Efficient Conjugation and Characterization of Distamycin-Based

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Bioconjugate Chem. 2004, 15, 520−529

Efficient Conjugation and Characterization of Distamycin-Based Peptides with Selected Oligonucleotide Stretches Sumana Ghosh,† Eric Defrancq,‡ Jean H. Lhomme,† Pascal Dumy,‡ and Santanu Bhattacharya*,† Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India and LEDSS, UMR CNRS 5616, Universite´ Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France. Received September 21, 2003; Revised Manuscript Received December 10, 2003

Selected sequences of oligodeoxyribonucleotides (ODNs) have been conjugated efficiently with distamycin-based peptides containing reactive cysteine and oxyamine functionalities at the C-terminus. The conjugation was performed easily within 30-60 min, using individual modified oligonucleotide stretches having sequences of 5′-d(GCTTTTTTCG)-3′, 5′-d(GCTATATACG)-3′, and 5′-AGCGCGCGCA3′. Two types of linkages were used for making the covalent connection: (i) a five-membered thiazolidine ring and (ii) an oxime. These distamycin-like polyamide-ODN conjugates were then converted to the corresponding DNA duplexes using complementary oligonucleotide sequences. To elucidate the binding specificity of the distamycin-oligonucleotide conjugates, UV-melting temperature measurements were performed. These studies indicated that the distamycin-ODN conjugate favored binding with the duplex with sequence 5′-d(GCTTTTTTCG)-3′ rather than 5′-d(GCTATATACG)-3′. On the other hand, no stabilization of the duplex with sequence 5′-d(AGCGCGCGCA)-3′ was observed. UV results also suggest that the thiazolidine and oxime linkages do not significantly influence the process of distamycin binding to the minor groove surface of the DNA duplex. The results obtained from duplex UV-melting studies were further corroborated by a temperature-dependent study of the circular dichroism spectra of the conjugates and a fluorescence displacement titration assay using Hoechst 33258 fluorophore as a competitive binder for the minor groove. All these studies reinforce the fact that the specific stabilization of A/T rich DNA-DNA duplexes by distamycin was preserved upon conjugation with oligonucleotide stretches.

INTRODUCTION

The complete sequencing of the human genome has a tremendous impact on biological and biomedical research (1). The intricate and precise regulation of the expression of various genes encoded in the DNA nucleotide sequence was achieved by influencing transcription (using groove binder, intercalator, or antigene strategy), translation (antisense strategy), or inhibiting the function of proteins (aptamer strategy) (2-6). Among all of these, the crucial factor involves reading and recognition of specific nucleotide bases on DNA. This specificity is important for the synthesis of any drug or inhibitor that can mimic the function of DNA binding proteins or enzymes. Among the naturally occurring drugs with low molecular mass, distamycin and netropsin are well-known. They bind selectively to A/T rich sequences of the B-DNA minor groove (7-11). Hydrogen donor groups located on the concave side of these molecules establish contacts with the hydrogen bond acceptor atoms such as N-3 nitrogen of adenine and C-2 carbonyl oxygen of thymine bases in the minor groove of DNA (12). Such oligopeptides, by virtue of their crescent shape, adopt nearly isohelical orientation with the B-form of the duplex. Functional groups that protrude into the minor groove, such as the 2-amino group of dG-dC base pairs, (13, 14) * Corresponding author and Swarnajayanti Fellow (DST, Government of India). E-mail: [email protected]. Also at the Chemical Biology Unit, JNCASR, Bangalore 560 012, India. Fax: +91-080-3600529. † Indian Institute of Science. ‡ LEDSS.

interfere with optimal binding and give rise to the noted sequence preferences. Tight binding within the minor groove by these ligands typically results in increased duplex stability (15). By combining the minor groove-based selectivity with the Hoogsteen binding of an ODN in the major groove, a variety of groove-binding agents have been tethered to DNA sequences to further improve the anti-sense and anti-gene activities and to achieve greater stabilization of the duplex and triplex structures (16-25). It has been found that the affinity of some of these conjugates for the target site is enhanced by over 100-fold than that of the unlinked subunits (26). In an early communication (24), the stabilization of certain DNA/DNA duplexes by N-methylpyrrolecarboxamide peptide-tethered ODN has been reported. These derivatives were required to possess a spacer at the C-terminal tagged with a 2-[[4-(phenylazo)benzyl]thio]ethyl dye to facilitate the monitoring of the covalent coupling between the peptide and ODN. It was not ascertained how the presence of such an interfering spacer affected the DNA recognition event. Moreover, the synthesis of these molecules was never described. Unfortunately, however, the methods of such tethering are often not available and sometimes not reproducible. Therefore, there is a necessity to develop efficient procedures for conjugation. In recent years, new strategies for the conjugation of oligonucleotide with a reporter group have focused on the utility of the oxime linkage. This linkage has been used efficiently for the chemical ligation of peptides (27, 28), conjugation of peptides with carbohydrates (29-31), and oligonucleotides (32-34).

10.1021/bc0341730 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/12/2004

Conjugation of Distamycin-Based Peptides with Oligonucleotides

Bioconjugate Chem., Vol. 15, No. 3, 2004 521

Scheme 1. Synthesis of Distamycin Analogues 7 and 9a

a Reagents: (a) (Boc) O, K CO , H O/dioxane (1:1, v:v), rt, 48 h, 60%; (b) HOSu, DCC, DMF, 0 °C for 1 h, then rt for 6 h, 80%; (c) 2 2 3 2 3, CHCl3, rt., 2 h, 50%; (d) CH2Cl2, TFA, EDT (1:1:0.2, v:v:v), rt, 2 h, 80%; (e) 4, CHCl3, rt., 2 h, 50%; (f) CH2Cl2:TFA (1:1, v:v), rt, 2 h, 80%.

The major advantage of this ligation technique is that it requires neither a coupling reagent nor chemical manipulations except mixing of the two components, namely the oxyamine and the aldehyde moieties. Adopting this highly chemoselective ligation procedure, we have now accomplished a convenient and efficient synthesis of five novel distamycin-ODN conjugates. In these conjugates, the C-terminus of a distamycin derivative (lacking the N-methylformamide unit) has been covalently attached to the 5′-end of selected ODN stretches. We describe herein the method of conjugation in detail. We also discuss the results of investigation on the specificity of distamycin binding and the duplex stabilizing properties resulting from the hybridization of these ODN-distamycin conjugates to sequences of appropriate ODN stretches by UV-melting temperature measurements, temperature-dependent circular dichroism studies, and a fluorescence displacement assay using Hoechst 33258 as a minor groove competitor. RESULTS AND DISCUSSION

