Bioconjugate Chem. 1995, 6, 578-586
578
Self-ComplementaryOligodeoxyribonucleotides Containing 2’-0-(Anthraquinone-2-methyl)adenosine Hemant M. Deshmukh, Seema P. Joglekar, a n d Arthur D. Broom” Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84112. Received May 15, 1995@
Incorporation of an intercalating agent into an oligodeoxynucleotide (ODN)has the potential to enhance binding affinity upon duplex formation, to increase ODN hydrophobicity, and to enhance resistance to nuclease hydrolysis. Site-specific intercalation has been achieved through the synthesis of 2’-0(anthraquinone-2-methyl)adenosine(rA*) and its incorporation into the palindromic dodecanucleotide d(CGCrA*CATGTGCG). Melting temperature, CD spectra, 1D and 2D (DQF-COSYand NOESY) NMR spectra, and molecular models were obtained and compared with the unmodified dodecamer. The data clearly establish that intercalation of the anthraquinone ring into a predominantly B-type helix occurs between the A4-T9 and C5-G8 base pairs, significantly stabilizing the duplex and enhancing the hydrophobicity of the ODN.
INTRODUCTION The chemistry, structural biology, and potential diagnostic and therapeutic applications of oligonucleotides are subjects of great current interest. The exquisite specificity of the genetic code and the relative ease of assembling sequences, which in principle, may inhibit the expression of a single gene in the entire human genome, have given great impetus to investigations intended to define the parameters required for the application of this technology to biologically relevant problems. A number of recent reviews document the rapid progress toward this goal (16).
Since the original demonstration by Zamecnik (7) of the feasibility of the antisense oligonucleotide concept in the inhibition of gene expression, much effort has gone into defining molecular modifications which may overcome the inherent limitations of oligodeoxynucleotides (ODN) as in vivo therapeutic agents. Thus, in order to take advantage of specificity intrinsic in the primary sequence, ODN must be able to penetrate membranes of target cells, be sufficiently stable to reach their targets without degradation in biological media, and have a high affinity for the target polynucleotide (8). Attempts to achieve goals of increased lipophilicity and stability to nucleases have included modification of ODN with intercalating (9, IO), cleaving (11-13), alkylating (14, 151, or photocrosslinking (16) modifications a t the 3’- andlor 5‘-termini. Recently, considerable attention has been given to incorporation of 2’-O-alkyl-or aralkylsubstituted ribonucleosides into homooligomers or mixed (chimeric) deoxyribohibooligomers (17-19). Two recent publications described the attachment of an anthraquinone moiety to the 2’-hydroxyl of ribonucleosides, their incorporation into ODN, and assessment of the resulting stabilization of the duplex by melting experiments. In both cases, the stabilization was said to result from intercalation, although little direct evidence was presented in support of that hypothesis. In the first of these studies (20), the anthraquinone moiety was attached through a methylene group. In more recent work (21), the linker was six atoms in length. The methylene-linked
* To whom correspondence should be addressed. Phone: (801) 581-7063. Fax: (801) 581-7087. “Abstract published in Advance ACS Abstracts, July 15, 1995.
anthraquinone was said to cause significant global distortion of the duplex based on CD spectropolarimetry (201, but no further characterization of the presumed intercalation complex was provided. A number of years ago we demonstrated that 2’-0benzyladenosine in DzO solution exists primarily in a conformation in which the purine and benzene rings are stacked, with the two rings overlapping and their planes nearly parallel (22). Such a conformation in the anthraquinone case would, indeed, favor intercalation in a duplex and would, by virtue of the constraints imposed by the methylene linker, permit intercalation only on the 3’-side of the nucleotide bearing the 2’-substituent. The present report describes the synthesis of 2’-0(anthraquinone-2-methylladenosine (Scheme 11, its incorporation into a self-complementary oligodeoxyribonucleotide, and characterization of the resulting duplex by NMR (lD, DQF-COSY, and NOESY), CD, U V , melting temperatures, and molecular modeling. In order to combine the properties of reasonable duplex stability, relative simplicity of NMR spectra, and a size compatible with NOESY experiments, self-complementary dodecanucleotide d(CGCACATGTGCG) and the modified oligo d(CGCrA”CATGTGCG1were synthesized; they will be referred to as “standard 12-mer” and “anthra 12-mer,” respectively, where rA* is nucleoside 1 (Scheme 1). EXPERIMENTAL PROCEDURES
