]-endo. Shifting the conformation of the DNA strand to 3[prime]-endo will put it in a more RNA-like conformation and will cause the hybridization properties to be more like those of an RNA/RNA geometry. The ODNs that contained 2 stabilized duplex formation with the complementary RNA more than those that contained 1, although the group electronegativity of OCF3 is smaller than that of OCH3. Accordingly, factors other than the gauche effect also seemed to contribute to the thermal stability of duplexes containing 2.

Duplex formation by heptadecamers 11-21 with the complementary DNA, 5[prime]-d[TTG TAT TTG TTT TTC T]-3[prime] (23), was next studied by thermal denaturation in a buffer of 5 mM sodium phosphate (pH 7.0) containing 140 mM KCl and 1 mM MgCl2. One transition was observed in the melting profile of each duplex. Tm values are listed in Table 1. All of the Tm values for duplexes containing 1 or 2 were smaller than that of the control duplex (42.6°C). Therefore, ODNs containing 1 and 2 decreased the thermal stability of duplexes with the DNA. The stability of duplexes was dependent on the number of modified nucleosides. The duplexes became less stable as the numbers of 1 and 2 increased. The [Delta]Tm1 values for ODNs 14 and 21 were -5.4 and -12.6°C, respectively. [Delta]Tm2 values also became smaller as the number of 2 increased. Therefore, nucleoside 2 was found to destabilize ODN/DNA duplex more than nucleoside 1.

When an ODN analog containing 1 or 2 forms a duplex with a complementary DNA, methoxy and trifluoromethoxy groups of 1 and 2 should be accommodated in the minor groove, since the 2[prime] position of a nucleoside is in the minor groove. The minor groove of a DNA/DNA duplex is known to be narrow and deep (39). Therefore, methoxy and trifluoromethoxy groups of 1 and 2, which are partially incorporated into the ODNs instead of dAdo, may not be well accommodated in the minor groove of the ODN/DNA duplex. Furthermore, since a trifluoromethoxy group is larger than a methoxy group, ODNs containing 2 might reduce the thermal stability of the ODN/DNA duplex more than ODNs containing 1.

Circular dichroism

To study the global conformation of duplexes, CD spectra of duplexes composed of ODNs containing 1 or 2 and either the complementary DNA (23) or RNA (22) were measured at 15°C. With DNA (23) as a complementary strand, the spectra of the duplexes (11-23, 15-23, and 20-23) showed positive CD bands at ~282 nm and negative CD bands at ~248 nm, which were attributable to B-like DNA conformation (Fig. 2a). Although the shapes of the spectra were similar to each other, the intensity of the positive and negative CD bands in the spectrum of the duplex containing 2 was slightly less than that of the unmodified duplex (11-23) and the intensity of the positive CD band at ~282 nm in the spectrum of the duplex containing 1 was slightly greater than that of the unmodified duplex.


Figure 2. CD spectra of ODN/DNA and ODN/RNA duplexes at 15°C in 5 mM sodium phosphate (pH 7.0) containing 140 mM KCl and 1 mM MgCl2 for ODN/DNA duplexes and 10 mM sodium phosphate (pH 7.0) containing 10 mM NaCl for ODN/RNA duplexes. (a) ODN/DNA duplexes: (-) 11-23; (- - -) 15-23; (- -) 20-23. (b) ODN/RNA duplexes: (-) 11-22; (- - -) 15-22; (- -) 20-22.

When RNA (22) was used as a complementary strand, the spectra of duplexes (11-22, 15-22 and 20-22) showed positive CD bands at ~220 and 268 nm and negative CD bands at ~207 and 246 nm (Fig. 2b). Although the intensity of CD bands in the spectra of the duplex containing 2 was slightly less than that in the duplex containing 1 and the unmodified duplex, the shapes of the spectra were similar to each other. Based on these results, nucleoside 2 did not dramatically change the global conformation of ODN/DNA and ODN/RNA duplexes.