Synthesis of Distamycin-Oligopeptide Derivatives 7 and 9 (Scheme 1). The N-methylpyrrole-based tetrapeptide 5 containing a free amine at the C-terminus represents the key intermediate for the introduction of

the 1,2-aminothiol and the oxyamine moieties. The tetrapeptide 5 was synthesized in solution phase as described previously (35, 36). Covalent attachment of the 1,2-aminothiol moiety to 5 was achieved upon coupling with the N-hydroxysuccinimide-activated ester of the cysteine derivative 3, in which the amino terminus and the thiol residue were protected with a t-Boc group. Deprotection of the t-Boc group was carried out by treating 6 with TFA/CH2Cl2 solution (1:1, v/v) at room temperature for 1 h. To prevent the oxidation of thiol to the corresponding disulfide, 2% dithiotheritol (DTT) was also included in the solution. Introduction of the oxyamino group to the oligopeptide was performed via the coupling of the activated ester of N-t-Boc-O-(carboxymethyl)-hydroxylamine 4 with the tetrapeptide 5. Subsequent acidic treatment with TFA/CH2Cl2 solution (1:1, v/v) for 1 h at room temperature of the protected intermediate 8 led to distamycin analogue 9. The distamycin analogues 7 and 9 containing cysteine and oxyamine functionalities, respectively, were then used for conjugation with ODN stretches without further purification. Oligonucleotides Synthesis. Aldehyde-Containing Oligonucleotides. The preparation of the oligonucleotides containing an aldehyde linker at the 5′-extremity was

522 Bioconjugate Chem., Vol. 15, No. 3, 2004

Ghosh et al.

Scheme 2. Synthesis of Different Oligonucleotides 13a,b and 14a-ca

a Reagents: (a) Incorporation of phosphoramidite 10 at the last stage of the oligonucleotide synthesis and then NH OH, 50 °C for 4 16 h; (b) aq 80% AcOH; (c) polymer-supported NaIO4, rt., 1 h; (d) 7, CH3COONa buffer (0.2 M, pH 5.4), CH3CN, 1 h, rt.; (e) 9, CH3COONa buffer (0.2 M, pH 5.4), CH3CN, 30 min, rt.

accomplished upon minor modification of a previously reported procedure (32). Briefly, the strategy included the preparation of the oligonucleotides 11a-c containing the 1,2-diol moiety using the corresponding phosphoramidite 10 (Scheme 2). Peroxidation of the diol upon reaction with 10-fold excess of polymer-supported NaIO4 for 1 h gave the corresponding aldehydes 12a-c. Subsequent conjugations were carried out without further purification of the aldehyde in the case of the preparation of the oxime conjugate. However, for the synthesis of the thiazolidine conjugate, it was necessary to purify the aldehyde from excess of periodate by reverse phase chromatography to avoid thiol oxidation (33). Conjugation via Thiazolidine and Oxime Linkage. The ligation involved the reaction between aldehyde-containing ODN at the 5′-terminus with a 1,2-aminothiol for thiazolidine formation and with an oxyamino moiety for oxime bond formation as depicted in Figure 1. For both type of linkers, the conjugates were obtained in good yields under mild conditions within 1 h and without the need for a protection/deprotection step. Thiazolidine conjugation was carried out by reacting the peptide 7 with either of the oligonucleotides 12a or

Figure 1. Strategy of efficient and facile synthesis of distamycin-ODN conjugates by using oxime and thiazolidine linkages.

12b at room temperature in sodium acetate buffer (0.2 M, pH ) 5.4). The progress of the reaction was followed by C18 reverse-phase HPLC. The reaction was completed in 1 h when 4 equiv of distamycin derivative 7 were used. Figure 2A shows the evidence of clear separation of the conjugate from the crude mixture containing 12a and 7. After purification by RP-HPLC, the conjugates 13a,b were obtained in ∼20-30% isolated yields. Formations of 13a,b were confirmed by ESI-MS analysis (Table 1).



Figure 2. HPLC profiles (detection at 260 nm) of the crude reaction mixture, after 30 min of reaction of the peptide 7 (thiazolidine conjugation) (A) and 9 (oxime conjugation) (B) with the corresponding aldehyde-containing ODN 12a. (A) Retention times 2.2, 15.3, and 16.6 min correspond to 7, modified ODN 12a, and the thiazolidine-conjugated distamycin-ODN 13a respectively. (B) Retention times 3.3, 16.1 min correspond to 9 and the N-oxime-conjugated distamycin-ODN 14a, respectively. Modified ODN 12a has already been consumed within 30 min of the reaction with the peptide 9. Table 1. Analytical Data for Peptides 7 and 9, Oligonucleotides 11a-c and 15a-c, and Conjugates 13a,b and 14a-c

Table 2. Melting Temperaturesa Determined at Different Concentrations of Sodium Chloride

compounda

tRb (min)

massc (calcd)

massc (found)

duplex sequences

7 9 5′-XGCTTTTTTCG- 3′ 11a 5′-XGCATATATCG-3′ 11b 5′-XAGCGCGCGCA-3′ 11c 5′-dis-th-GCTTTTTTCG-3′ 13a 5′-dis-th-GCATATATCG-3′ 13b 5′-dis-ox-GCTTTTTTCG-3′ 14a 5′-dis-ox-GCATATATCG-3′ 14b 5′-dis-ox-AGCGCGCGCA-3′ 14c 3′-CGAAAAAAGC-5′ 15a 3′-CGTATATAGC-5′ 15b 3′-TCGCGCGCGT-5′ 15c

2.1 3.5 10.3 13.4 11.4 16.0 16.3 16.9 17.1 16.5 11.8 10.2 14.8

694.8 663.7 3196.02 3222.06 3234.05 3840.02 3866.06 3809.02 3835.06 3848.05 3054.1 3027.06 3020.02

694.2 664.3 3195.4 3222.1 3232.9 3838.9 3866.6 3811.1 3835.8 3847.7 3054.0 3026.6 3019.3

11a/15a 13a/15a 11a/15a + 7 11b/15b 13b/15b 11b/15b + 7 14a/15a 14b/15b 14c/15cc 11c/15c

a X ) 5′-diol linker, dis ) distamycin analogue, ‘th’ ) thiazolidine linkage, ‘ox’ ) oxime linkage. b Retention time (tR) by analytical C18 reverse-phase HPLC using a gradient of 0 to 30% of acetonitrile over 20 min, at a flow rate of 1 mL min-1. c Electrospray ionization MS.

