Bioconiugate Chem. 1990, 1, 319-324
319
Synthesis and Interactive Properties of an Oligonucleotide with Anthraquinone at the Sugar Fragment Kazushige Yamana,’ Yoshitaka Nishijima, Tadashi Ikeda, Tadao Gokota, Hiroaki Ozaki,+ Hidehiko Nakano, Osamu Sangen, and Takeo Shimidzut Department of Applied Chemistry, Himeji Institute of Technology, 2167 Shosha, Himeji, Hyogo 671-22, Japan, and Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan. Received May 30, 1990
The synthesis of a self-complementary oligonucleotide possessing an anthraquinonylmethyl substituent a t the designated sugar fragment, 5’-CCU(B’AQ)AGCTAGG (I),is described. The anthraquinonylmethyl group was introduced to 2’-hydroxyl moiety of uridine, which was then converted to the protected phosphorobisdiethylamidite derivative. This reagent was used for the solid-phase synthesis of the modified oligonucleotide 1. The UV and CD melting behaviors indicate that the modified oligonucleotide 1 can form a duplex in aqueous buffer solution similar to the unmodified strand 5’XCTAGCTAGG (7). The observed melting temperatures for the duplexes 1 and 7 were 57.4 and 40.0 “C, respectively. The temperature-dependent change in the intensity of the induced CD a t around 335 nm reflected directly to the melting behaviors of duplex 1, indicating that the anthraquinone groups intercalate into the base pairs in the duplex. The intercalation-induced stability of the duplex translates into a free energy cost of 5.2 kcal/mol. The present work provides a novel method for enhancing the affinity of oligonucleotides for their complementary sequences.
There is current interest in conjugation of oligonucleotides with intercalating agents that stabilize the complexes with the complementary sequences without losing the specificity of recognition (1-11). It has been previously shown that oligonucleotide-acridine or -anthraquinone conjugates can be used for inhibiting mRNA translation or viral expression (5-8). The synthesis of oligonucleotideintercalator conjugates has been accomplished by linking oligonucleotides via linker arms to intercalating agents a t a terminal position (1-8), an internal phosphorus (9, IO), or a pyrimidine C-5 (11). We describe here the synthesis of an oligonucleotide with an anthraquinone selectively incorporated to the sugar at a specific residue. Our strategy involves the preparation of a 2’-anthraquinone-modifieduridine derivative that is suitable for a solid-phase synthesis of oligonucleotides. Molecular models allowed us to consider that an anthraquinone group could be incorporated via a relatively short linker to the 2’-sugar position in an oligonucleotide, the resulting oligonucleotide binds to complementary sequence, and the anthraquinone intercalates between the base pairs adjacent to the sugar in the duplex. To test the validity of our consideration, we synthesized selfcomplementary oligonucleotide 5’-CCU(2’AQ)AGCTAGG ( l ) , I containing the anthraquinone-modified uridine (Figure 1). Spectroscopicand thermodynamic studies indicate that a single oligonucleotide-anthraquinone conjugate 1 binds to another in aqueous buffer solution forming a duplex with an enhanced thermal stability by intercalation. + Kyoto University.
* Abbreviations: U(2’AQ), 2’-0-(2-anthraquinonylmethyl)uridine; BzU(2’AQ), 2’-0-(2-anthraquinonylmethy1)-N3benzoyluridine; DMT, 4,4’-dimethoxytrityl; BzC, N4-benzoyldeoxycytidine; ibG, W-isobutyryldeoxyguanosine;CPG, controlledpore glass; oligonucleotide-anthraquinone conjugate, 5’-CCU(2’AQ)AGCTAGG [an oligodoxyribonucleotide containing an anthraquinone-modified uridine [U(2’AQ)] at position 71; tm,
melting temperature, poly(rA), poly(riboadeny1ic acid). 1043-1802/90/290 1-0319$02.50/0
0
I O=P-0
5’-CCU(AQ)AGCTAGG 1 Figure 1. The oligonucleotide containing an anthraquinone at the sugar fragment.
