Synthesis of a Hoechst 32258 Analogue Amino Acid Building Block for

Nov 2, 2001 - The synthesis of a new versatile “Hoechst 33258-like” Boc-protected amino acid building block for peptide synthesis is described. It...
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Bioconjugate Chem. 2001, 12, 1021−1027

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Synthesis of a Hoechst 32258 Analogue Amino Acid Building Block for Direct Incorporation of a Fluorescent, High-Affinity DNA Binding Motif into Peptides Carsten Behrens,†,‡ Niels Harrit,† and Peter E. Nielsen*,§ Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100, Copenhagen, Denmark, and Center for Biomolecular Recognition, Department of Biochemistry B, The Panum Institute, Blegdamsvej 3c, DK-2200, Copenhagen, Denmark. Received May 9, 2001; Revised Manuscript Received July 15, 2001

The synthesis of a new versatile “Hoechst 33258-like” Boc-protected amino acid building block for peptide synthesis is described. It is demonstrated that this new ligand is an effective mimic of Hoechst 33258 in terms of DNA affinity and sequence specificity. Furthermore, this minor groove binder was conjugated to a DNA condensing peptide (KSPKKAKK) by continuous solid-phase peptide synthesis, and the conjugate exhibited increased DNA affinity (ca. 10-fold), but similar sequence preference compared to Hoechst 33258 as analyzed by DNaseI footprinting. Finally, the fluorescence quantum yield of the new chromophore is found to increase 30% upon binding to double stranded DNA.

INTRODUCTION

Sequence-specific recognition of DNA by proteins such as transcription factors and repressors play a central role in cellular gene regulation at the transcriptional level and is also crucial in DNA replication and recombination. One long-term objective of structural and biophysical studies of protein DNA interactions is to understand the molecular basis for these processes (1, 2). Such understanding can provide valuable input to the development of new principles for design of DNA recognizing drugs for direct modulation of gene expression, e.g., for use as gene therapeutic drugs (3). Protein DNA recognition relies on hydrogen bonds, electrostatic interactions, and van der Waals contacts between individual amino acid residues of the protein and nucleotides in the cognate DNA target. However, no simple “amino acid-nucleotide” recognition code has been identified, and the sequence specificity of protein binding to DNA is therefore not a “digital readout” but rather an “analogue readout” of the DNA base sequence in which the entire binding region of the protein exhibit “molecular complementarity” to the major (or more rarely the minor) groove of the DNA target (1, 2, 4). Most success so far in developing small synthetic ligands for sequence specific recognition of double stranded DNA has come from using major groove binding, triple helix forming oligonucleotides (5), helix-invading peptide nucleic acids (PNA) (6), and minor groove binding, hairpin polyamides (7). Furthermore, it has been possible to use “zink-finger” peptide modules (each recognizing three base pairs) to construct sequence-targeted proteins (8). Finally, several attempts without major breakthroughs have been described to select small peptides from chemical libraries that sequence selectively bind * To whom correspondence should be addressed. Fax: +45 35396042. E-mail: [email protected]. † University of Copenhagen. ‡ Present address: Novo Nordisk A/S, Novo Nordisk Park 1, DK-2760 Måløv, Denmark. § The Panum Institute.

double-stranded DNA, to discover novel recognition motifs and/or principles (9-10). We recently described such a combinatorial method for probing small peptides for DNA minor groove recognition potential (9). Bead-supported tripeptides, each placed between N-methylpyrrole amino acid containing polyamides operating as DNA minor groove binding anchoring points, were synthesized in a library format and subsequently screened against double-stranded DNA probes (9). Although reliable procedures are available for synthesising polyamides of N-methylpyrrole amino acids by peptide coupling of protected N-methyl-4-aminopyrrole-2-carboxylate units (11), their synthesis is resourceful as three or more interlinked units are required for obtaining strong DNA minor groove binding. To improve our methodology, we aimed to minimize the number of synthetic operations needed for library production and therefore required a monomer building block that in a single synthesis step introduces a DNA minor groove binding anchoring point into peptides. The fluorescent chromosomal stain Hoechst 33258 is a well-known DNA binding agent made of two linked benzimidazoles, with a phenol and a N-methylpiperazine substituent in either end. The interaction between Hoechst 33258 and double-stranded DNA has been extensively studied using DNase I footprinting (12), electric linear dicroism, and solution-phase NMR techniques (13) as well as X-ray crystallography (14-16). Studies have shown that Hoechst 33258 binds with high affinity to the minor groove of double stranded B-DNA with a strong preference for AT-rich regions. As a part of our continuous studies on peptide DNA interactions, we now report the synthesis of a novel protected amino acid based on Hoechst 33258. The building block is suitable for use in solid-phase peptide synthesis following Boc strategy and designed to allow for direct incorporation of one or multiple DNA minor groove-binding motifs into any arbitrary position of a peptide sequence. We illustrate the potential of this novel Hoechst 33258 amino acid analogue by preparing a small series of peptides and 9-amino acridine conjugates and