General Methods. The ‘H-NMR spectra were recorded on either an IBM AF200 FT-NMR spectrometer a t 200 MHz or a Varian Unity 500 MHz spectrometer. NMR samples of nucleosides were prepared using (CD3)ZSO or CDC13 with TMS or solvent peak as internal standard. The W spectra and melting curves were recorded on a Hewlett-Packard diode array spectrophotometer. Low-resolution mass spectra were recorded on either a Finnegan MAT 95 or a VG 7050E mass spectrometer. Electrospray mass spectra were recorded on a Vestec 201 ionization instrument with a quadrupole mass analyzer. Thin-layer chromatography (TLC) was performed on Kieselgel 6OFZb4 aluminum-backed silica gel sheets. CD spectra were recorded on a JASCO 5-720 spectropolarimeter. The oligomers were synthesized on a n Applied Biosystems 380B instrument. T,,, Determination. All T, values were determined on a Hewlett-Packard diode array spectrophotometer
1043-1802/95/2906-0578$09.00/0 0 1995 American Chemical Society
ODN Containing 2'-O(Anthraquinone-2-methyl)ADO equipped with an electronic temperature controlling device. Absorbance was measured at 1"C intervals, and the cuvette was equilibrated a t each temperature for 2 min. The oligomers were annealed by heating to approximately 70 "C in a water bath and then cooled slowly. Duplexes were kept in the refrigerator overnight before T, measurements were carried out. Duplexes were M concentration in 0.1 M NaCl formed a t 1.58 x containing 10 mM phosphate buffer (pH 7). An aliquot of each solution was diluted 50-fold a t 4 "C with the same buffer, and the T, of the diluted solution was measured. HPLC Purification and Analysis of Oligomers. All HPLC purification and analysis was performed on a Hitachi D-6200 HPLC system equipped with a n L-3000 diode array spectrophotometer. DNA synthesized on a 1 pmol scale was purified on a preparative Whatman Partisil ODS reversed-phase column. Purified DNA was analyzed on a Rainin Microsorb C18 analytical column (25 mL x 4.4 mm). The solvents used for elution in both purification and analysis wereas follows. Solvent A: 95% 50 mM ammonium bicarbonate buffer (pH 7):5% acetonitrile. Solvent B: 80% 50 mM ammonium bicarbonate buffer (pH 7):20% acetonitrile. The gradient used for purification was solvent A (0-4 min), linear gradient ofA to B (4-15 mid; solvent B (1522 min), linear gradient B to A (22-32 min); solvent A (32-40); for analysis the gradient was solvent A (0-10 m i d ; solvent Nsolvent B, 1:l (10-20 min); solvent A (20-30 m i d . The flow rate for purification was 2 mL/ min, whereas that for analysis was 1 mumin. The oligomers synthesized on a 10 pmol scale were purified by size exclusion chromatography. A Sephadex G25 column (100 x 2.5 cm) was used to purify the standard 12-mer, whereas a G50 was used to purify the anthra 12-mer. Elution was performed with distilled water a t a flow rate of 20 mL/h; fractions for the standard DNA were checked by HPLC on a BioRad SEC-125 gel filtration column; elution was performed by 50 mM phosphate buffer (pH 6.8) containing 0.1 M NaCl and 10 mM sodium azide. The fractions for the anthra 12-mer were checked by reversed-phase HPLC. Nuclease Digestion and Analysis of Modified and Unmodified Oligomers. A mixture of oligonucleotide (0.5 OD units) with 50 mM Tris HC1 (pH 8), 10 mM MgC12, snake venom phosphodiesterase (3 units), and bovine alkaline phosphatase (3 units) in 100 pL of distilled water was incubated a t 37 "C for 12 h. Sodium acetate (0.05 M, pH 5 , 1 5pL) was added to the hydrolysis mixture followed by the addition of 230 pL of absolute ethanol. This mixture was chilled a t -70 'C for 30 min and centrifuged a t 30000g for 5 min. The supernatant was diluted with 1 mL of 95% ethanol, chilled, spun, decanted, and evaporated to dryness. The residue was redissolved in distilled water for HPLC analysis. HPLC analysis for the hydrolysate of the unmodified oligomer was performed on a C18 column. A C8 column was used for analysis of the hydrolysate containing the more hydrophobic modified nucleoside. Elution was performed with a 50 mM potassium phosphate buffer (pH 4Ymethanol gradient for the standard 12-mer hydrolysate; the anthra 12-mer hydrolysate was analyzed using 50 mM potassium phosphate buffer (pH 4) containing 30% acetonitrile. NMR Experiments on Oligonucleotides. One- and two-dimensional proton data sets were collected on a Varian Unity 500 MHz spectrometer. One-dimensional temperature-dependent 500 MHz IH data sets were collected in 90% H20 bufferllO% D2O. For NOESY and DQF-COSY experiments, the oligomers were lyophilized
Bioconjugb'te Chem., Vol. 6,No. 5, 1995 579