EXPERIMENTAL PROCEDURES Melting and boiling points were uncorrected. Elementary analyses were performed a t the Analytical Center of Kyoto University. ‘H NMR and 31PNMR spectra were obtained on a JEOL-JNM-GX400 spectrometer, using tetramethylsilane as internal standard and 85 96 H3P04 as external standard, respectively. Assignment of IH NMR signals was done by analysis of 2D NMR spectra. Highperformance liquid chromatography (HPLC) was performed on a Waters ALC/GPC 600E model equipped with a 254 nm fixed-wavelength detecter, using a reversedphase Cosmosil5Cl~-300column (0.46 X 15 cm). Column chromatography and thin-layer chromatography (TLC) were carried out on Wako silica gel C-200 and Merck 60 PFm, respectively. Ultraviolet (UV) spectra were recorded with a Shimadzu UV-300 spectrophotometer equipped with 0 1990 American Chemical Society
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6/oconJugareChem., Voi. 1, NO. 5, 1990
a thermoelectrically controlled cell holder. Circular dichroism (CD) spectra were obtained on a JASCO CD 5-600 spectrometer equipped with a thermoelectrically controlled cell holder. Materials and Solvents. N3-Benzoyluridine (12), 2-(bromomethy1)anthraquinone (13),and N-(trimethylsi1yl)diethylamine (14) were prepared by the literature procedures. Protected deoxyribonucleoside P-cyanoethyl phosphoramidites and CPG support were purchased from Applied Biosystems. The oligodeoxyribonucleotide 5’-CCTAGCTAGG was synthesized by using a DNA synthesizer (Applied Model 380A). Snake venom phosphodiesterase and alkaline phosphatase were obtained from Boehringer-Mannheim. Dimethylformamide (DMF) was stirred in the presence of CaHz, distilled, and stored over CaHz. Dichloromethane, acetonitrile, and diethylamine were dried by refluxing with CaHz a t least for 5 h and then distilled and stored over CaHz or molecular sieves. Synthesis of 5’-DMT-U(2’AQ) (5). N3-Benzoyluridine (2; 2.1 g, 6 mmol) was allowed to react with di-nbutyltin oxide (1.5 g, 6 mmol) in methanol (300 mL) under reflux for 2 h (15). The solvent was removed to dryness, giving 2‘,3‘-0-dibutylstannylene-N3-benzoyluridine (3). Dibutylstannylene derivative 3 was dried over PzO5 under vacuum for 12 h. To a solution of 3 in dry DMF (36 mL) were added 2-(bromomethy1)anthraquinone (4.5 g, 15 mmol) and CsF (1.8 g, 12 mmol), and the mixture was stirred at room temperature for 45 h. Ethyl acetate (200 mL) was added to the reaction mixture and the resulting solution was washed with water (50 mL X 3) and then dried over NazS04. The solution was concentrated to a minimum volume and then applied to a silica gel column (4.5 X 30 cm); elution with CHzClz-MeOH (20:1, v/v) gave fractions which contain a mixture of BzU(2’AQ) (4) and its 3’isomer (1.9 g, 55% combined yield). The mixture (1.9 g, 3.3 mol) was allowed to react with DMT chloride (1.2 g, 3.6 mmol) in dry pyridine (20 mL) a t room temperature for 6 h. Concentrated ammonium hydroxide ( 5 mL) was added to the solution and the solution was stirred at room temperature for 16 h. The solution was concentrated to dryness, dissolved in CHzClz (50 mL), and then washed with water (20 mL). The organic solution was dried over NazS04, the solvent was removed, and then the residue was applied to a silica gel column (3 X 45 cm); elution with CHzClz-MeOH (20:1, v/v) gave two major fractions which were separated to 5’-DMT-U(2’AQ) (5; 0.8 g, 31 % ) and its 3’-isomer (1.1 g, 5 4 % ) . 5: mp 138-139 “C; TLC (CHzClz-MeOH 9:1, v/v) Rf 0.69; ‘H NMR (DMSO-ds) 6 3.30 (m, 2 H, H ~ P 3.72 ) , (s, 6 H, CH30 of DMT), 4.09 (m, 1 H, H4’), 4.10 (dd, 1 H, Hz’), 4.28 (ddd, 1 H, becoming dd with D20, H3’), 4.92 (d, 2 H, J g e m = 13.68 Hz, ArCHz), 5.24 (d, 1H, uracil H5), 5.47 (d, 1H, diminished with DzO, 3-OH), 5.95 (d, 1 H, J l t , ~= ! 3.66 Hz, HI’), 6 7.26 (d, 1 H, uracil HG),7.80, 7.94, and 8.20 (m, total 7 H, Ar of anthraquinone). Anal. Calcd for C45H38N2010: C, 70.49; H, 4.99; N, 3.65. Found: C, 69.97; H, 4.90; N, 3.58. 3’isomer of 5: mp 140-141 “C; TLC (CHzClz-MeOH 9:1, v/v) R f 0.55; ‘H NMR ( D M s 0 - d ~6) 3.27 (m, 2 H, H5’),3.68 (s, 6 H, CH30 of DMT), 4.13 (m, 1H, H4’),4.14 (dd, 1H, H i ) , 4.42 (ddd, 1H, becoming dd with D20, Hz’), 4.82 (d, 2 H, J,,, = 13.19 Hz, ArCHZ), 5.24 (d, 1H, uracil H5), 5.72 (d, 1H, diminished with DzO, 2’-OH), 5.80 (d, 1H, JIT,~/ = 4.15 Hz, HI’), 7.78 (d, 1 H, uracil He), 7.78, 7.95, and 8.20 (m, total 7 H , Ar of anthraquinone). Anal. Calcd for C45H38N2010: C, 70.49; H, 4.99; N, 3.65. Found: C, 69.40; H, 4.77; N, 3.52. Conversion of 5 to U(2’AQ). 5’-DMT-U(2’AQ) (76.6 mg, 0.1 mmol) was treated with 10 mL of 80% CH3COOH
Yamana et al.