10.1021/bc0100556 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/02/2001

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analyzing their DNA binding properties by DNaseI footprinting. EXPERIMENTAL SECTION

General Comments. All reagents (Aldrich, Fluka, or NovaBiochem) and solvents (LabScan) were of standard quality and used without further purification unless indicated. NMR spectra were recorded on either a Bruker 250 MHz or a Varian 400 MHz spectrometer with tetramethylsilane (1H and 13C) as internal standard. Melting points were recorded on a Bu¨chi melting point apparatus and are uncorrected. Microanalyses were performed at The Microanalytical Laboratory, Department of Chemistry, University of Copenhagen. 2-(4-Cyanophenyl)benzimidazole-5-carboxylic Acid (3). 3,4-Diaminobenzoic acid (4.00 g, 26 mmol) and p-cyanobenzaldehyde (3.45 g, 26 mmol) were dissolved in nitrobenzene (120 mL) and heated to 150 °C. After 1 h, a turbid solution was obtained. DMF (20 mL) was added to dissolve the precipitate, and heating was resumed for an additional 5 h (TLC1 monitoring in CHCl3/ NEt3/MeOH (7:1:2 v/v/v)). The mixture was cooled on an ice bath, and the crystals that separated out were collected by filtration and washed several times with cold petroleum ether to remove traces of nitrobenzene. Recrystallization from ethanol/water afforded 5.02 g (73%) of the title material. Rf ) 0.45 in CHCl3/NEt3/MeOH (7: 1:2 v/v/v). Mp > 300 °C. 1H NMR (DMSO-d6) δ: 7.70 (d, 1H); 7.86 (dd, 1H); 8.04 (d, 2H); 8.22 (d, 1H); 8.37 (d, 2H), 12.75 (bs, 1H). 13C NMR (DMSO-d6) δ: 112.78; 115.17; 117.06; 117.82; 124.33; 125.94; 127.49; 133.19; 133.90; 139.59; 142.23; 151.82; 168.13. MS-FAB+: 264.09 [M + H]+. Anal. Calcd for C15H9N3O2‚H2O: C, 64.05; H, 3.94; N, 14.93. Found: C, 64.11; H, 3.96; N, 14.78. Ethyl N3-(2-(4-Cyanophenyl)benzimidazole-5-carbonyl)-3,4-diaminobenzoate (4). 2-(4-Cyanophenyl)benzimidazole-5-carboxylic acid (3, 5.02 g, 19 mmol) was suspended in DMF (120 mL). HBTU (7.22 g, 19.0 mmol) was added followed by DIEA (5.0 mL). The mixture was stirred at room temperature for 10 min before the addition of ethyl 3,4-diaminobenzoate (3.43 g, 19 mmol). After the mixture was stirred for 24 h at room temperature, water (400 mL) was added. The precipitated product was collected by filtration, washed with water three times, and dried in a vacuum oven overnight. Yield: 5.75 g (71%). Rf ) 0.4 in MeOH/CH2Cl2 (1:9, v/v). MS-FAB+: 426.16 [M + H]+. Mp ) 222-225 °C. 1H NMR (DMSO-d6) δ: 1.28 (t, 3H); 4.22 (q, 2H); 5.83 (bs, 2H); 6.81 (d, 1H); 7.58 (d, 1H); 7.70 (m, 1H); 7.83 (d, 1H); 7.96 (d, 1H); 8.06 (d, 2H); 8.36 (m, 1H); 8.40 (d, 2H); 9.73 (bs, 1H); 13.46 (bs, 1 H). 13C NMR (DMSO-d6) δ: 14.45; 59.82; 111.84; 112.38; 114.72; 116.62; 118.63; 119.21; 119.46; 122.21; 123.57; 127.33; 128.38; 129.00; 133.12; 133.96; 144.98; 146.29; 148.47; 151.78; 165.76; 166.12. Anal. Calcd for C24H19N5O3: C, 67.76; H, 4.50; N, 16.46. Found: C, 66.57; H, 4.31; N, 16.56. Ethyl 2-(2-[4-Cyanophenyl]benzimidazol-5-yl)benzimidazole-5-carboxylate (5). Ethyl N3-(2-(4-cyanophenyl)benzimidazole-5-carbonyl)-3,4-diaminobenzoate (4, 5.75 g, 13.5 mmol) was suspended in nitrobenzene (120 mL), and concentrated H2SO4 (0.5 mL) was added. The mixture was heated to 190 °C for 12 h during which time complete ring closure took place according to TLC 1 Abbreviations: CT-DNA (calf thymus DNA), DIEA (diisopropylethylamine), DMF (dimethylformamide), DMSO (dimethyl sulfoxide), HBTU (benzotriazol-1-yl 1,1,3,3-tetramethyluronium hexafluorophosphate), TFMSA (trifluoromethansulfonic acid), TLC (thin-layer chromatography).