Oxime conjugation reaction was similarly performed by reacting the oligonucleotides 12a-c with the peptide 9 at room temperature in sodium acetate buffer (0.2 M, pH ) 5.4). This reaction was found to be quite rapid and was completed within ∼30 min when 4 equiv of peptide 9 was used. Figure 2B shows the HPLC profile of the crude reaction mixture with oligonucleotide 12a. The yields after purification by RP-HPLC ranged from 38 to 48% and each of the conjugates 14a-c were characterized by ESI-MS. In all cases, the experimentally determined molecular weights were in excellent agreement with the calculated values (Table 1). Although in principle, both thiazolidine- and oximebased oligopeptide-linked ODN should have two diastereomeric forms, but they could not be identified as two separate peaks during RP-HPLC purification process. The conjugated products (13a,b, 14a-c) exhibited UVvis spectra having characteristics of both DNA (λmax ) 260 nm) and the distamycin (λmax ) 316 nm) band. Duplex-Stabilizing Properties. Melting Temperature Analysis of Hybridization with a Target Oligonucleotide Stretch (Table 2). The hybridization properties of the conjugates 13a,b and 14a-c were investigated by UV-melting (thermal denaturation) temperature measurements (Tm). The study was carried out with the following objectives. First, to evaluate the influence of covalent coupling of distamycin-like polyamide with the

Tm Tm ∆Tm ∆Tm ∆Tm Tm (°C), (°C), (°C), (°C), (°C), (°C), 0.04 Mb 0.1 Mb 0.5 Mb 0.04 Mb 0.1 Mb 0.5 Mb 35.7 47.2 41.6 34.8 39.2 37.8 46.6 40.1 63.9 64.3

40.0 51.1 46.5 38.3 43.8 41.3 51.4 43.0 -----

46.5 54.6 50.3 44.1 47.7 44.8 59.4 48.2 -----

--11.5 5.9 --4.4 3.0 10.9 5.3 0.3 ---

--11.1 6.5 --5.5 3.0 11.4 4.7 -----

--8.1 3.8 --3.6 0.7 12.9 4.1 -----

a Optical melts were generated in 0.01 M sodium phosphate buffer/0.001 M EDTA containing the relevant concentrations of NaCl, pH 7.4, with [duplex] ) [7] ) 9.31 × 10-6 M. ∆Tm correspond to the difference in Tm between the modified and unmodified duplex. b NaCl concentrations (40, 100, 500 mM). c Duplex melting was measured only at 0.04 M NaCl concentration.

oligonucleotides and second, to examine the effects of the two kinds of linkages used to tether distamycin with ODN stretches and also their impact on the stability of the corresponding duplex oligonucleotides. The thiazolidine conjugates 13a,b and the oxime conjugates 14a-c were individually hybridized with the corresponding complementary oligonucleotide sequences, 15a-c 3′-d(CGAAAAAAGC)-5′, 3′-d(CGATATATGC)-5′, and 3′-d(TCGCGCGCGT)-5′, respectively. The melting temperatures of the resulting duplexes were determined by following changes in absorbance at 260 nm as a function of temperature from 20 to 80 °C. There was no hysteresis observed during heating and cooling processes of the duplex melting, suggesting a reversible binding process of tethered distamycin to the minor groove surface of duplex. The ODNs, 11a-c, containing the diol linker at the 5′-ends were also compared. Furthermore, the effect of free distamycin analogue was verified with the corresponding unconjugated duplex. It was found that distamycin-conjugated oligonucleotides with A/T rich sequences (13a,b and 14a,b) were significantly stabilized in their duplex forms. In contrast, the distamycin-linked ODNs possessing alternating G/C base pairs (14c) did not enhance the duplex melting temperature. This suggests that the hybridization of the DNA-distamycin conjugates with the target sequences generates a DNA minor groove surface and facilitates the binding event by the tethered distamycin unit. Besides, continuous


stretches of A/T (13a and 14a) were stabilized to a greater extent than their counterparts with alternating AT base pairs (13b and 14b). Earlier studies based on NMR and chemical nucleasemediated footprinting experiments suggest that distamycin induces a ‘collapse’ of a normal minor groove into a narrower groove (37, 38). This should enhance the hydrogen bonding and van der Waals interactions. Such deep penetration of the drug to the walls of the minor groove in turn should favor enthalpic contribution for the binding. The ATAT sequence in the decamer d(5′-GCTATATACG-3′)2 has a low intrinsic propeller twist angle, which may not facilitate the formation of a narrower minor groove for favorable binding of the distamycin (37). Thus the readjustment of the backbone in case of alternating A/T stretches would be difficult. This may account for the low affinity of ODN-conjugated distamycin to the alternating A/T sequence in comparison to that of a contiguous A or T sequence. The enhancement of melting temperature (∆Tm) was almost comparable for both thiazolidine- and N-oximelinked distamycin conjugates. This suggests that the nature of linker (five-membered thiazolidine ring vs linear oxime bond) does not significantly influence and contribute to the duplex stabilization. The stabilization upon duplex formation between a complementary oligonucleotide sequence with the oligonucleotide-conjugated distamycin was also compared with respect to free distamycin binding to the corresponding DNA duplex. In each case, the stabilization was found to be more pronounced for the covalently conjugated distamycin than that of the free duplex. Effect of Salt on Hybridization to the Target Oligonucleotides. Most of the ligands including distamycin or netropsin that bind to DNA are cationic. Therefore, experiments at varying salt concentrations provide useful information pertaining to the relative importance of nonspecific (electrostatic) and specific (hydrogen bonding, van der Waals interactions) factors on the DNA recognition process. Salt-induced ligand-DNA complex dissociation experiments are often useful for comparing the relative affinities and mode of ligand binding toward DNA (39). Generally for compounds with similar binding modes, the higher the binding affinity, the lower should be the dissociation of the ligand-DNA complex with increasing salt concentration. Additionally, DNA is known to undergo structural transition in the presence of high salt concentration (40, 41). Strong precomplexation of a ligand with DNA should also resist such structural transition. Duplex melting was examined at various NaCl concentrations (40, 100, 500 mM) to analyze the electrostatic effect of distamycin binding on Tm enhancement. It was observed that on increase in salt concentration, the Tm value for a given duplex was increased (Table 2). This is in agreement with known salt effects on polyelectrolyte solutions, explained on the basis of the counterion condensation theory (42-45). According to this concept, an increase in bulk salt concentration should lead to the stabilization of the duplex state of DNA as having a higher charge density as compared with the singlestranded form. This would result in an increase in Tm for the helix-to-coil transition. However, at high salt concentration such as in 500 mM, although the Tm increased to the highest extent, the ∆Tm value was found to be less compared to the ∆Tm in low salt conditions (40, 100 mM) in case of both free and covalently conjugated distamycin except for 14a/15a duplex. This fact is correlated with the energetic contribution (large negative

Ghosh et al.