in water at room temperature for 2 h. The solvent was removed to dryness. The residue was dissolved in CHzClz (10 mL) and the solution was washed with aqueous 5 % NaHC03. A pale yellow crystalline solid appeared from the organic solution. The product was filtered and then dried in vacuo over PzO5 to yield 38 mg (82%): TLC (CHzClz-MeOH 9:1, v/v) Rf 0.42; ‘H NMR (DMSO-ds) 6 3.63 (m, 2 H, H59, 3.96 (m, 1H, H4’), 4.10 (dd, 1 H, Hz’), 4.20 (ddd, 1H, H i ) , 4.85 (d, 2 H, Jgem = 13.19 Hz, ArCHz), 5.56 (d, 1H, uracil H5), 5.97 (d, 1H, J I T ,=~ 4.87 ! Hz, HI’), 7.90, 7.94, and 8.20 (total 8 H, Ar of anthraquinone and uracial Hs); €260 (25 “C, Hz0-EtOH 1:1, v/v) 3.50 X lo5, €335 (25 “C, H20-EtOH 1:1, v/v) 0.46 X lo5. Synthesis of Bis(diethy1amino)phosphorochloridite. To PCl3 (11.6 mL, 0.13 mol) cooled a t 0 “C with stirring was added N-(trimethylsily1)diethylamine (53.6 mL, 0.28 mol). The reaction was carried out for 60 min. The produced trimethylsilyl chloride was removed by distillation at atmospheric pressure. The remaining liquid was distilled under reduced pressure to give bis(diethy1amino)phosphorochloridite as a colorless liquid (20 g, 72%): bp 68-71 “Cat 3 mmHg; dw 1.018; 31PNMR 6 161.5. Anal. Calcd for C4HloNzPCl: C, 45.16; H, 9.57; N, 13.30; P , 14.70. Found C, 45.19; H, 9.92; N, 13.09; P, 14.78. Synthesis of 5’-DMT-U(2’AQ)Phosphorobisdiethylamidite (6). To a solution of 5 (35.2 mg, 0.046 mmol) in dry CHzClz (0.25 mL) containing triethylamine (1equiv) was added a solution (92 pL) of bis(diethy1amino)phosphorochloridite (1equiv vs 5) in CHzClz. The solution was stirred at room temperature for 15 min. Diethylamine (0.23 mmol) in CHzClz (0.7 mL) was added to the solution and the solution was washed with aqueous, saturated NaCl (1mL). The organic solution was dried over Na2S04 and then the solvent was removed in vacuo to dryness. The residual powder was dissolved in dry CH3CN and then analyzed by 3’P NMR. The one major peak appeared a t 134 ppm, indicating that bisamidite 6 thus obtained could be used for the solid-phase oligonucleotide synthesis. 5’-DMT-BzC phosphorobisdiethylamidite was synthesized by essentially the same procedure described above. Synthesis of Oligonucleotide-Anthraquinone Conjugate 5’-CCU(2’AQ)AGCTAGG(I). The synthesis of oligonucleotide-anthraquinone conjugate 1 was accomplished by the manual solid-phase phosphoramidite (16,17)and phosphorobisamidite (18,19)methods, beginning with 5’-DMT-ibG (0.2 pmol) bound to a CPG support. The fully protected 5’-DMT-AGCTAGGCPG was synthesized by using deoxyribonucleoside P-cyanoethyl phosphoramidites (16,17). Bisamidite 6 was coupled in the seventh addition cycle as follows: 6 (0.046 mmol) was activated with 0.5 equiv of @-nitropheny1)tetrazole in CH3CN (0.2 mL) and coupled (15 min) to the protected, CPG-bound, 5’-detritylated AGCTAGG. After the hydrolysis with aqueous tetrazole (181,DMT cation released from the CPG was monitored spectroscopically,indicating a coupling yield of 87% for U(2’AQ). The remainder of the oligonucleotide synthesis cycles using 5’-DMT-BzC phosphorobisdiethylamidite were as previously described ( I @ , resulting in each coupling yield of 96%. Thus an overall yield for the 10-mer 1 based on DMT cation was approximately 70%. The remaining H-phosphonate linkage was oxidized with iodine-water (18)and then CPGbound 10-mer 1 was treated with concentrated ammonium hydroxide a t 55 “C for 12 h. The purification of oligomer 1 was performed with reversed-phase HPLC eluting with a CH3CN linear gradient (1% / m i d from 3% CH3CN in 0.1 M triethylammonium acetate (pH 7.0) a t a flow rate