twice from 99.996% DzO and then dissolved in 99.996% D20 containing 100 mM NaCl and 20 mM phosphate buffer (pH 7) with (anthra 12-mer) or without (standard 12-mer) 10 mM MgC12. Two-dimensional phase-sensitive NOESY spectra of all the oligomers were collected using a 250 ms mixing time. All the data sets were acquired in the hypercomplex mode, with 256 increments in the tl dimensions, 32 or 64 scans per fid, and 2048 complex points in t2. The t2 dimension was processed with a Gaussian filter without line broadening. The t l increments were zero-filled to 2048 points and transformed with a Gaussian apodization function. Two-dimensional proton phase-sensitive double quantum filtered COSY (DQF-COSY) data were collected using the standard pulse sequence with a 2 ps pulse repetition time and homospoil of 1 ms. The data sets were collected in the hypercomplex mode with 256 t l increments, 32 or 64 scanslfid, and 2048 complex points to t2. The t2 dimension was processed with a Gaussian filter. The tl increments were zero-filled to 2048 points and transformed with a Gaussian apodization function. Molecular Modeling. The molecular modeling studies were performed on Silicon Graphics Iris or Indigo work stations. The molecules were visualized using Biosym (San Diego, CA) software. Minimizations and dynamics were performed using DISCOVER software. For simulation of nucleosides, CVFF potentials were used. All the nucleosides were first minimized by using the steepest gradient for 500 iterations and then by conjugate gradient for 2000 iterations. To obtain a global minimum, molecular dynamics simulations were used. All the dynamics were performed a t 300 K for 50 ps including a 10 ps equilibration period. Lower energy conformers obtained in the molecular dynamics stimulation were further minimized to obtain a family of low energy conformers. The protocol used for initial minimization was used for these minimizations also. Oligonucleotides were visualized using the biopolymer module of the Insight I1 software, and AMBER potentials were used during minimization of the oligonucleotides. The software was unable to assign a potential for C5 of adenosine when anthraquinone groups were attached on the 2'-hydroxyl. Potential "CB" was assigned to this carbon manually. The anthraquinone was manually intercalated inside the helix by torsioning the bonds between the anthraquinone ring and the 2'-hydroxyl. The torsions were removed, and the oligonucleotide was minimized first by 500 iterations of the steepest gradient minimizer and then by 2000 iterations of the conjugate gradient minimizer. Partial charges on the backbone phosphorus nuclei were reduced to 0.8, and those on the oxygen were reduced to -0.5 in some instances. All the hydrogen bonded atoms of the first three base pairs were fixed in space before minimizations. 2'-O-(Anthraquinone-2-methyl)adenosine(1). Synthesis of the title compound was performed generally according to the method of Yamana et a1.(20) 2',3'(Dibutylstanny1ene)adenosine (2.0 g, 4 mmol), 2-(bromomethy1)anthraquinone (2.40 g, 8 mmol), and cesium fluoride (1.20 g, 8 mmol) were mixed in 100 mL of anhydrous DMF. The reaction mixture turned black immediately but converted to a brown suspension after 1h. The reaction was allowed to proceed for 48 h. The suspension was filtered, and the solvent was evaporated in vacuo to afford a residue which was dissolved in ethyl acetate (1L). The ethyl acetate layer was washed with water, dried over sodium sulfate, and evaporated to dryness, and the compound was purified by silica gel chromatography using a gradient of methanol (0-2.5%) in chloroform: yield 1.9 g (19%); IH NMR [(CD&SO] 6
Deshmukh et al.
580 Bioconjugate Chem., Vol. 6, No. 5, 1995
Scheme 1. Synthesis of 2‘-O-(Anthraquinone-28.34 (s, l H , Hs), 8.18 (m, 2H, Ar),8.05 (s, Hz), 8.03 (m, methy1)adenosineNucleosides Using a 2,3’-Dibutyl2H, A r j 7.91 (m, 2H, Ar),7.69 (d, l H , Ar),7.25 (broad s, stannylene Derivative 2H, NH2), 6.10 (d, J11,2. = 6.0 Hz, lH, l’H), 5.45 (m, 2H, 80 0 dcnine 5’-OH and 3’-OH), 4.88 (d, J,,, = 13.2 Hz, l H , 0-CHZv3 Anth), 4.68 (m, 2H, OCHz-Anth and 2’H), 4.42 (m, l H , 3’H), 4.04 (m, l H , 4’H), 3.65 (m, 2H, 5’H,5”H); MS m l z ad bu 488.154 725 (M l)+ (calcd 488.157 008, diff = 2.3 mmu). R The purity of the compound was checked by normalphase HPLC. Ne-Benzoyl-2’-0-( anthraquinone-2-methy1)adenosine (2). The title compound was synthesized according to the general procedure of Ti et a1.(23). 2’-0-(An8 0 0 Adenine thraquinone-2-methy1)adenosine(840 mg, 1.72 mmol) was dried by coevaporation (3x) with dry pyridine and % H dissolved in 25 mL of dry pyridine. Trimethylsilyl chloride (TMS chloride, 1.67 mL, 12.04 mmol) was added to the reaction. The mixture was stirred for about 1 h and 1 mL (8.6 mmol) of benzoyl chloride was added. 2’-isomer After 5 min, 10 mL of 30% aqueous ammonia were added rial. More phosphorodiamidite (0.01 mL) was added, and and the reaction was stirred for 45 min. The solvent was the reaction was continued for 12 more h. The reaction evaporated to dryness, and the residue was suspended was diluted with saturated sodium bicarbonate (50 mL) in 25 mL of 1N HC1. The suspension was extracted with and extracted with dichloromethane (2x, 50 mL). Comethyl acetate (3x). Solvent was evaporated to yield an bined organic extracts were washed with brine (3x), dried oil. The oil was purified by silica gel chromatography over sodium sulfate, and concentrated in vacuo to obtain using a gradient of methanol (0-2%) in chloroform: yield a dry foam. The foam was purified by silica gel chroma62.8%;‘H NMR [(CD&SO] 6 11.12 (s, lH, NH), 8.68,8.66 tography using 20% hexane in chloroform containing 1% (29, 2H, HE and Hz), 8.31-7.49 (complex multiplets, 12 triethylamine: yield 500 mg (81.7%). 