enthalpy and positive entropy value) in the distamycinDNA binding process described earlier (46, 47). The favorable entropic gain arises because of the release of ordered water molecules from the hydration shell of the DNA backbone during any ligand binding. At high salt concentration, the release of water molecules is expected to be less. This in turn would reduce the entropy gain as well as the binding energy. This may result in low ∆Tm value at higher salt concentration. Thus, for the peptideODN conjugates, there may be two opposite influences on the Tm of duplex formation as the concentration of salt increases, namely increased stability of the doublestranded DNA region and decreased association of the peptide portion with the oligonucleotide target. CD Spectral Analysis of the Distamycin-ODN Conjugates. Considerable information on the binding of distamycin to nucleic acid duplex has been obtained from circular dichroism measurements (48, 49). Since free distamycin does not exhibit optical activity and is therefore CD-silent. However, its binding to double-stranded nucleic acid induces a CD signal that can be used to monitor the binding process. In the present case, the conjugates 13a,b and 14a-c alone exhibited two positive peaks at 280 nm (DNA-CD) and 334 nm (distamycin-CD) that were characteristics of the single-stranded ODN and conjugated distamycin moiety, respectively. Efficient intraduplex binding of the covalently attached distamycin to the minor groove of the duplexes (13a/15a, 13b/15b) intensifies the magnitude of molar ellipticity at 334 nm compared to their corresponding unmodified duplexes (11a/15a, 11b/15b) bound with free distamycin. Between these two duplexes (13a/15a, 13b/15b), the former one (with continuous A/T stretch) showed a more intense CD band than the latter (with alternating A/T stretch). These results are consistent with the data obtained from the UV-melting temperature study, suggesting that covalently conjugated distamycin stabilizes the duplex ODN more than the ODN bound noncovalently with distamycin, and it has greater sequence selectivity toward the continuous stretch of A/T rich tract than the one with alternating A/T stretch. The nature and specificity of distamycin binding to the minor groove surface of different A/T stretch DNA duplex was further examined by duplex melting at 334 nm (distamycin-CD) as a function of temperature from 20 to 80 °C (Figure 3). The sequence specificity and mode of distamycin binding to the different ODN stretches were analyzed from the peak height and the magnitude of the peak minima of such derivative plot of d(CD)/dT vs T at 334 nm. The CD intensity (peak height) at 334 nm was higher in both ODN-distamycin conjugates (e.g. 14a/ 15a) than the one with unmodified duplex (e.g. 11a/15a) containing free distamycin. This might be due to a favorable binding of the covalently attached distamycin to an ODN stretch, owing to its higher effective concentration at the DNA minor groove. Moreover, the peak height was higher in case of continuous A/T stretch than alternating A/T sequence. Magnitude of peak minima was almost same for continuous A/T stretch duplexes (14a/ 15a and 11a/15a + 9). In this case, it is possible that more efficient binding of distamycin to a continuous A/T tract (as in 11a/15a) probably does not allow any further conformational change of distamycin in ODN irrespective of whether it is covalently conjugated with the distamycin or not. This is in contrast to what was observed with the ODN bearing alternating A/T tract, where peak magnitude was differed by ca. 4 °C between (14b/15b and 11b/ 15b + 9). It may be that covalent attachment of distamycin induced a conformational change of ODN that


Figure 3. First derivative plot of molar ellipticity vs temperature during duplex melting at 334 nm. CD melting curves of the duplexes, 5′-dis-ox-GCATATATCG-3′ 14b/3′-CGTATATAGC-5′ 15b (a, 9), 5′-XGCATATATCG-3′ 11b/3′-CGTATATAGC-5′ 15b + 9 (b, 2) 5′-XGCTTTTTTCG-3′ 11a/3′-CGAAAAAAGC-5′ 15a + 9 (c, [), 5′-dis-ox-GCTTTTTTCG-3′ 14a/3′CGAAAAAAGC-5′ 15a (d, 1) with change in temperature at the rate of 1 °C/min from 20 to 80 °C at 334 nm. In each case CD spectra were recorded in 0.01 M Na2HPO4 buffer, 0.04 M NaCl, 0.001 M EDTA, pH 7.4, with [duplex] ) [9] ) 1.3 × 10-5 M. (X ) diol-modified oligonucleotide, dis ) distamycin, and ‘ox’ corresponds to oxime-conjugated ODN-distamycin hybrid).

enhanced hydrogen-bonding interaction at its minor groove surface compared to the unmodified ODN where distamycin was not covalently attached. It had been observed that the equilibrium between free distamycin and DNA in the binding process was affected by addition of salt to the preformed noncovalent distamycin-DNA complex. This complex dissociation process as a function of salt concentration can be followed by examining the changes in magnitude and the nature of the CD band at 334 nm. A gradual decrease in the CD band at 334 nm with a minute change at 280 nm was observed for 5, noncovalently bound to duplex with increasing salt concentration up to ca. 1 M (50). But no significant change in the corresponding bands at 280 and 334 nm was observed for 14a/15a and 14b/15b duplexes. In both cases, addition of salt (NaCl, up to ca. 1 M) to the preformed distamycin-DNA complex and also the formation of such complexes under different salt concentrations did not lead to any significant change in the 334 nm CD band. Therefore, high salt concentration could not dissociate the tightly bound, covalently attached distamycin arm from the DNA surface, preserving the structural integrity of both the DNA and the distamycin moiety. Competition with Hoechst 33258 Binding. In this study Hoechst 33258 fluorophore was used as a minor groove competitor, which has the same sequence selectivity but a higher DNA binding constant (ca. 108 mol-1) (51) than that of distamycin (ca. 105 to 106 mol-1). To determine the accessibility of strong groove binding ligands such as Hoechst 33258 to the minor groove of the distamycin-ODN conjugates, we performed fluorescence titration using the above probe. For this purpose, we compared the binding of Hoechst 33258 with unmodified ODN in the presence and absence of distamycin and also with the corresponding distamycin-ODN conjugates having two different A/T stretches. The fluorescence intensity of Hoechst 33258 is very low in its free state in solution. However, the emission due to Hoechst 33258 increases dramatically upon binding to duplex DNA (52). Fluorescence titrations were per-


Figure 4. Fluorescence titration of corresponding duplexes with minor groove competitor Hoechst 33258, 5′-XGCTTTTTTCG3′ 11a/3′-CGAAAAAAGC-5′ 15a (9), 5′-XGCTTTTTTCG-3′ 11a/ 3′-CGAAAAAAGC-5′ 15a + 9 (b), 5′-dis-ox-GCTTTTTTCG-3′ 14a/3′-CGAAAAAAGC-5′ 15a (2), and 5′- XGCATATATCG-3′ 11b/3′-CGTATATAGC-5′ 15b (0), 5′- XGCATATATCG-3′ 11b/ 3′-CGTATATAGC-5′ 15b + 9 (O), 5′-dis-ox- GCATATATCG-3′ 14b/ 3′-CGTATATAGC-5′ 15b (4) in 0.01 M Na2HPO4 buffer, 0.04 M NaCl, 0.001 M EDTA, pH 7.4 with [duplex] ) [9] ) 3 × 10-6 M. Spectra were recorded at λex ) 350 nm, λem ) 465 nm, slit width Ex/Em ) 10 nm/10 nm. (X) 5′-diol modified oligonucleotide, dis ) distamycin and ‘ox’ corresponds to oximeconjugated ODN-distamycin hybrid).