Bioconjugate Chem., Vol. 1, No. 5, 1990
Oligonucleotides with Anthraquinone at Sugar Fragment
321
+ 3’-ismer HO OH
0 0
BX
2
/ ‘
Bu
Bu
3
4
0
0
0
5
0
Figure 2. Synthesis of the anthraquinone-modifieduridine derivative. of 1.0 mL/min. The major peak ( t =~23.2 min) was collected and lyophilized, affording 8.0 A m units of purified oligomer 1. unit) was The purified oligonucleotide 1 (ca. 0.1 subjected t o digestion with snake venom phosphodiesterase (0.3 unit/mL) and alkaline phosphatase (100 unit/ mL) in 50 pL of Tris-HC1 buffer (pH 7.2) a t 37 “C for 2 h (20). The reaction mixture was analyzed by reversedphase HPLC; elution with 0.05 M ammonium formate containing a 15% CH3CN gradient (20 min) a t a flow rate of 1.5 mL/min gave deoxyribonucleosides in an expected molar ratio of dG:dC:dA:T = 3.29:3.15:1.95:1.00, The nucleoside U(2’AQ) was detected from the mixture with 30% CH3CN in the buffer as the eluent. The purified oligomer 1 exhibits the absorption band a t around 335 nm similar to U(2’AQ), indicating that U(2’AQ) is present in this sequence. Preparation of Oligonucleotide Solutions for Physical Measurements. All solutions were prepared by using a buffer containing 0.01 M sodium phosphate and 0.1 M NaC1, adjusted to pH 7.0. Oligonucleotide concentrations were determined based on the measured absorbance a t 260 nm and the following single-strand M-’ cm-l at 25 “C): oliextinction coefficients ( E X gonucleotide-anthraquinone conjugate 1, 1.23; oligonucleotide 5’XCTAGCTAGG (7), 0.95. Each t value was determined by calculations based on a nearest neighbor model (22). Since oligomers studied here have a selfcomplementary sequence, the calculations for oligomer concentrations were carried out by taking account of the hypochromic effect on the absorbance a t 25 “C: for oligomer 1, 11%; for oligomer 7, 14%. Except for thermodynamic studies, all duplex melting curves by UV and CD spectra were measured a t a common strand concentration (1.5 X M). Temperaturedependent induced CD curves were obtained a t the same concentration. T h e thermodynamic data for duplex formation were obtained from UV melting curves by plotting inverse melting temperature versus log C, where C is the total strand concentration (22). RESULTS
Synthesis of 5’-DMT-U(2’AQ) Phosphorobisdiethylamidite (6). The synthesis of 5’-DMT-U(2’AQ) phosphorobisdiethylamidite (6) is shown in Figure 2. W Benzoyluridine (2) was converted according to Moffatt’s