31P-NMRgave the H, Arj, 6.25 (d, J y , y = 5.8 Hz, lH, l’H), 5.53 (d, l H , ?OH), expected two signals (diastereoisomers) a t 150.3 and 5.18 (t, lH, 5’OH), 4.94 (d, J,,, = 13.3 Hz, l H , OCHz151.6 ppm relative to ortho phosphoric acid. Anth), 4.73 (m, 2H, OCHz-Anth and 2’H), 4.45 (m, l H , 3’H), 4.08 (m, lH, 4’H), 3.34-3.28 (m, 2H, 5’H, 5”H); MS RESULTS m l z 592.181 744 (M 1)-(calcd 562.183 223, diff = 1.5 mmu). Purity of the compound was checked by normalChemistry. Synthesis of 2’-O-(anthraquinone-2phase HPLC. methy1)adenosine was attempted by the sodium hydride W-Benzoyl-5’-0-( dimethoxytrityl)-2’-0-(anthra- procedure of Ts’o and colleagues (26). However, this approach led to a complex mixture, probably due to quinone-2-methy1)adenosine(3). The title compound miscellaneous reactions of the anthraquinone under was synthesized according to the procedure given by Wu et al. (24). NG-Benzoyl-2’-O-(anthraquinone-2-methyl)- strongly basic conditions. Consequently, 2’-O-(anthraquinone-2-methy1)adenosinewas synthesized using 2’,3’adenosine (640 mg, 1.08 mmol) was dried by coevapora(dibutylstanny1ene)adenosine as the starting material tion with pyridine and was dissolved in 25 mL of (20). The alkylation reaction proceeded very slowly even pyridine. Dimethoxytrityl chloride (4.14 mg, 1.5 mmol) a t high temperatures. The reaction rate was improved was added to the solution, and the reaction was allowed by adding cesium fluoride, which appears to act as a to stir for 12 h a t 4 “C. Thin-layer chromatography catalyst by forming a complex with the tin of 2‘,3‘indicated the presence of starting material; 100 mg of dimethoxytrityl chloride was added, and the reaction was (dibutylstanny1ene)adenosine; this complexation incontinued for 12 more h. Methanol (5 mL) was added to creases the nucleophilicity of the 2‘ and 3‘ hydroxyls. The yield of the reaction (Scheme 1)was approximately 19%. quench the reaction. The solvent was evaporated under Synthesis and Purification ofAnthra 12-mer. The vacuum, and the residue was dissolved in ethyl acetate 2’-0-(anthraquinone-2-methyl)adenosine was protected (50 mLj. The ethyl acetate layer was washed with 5% by standard procedures and incorporated into the anthra NaHC03 (3x), water (75 mL), and brine and dried over 12-mer defined above on a n automated DNA synthesizer. sodium sulfate. The product was purified by silica gel The coupling reaction using the modified nucleoside chromatography with a gradient of hexane in chloroform (20% to 0%). Triethylamine (0.1%) was added to the phosphoramidite was allowed to proceed for 10 min to ensure maximal coupling. The coupling yield was more solvent to prevent degradation of tritylated product: yield than 90% for the modified nucleoside as judged by 51%; IH NMR [(CD3)2SOl6 8.59 (2s, 2H, HZ and HE), dimethoxytrityl release. 8.32-6.79 (complex multiplets, 25 H, Ar) 6.29 (d, J I J ,= ~, 4.9 Hz, l H , l’H), 5.57 (d, l H , 3’OH), 4.96 (d, 12.8 Hz, The anthra 12-mer was purified by size exclusion chromatography. Since the size separation range for l H , OCHz-Anth),4.85 (m, 2H, OCHz-Anth and 2’H), 4.54 G-25 is 1000-3500 and the molecular weight of the (m, l H , 3’H), 4.20 (m, lH, 4‘Hj, 3.70 (s, 6H, -OCH3), 3.32 (m, 6H, 5’H, 5”H and HzO). anthra DNA was slightly above this range, G-50 (molec~-Benzoyl-5’-O-(dimethoxytrityl)-2’-0-(anthra-ular weight range 5000-10 000) was used for the purification. Adequate resolution was ensured by using a quinone-2-methy1)adenosine 3’-O-(CyanoethylNJVlong column length (100 x 2.5 cm), slow elution rate (20 diisopropylphosphoramidite) (4). The title comm u ) , and small fraction size. Initially, attempts were pound was synthesized according to the procedure given made to analyze the fractions by size exclusion HPLC. by Kierzek et al. (25). To /?-cyanoethyl N,N,N’,N’However, very broad peaks were obtained, suggesting the tetraisopropylphosphorodiamidite (0.20 mL, 1.4 mmol) in anthra 12-mer was equilibrating between two or more acetonitrile (15 mL) was added 3 (490 mg, 0.548 mmol) conformations a t room temperature in the buffer used and diisopropylammonium tetrazolide (467 mg, 0.27 for elution. Consequently, reversed-phase HPLC was mmol, dried in vacuo for 3 h). After 12 h, thin-layer used to analyze the fractions. chromatography indicated the presence of starting mate-
+
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ODN Containing 2'-O(Anthraquinone-2-methyl)ADO
Bioconjugate Chem., Vol. 6,No. 5, 1995 581 290,800
7
1
Peak 775.80 969.80
lntenalty 290,800 188,800
Charge 5.00000 4.00416
Mara Ertlmate 3884.04 3863.23
Flnal Esllmaled Mass: 3883.64 Std. Devlallon: 0.57 2 of 2 Esllmales Used.