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(MeOH/CH2Cl2 (1:9, v/v)). After being cooled to room temperature, the product was precipitated as an oil by addition of petroleum ether. The adhesive oil turned into a fine powder after repeated addition and decanting of petroleum ether. The powder, which contained traces of hydro sulfate salts, was dissolved in a mixture of DMF (120 mL), saturated Na2CO3 (50 mL), and water (150 mL) by heating. The clear dark solution thus obtained was allowed to cool slowly, and the precipitating product was collected by filtration. Rf ) 0.45 in MeOH/CH2Cl2 (1:9, v/v). The yield after oven-drying overnight was 4.15 g (75%). Mp ) 257-259 °C. 1H NMR (DMSO-d6) δ: 1.35 ppm (t, 3H); 4.33 (q, 2H); 7.67 (d, 1H); 7.80 (d, 1H); 7.83 (d, 1H); 8.04 (d, 2H); 8.14 (d, 1H); 8.19 (s, 1H); 8.37 (d, 2H); 8.45 (s, 1H). 13C NMR (DMSO-d6) δ: 14.28 ppm.; 60.92; 112.52; 114.24; 115.45; 118.52; 119.84; 122.33; 123.39; 124.86; 125.41; 127.34; 129.95; 132.89; 135.08; 135.39; 138.40; 139.72; 141.21; 151.75; 152.83; 165.55. MS-FAB+: 408.09 [M + H]+. Anal. Calcd for C24H17N5O2‚ H2O: C, 67.75; H, 4.50; N, 16.46. Found: C, 67.49; H, 4.32; N, 16.40. Ethyl 2-[2-[4-(tert-Boc-aminomethyl)phenyl]benzimidazol-5-yl]benzimidazole-5-carboxylate (6). Ethyl 2-(2-[4-cyanophenyl]benzimidazol-5-yl)benzimidazole5-carboxylate (5, 4.00 g, 9.8 mmol) was dissolved in DMF (120 mL) by heating. After the solution was cooled to room temperature, di-tert-butyl pyrocarbonate (2.50 mL, 10.8 mmol) and DIEA (6.0 mL, mmol) were added, followed by palladium catalyst (10% on charcoal, 520 mg). The solution was then hydrogenated at room temperature for 48 h (1 atm hydrogen pressure). The catalyst was removed by filtration and the solvent removed by rotary evaporation, in vacuo, to leave a dark tar. This was redissolved in ethanol (100 mL), and the product was precipitated as a solid by addition of water (200 mL). Pure title material was obtained after recrystallization from ethanol/water. Yield: 4.20 g (83%). Rf ) 0.4 in MeOH/CH2Cl2 (1:9, v/v). Mp: 195-200 °C dec. 1H NMR (DMSO-d6) δ: 1.36 ppm (t, 3H); 1.41 (s, 9H); 4.22 (d, 2H); 4.34 (q, 2H); 7.44 (d, 2H); 7.51 (t, 1H); 7.68 (d, 1 H); 7.75 (d, 1H); 7.85 (d, 1H); 8.10 (d, 1H); 8.17 (d, 2H); 8.20 (s, 1H); 8.42 (s, 1H). 13C NMR (DMSO-d6) δ: 14.36; 28.34; 43.34; 60.58; 78.06; 121.62; 123.31; 123.54; 123.65; 126.82; 127.60; 128.11; 142.78; 153.15; 154.83; 155.96; 166.35; 197.16 (only the strong signals are reported). MSFAB+: 512.26 [M + H]+. Anal. Calcd for C29H29N5O4 ‚ 3 H2O: C, 61.58; H, 6.23; N, 12.38; found: C, 61.49; H, 6.01; N, 12.52. 2-[2-[4-(tert-Butoxycarbonylaminomethyl)phenyl]benzimidazol-5-yl]benzimidazole-5-carboxylic Acid (7). Ethyl 2-[2-[4-(tert-Boc-aminomethyl)phenyl]benzimidazol-5-yl]benzimidazole-5-carboxylate (6, 2.98 g, 5.83 mmol) was dissolved in ethanol (80 mL), and aqueous sodium hydroxide solution (1 N, 50 mL) was added. The clear solution was heated to reflux for 2 h. The solution was cooled to room temperature and the reaction volume reduced to one-third by rotary evaporation, in vacuo. Water was added to a total volume of 150 mL and the clear solution extracted with diethyl ether (2 × 100 mL). The solution was acidified to pH 3.5 with 4 N HCl and extracted twice with n-butanol. The organic phase was dried with Na2SO4, filtered, and taken to dryness. The oil thus obtained was dissolved in a 5% ethanolic water solution by gentle heating, and after lyophilization of this solution, a fine white powder was obtained. Yield: 1.98 g (70%). Rf ) 0.3 in CHCl3/NEt3/MeOH (7:1:2, v/v/v). Mp > 300 °C. 1H NMR (DMSO-d6) δ: 1.28 (t, 3H); 4.07 (d, 2H); 7.41 (d, 2H); 7.48 (d, 1H); 7.70 (d, 1H); 7.84 (d, 1H); 8.08 (d, 1H); 8.15 (s, 1H); 8.23 (d, 2H); 8.42 (s, 1H). 13C