procedure (15) to 2’,3’-O-dibutylstannylenederivative 3, which was allowed to react directly with 2-(bromomethy1)anthraquinone in the presence of CsF as a catalyst, affording a mixture of BzU(2’AQ) (4) and its 3’4somer. These nucleosides were treated with DMT chloride followed by ammonium hydroxide, giving two products which were separated by a silica gel chromatography, 5’DMT-U(2’AQ) (5) and its 3’-isomer. Nucleoside 5 was phosphitylated with bis(diethy1amino)phosphorochloridite, yielding 5’-DMT-U(2’AQ) phosphorobisdiethylamidite (6). Synthesis of Oligonucleotide-Anthraquinone Conjugate (1). The oligonucleotide S-CCU(2’AQ)AGCTAGG (1) was synthesized by the manual solidphase phosphoramidite and phosphorobisamidite methcds (16-19). Phosphorobisdiethylamidite 6 was used directly for introduction of U(2’AQ) into the sequence of 1. DMT cation measurements indicated coupling efficiencies of 9 6 98 % for the phosphoramidites and the phosphorobisamidite, although a slightly lower efficiency (87 %) was observed in the coupling of DMT-U(2’AQ) phosphorobisdiethylamidite (6). The protected oligonucleotide 1 bound to the support was treated with concentrated ammonium hydroxide in a usual manner to give deprotected oligomer 1. Final purification of 1was effected by reversed-phase HPLC. The nucleoside composition of oligonucleotide 1was verified by enzymatic digestion analysis. The purified oligonucleotide 1 exhibits a UV absorption band a t around 335 nm due to the 2-substituted anthraquinone. Oligonucleotide Duplex. Figure 3 shows the UV melting curves for the anthraquinone-modified duplex 1 and the corresponding unmodified oligonucleotide duplex 5’-CCTAGCTAGG (7). Analyses of the UV melting curves provide the melting temperature (tm) values which are listed in Table I. Table I also summarizes the thermodynamic data derived from the analysis of dependency of inversed tm values on logarithm of the total oligonucleotide concentrations. Figure 4 indicates the two families of temperature-dependent CD spectra for duplex 1 and 7. The CD melting curves for duplexes 1and 7 are shown in each box in Figure 4. The tm values yielded from the CD melting curves were identical with those derived from the UV melting profiles. The families of the induced CD spectra for duplex 1 between 300 and 400 nm at different temperatures are presented in Figure 5. The intensity of
Yamana et al.
322 Bioconjugate Chem., Vol. 1, No. 5, 1990
Table I. Spectroscopically Measured Tm Values and Thermodynamic Parameters for Duplexes 5’-CCU(2‘-AQ)AGCTAGG (1) and 5I-CCTAGCTAGG (7). duplex tm Atm AGO AAGO AHo AAHO ASo AASO 1 7
57.4 40.0
17.4
-5.3
-15.1 -9.8
a Tm was measured at a strand concentration of 1.5 X cal/mol.K. AGO and AAGO are at 27 O C .
20
10
30
40
50
60
-219.2 -133.7
-85.5
5000
I
0
-31.0
M. The free energy and enthalpy data are in kcal/mol; the entropy data are in
I
I
-80.9 -49.9
70
.2o*c
I
80 300
T(’C)
Figure 3. UV melting curves measured at 260 nm for duplexes [O,5’-CCU(P’-AQ)AGCTAGG (1); O , 5’CCTAGCTAGG (7)] at a common strand concentration (1.5 X lW5 M). The buffer used contained 0.1 M NaCl and 0.01 M sodium phosphate, adjusted to pH 7.0.
350 Wavelength(nm)
400
Figure 5. Expanded CD spectra for duplex 1 between 300 and 400 nm as a function of the indicated temperature. The measurements were carried out at a strand concentration of 1.5 X 10” M in a pH 7.0 buffer containing 0.1 M NaCl and 0.01 M sodium phosphate.
3.0
2.0
1.0 m 0
x
0
m
I
-2.0
1
220
.
.
I
. 250
300
350
Wavelength(nm)
Figure 6. Model of the duplex of oligonucleotide-anthraquinone conjugate. Dotted lines indicate hydrogen bondings between Watson-Crick base pairs. 2.0
.
1.0
.
Y)
0 X I
0 .