0 500
600
700
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900
m 1000
1100
1200
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Figure 1. Electrospray mass spectrum for the anthra 12-mer
The presence of 2'-0-(anthraquinone-2-methyl)adenosine was confirmed by hydrolyzing the oligonucleotide with snake venom phosphodiesterase and bacterial alkaline phosphatase. The nucleosides were freed from the enzyme and injected on the HPLC. 2'-O-(Anthraquinone2-methylladenosine is very hydrophobic and is indefinitely retained on a column. Consequently, a c8 column was used to analyze the nucleosides. The nucleosides were eluted by 30% acetonitrile in potassium phosphate buffer (pH 4.0). The relatively high polarity of this system prohibited base-line separation of the normal nucleosides. The ratio of peak areas of 2'-0(anthraquinone-2-methy1)adenosine peak to the normal nucleoside cluster was 10.3:1, very close to the expected 11:l. The anthra 12-mer was subjected to electrospray mass spectral analysis to confirm the presence of all nucleotides. The electrospray mass spectrum shows multiple peaks for a single molecule since multiple charges are introduced in the ionization process. The mass spectrum for the anthra 12-mer is shown in Figure 1. The peak a t 775.8 m I z has five negative charges; the mass derived from this spectrum is 3883.2, whereas the calculated mass equals 3882.3. W Melting Studies. Melting temperature curves (T,) were taken in 0.1 M NaCl containing 10 mM phosphate buffer. The anthra 12-mer melted cooperatively a t approximately 48 "C compared to 41.6 "C for the standard 12-mer. Thus, incorporation of each anthraquinone moiety stabilized the duplex by 3 to 3.5 "C. The melting curve consisted of a single smooth transition as would be expected for formation of a single duplex M) conditions. structure under these very dilute (3 x Circular Dichroism (CD) Studies. CD studies were performed by dissolving the anthra 12-mer in 0.1 M NaCl containing 10 mM phosphate buffer. Figure 2 shows the CD curves for the anthra 12-mer a t various temperatures. The shape of the long wavelength maximum a t lower temperatures (35-45 "C) seems to imply the
presence of two peaks, one a t 290 nm and the second a t 280 nm, which are very poorly resolved. As temperature was increased, the first peak merged into the second. The CD spectrum a t 45 "C (near the T,) indicates the conformation of the anthra DNA is close to B-form. The presence of the unusual shape a t low temperatures may indicate the presence of multiple conformations at low temperature, consistent with both the HPLC data noted above and the NMR studies described below. As the temperature is increased the peak shape becomes that of normal B-DNA, indicating melting of these conformations. High Resolution NMR Studies. Alkylation of adenosine as described above gives rise to some 3'-O-aralkyl product as well as 1. In order to establish unequivocally that 1 is indeed 2'-substituted, a combination of 1D NMR with 2D COSY and NOESY was used. The 5'- and (presumed) 3'-OH signals formed an overlapped multiplet centered a t 6 5.45. COSY was used to establish the position of 2'-CH as 6 4.68 and 3'-CH as 6 4.42 by means of Hl'-H2' and H2'-H3' cross-peaks. Both COSY and NOESY spectra reveal strong cross-peaks with the OH doublet signal a t 6 5.45 and the 6 4.42 peak and no crosspeak with that a t 6 4.68, establishing unequivocally that the aralkyl substituent must be a t the C-2' oxygen. Oligonucleotideswere dissolved in 0.1 M NaCl containing 20 mM phosphate buffer (pH 6.8) a t a concentration of about 5 mM. The first experiment performed on the anthra 12-mer was observation of hydrogen-bonded imino protons. The 15 "C spectrum revealed more than the six hydrogen-bonded imino protons expected from the 2-fold symmetry of the duplex (data not shown), suggesting the presence of additional DNA conformers. As the temperature is increased to 25 "C, sharpening of the peaks is observed and some of the peaks disappear, consistent with melting of the less stable conformations. It was reasoned that the addition of magnesium ion might stabilize one conformer and simplify the NMR spectrum. Hence, the anthra 12-mer was dialyzed to
Deshmukh et al.