Nuclease resistance

Several studies have demonstrated that 3[prime]-exonuclease activities were the major cause of the degradation of unmodified ODNs in serum. We examined the stability of ODNs containing 2 against snake venom phosphodiesterase, a 3[prime]-exonuclease. ODNs 11, 14, and 19 were labeled at the 5[prime]-end with 32P and incubated with the nuclease and the reactions were analyzed by polyacrylamide gel electrophoresis under denaturing conditions (40) (Fig. 3). In ODNs 14 and 19, the second nucleosides from the 3[prime]-ends of the strands are 1 and 2, respectively. The phosphodiester linkages at the 5[prime]-sides of 1 (5,6) and 2 were slightly more resistant to hydrolysis by the enzyme than that of dAdo (Fig. 3, lanes 5, 6, 11, 12, 17 and 18).


Figure 3. Polyacrylamide gel electrophoresis of 5[prime]-32P-labeled ODNs hydrolyzed by snake venom phosphodiesterase: 11 (lanes 1-6), 14 (lanes 7-12) and 19 (lanes 13-18). ODNs were incubated with snake venom phosphodiesterase for 0 (lanes 1, 7 and 13), 10 (lanes 2, 8 and 14), 20 (lanes 3, 9 and 15), 30 (lanes 4, 10 and 16), 60 (lanes 5, 11 and 17) and 120 min (lanes 6, 12 and 18). Experimental conditions are described in Materials and Methods.


Conclusions

In this paper, we described the synthesis and properties of ODNs containing 2[prime]-O-(trifluoromethyl)adenosine (2). 2[prime]-O-(Trifluoro-methyl)adenosine (2) or N6-(benzoyl)-2[prime]-O-(trifluoromethyl)adenosine (6) was obtained in 22 or 32% yield by treating 2[prime]-O-[(methylthio)thiocarbonyl]-3[prime],5[prime]-O,-(TIPDS)adenosine (4) or N6,N6-(dibenzoyl)-2[prime]-O-[(methylthio)thiocarbonyl]-3[prime],5[prime]-O-(TIPDS)adenosine (5), respectively, with HF/pyridine in the presence of DBH. The nucleoside 2 was incorporated into DNA hexadecamers. ODNs containing 2 decreased the thermal stability of duplexes with their complementary DNAs but increased the thermal stability of duplexes with their complementary RNAs. Furthermore, ODNs containing 2 were slightly more resistant to snake venom phosphodiesterase than an unmodified ODN. These properties seem to make ODN containing 2 a candidate as a novel antisense molecule.

MATERIALS AND METHODS

NMR spectra were recorded at 270 or 500 (1H), at 125 (13C) and at 202 MHz (31P) and are reported in p.p.m. downfield from TMS or 85% H3PO4. J values are given in Hertz. Mass spectra were obtained by the fast atom bombardment (FAB) method. Thin layer chromatography was done on Merck coated plates 60F254. The silica gel or the neutralized silica gel used for column chromatography were Merck silica gel 5715 or ICN silica 60A, respectively.

2[prime]-O-[(Methylthio)thiocarbonyl]-3[prime],5[prime]-O-(TIPDS)adenosine (4)

n-BuLi (1.68 M in hexane, 0.11 ml, 1.8 mmol) was added to a solution of 3,5-O-(TIPDS)adenosine (200 mg, 0.4 mmol) in THF (5 ml) at -78°C and the resulting mixture was stirred at -78°C. After 10 min, CS2 (0.23 ml, 3.9 mmol) was added to the solution and the resulting mixture was sirred at -78°C. After 1 h, MeI (73 µl, 1.2 mmol) was added to the solution and the resulting mixture was stirred at room temperature for 15 h. The mixture was diluted with AcOEt and washed with H2O and then brine. The separated organic layer was dried (Na2SO4) and evaporated. The residue was purified by column chromatography (SiO2, hexane:AcOEt = 1:1) to give 4 (171 mg, 73% as a white solid). 1H NMR (CDCl3) [delta] 8.28 (s, 1H), 7.94 (s, 1H), 6.53 (dd, 1H, J = 2.3 and 5.4 Hz), 6.08 (d, 1H, J = 2.3 Hz), 5.87 (br s, 2H, NH2), 5.37 (dd, 1H, J = 5.4 and 8.6 Hz), 4.20-4.03 (m, 3H), 2.61 (s, 3H), 1.13-0.94 (m, 28H); 13C NMR (CDCl3) [delta] 215.1, 155.5, 153.2, 149.3, 139.7, 120.3, 87.5, 87.4, 83.3, 82.2, 69.9, 60.9, 19.1, 17.4, 17.3, 17.1, 16.9, 13.2, 13.0, 12.8; EI-LRMS m/z 599 (M+).