formed at a fixed concentration of ODNs while varying the Hoechst 33258 concentration till saturation, as indicated from emission spectroscopy. In all the cases, the enhanced fluorescence intensity was measured after each addition (Figure 4) and the binding efficiency was compared from Hoechst 33258 concentration required to saturate its fluorescence intensity. A significantly higher concentration of Hoechst 33258 was required to saturate its fluorescence intensity in either free or covalently bound distamycin, in comparison to the duplex itself. Duplexes 11a/15a and 11b/15b containing noncovalently bound distamycin-like polyamide at its minor groove surface required 1 and 0.7 µM greater Hoechst 33258, respectively, to saturate its binding with respect to the duplex ODN without distamycin. In the case of duplex ODN bearing covalently tethered distamycin derivatives such as 14a/15a and 14b/15b, respectively, a greater Hoechst 33258 concentration was required (0.3-0.4 µM) with respect to the unconjugated duplex ODN. In both cases it was found that more Hoechst 33258 was required to saturate its fluorescence intensity in duplexes containing a continuous A/T stretch (14a/15a, 11a/15a + 9) rather than an alternating A/T stretch (14b/15b, 11b/ 15b + 9). It is known that fluorescence intensity of the Hoechstlike fluorophore is quenched after it saturates its binding to the DNA minor groove surface (52). This was due to the nonspecific binding of excess Hoechst 33258 molecules to the phosphate backbone of DNA. In our displacement assay, there was less quenching observed for duplexes containing free distamycin, and no such quenching was observed for the covalently bound distamycin analogue (Figure 4). When distamycin is noncovalently complexed with the duplex ODN, addition of Hoechst 33258 releases distamycin molecules from its minor groove. Such released distamycin molecules then compete for the nonspecific binding with the phosphate backbone, impeding further approach of Hoechst molecule to these sites. Interestingly, when distamycin is covalently conjugated, nonspecific binding to the phosphate sites by Hoechst was completely inhibited. Importantly, this study reinforces the fact that the duplex formation is facilitated by a covalently attached distamycin analogue


and also supports its sequence preference for a continuous A/T stretch rather than an alternating A/T sequence. CONCLUSIONS

In summary we have reported two chemical methods for the conjugation of oligonucleotides to a minor groovebinding (MGB) polyamide. Although conjugates of MGB polyamides have been prepared in the past, the utility of the present approach is that it would be possible to prepare a selection of appropriately derivatized polyamides and oligonucleotides and ligate together in a modular fashion. Thus, if the goal is to prepare a library of compounds, then this would be a time-saving approach. The chemistry employed here for the conjugation of an ODN to a distamycin-like polyamide is an extension of existing techniques based on the reaction of an aldehyde to form an oxime linkage or a thiazolidine ring. After HPLC purification and characterization of these conjugates, we investigated the influence of tethered distamycin on duplex formation and assessed the stability with the complementary strand through interactions mediated by an A/T stretch. Hybridization by the conjugate to a target sequence converts a single-stranded species to a double-stranded complex and generates a secondary structure with both major and minor grooves. The hybridization event triggers a binding event by the 5′-end ODN-tethered distamycin within the generated minor groove structure. This was proved by the enhanced thermal denaturation temperature (∆Tm) at 260 nm, increased CD intensity at 334 nm, and a requirement of more Hoechst 33258 molecules during the fluorescence displacement assay. Taken together, it is evident that the distamycin conjugates are more selective in binding to ODNs containing a continuous stretch of A/T base pairs rather than the one having alternating A/T tract. CD studies at different salt concentrations reinforce the specific interaction of distamycin conjugates to the corresponding duplexes. No significant binding preference was observed for the five-membered thiazolidine linker over the linear N-oxime-conjugated distamycin to the duplex. This could be due to long linker length between the ODN and the peptide, allowing the linker part to loop during the distamycin-DNA binding process. The combination of such hybridization-triggered binding events should provide the basis for the development of DNAbased diagnostic properties of antigens or therapeutic ODNs by enhancing its target-binding properties, cellular uptake, or exonuclease stability or can be used as an in situ hybridization probe. Since distamycin is known to form a stacked, antiparallel dimer upon binding to AT tracts of DNA (53, 54), it is possible to employ distamycin-ODN conjugates for pseudo-cyclized duplex DNA and triple helix formation. Work is currently underway in this direction. EXPERIMENTAL SECTION

Materials and Methods. All chemical reagents were of the highest purity available and were used without purification. Solid-supported NaIO4 (polystyrylmethyl)trimethylammonium metaperiodate, loading 2.5 mmol/ g, was purchased from Novabiochem and 2-cyanoethyl diisopropylchlorophosphoramidite from Aldrich. The phosphoramidite 10 was prepared by analogy with the previously described protocol (29). The activated ester 4 of N-Boc-O-(carboxymethyl)hydroxylamine was prepared as described earlier (55). Thin-layer chromatography (TLC) was performed using silica gel-G, and preparative column chromatography was performed on silica gel (60-120

Ghosh et al.