582 Bioconjugate Chem., Vol. 6,No. 5, 1995
200.0
3: -.-.
I: 2: 4:
a n t h 3~ 8 n t h 40
-
enth 4 5 a n t h 10
5: 8: 7:
400.0
---- 8 n t h ----
----
89 a n t h 60 a n t h 65
Figure 2. Circular dichroism (CD) spectra for the anthra 12-mer at various temperatures. remove all the salts and then redissolved in 20 mM phosphate buffer (pH 7) containing 0.1 M NaCl and 0.01 M MgC12; all subsequent studies were carried out in this medium. The anthra 12-mer was first subjected to imino proton analysis. Figure 3 shows imino proton spectra for the anthra DNA a t various temperatures. The predominant conformation (35-55 "C) shows only five hydrogenbonded imino protons instead of the six predicted for a palindromic duplex. It is likely this conformation is a duplex with the end base pair melted, which would explain the presence of only five imino protons. It is also plausible, albeit less likely, that the presence of anthraquinone is causing some conformational change in the molecule which would result in breaking one of the interior hydrogen bonds or that a hairpin with a two base-pair loop was the predominant conformer. To resolve these speculations and to determine whether intercalation occurs, the anthra 12-mer was subjected to more extensive NMR analysis. The one-dimensional spectrum of the anthra 12-mer was examined a t various temperatures. These spectra also show considerable overlap a t lower temperatures, confirming the presence of multiple conformations. The one-dimensional spectrum a t 55 "C (Figure 4, aromatic region) is well-resolved, probably indicating the presence of a single conformation. Since the best resolution was observed a t 55 "C, the two-dimensional analysis was performed a t this temperature. The higher temperature required to melt unwanted conformers relative to the T, observed by W and CD results from the far greater oligonucleotideconcentration required for NMR analysis. Two-Dimensional NMR Spectroscopy. The unmodified d(CGCACATGTGCG)duplex was characterized by a combination of NOESY and DQF-COSY NMR according to the standard procedure developed by Hare et al. (27). As expected, the data were fully consistent with B-DNA geometry; chemical shift data for aromatic, methyl, and selected sugar protons are presented in Table
_.-, . A
75
A
oc
65 O C
55
oc
45 o c
35 o c
25 OC
15
oc
5 oc 14.5
14.0
13.5
13.0
12.5
12.0
11.5
11.0
10.5
ppm
Figure 3. Imino proton spectra for the anthra 12-mer at various temperatures. These spectra were taken of the duplex (2.85 mM) in 99.996% DzO, 160 mM NaCl, 20 mM phosphate buffer (pH 71, 10 mM MgC12.
1. The anthra 12-mer also was studied in detail by twodimensional (NOESY and DQF-COSY) NMR spectroscopy. Figure 5 shows the expanded contour plot (aromaticH1' region) of the NOESY spectrum for the anthra 12mer. Most of the peaks are well resolved and were used for the sequential assignments given in Table 2. How-
Bioconjugate Chem., Vol. 6,No. 5, 1995 583
ODN Containing 2'-O(AnthraquinoneB-methyl)ADO
w Figure 4. Aromatic region of the anthra 12-mer.
-
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Figure 5. Expanded contour plot (aromatic-HI region) of the NOESY spectrum for the anthra 12-mer. Table 1. Nonexchangeable lH Chemical Shifts (ppm) of d(CGCACATGTGCGh in D20a
c1 G2 c3 A4 c5 A6 T7 G8 T9 G10
c11
G12
H1'
H8/H6
H5lmethyl
5.43 5.59 5.27 5.91 5.23 5.89 5.48 5.61 5.51 5.53 5.43 5.44
7.34 7.67 7.09 7.98 7.00 7.92 6.84 7.48 6.88 7.60 7.07 7.65
5.57 5.14 5.01 1.05
1.00
5.12
H2'i"" 2.11, 1.68 2.42,2.09 2.09, 1.75 2.62, 2.40 2.10, 1.75 2.63, 2.30 2.16, 1.80 2.44, 2.25 2.16, 1.77 2.36, 2.32b 2.04, 1.62 2.34, 2.05
The solution was ?? M in ODN prepared in 99.996% DzO containing 0.10 M NaCl and 0.02 M phosphate buffer (pH 7.0). Values are estimates because of severe peak overlap.
ever, the intramolecular H8-H1' connectivity of A4 is very weak, and the H8-H1' connectivity of G8 is missing. These missing or weak connectivities are best explained on the basis of intercalation.