Synthesis of a Hoechst 32258 Analogue

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Scheme 1a

a Key: (a) nitrobenzene/DMF, 190 °C, 6 h (73%); (b) HBTU, DIEA in DMF, 10 min, rt., then ethyl 3,4-diaminobenzoate in DMF, 24 h, rt (71%); (c) cat. H2SO4, nitrobenzene, 150 °C, 24 h (75%); (d) H2/Pd-C, Boc2O, DIEA, DMF rt, 24 h (83%); (e) NaOH in water, then HCl (70%).

NMR (DMSO-d6) δ: 28.34; 43.33; 77.99; 120.97; 123.74; 124.21; 126.78; 127.44; 128.97; 134.41; 142.15; 153.01; 153.41; 155.94; 171.36 (only the strong signals are reported). HRMS: calcd for C27H25N5O4 484.1985, found 484.1978. 3-Dimethylaminopropyl 2-[2-[4-(Aminomethyl)phenyl]benzimidazol-5-yl]benzimidazole-5-carboxamide (8). 2-[2-[4-(tert-Butoxycarbonylaminomethyl)phenyl]benzimidazol-5-yl]benzimidazole-5-carboxylic acid (7, 250 mg; 0.51 mmol) was dissolved in dimethylformamide (8 mL), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 195 mg; 0.51 mmol) and diisopropylethylamine (DIEA, 500 µL; 2.87 mmol) were added. The mixture was stirred at room temperature for 15 min, and then 3-(dimethylamino)propylamine (500 µL; 3.97 mmol) was added. After being stirred for a further 2 h at room temperature, the solvent was removed by rotary evaporation to leave an oil. The oil was dissolved in saturated aqueous sodium hydrogen carbonate (20 mL). Aqueous sodium hydroxide (1 M, 4 mL) was added, and the solution was extracted twice with n-butanol (20 mL). The combined organic phases were dried with anhydrous sodium sulfate and taken to dryness to leave a colorless foam. The foam was suspended in 2 N aqueous hydrochloric acid (40 mL), and the suspension was refluxed until a clear solution was obtained. Solvent was removed, in vacuo, and the crystalline residue recrystallized from ethanol to leave the title material as a crystalline slightly hygroscopic powder. Yield: 130 mg (68%). Mp ) 283-286 °C dec. 1H NMR (D2O, 250 MHz) δ: 1.97 ppm (d, 2H); 2.88 (s, 6H); 3.14 (d, 2H); 3.26 (t, 2H); 3.95 (s, 2H); 7.36-7.40 (m, 4H); 7.52 (s, 2H); 7.59 (s, 2H); 7.61 (s, 1H); 7.67 (s, 1H). Anal. Found: C, 51.26; H, 6.62; N, 15.48 (agree with the dihydrochloride pentahydrate). MS found 468.29; calcd for [C27H29N7O + H]+ 468.58. Spectroscopic Methods. Absorption spectra were recorded on a Perkin-Elmer UV-vis Lambda 16 spectrophotometer in 1 cm cells. Fluorescence spectra were recorded on an LS 50B Perkin-Elmer luminescence spectrometer in perpendicular geometry. In all excitation and fluorescence spectra shown, excitation and emission band-pass were 1 nm. All excitation spectra are corrected. However, no correction was performed for the wavelength-