mesh size). DMF, CH2Cl2, and CHCl3 were dried over P2O5, Et3N was dried over KOH, and THF was dried using sodium and benzophenone ketyl. HPLC purification as well as HPLC analysis of oligonucleotides and conjugates was performed on a water system equipped with two M510 pumps, a M490E detector, and an M680system controller. The oligonucleotides and the conjugates were purified on a µ-bondapak C-18 column (Macherey-Nagel Nucleosil: 10 × 250 mm, 7 µm) using the following system of solvent: solvent A, 20 mM ammonium acetate/CH3CN, 95:5 (v:v); solvent B (CH3CN); flow rate, 4 mL/min; a linear gradient from 0 to 30% B in 20 min was applied. 1H NMR and 13C NMR spectra were recorded on JEOL JNM λ-300 or using a Bruker AMX 400 (400 MHz for 1H and 100.6 MHz for 13C) NMR spectrometer. Chemical shifts (δ) are reported in ppm downfield from the internal standard (TMS). Electrospray ionization (ESI) mass spectra were measured on an Esquire spectrometer (Bruker). Analysis was performed in the negative mode for both the oligonucleotides and conjugates and in the positive mode for the peptides. The oligonucleotides and the conjugates were dissolved in 50% aqueous acetonitrile, and 1% of NEt3 was added. The peptides were dissolved in 50% aqueous acetonitrile containing 1% of TFA. 2-tert-Butoxycarbonylamino-3-tert-butoxycarbonylsulfanylpropionic Acid 2. To a cooled aqueous/ dioxane solution (1:1, v/v, 1.2 mL) of L-cysteine, 1 (0.4 g, 3.3 mmol), were added potassium carbonate (2.4 g, 18.3 mmol) and di(tert-butyl) dicarbonate (2.8 g, 13.2 mmol), and the mixture was stirred for 48 h at room temperature. The resulting mixture was then washed with ether to remove excess of di(tert-butyl) dicarbonate and acidified with 0.2 N aq HCl. The aqueous layer was then extracted with EtOAc. The organic layer was washed with brine solution, dried over anhydrous Na2SO4, and evaporated in a vacuum. Purification by column chromatography over silica gel (MeOH/CHCl3 1:49) afforded a white solid compound. Yield: 0.28 g, 60%; 1H NMR (300 MHz, CDCl3): δ 1.43 (s, 9H, C(CH3)3), 1.47 (s, 9H; C(CH3)3), 3.16-3.23 (dd, 1J (H, H) ) 14.4 Hz, 2J (H, H) ) 7.8 Hz, 2J(H, H) ) 6.6 Hz, 1H; CH2S(Boc)), 3.3-3.4 (dd, 1J(H, H) ) 14.1 Hz, 2J(H, H), ) 4.5 Hz, 2J(H, H) ) 3.3 Hz, 1H; CH2S(Boc)), 4.5 (m, 1H; CH-CH2), 5.4 (m, 1H; NH); 13C NMR (100.6 MHz,CDCl3): δ 28.1, 28.3, 32.6, 54.4, 80.5, 85.7, 155.6, 168.7, 174.5 ppm; Elemental analysis: calcd (%) for C13H23O6NS (321.4): C 48.58, H 7.21, N 4.36; found: C 49.0, H 7.3, N 4.3. 2-tert-Butoxycarbonylamino-3-tert-butoxycarbonylsulfanylpropyl 2,5-Dioxopyrrolidin-1-yl Ester, 3. 2-tert-Butoxycarbonylamino-3-tert-butoxycarbonylsulfanylpropionic acid 2 (0.1 g, 0.3 mmol) was dissolved in DMF (1 mL) and cooled in an ice bath. N-Hydroxysuccinimide (HOSu, 0.04 g, 0.4 mmol) was added to the above solution, followed by dicyclohexylcarbodiimide (0.08 g, 0.4 mmol). The resulting solution was stirred for 1 h at 0 °C and then at room temperature for another 6 h. The precipitated dicyclohexylurea was filtered off, and the filtrate was diluted with EtOAc. The EtOAc layer was washed successively with NaHCO3 and brine, dried over anhydrous Na2SO4, and evaporated to obtain the succinimide ester 3 as practically pure solid. Yield: 0.16 g, 80%. This was directly used for the next step without further purification; 1H NMR (300 MHz, CDCl3): δ 1.41 (s, 9H; C(CH3)3), 1.46 (s, 9H; C(CH3)3), 2.8 (s, 4H; CO(CH2CH2)CO), 3.17-3.25 (dd, 1J(H, H) ) 14.1 Hz, 2J(H, H) ) 8.1 Hz, 1H; CH2S(Boc), 3.4-3.45 (dd, 1J ) 14.1 Hz, 2J ) 4.6 Hz, 1H; CH2S(Boc)), 4.82 (m, 1H; CH(CO)NH), 5.4 (m, 1H; NH).


N2-2-[[2-(2-tert-Butoxycarbonylamino-3-tert-butoxycarbonylsulfanylpropylamino)ethyl](methyl)amino]ethyl-1-methyl-4-[(1-methyl-4-[(1-methyl-4[(1methyl-1H-pyrrol-2-yl)carbonyl]amino-1H-pyrrol-2yl)carbonyl]amino-1H-pyrrol-2-yl)carbonyl]amino1H-2-pyrrolecarboxamide, 6. To a solution of N2-2[(2-aminoethyl)(methyl)amino[(1-methyl-4-[(1-methyl-4[(1-methyl-4[(1-methyl-1H-pyrrol-2-yl)carbonyl]amino1H-pyrrol-2-yl)carbonyl]amino-1H-pyrrol-2-yl)carbonyl]amino-1H-2-pyrrolecarboxamide 5 (0.2 g, 0.3 mmol) in CHCl3 (5 mL) was added activated N-hydroxysuccinimide ester 3 (0.15 g, 0.4 mmol), and the resulting solution was stirred at room temperature for 2 h. At the end of this period, the reaction mixture was filtered off and the filtrate evaporated under vacuum. The resulting mixture was purified by column chromatography over silica gel using MeOH/CHCl3 (3/47) as eluent. This furnished 6 as a yellow powder. Yield: 0.14 g, 50%, 1H NMR (300 MHz, CDCl3): δ 1.34 (s, 9H; C(CH3)3), 1.42 (s, 9H; C(CH3)3), 2.23 (s, 3H; N(CH3)), 2.56 (s, 4H; CH2N(Me)CH2), 2.882.97 (m, 4H; 2CH2NH(CO)), 3.12-3.47 (m, 2H; CH2S(Boc)), 3.79, 3.85, 3.89, 3.97 (s, 12H; 4 Py-N(CH3)), 4.26 (m, 1H; CHNH(Boc)), 5.69 (m, 1H; NHBoc), 6.1 (dd, J(H, H) ) 1.8 Hz, 2J(H, H) ) 3.8 Hz, 1H; ArH), 6.5 (d, J(H, H) ) 1.8 Hz, 1H; ArH), 6.69 (d, J ) 1.8 Hz, 1H; ArH), 6.74 (s, 1H; ArH), 6.83 (s, 1H; ArH), 6.9 (s, 1H; ArH), 7.22 (s, 2H; ArH), 7.4(s, 1H; ArH), 7.73, 8.41, 8.89, 9.03 (br s, 4H; NH); 13C NMR (100.6 MHz, CDCl3): δ 28.3, 28.1, 35.8, 36.6, 36.9, 41.3, 56.1, 56.8, 80.3, 85.8, 103.5, 103.9, 104.02, 107.4, 112.5, 119.3, 119.6, 119.8, 121.8, 122.1, 122.4, 122.7, 122.8, 125.6, 128.3, 159.0, 159.2, 159.5, 162.2, 171.3, 175.4 ppm; MS (ESI): m/z (%): 894.6 (100) [M + H]+; elemental analysis calcd (%) for C42H59O9N11S. CHCl3: C 50.96, H 5.97, N 15.2; found: C 50.8, H 6.1, N 15.6. N2-2-[[2-(2-tert-Butoxycarbonylaminooxyacetylamino)ethyl](methyl)amino]ethyl-1-methyl-4-[(1methyl-4-[(1-methyl-4[(1-methyl-1H-pyrrol-2-yl)carbonyl]amino-1H-pyrrol-2-yl)carbonyl]amino-1H-pyrrol-2-yl)carbonyl]amino-1H-2-pyrrolecarboxamide, 8. To a solution of 4 (15 mg, 56 µmol) in chloroform was added tetrapeptide 5 (30 mg, 51 µmol) under argon. The reaction mixture was stirred at room temperature for 2 h. On completion, the reaction mixture was filtered off and the filtrate evaporated under vacuum. The resulting mixture was adsorbed in silica, and it was purified by column chromatography using MeOH/CHCl3 (5/45) as eluent. The product, 8, was obtained as a yellow powder. Yield: 20 mg, 50%; 1H NMR: (300 MHz, CDCl3): δ 1.4 (s, 9H; C(CH3)3), 2.4 (br s, 3H; N(CH3)), 2.7 (br s, 4H; CH2N(Me)CH2), 3.4 (br s, 4H; 2CH2NH(CO)), 3.82, 3.87, 3.89, 3.96 (s, 12H; 4 Py-N(CH3)), 4.28 (br s, 2H; CH2(ONH)(Boc)), 6.1 (dd, J(H,H) ) 1.8 Hz, 2J(H,H) ) 3.8 Hz, 1H; ArH), 6.6 (d, J(H,H) ) 1.8 Hz, 1H; ArH), 6.7 (d, J ) 1.8 Hz, 1H; ArH), 6.8 (s, 1H; ArH), 6.9 (s, 1H; ArH), 7.0 (s, 1H; ArH), 7.2 (s, 2H; ArH), 7.3 (s,1H; ArH), 7.7, 8.4, 8.9, 9.03 (br s, 4H; NHs); 13C NMR (100.6 MHz, CDCl3): δ 28.2, 36.56, 36.6, 36.7, 36.9, 40.5, 56.1, 57.1, 65.9, 82.5, 103.7, 104.0, 104.5 107.4, 112.6, 119.5, 119.7, 119.9, 121.9, 121.96, 122.0, 122.3, 122.8, 125.6, 128.4, 159.1, 159.2, 159.6, 162.6, 170.7, 174.2 ppm; MS (ESI): m/z (%) ) 764.3 (100) [M + H]+. N2-2-[[2-(2-Amino-3-sulfanylpropylamino)ethyl](methyl)amino]ethyl-1-methyl-4-[(1-methyl-4-[(1methyl-4[(1-methyl-1H-pyrrol-2-yl)carbonyl]amino1H-pyrrol-2-yl)carbonyl]amino-1H-pyrrol2yl)carbonyl]amino-1H-2-pyrrolecarboxamide, 7. To a solution of 6 (10 mg, 0.011 mmol) in dichloromethane (0.2 mL) was added ethane dithiothreitol (EDT, 8 µL, 2%)