m
I
-2.0 220
250
300
350
Wavelength (nm)
Figure 4. Temperature-dependent CD spectra and CD melting curves at 266 nm for duplexes (upper, 1; lower, 7) at a common strand concentration (1.5X 10+ M).The buffer used contained 0.1 M NaCl and 0.01 M sodium phosphate, adjusted pH 7.0. the ellipticity a t around 335 nm decreased with increase in temperature. For unmodified duplex 7, no induced CD can be detected. DISCUSSION Synthesis of Oligonucleotide-Anthraquinone Conjugate (1). Our strategy for introduction of an an-
thraquinone group into the specific sugar residue of an oligonucleotide was based on the preparation of a modified ribonucleoside with anthraquinonylmethyl group a t 2’hydroxyl moiety. Benzyl substitution a t the 2’- or 3’hydroxyl moiety of the ribonucleoside can be achieved by reaction of 2’,3’-0-dibutylstannyleneribonucleoside with benzyl bromide (15). This reaction has proved to be useful for introduction of the anthraquinonylmethyl group into the uridine 2‘- or 3’-hydroxyl moieties, yielding 4 and its 3’4somer. It should be noted that use of an N-acyl group as a protecting group of uridine and CsF as a catalyst is important for obtaining the desired product by the above reaction. After 5’-dimethoxytritylation and debenzoylation, 5’-DMT-U(2’AQ) (5) can be isolated in a pure form by a silica gel chromatography. 5’-DMT-U(2’AQ) phosphorobisdiethylamidite (6), prepared by phosphitylation of 5 with bis(diethy1amino)phosphorochloridite, is a suitable monomer for phosphorobisamidite oligonucleotide synthesis procedures that we have recently developed (1419). Coupling reactions of deoxyribonucleoside phosphorobisdiethylamidites are almost equally effective as standard deoxyribonucleoside phosphoramidite reagents, although 6 was found to
Oligonucleotides with Anthraqulnone at Sugar Fragment be slightly less efficient in the coupling probably because of the bulky 2’-substituent. Duplex Stability. Model-building studies show that the duplex formation of oligonucleotide-anthraquinone conjugate (1) by Watson-Crick base pairing should allow two anthraquinone moieties to intercalate into both the base pairs of -*.3LA-T-5*’**
..-
I, I,
I,
0 ,
0I ,,
5’-U-A-3’.--
in the resulting duplex (Figure 6). The UV and CD melting behaviors, where the shapes of the melting profiles for duplex 1 exhibit sigmoidal curves similar to those for 7, indicate the formation of oligonucleotide duplex for 1. The temperature-dependent change in the intensity of the induced CD at around 335 nm reflects directly the melting behaviors of duplex 1. These observations suggests that the intercalation of the anthraquinone moieties occurs in a cooperative manner with Watson-Crick base pairing. Inspection of the melting profiles in Figure 3 and the t m values in Table I reveals that the modification with anthraquinonylmethyl at the 2’-sugar hydroxyl moiety of the oligonucleotide double helix increases the duplex melting temperature by 17 “C relative to the corresponding unmodified duplex. The thermodynamic data in Table I show that this modification-induced increase in thermal stability translates into a free energy cost (AA GO) of 5.3 kcal/mol for duplex at 27 “C. The magnitude of AAGO per one anthraquinone molecule can be estimated to be ca. 2.7 kcal/mol, which is larger than that reported for the oligonucleotide duplex with anthraquinone at the thymidine c-5 (7). Close examination of the thermodynamic data reveals that the stabilizing influence (AAGO) results from negative reductions in both the transition enthalpy ( A M o ) and the transition entropy (AASO). Anthraquinone-modified duplex 1 exhibits different CD spectral properties from the unmodified duplex 7 in the region between 200 and 300 nm, indicating that the modification with the anthraquinone at the sugar alters the global conformation of the double helix. This alteration must be considered for interpreting the enthalpy and the entropy effects that we have obtained for the duplex formation of oligonucleotide-anthraquinone conjugate 1. Nevertheless, the overall gain in the transition enthalpy may be derived mainly from intercalation between anthraquinone and the adjacent base pairs. Similar enthalpy effects have been reported on stabilizing complexes of oligonucleotides containing 5’linked anthraquinone with poly(rA) (11). Further studies may be necessary to elucidate more detailed effects of the anthraquinone intercalation on the duplex stability. A general procedure has been developed for incorporation of an anthraquinone to a specific sugar residue in oligonucleotide sequences. T h e oligonucleotideanthraquinone conjugate has an increased affinity for the complementary sequence. The anthraquinone moiety should locate in between the base pairs adjacent to the designated sugar in the duplex. The present procedure would provide a useful method for preparation of oligonucleotide derivatives to deliver an anthraquinone group specifically to a given nucleotide sequence in DNA or RNA as well as for enhancement of the affinity of oligonucleotides for their complementary sequences. ACKNOWLEDGMENT We are grateful to Dr. H. Sugiyama for instruction in enzyme digestion analysis and to Dr. Y. Kuroda for the use of the CD spectrometer. A part of this work was
Bloconjugate Chem., Vol. 1, No. 5, 1990 323
supported by a Grant from the Ministry of Education, Science, and Culture of Japan. LITERATURE CITED (1) Asseline, U., Delarue, M., Lancelot, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T., and Helene, C. (1984) Nu-
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