2[prime]-O-(Trifluoromethyl)adenosine (2) and 2[prime],3[prime]-O-(difluoromethylidene)adenosine (9)

A solution of 4 (780 mg, 1.3 mmol) and HF/pyridine complex (12 ml, Aldrich) in CH2Cl2 (10 ml) was added to a suspension of DBH (1.5 g, 5.2 mmol) in CH2Cl2 (10 ml) at -78°C and the resulting mixture was stirred at 0°C for 3 h. The reaction mixture was poured into an aqueous saturated solution of NaHCO3 (50 ml) and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, 12% EtOH in CHCl3) to give 2 (96 mg, 22% as a white solid) and 9 (115 mg, 28% as a white solid). The physical data of 2: 1H NMR (DMSO-d6) [delta] 8.41 (s, 1H), 8.15 (s, 1H), 7.47 (br s, 2H, NH2), 6.23 (d, 1H, J = 6.1 Hz), 5.99 (d, 1H, OH, J = 5.3 Hz), 5.53-5.48 (m, 2H), 4.42 (m, 1H), 4.06 (m, 1H), 3.71 (ddd, 1H,J = 3.6, 4.6 and 12.3 Hz), 3.59 (ddd, 1H, J = 3.6, 7.0 and 12.3 Hz); FAB-LRMS m/z 336 (MH+); FAB-HRMS calculated for C11H13F3N5O4 336.0919, found 336.0904. The physical data of 9: 1H NMR (DMSO-d6) [delta] 8.33 (s, 1H), 8.17 (s, 1H), 7.39 (br s, 2H, NH2), 6.40 (d, 1H, J = 2.4 Hz), 6.06 (m, 1H), 5.60 (m, 1H), 5.27 (t, 1H, OH, J = 5.4 Hz), 4.38 (m, 1H), 3.56-3.52 (m, 2H); FAB-LRMS m/z 316 (MH+); FAB-HRMS calculated for C11H12F2N5O4 316.0857, found 316.0837.

N6,N6-(Dibenzoyl)-2[prime]-O-[(methylthio)thiocarbonyl]-3[prime],5[prime]-O-(TIPDS)adenosine (5)

BzCl (72 µl, 0.6 mmol) was added to a solution of 4 (100 mg, 0.2 mmol) in pyridine (5 ml) at 0°C and it was stirred at room temperature for 12 h. Ice-water and AcOEt were added, the resulting mixture was partitioned and the organic layer was washed with H2O and brine, dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, hexane:AcOEt = 2:1) to give 5 (150 mg, 93% as a white foam). 1H NMR (CDCl3) [delta] 8.75 (s, 1H), 8.21 (s, 1H), 7.88-7.35 (m, 10H), 6.55 (m, 1H), 6.13 (s, 1H), 5.36 (dd, 1H, J = 5.5 and 8.5 Hz), 4.19-4.04 (m, 3H), 2.62 (s, 3H), 1.14-0.92 (m, 28H); FAB-LRMS m/z 808 (MH+); FAB-HRMS calculated for C38H50N5O7S2Si2 808.2690, found 808.2704.