first to prevent thiol oxidation. An equal volume of TFA (0.2 mL, 2.6 mmol) was added into it under cold conditions, and the reaction was continued for 2 h at room temperature under an argon atmosphere. After completion of the reaction, the excess TFA and solvent were removed in vacuo. To the residue were added CHCl3 and H2O (500 µL:500 µL) and the biphasic solution was mixed thoroughly. The excess ethane dithiothreitol (EDT) was removed in organic phase, and the aqueous phase was lyophilized to obtain 7. It was directly used without further purification due to its instability and high reactivity. Yield: 6.1 mg, 80%; MS (ESI): m/z (%) ) 694.22 (50) [M + H]+, 347.76 (100) [(M + H)/2]+ N2-2-[[2-(2-Aminooxyacetylamino)ethyl](methyl)amino]ethyl-1-methyl-4-[(1-methyl-4-[(1-methyl-4[(1methyl-1H-pyrrol-2yl)carbonyl]amino-1H-pyrrol-2yl)carbonyl]amino-1H-pyrrol-2yl)carbonyl]amino1H-2-pyrrolecarboxamide, 9. To a solution of 8 (10 mg, 0.013 mmol) in dichloromethane (0.2 mL) was added an equal volume of TFA (0.2 mL, 2.6 mmol), and the reaction mixture was stirred at room temperature for 2 h. The solvent and excess TFA was removed under vacuum, and the residue was directly used for the conjugation reaction without further purification due to its high reactivity. Yield: 6.9 mg, 80%; MS (ESI): m/z (%) ) 664.22 (100) [M + H]+. Oligonucleotide Synthesis. Automated DNA synthesis was performed on a Perkin-Elmer model Expedite DNA synthesizer using standard cyanoethyl nucleoside phosphoramidite chemistry on a 1 µM scale. After cleavage from the solid support and deprotection by treatment with concentrated ammonia (28%) for 16 h at 55 °C, the oligonucleotides were purified by RP-HPLC. Diol-Containing Oligonucleotides 11a-c. The 5′protected oligonucleotides were treated with 80% aq AcOH for 1 h. The residue obtained after lyophilization was dissolved in water, and the aqueous layer was extensively washed with Et2O to remove the benzylidene byproduct. Subsequent lyophilization afforded the oligonucleotides 11a-c, which were characterized by ESI-MS (Table 1). Aldehyde-Containing Oligonucleotides 12a-c. To a solution of the oligonucleotide 11a (1 mg, 0.3 µmol) in water (1 mL) was added solid-supported NaIO4 (0.67 mg, 3 µmol), and the mixture was shaken gently for ∼1 h. The solution was then decanted, and the polymer was washed twice with H2O. Washings were added to the solution, and the resulting solution was evaporated under vacuum to remove volatiles. The product was then directly used for oxime conjugation without further purification. For thiazolidine-containing oligonucleotides, it was purified by RP-HPLC to remove excess periodate. The decamers 12b,c were prepared in the same manner. Conjugation through Thiazolidine and Oxime Linkages. Preparation of Oligonucleotides 13a,b and 14a-c. The cysteine-tethered peptide 7 (0.4 mg, 0.64 µmol) was dissolved in 0.2 M NaOAc buffer (pH ) 5.4, 30 µL) and was added into 0.2 M NaOAc buffer solution (130 µL) containing the aldehyde-containing oligonucleotide 12a (500 µg, 0.16 µmol). The mixture was stirred at room temperature, and the progress of the reaction was followed by RP-HPLC. Disappearance of the starting material 12a was achieved in 1 h (Figure 2A). In Figure 2A, 12a showed three peaks in HPLC profile, which correspond to the imine formation of some part of the aldehyde-containing ODN with ammonium acetate buffer used as a solvent during the HPLC elution period. Purification by HPLC afforded the conjugate 13a in 30%