dependent response function of the detection system. Concentration of CT-DNA was calculated using 258 ) 16 800 M-1 cm-1 pr. base-pair from the absorption at 256 nm (Figure 3, trace b) corrected for absorption of 8 (using the unbound absorption coefficient). Oligomer Synthesis. Peptides and peptide conjugates were synthesized by standard Boc protocol on an MBHA resin at a loading of 0.1 mmol/g. Oligomers were cleaved off the resin using the low/high TFMSA procedure, purified by reversed-phase HPLC, and were characterized by MALDI-TOF mass spectrometry recorded on a Kratos Compact MALDI II instrument operating in the positive-ion mode, using 3,5-dimethoxy-4-hydroxycinnamic acid as the matrix (17). DNA Footprinting. Footprinting with DNaseI was performed in 100 µL of buffer (50 mM Tris-HCl, pH 7.4, 1 mM MgCl2) using a 3′-32P-end labeled EcorRI-PvuII fragment of plasmid PUC19. The DNaseI was titrated to yield ca. 20% cleavage after 30 min incubation at room temperature (20 °C). The DNA was precipitated with 66% ethanol, 0.1 M K-acetate, washed, dried, and analyzed by electrophoresis in 10% polyacrylamide sequencing gels run in standard Tris-Borate-EDTA buffer, pH 8.3. Radioactive DNA bands were visualized by autoradiography (18). RESULTS AND DISCUSSION

Chemistry. The construction of the 2-(2-arylbenzimidazol-5-yl)benzimidazole framework is outlined in Scheme 1 and essentially followed previously described strategies (19). Initially, 4-cyanobenzaldehyde (1) was condensed with 3,4-diaminobenzoic acid (2) under oxidative conditions, leaving 2-(4-cyanophenyl)benzimidazol-5-carboxylic acid (3) in 73% yield. Installation of the second benzimidazole moiety was accomplished in three steps: (1) conversion of 3 to its 1,2,3-hydroxybenzotriazole ester (HOBT ester) by reaction with HBTU, (2) condensation with ethyl 3,4-diaminobenzoate to form amide 4, and finally, (3) acid-catalyzed ring closure using sulfuric acid in nitrobenzene (53% overall for the three steps). Reduction of the cyano group in 5, in the presence of di-tert-butyl pyrocarbonate and DIEA, directly afforded the Bocprotected benzylic amine (6), which after saponification provided the target building block 7 in 70% yield.

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Scheme 2a

a Key: (a) HBTU, DIEA in DMF, 10 min, rt, then 3-dimethylaminopropylamine in DMF, 2 h, rt; (b) 2 N HCl, reflux 10 min (68% overall).

Scheme 3

DNA Recognition of Conjugates. To evaluate the potential of this new amino acid, we used it to prepare a simple Hoechst 33258 analogue (8, Scheme 2), a conjugate to a DNA binding (condensing) peptide motif, that is found in histone H1 (20) (9), and a further conjugation of 9 to the DNA intercalator 9-amino acridine (10) (Scheme 3). All conjugates were synthesized on solid support starting with the peptide, and were characterized by MALDI-TOF mass spectrometry. The DNA recognition properties of these compounds were evaluated by DNaseI footprinting (Figure 1) using a 32P-end labeled plasmid DNA restriction fragment (Figure 2). Comparing the footprint produced by 8 with that of Hoechst 33258 shows close to identical (AT target) sequence preference of the two compounds. Concordant with this conclusion, an NMR study of a DNA-8 complex shows AT-minor groove binding (21) fully analogous to that typically observed for Hoechst 33258. The present results (Figure 1a, e.g., compare lanes 4 and 10-11) also indicate a 4-8-fold higher affinity of 8 as compared to Hoechst 33258. In terms of sequence specificity, the binding of the conjugates 9 and 10 is almost identical to that of Hoechst 33258 (and thus also identical to that of 8). All major binding sites contain (A/T)4 motifs (cf. Figure 2), exhibiting the following order of potency (5′-3′-orientation) as