Table 2. Nonexchangeable lH Chemical Shifts (ppm) of d(CGCA*CATGTGCG)zwhere A* = 2'-O-(Anthraquinone-2-methyl)adenosine H1' H8M6 H5/methyl 6.07 c1 5.98 7.77 5.98 8.07 G2 7.27 5.43 c3 5.74 A4 5.75 8.17 c5 5.32 7.67 5.79 A6 6.33 8.42 1.48 T7 5.71 7.00 G8 5.93 7.84 2.00 T9 5.94 7.59 G10 6.01 8.04 5.60 6.02 7.52 c11 G12 6.03 8.10
H2'/H2" 2.54, 2.07 2.81. 2.77 2.43; 1.79 4.81 2.56, 2.40 2.96, 2.82 2.08, 1.53 2.83, 2.69 2.68, 2.35 2.82, 2.72 2.51; 2.06 2.77, 2.57
Figure 6 shows the molecular stereo model for the intercalation of the anthraquinone ring. One can easily observe that the intercalation of the anthraquinone ring has expanded the space between A4 and C5 as well as between G8 and T9. The intercalation of the anthraquinone inside the helix forces the sugars of A4 and G8 to
Deshmukh et al.
584 Bioconjugate Chem., Vol. 6,No. 5, 1995
--
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Figure 6. Stereo view of the anthra 12-mer. One-half the palindromic dodecamer is shown with the anthraquinone intercalated between the A4-T9 and C5-G8 base pairs. 2 . (pp"
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Figure 8. Expanded contour plot (Hl'-Hz' region) of the DQF-COSY spectrum for the anthra 12-mer.
move away from the bases, accounting for the loss of connectivities between sugar and aromatic protons in the cases of A4 and G8. The aromatic-H1' region provides more evidence for intercalation. The cross-peaks a t 7.27 and 7.37 ppm (marked by asterisks in Figure 5) show connectivity between protons of the anthraquinone ring and H1' of A4. A cross-peak observed a t 7.36 ppm represents through-space connectivity between aromatic protons of anthraquinone and H1' of T9, confirming that the anthraquinone ring stacks directly below the AT base pair.
Additional evidence for intercalation comes from the aromatic H2' region of the NOESY spectrum (Figure 7). This region shows complete absence of cross-peaks between H2' and H2" of G8 with its own H8. It also shows aromatic methyl cross-peaks for thymidines. The aromatic methyl group normally shows connectivities with its own H6 and H8/H6 of the preceding base; these cross-peaks are clearly visible in the aromatic-methyl region of the standard 12-mer duplex (data not shown). The T7-methyl in the anthra oligo shows clear connectivity between its own H6 and H8 of A6. However, the
ODN Containing 2’-O(AnthraquinoneB-methyl)ADO
methyl group of T9 fails to show through-space connectivity with H8 of G8. Two-dimensional NMR studies also provide important information about the sugar conformation of 2’-0-(anthraquinone-2-methy1)adenosinein the duplex. Figure 8 shows the expanded contour plot of the DQF-COSY spectrum for anthra DNA. The label indicates the crosspeak between H1’ and H2’ of A4. If the C3’-endo sugar conformation characteristic of ribonucleotides in RNA helices (28)or RNA-DNA hybrids (29) was found in A4, J1,,2, would be undetectably small. Clearly, the observed cross-peak, albeit less intense than some of the deoxynucleotide signals, confirms a sugar conformational shift toward C2’-endo. This sugar conformation is required to enable intercalation of the anthraquinone ring below A4, as if it entered the helix through the major groove. Molecular modeling studies are in agreement with this observation. The entry of the anthraquinone from the major groove explains the chemical shifts of anthraquinone. The proton a t the 1 position on anthraquinone exhibited a chemical shift of 6 7.26, considerably upfield despite its proximity to the oxygen present in the center ring of the anthraquinone. This proton is completely within the shielding cone of adenine, whereas the other two (H3 and H4) are on the periphery. DISCUSSION
The two-dimensional NMR studies described above conclusively prove intercalation of the anthraquinone ring. Thus, the stabilization provided by the anthraquinone is a result of intercalation and not hydrogen bonding between anthraquinone oxygen and amino group(s) in the minor groove. Intercalation forces the sugar conformation of 2’-O-(anthraquinone-2-methyl)adenosine toward C2’-endo. Although this is normally an unfavorable conformation for a ribonucleoside, the energy lost due to this conformational change must be significantly less than that gained by intercalation. These studies establish that even a methylene linker is adequate for inserting the intercalating agent inside the helix. They also show that the structure of the group conjugated a t the 2’-hydroxyl plays a very important role in duplex stability. These studies are in agreement with Yamana et al. (20) in observing stabilization of the DNA duplex. However, the stabilization is considerably less than that observed by Yamana et al. The shape of the CD