N6-(Benzoyl)-2[prime]-O-(trifluoromethyl)adenosine (6) and N6-(benzoyl)-2[prime],3[prime]-O-(difluoromethylidene)adenosine (10)

A solution of 5 (250 mg, 0.4 mmol) and HF/pyridine complex (5 ml; Aldrich) in CH2Cl2 (10 ml) was added to a suspension of DBH (430 mg, 1.5 mmol) in CH2Cl2 (5 ml) at -78°C and the resulting mixture was stirred at 0°C for 3 h. The reaction mixture was poured into an aqueous saturated solution of NaHCO3 (100 ml) and the mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, 5% MeOH in CHCl3) to give 6 (56 mg, 32% as a white foam) and 10 (60 mg, 36% as a white foam). The physical data of 6: 1H NMR (CDCl3) [delta] 9.09 (br s, 1H), 8.82 (s, 1H), 8.07 (s, 1H), 8.05-7.53 (m, 5H), 6.13 (d, 1H, J = 7.6 Hz), 5.99 (br d, 1H), 5.63 (dd, 1H, J = 4.4 and 7.6 Hz), 4.71 (d, 1H, J = 4.4 Hz), 4.41 (s, 1H), 4.00 (br d, 1H), 3.79 (br m, 1H), 2.72 (br s, 1H); FAB-LRMS m/z 440 (MH+); FAB-HRMS calculated for C18H17O5N5F3 440.1182, found 440.1189. The physical data of 10: 1H NMR (DMSO-d6) [delta] 11.30 (s, 1H), 8.83 (s, 1H), 8.69 (s, 1H), 8.16-7.58 (m, 5H), 6.59 (d, 1H, J = 2.3 Hz), 6.21 (m, 1H), 5.69 (m, 1H), 5.30 (m, 1H), 4.51 (m, 1H), 3.64-3.61 (m, 2H); FAB-LRMS m/z 420 (MH+); FAB-HRMS calculated for C18H16F2N5O5 420.1119, found 420.1120.

N6-(Benzoyl)-5[prime]-O-(dimethoxytrityl)-2[prime]-O-(trifluoromethyl)-adenosine (7)

A mixture of 6 (145 mg, 0.4 mmol) and DMTrCl (305 mg, 0.9 mmol) in pyridine (5 ml) was stirred at room temperature. After 12 h, ice-water and AcOEt were added, the resulting mixture was partitioned and the organic layer was washed with H2O and brine, dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography (SiO2, hexane:AcOEt = 1:1) to give 7 (246 mg, 83% a pale yellow foam). 1H NMR (CDCl3) [delta] 8.99 (br s, 1H, NH), 8.68 (s, 1H), 8.11 (s, 1H), 8.04-6.78 (m, 18H), 6.31 (d, 1H, J = 5.9 Hz), 5.81(m, 1H), 4.74 (m, 1H), 4.30 (m, 1H), 3.78 (s, 6H), 3.57 (dd, 1H, J = 3.6 and 10.8 Hz), 3.42 (dd, 1H, J = 3.8 and 10.8 Hz), 2.61(d, 1H, OH, J = 4.3 Hz).

N6-(Benzoyl)-5[prime]-O-(dimethoxytrityl)-2[prime]-O-(trifluoromethyl)-adenosine 3[prime]-O-(2-cyanoethyl N,N-diisopropyl-phosphoramidite) (8)

After successive co-evaporation with pyridine, 7 (463 mg, 0.63 mmol) was dissolved in CH2Cl2 (5 ml) containing N,N-diisopropylethylamine (313 µl, 1.85 mmol). Chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine (280 µl, 1.24 mmol) was added to the solution and the reaction mixture was stirred at room temperature. After 30 min, aqueous saturated NaHCO3 and AcOEt were added to the mixture, the separated organic layer was washed with aqueous saturated NaHCO3 and brine, dried (Na2SO4) and concentrated. The residue was purified by column chromatography (neutralized SiO2, hexane:AcOEt = 1:1) to give 8 (480 mg, 81% as a white foam). 31P NMR (CDCl3) [delta] 152.5, 152.2; FAB-LRMS m/z 942 (MH+); FAB-HRMS calculated for C48H52F3N7O8P 942.3567, found 942.3569.