judged from the concentration resulting in ca. 50% protection from DNaseI cleavage: AATTAAT ∼ AATTT > AATT > ATTA > TTTAT > ATAA > TTTA. Furthermore, as estimated from the binding to the most 3′ ATTA target (and also the TTAA target), similar protection by 9 is achieved at 60-fold lower concentration than that required for Hoechst 33258 (compare, e.g., lanes 3 and 7 of Figure 1). Therefore, 9 binds to the DNA targets with ∼60-fold higher affinity than Hoechst 33258, and thus with ca. 10-fold higher affinity than the nonconjugated 8. Finally, no sequence-specific binding of the peptide alone (11, H-KSPKKAKK-NH2) was detected by DNaseI footprinting. Therefore, conjugation of the cationic peptide, KSPKKAKK increases the DNA affinity of the minor groove binder by approximately 10-fold without affecting the sequence preference. Attachment of a DNA intercalating acridine to 9 yielding 10, surprisingly only seem to contribute little additional affinity. As would be expected from the very limited sequence prefence found for DNA binding of 9-amino acridines (22), the sequence preference of 10 was not changed either as compared to 9. Fluorescence Properties. Hoechst 33258 is widely used as a DNA stain in molecular biology and in cytology based on the great increase in quantum yield from 0.01 to 0.6 of its bright blue fluorescence upon binding to DNA

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Figure 1. Autoradiograms of DNaseI footprinting experiments. The compound used is indicated above the lanes, while the concentrations are indicated below the lanes. The putative binding sites are indicated by bars between the autoradiograms. S is an A/G sequence reaction, while C is a control without reagent.

Figure 2. Base sequence of the EcoRI-PvuII restriction fragment used in the DNaseI footprinting experiments. The binding sites detected by the footprinting are indicated.

(23). The dye shows pronounced preference for A/T-rich regions, but also exhibits multiple binding modes and distinct fluorescence emission spectra depending on dye/ base pair ratios (24). As the benzimidazole derivative 8 is closely related to the Hoechst chromophore, we decided to investigate the fluorescence properties of the compound. The electronic spectra of 8 and its complex with CTDNA are displayed in Figure 3. The absorption spectrum (trace a) displays two absorption maxima in the range 250-400 nm. The long-wavelength absorption undergoes

a blue shift from 332 to 340 nm upon complexation with DNA (traces a and b). Upon excitation into the long wavelength band, unbound 8 fluoresces with a maximum at 390 nm (trace e). The spectral profile features an unresolved vibronic structure. However, the fluorescence from the 8-DNA complex (trace f) is more intense (integrated intensity is up by 31% relative to unbound) and appears with three clearly resolved vibronic bands at 372, 392 (max), and 412 nm, corresponding to an average progression of 1300 cm-1. The excitation spectra derived from the fluorescence of compound 8 are seen to comply completely with the long wavelength absorption bands, whether in the free (traces a and c) or in the DNA-bound state (traces b and d). Furthermore, excitation spectra recorded with emission wavelengths set at the three vibronic bands at 372, 392 and 412 nm of trace f were found to be completely identical (spectra not shown), which implies that only one fluorescing species is present when 8 binds to DNA. The maximum observed at 280 nm in the excitation spectrum of DNA-bound 8 (trace d) indicates a small contribution of resonance energy transfer from DNA, the DNA absorption itself is seen going off-scale in trace b.

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Figure 3. Electronic spectra of compound 8 and its complex with CT-DNA in water containing NaCl (0,1 M), KH2PO4 (10 mM), and EDTA (0.1 mM). In all spectra [8] ) 2.2 µM. In traces b, d, and f [basepair CT-DNA] ) 22 µM (see the Experimental Section): (a) absorption, compound 8 (256 ) 2.8 × 104 M-1 cm-1, 332 ) 3.3 × 104 M-1 cm-1); (b) absorption, compound 8 (λmax) 340 nm) bound to CT-DNA; (c) excitation (λem) 400 nm) of compound 8 (λmax) 331 nm); (d) excitation (λem) 400 nm) of compound 8 (λmax) 339 nm) bound to CT-DNA; (e) fluorescence (λex) 338 nm, same intensity scale as spectrum f) of compound 8 (λmax) 390 nm); (f) fluorescence (λex) 338 nm, same intensity scale as spectrum e) of compound 8 bound to CT-DNA.