curve observed by Yamana et al. is quite different than that exhibited by the standard B-form DNA. The shape of the CD spectrum depends upon the sequence of the oligonucleotide (301, which may explain the difference. We clearly demonstrate intercalation using two-dimensional NMR,whereas Yamana et al. based their conclusions on a narrow region of the CD curve (300-400 nm). The CD studies of Yamana et al. were interpreted as reflecting global changes in the geometry of the duplex. However, the CD, NMR and molecular modeling data presented here are consistent with a conformation altered only locally by intercalation of the planar anthraquinone system a t temperatures high enough to eliminate nonspecific complex interactions while retaining a duplex structure. The presence of five exchangeable imino proton signals a t 55 “C also strongly supports minimal disruption of B-DNA geometry, since the terminal G-C base pairs will certainly be frayed with loss of signal for that imino proton. Thus, site-specific intercalation into a B-DNA duplex can be achieved by linking a planar hydrophobic functional group to the 2’-OH of an internally situated (or, presumably, 3‘ or 5’-terminal) ribonucleoside. Since the energetics of the system lead to a ribose sugar confor-
Bioconjugate Chem., Vol. 6,No. 5, 1995 585
mational change approaching a B-DNA-like sugar pucker, the overall B-DNA geometry is only minimally affected. It will be of considerable interest to apply this approach to the study of DNA-RNA hybrids, which tend to assume a global conformation resembling A-type geometry. Those studies are in progress and will be reported elsewhere. ACKNOWLEDGMENT
These studies were supported in part by NIH grant R01 AI27692 and by a research fellowship (H.M.D.) from the University of Utah Research Committee. Facility support (mass spectrometry, NMR, and DNA synthesis) was partially provided through the Cancer Center grant IP30 CA42014. The authors are very grateful to Dr. Darrell R. Davis for many helpful discussions. LITERATURE CITED (1) Beaucage, S. L., and Iyer, R. P. (1993) The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 49, 6123-6194. (2) Uhlmann, E., and Peyman, A. (1990) Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90, 543-584. (3) Beaucage, S. L., and Iyer, R. P. (1993) The synthesis of specific ribonucleotides and unrelated phosphorylated biomolecules by the phosphoramidite method. Tetrahedron 49, 10441-10488. (4) Tonkinson, J. L., and Stein, C. A. (1993) Antisense nucleic acids-Prospects for antiviral intervention. Antiviral Chem. Chemother. 4, 193-200. (5) Stein, C. A., and Cheng, Y.-C. (1993) Antisense oligonucleotides as therapeutic agents-Is the bullet really magical? Science 261, 1004-1012. ( 6 ) Beaucage, S.L., and Iyer, R. P. (1992) Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48, 2223-2311. (7) Zamecnik, P., and Stephenson, M. (1978) Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sei. U.S.A. 75, 280284. (8) Milligan, J. F., Matteucci, M. D., and Martin, J. C. (1993) Current concepts in antisense drug design. J . Med. Chem. 36, 1923-1937. (9) Sun, J.-S.,Francois, J.-C., Montenay-Garestier, T., SaisonBehmoaras, T., Roig, V., Thuong, N. T., and Helene, C. (1989) Sequence-specific intercalating agents: Intercalation a t specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates. Proc. Natl. Acad. Sci. U S A . 86, 9198-9202. (10) Mann, J. S., Shibata, Y., and Meehan, T. (1992) Synthesis and properties of a n oligodeoxynucleotide modified with a pyrene derivative at the 5‘-phosphate. Bioconjugate Chem. 3, 554-558. (11) Iverson, B. L., and Dervan, P. B. (1987) Nonenzymatic sequence-specific cleavage of single-stranded DNA to nucleotide resolution. DNA methyl thioether probes. J . Am. Chem. SOC.109, 1241-1243. (12) Boidot-Forget, M., Chassignol, M., Takasugi, M., Thuong, N. T., Helene, C. (1988) Site-specific cleavage of singlestranded and double-stranded DNA sequences by oligodeoxyribonucleotides covalently linked to a n intercalating agent and an EDTA-Fe chelate. Gene 72, 361-371. (13) Lin, S.-B., Blake, K. R., Miller, P. S., and Ts’o, P. 0. P. (1989) Use of EDTA derivatization to characterized interactions between oligodeoxyribonucleoside methylphosphonates and nucleic acids. Biochemistry 28, 1054- 1061. (14) Vlassov, V. V., Gaidamakov, S. A., Zarytova, V. F., Knorre, D. G., Levina, A. S., Nikonova, A. A., Podust, L. M., and Fedorova, 0. S. (1988) Sequence-specific chemical modification of double-stranded DNA with alkylating oligodeoxyribonucleotide derivatives. Gene 72, 313-322. (15) Boutorin, A. S., Gus’kova, L. V., Imanova, E. M., Kobetz, N. D., Zarytova, V. F., Ryte, A. S., Yurchenko, L. V., and
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