Synthesis of ODNs

ODNs were synthesized on a DNA synthesizer (Applied Biosystem Model 392) by the phosphoramidite method (34). The fully protected ODNs were then deblocked and purified by the same procedure as for the purification of normal ODNs (24), i.e. each ODN linked to the resin was treated with concentrated NH4OH at 55°C for 16 h and the released ODN protected by a DMTr group at the 5[prime]-end was chromatographed on a C-18 silica gel column (1 × 10 cm; Waters) with a linear gradient of MeCN from 0 to 30% in 0.1 M TEAA buffer (pH 7.0). The fractions were concentrated and the residue was treated with aqueous 80% AcOH at room temperature for 20 min, then the solution was concentrated and the residue was co-evaporated with H2O. The residue was dissolved in H2O and the solution was washed with Et2O, then the H2O layer was concentrated to give the deprotected ODNs 17 (49), 18 (20), 19 (77), 20 (74) and 21 (31) (the yields are indicated in parentheses as OD units at 260 nm starting from the 1 µmol scale).

Electrospray ionization mass spectrometry

Spectra were obtained on a Quattro II (Micromass, Manchester, UK) triple quadrupole mass spectrometer equipped with an ESI source in the negative ion mode. The HPLC-purified ODN samples were dissolved in aqueous 50% 2-propanol containing 1% triethylamine (10 pmol ODN/µl) and introduced into the ion source through a loop injector with a carrier solvent, 33% aqueous MeOH, flowing at 10 ml/min flow rate. About 15 scans were acquired in an ~1 min period and combined to obtain smoothed spectra. All molecular masses of the ODNs were calculated from the multiple charge negative ion spectra. The observed average molecular masses of 17 and 19 were 5076.00 and 5244.25, respectively, and fit the calculated molecular weights (theoretical average molecular masses) for these compounds, i.e. 5076.31 (for 17, C160H192N73O85F6P15) and 5244.31 (for 19, C162H190N73-O87F12P15) within a commonly accepted error range for ESI MS, 0.01%.

Thermal denaturation and CD spectroscopy

Each solution contains each ODN (3 µM) and the complementary RNA 22 (3 µM) or DNA 23 (3 µM) in an appropriate buffer. The solution containing each ODN was heated at 100°C for 3 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 Elmer Lambda2S. Sample temperature was increased by 0.5°C/min. Samples for CD spectroscopy were prepared by the same procedure used in the thermal denaturation study and spectra were measured on a JASCO J720 Spectropolarimeter at 15°C. The ellipticities of duplexes were recorded from 200 to 320 nm in a cuvette with a path length of 1 mm. CD data were converted into mdeg/mol strands/cm.

Partial hydrolysis of ODNs with snake venom phosphodiesterase

Each ODN labeled with 32P at the 5[prime]-end (10 pmol) was incubated with snake venom phosphodiesterase (0.4 µg) in the presence of Torula RNA (0.15 OD260 units) in a buffer containing 37.5 mM Tris-HCl (pH 8.0) and 7.5 mM MgCl2 (total 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), then the mixtures were heated at 100°C for 5 min. The solutions were analyzed by electrophoresis on 20% polyacrylamide gel containing 7 M urea (40).

ACKNOWLEDGEMENTS

This investigation was supported in part by a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00301). This paper constitutes part 181 of Nucleosides and Nucleotides; for part 180 in the series, see the following: Ogawa,A., Tanaka,M., Sasaki,T. and Matsuda,A. J. Med. Chem., in press.

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*To whom correspondence should be addressed. Tel: +81 11 706 3228; Fax: +81 11 706 4980; Email: matuda@pharm.hokudai.ac.jp

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T. Ito, Y. Ueno, Y. Komatsu, and A. Matsuda
Synthesis, thermal stability and resistance to enzymatic hydrolysis of the oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2'-O-methyluridines
Nucleic Acids Res., May 15, 2003; 31(10): 2514 - 2523.
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