In general, when comparing corresponding absorption and fluorescence bands, it is fairly common to see a vibrational fine structure in the absorption spectrum being lost in fluorescence (25). Far more unusual is the observation of fine structure in a fluorescence band that corresponds to a structureless absorption, as it can be seen for the 8-DNA complex by comparing traces b and f of Figure 3, respectively. To a lesser extent the same feature is found in the spectra of unbound 8 (traces a and e). The default example of this phenomenon is biphenyl for which a range of torsional conformers is present among ground-state molecules while the distribution narrows in on a more planar conformation in the excited singlet state (26). The same process seems to occur in case of 8 and is especially pronounced when it is bound to DNA. This conclusion is supported by the full-widthat-half-maximum (fwhm) values of the fluorescence spectra, being 4660 cm-1 for unbound 8 (trace e) and 3600 cm-1 for the 8-DNA complex (trace f). The conformational distribution is narrower among the molecules of 8 when it is bound to DNA. Furthermore, the fluorescence appear at virtually the same wavelength whether 8 is free (trace e) or DNA bound (trace f). Most likely, DNA-bound 8 is already forced into planar configuration in the ground state. Thus, the Stoke shift is smaller (3900 cm-1, traces b and f) when 8 is bound than in the unbound form (4490 cm-1, traces a and e) indicating that less reorientation occurs in the excited state of 8 in the complex relative to the unbound molecule. This conclusion is also consistent with minor groove binding of 8, which indeed requires (enforces) coplanarity of the two aromatic systems, as is also consistent with the NMR structure determination of the complex (21). CONCLUSION

The presently described Hoechst analogue represents a versatile and convenient building block for introducing a high affinity minor groove binder into peptides or other

oligoamides via Boc solid-phase chemistry. Thus the monomer should also be useful in combinatorial library approaches. Furthermore, the fluorescense properties of the ligand may be exploited to study DNA binding. ACKNOWLEDGMENT

This work was supported by the Danish Biotechnology Program. We thank Ms. Karin Frederiksen and Ms. Jolanta Ludwigsen for expert technical assistance. LITERATURE CITED (1) Rhodes, D., Schwabe, J. W., Chapman, L., & Fairall, L. (1996) Philos. Trans. R. Soc. London: B Biol Sci. 351, 5019. (2) Oda, M., & Nakamura, H. (2000) Genes Cells 5, 319-26. (3) Nielsen, P. E. (1997) Chem. Eur. J. 3, 505-508. (4) Bewley, C. A., Gronenborn, A. M., & Clore, G. M. (1998) Annu Rev Biophys Biomol Struct. 27, 105-31. (5) Fox, K. R. (2000) Curr Med Chem. 7, 17-37. (6) Nielsen, P. E. (2001) Methods Enzymol. (in press). (7) Dervan, P. B., & Burli, R. W. (1999) Curr. Opin. Chem. Biol. 3, 688-93. (8) Choo, Y., & Isalan, M. (2000) Curr. Opin. Struct. Biol. 10, 411-6. (9) Behrens, C., & Nielsen, P. E. (1998) Combin. Chem. High Throughput Screening 1, 127-134. (10) Zhang, Z., & Herdewijn, P. (2001) Curr. Med. Chem. 8, 517-31 (11) Baird, E. E., & Dervan, P. D. (1996) J. Am. Chem. Soc. 118, 6141-6146. (12) Harshman, K. D., & Dervan, P. D. (1985) Nucleic Acid Res. 13, 4825-4835. (13) Searle, M. S., & Embrey, K. J. (1990) Nucleic Acid Res. 18, 3753-3762. (14) Teng, M. K., Usman, N., Frederick, C. A., & Wang, A. H. J. (2000) Nucleic Acid Res. 16, 2671-2690. (15) Spink, N., Brown, D. G., Skelly, J. V., & Neidle, S. (1994) Nucleic Acid Res. 22, 1607-1612. (16) Quintana, J. R., Lipanov, A. A., & Dickerson, R. E. (1991) Biochemistry 30, 10294-10306.

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