yield. The conjugate 13b was obtained in the same manner from the oligonucleotide 12b. The oligonucleotides 14a-c were prepared in the same manner by adding 0.2 M NaOAc buffer (pH ) 5.4, 30 µL) containing oxyamine-containing peptide 9 into 0.2 M NaOAc buffer solution (130 µL) containing the reactive oligonucleotide 12a-c. Disappearance of the starting material 12a-c was achieved within ∼30 min, and purification by HPLC afforded the conjugate 14a-c in 40% yield (Figure 2B). Subsequent lyophilization afforded the distamycin-ODN conjugates 13a,b and 14a-c, which were characterized by ESI-MS (Table 1). Hybridization Experiments. Duplex formation and the melting Tm experiments were carried out by mixing equimolar amounts of the two decamer oligonucleotide strands in 10 mM Na2HPO4 buffer, 1 mM EDTA, and varying concentrations of NaCl (40, 100, 500 mM) at pH 7.4. All measurements were performed at 9.31 µM concentration of each strand, and the mixtures were heated at 90 °C for 6 min and then cooled slowly to room temperature to allow the formation of duplex. The melting curves (absorbance at 260 nm vs temperature) were generated using a Lambda 5 UV-vis spectrophotometer equipped with a Perkin-Elmer C570-070 temperature controller using a scan rate of 0.5 °C/min (from 20 to 70 °C) and 1 °C/min (from 70 to 80 °C). The data were recorded at each 0.1 °C interval. In all cases the buffer-corrected absorbance vs temperature curves were converted to relative absorbance, θ ) (At - A20)/(A80 A20) vs temperature (At correspond to the absorbance at any temperature t) to obtain the value of Tm. Circular Dichroism Measurements. CD spectra were recorded on a JASCO (J-715) model spectropolarimeter equipped with a Perkin-Elmer temperature controller. CD melting experiments were performed using a scan rate of 1 °C/min from 20 to 80 °C, and the data were recorded at each 0.2 °C interval. All CD spectra were averaged over two acquisitions, and the scan rate was maintained at 100 nm/min with optical cells of path length 1 cm in the CD melting study and 0.2 cm for recording other CD spectra. The CD values, expressed as molar ellipticity (θ), has been calculated from the equation, (θ) ) (100 × φ/l × c) deg cm2 dmol-1 where φ is the observed ellipticity in degrees, l is the path length of the cell in centimeters, and c is the concentration of DNA in base molarity. All the experiments including CD melting study were carried out in 0.01 M Na2HPO4 buffer and 0.04 M NaCl, pH 7.4, with 1.31 × 10-5 M duplex concentration. Fluorescence Measurements. Fluorescence spectra were recorded on a Hitachi model F-4500 spectrofluorimeter at 25 °C with excitation and emission bandwidths fixed at 10 nm (λex ) 350 nm, λem ) 464.8 nm). In each case duplex concentration was taken as 3 × 10-6 M and each time an aliquot (10 µL) of Hoechst 33258 (4.2 × 10-5 M) was added into the duplex solution in 0.01 M Na2HPO4 buffer, 0.04 M NaCl, pH 7.4. In the case of the duplex containing free distamycin, 3 × 10-6 M distamycin was added to the duplex and the solution was incubated for 10 min prior to the addition of Hoechst 33258. The same concentration was also maintained for the duplex ODN covalently conjugated with distamycin. The enhanced fluorescence intensity was measured at each point after 5 min of equilibration of added Hoechst 33258 into individual duplex solutions.

Ghosh et al. ACKNOWLEDGMENT

The Ministe`re des Affaires Etrange`res and the Re´gion Rhone-Alpes are gratefully acknowledged for a grant for S.G. LITERATURE CITED (1) Yager, T. D., Nickerson, D. A., and Hood, L. E. (1991) The human genome project: creating an infrastructure for biology and medicine. Trends Biochem. Sci. 16, 454. (2) Praseuth, D., Guieyse, A. L., and Helene, C. (1999) Triple helix formation and the antigene strategy for sequence specific control of gene expression. Biochim. Biophys. Acta. 1489, 181-206. (3) Hermann, T., and Patel, D. J. (2000) Adaptive recognition by nucleic acid aptamers. Science 287, 820-825. (4) Mapp, A. K., Ansari, A. Z., Ptashne, M., and Dervan, P. B. (2000) Activation of gene expression by small molecule transcription factors. Proc. Natl. Acad. Sci. U.S.A. 95, 39303935. (5) Trauger, J. W., Baird, E. E., and Dervan, P. B. (1998) Recognition of 16 base pairs in the minor groove of DNA by a pyrrole-imidazole polyamide. J. Am. Chem. Soc. 120, 35343535. (6) Gottesfeld, J. M., Neely, L., Trauger, J. W., Baird, E. E., and Dervan, P. B. (1997) Regulation of gene expression by small molecules. Nature 387, 202-205. (7) Bailly, C., and Charies, J. B. (1998) Sequence-specific DNA minor groove binders. Design and synthesis of netropsin and distamycin analogues. Bioconjugate Chem. 9, 513-538. (8) Iida, H., Jia, G., and Lown, J. W. (1999) Rational recognition of nucleic acid sequences. Curr. Opin. Biotechnol. 10, 29-33. (9) Neidle, S. (1997) Crystallographic insights into DNA minor groove recognition by drugs. Biopolymers 44, 105-121. (10) Patel, D. J. (1982) Antibiotic - DNA interactions: intermolecular nuclear Overhauser effects in the netropsin d(CGCGAATTCGCG) complex in solution. Proc. Natl. Acad. Sci. U.S.A. 79, 6424-6428. (11) Schultz, P. G., and Dervan, P. B. (1984) Distamycin and penta-N-methylpyrrolecarboxamide binding sites on native DNA. A comparison of methidiumpropyl-EDTA-Fe(II) foot printing and DNA affinity cleaving. J. Biomol. Struct. Dyn. 1, 1133-1147. (12) Lee, M., Hartley, J. A., Pon, R. T., Krowicki, K., and Lown, J. W. (1988). Sequence specific molecular recognition by a monocationic lexitropsin of the decadeoxyribonucleotide d[CATGGCCATG]2: Structural and dynamic aspects deduced from high field 1H NMR studies. Nucleic Acids Res. 16, 665685. (13) Kielkopf, C. L., White, S., Szewczyk, J. W., Turner, M. T., Baird, E. E., Dervan, P. B., and Ress, D. C. (1998) A structural basis for recognition of A-T and T-A base pairs in the minor groove of B-DNA. Science 282, 111-117. (14) White, S., Baird, E. E., and Dervan, P. B. (1997) Orientation preferences of pyrrole-imidazole polyamides in the minor groove of DNA. J. Am. Chem. Soc. 119, 8756-8765. (15) Bhattacharya, S., and Thomas, M. (2000) DNA binding properties of novel distamycin analogues that lack the leading amide unit at the N-terminus. Biochem. Biophys. Res. Commun. 267, 139-144. (16) Wiederholt, K., Rajur, S. B., Giuliano, J., Jr., O’ Donnell, M. J., and Mclaughlin, L. W. (1996) DNA-tethered Hoechst groove-binding agents: Duplex stabilization and fluorescence characteristics. J. Am. Chem. Soc. 118, 7055-7062. (17) Wiederholt, K., Rajur, S. B., O’Donnell, M. J., and Mclaughlin, L. W. (1997) Oligonucleotides tethering Hoechst 33258 derivatives: Effect of the conjugation site on duplex stabilization and fluorescence properties. Bioconjugate Chem. 8, 119126. (18) O’Donnell, M. J., Rajur, S. B., McLaughlin, L. W., Afonina, I., Kutyavin, I., Lukhtanov, E., Meyer, R. B., and Gamper, H. (1995) Synthesis and properties of a Hoechst-like minorgroove binding agent tethered to an oligodeoxynucleotide. Bioorg. Med. Chem. 3, 743-750.

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Efficient Conjugation and Characterization of Distamycin-Based

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