Conjugates of PNA with Naphthalene Diimide Derivatives Having a

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Bioconjugate Chem. 2003, 14, 877−883

877

Conjugates of PNA with Naphthalene Diimide Derivatives Having a Broad Range of DNA Affinities Andriy A. Mokhir and Roland Kraemer* Anorganisch-Chemisches Institut, Ruprecht-Karls-Universitaet Heidelberg, Im Neuenheimer Feld 270, Heidelberg 69120, Germany. Received October 31, 2002; Revised Manuscript Received June 23, 2003

Peptide nucleic acids (PNAs) are neutral DNA analogues, which bind single-stranded DNA (ssDNA) strongly and with high sequence specificity. However, binding efficiency is dependent on the purine content of the PNA strand. This property make more difficult application of PNA as hybridization probes in, e.g., PNA chips, since at a set temperature the hybridization of a fraction of the DNA targets to the PNA probes does not obey Watson-Crick binding rules. The polypurine PNAs, for example, bind the mismatch containing DNA targets stronger, than the pyrimidine rich PNAs their fully complementary targets. Herein we show that PNA-DNA binding efficiency can be finely tuned by the conjugation of derivatives of naphthalene diimide (NADI) to the N-terminus of PNA using polyamide linkers of different lengths. This approach can potentially be used for the design of PNA probes, which bind their DNA targets with similar affinity independently of the PNA sequence.

INTRODUCTION

Peptide nucleic acids are DNA analogues, in which the sugar-phosphate backbone has been substituted by N-(2aminoethyl)-glycine units. Therefore, PNA is characterized by high chemical stability and high nucleic acid binding affinity and specificity (1). Additional desirable properties of PNA can be introduced via terminal conjugation of small molecules. The majority of such modifications reported up-to-date facilitate detection of PNA, using for example fluorescence spectroscopy (2), electrochemistry (3), and vibrational spectroscopy (4). There is just a limited number of reports on terminal modifications increasing affinity of PNA toward ssDNA significantly (5, 6). This would be important, first, because PNA-DNA duplex stability is dependent on the purine content of PNA strand, which makes applications of PNA probes more difficult (7). In particular, at a set hybridization temperature binding of mismatch polypyrimidine DNA sequences will be as efficient as binding of match polypurine DNA sequence. Second, PNA probes with high binding affinity toward ssDNA may be able to disrupt dsDNA, by forming PNA-DNA duplex and leaving one DNA strand unbound, which was reported up to now only for polypurine PNA (8). Alternative mechanism of disruption of dsDNA was reported for pseudocomplementary PNA, which bind both DNA strands (9, 10). Sequence specific dsDNA recognition, which is not restricted to polypurine/polypyrimidine DNA sequences can potentially find many applications, including, e.g., artificial restriction enzymes, gene suppression, labeling, and identification. Although many small molecules stabilizing DNA duplexes are known and factors important for efficient ligand-dsDNA interactions are well understood (11-13), this knowledge cannot be directly applied for the design * Corresponding author’s address: Anorganisch-Chemisches Institut, Ruprecht-Karls-Universitaet Heidelberg, Im Neuenheimer Feld 270, Heidelberg 69120, Germany. Tel.: (0)6221548438. Fax: (0)6221-548599. E-mail: [email protected].

of ligands affecting PNA-DNA duplex stability, because the PNA-DNA duplex is structurally considerably different from DNA duplex and has a lower negative charge density. Common intercalators and groove-binders are normally poor ligands for PNA-DNA duplexes (14). Therefore, further studies on new modifications stabilizing PNA-DNA duplexes are warranted. N-terminal modifications affecting PNA-DNA duplex thermal stability substantially have been reported by Okamoto et al. for 8-methoxypsoralen and Harrison et al. for polycationic peptides (for both ∆Tm ) + 8.0 °C) (5). It should be noted that an alternative strategy including incorporation of nonnatural bases into the interior of PNA has proven to be successful for stabilization of PNA-DNA duplexes, but it often includes laborious synthesis (15, 16). For making PNA probes having sequence independent binding affinity toward DNA, N-terminal modifiers with different stabilizing power are needed. Our strategy for generation of such a set of modifications is based, first, on modulation of π-stacking interactions between a planar aromatic fragment and the PNA-DNA duplex using linkers of different length. Second, the conjugate with the best linker is modified by attaching DNA binding ligands (Figure 1B), which further stabilize PNA-DNA duplex. π-Stacking between the planar fragment and basepairs of PNA-DNA restricts motions of the ligands to the region close to DNA, which should entropically favor ligand-DNA interactions in comparison with a fully flexible system (Figure 1A). Herein we demonstrate that this two-step approach allows generation of N-terminal PNA modifications with a broad range of effects on stability of PNA-DNA duplexes. EXPERIMENTAL PROCEDURES

General. The best commercially available chemicals from Acros (Geel, Belgium), Aldrich/Sigma/Fluka (Deisenhofen, Germany), Advanced Chemtech (Louisville, KY) and Novabiochem (Weidenmattweg, Switzerland) were obtained and used without purification. The reagents for PNA synthesis were obtained from PerSeptive

10.1021/bc0256345 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/29/2003

878 Bioconjugate Chem., Vol. 14, No. 5, 2003

Figure 1. (A) Positively charged fragment conjugated to PNA via a flexible linker. (B) Internal motions of the modification are reduced due to π-π interaction of naphthalene diimide and basepairs of PNA-DNA. This favors interaction of modification (+) with DNA (-). (C) A terminal part of 19a:26 duplex obtained from molecular modeling using Hyperchem 5.0.2.

Biosystems (Hamburg, Germany) and fluorescent metal indicators from Molecular Probes (Leiden, The Netherlands). The N-allyloxycarbonylethylenediamine was synthesized as described (15). MALDI-TOF mass spectra were recorded on a Bruker BIFLEX III spectrometer. The solution of 3,5-dimethoxy-4-hydroxycinnamic acid (27 mM, TFA 0.1%, CH3CN 33% in water) was used as a matrix for MALDI-TOF analysis of the PNA conjugates. Samples for mass spectrometry were prepared on a Bruker MAP II probe preparation station by dried droplet method using 1/2 probe/matrix ratio for water and water/ CH3CN solutions (HPLC fractions) and 1/20 ratio for TFA/m-cresol (4/1) solutions. Mass accuracy with external calibration was 0.1% of the peak mass, i.e., ( 3.0 at m/z 3000. HPLC was performed at 22 or 49 °C on a Shimadzu liquid chromatograph equipped with UV-Vis detector, column oven and Advantec SF-2100W fraction collector. Macherey-Nagel Nucleosil C4 250 × 4.6 mm column with gradients of CH3CN (0.1% TFA, solvent B) in water (0.1% TFA, solvent A) was used (gradient A: 49 °C, 0% B for 5 min, in 30 min to 35% B, in 10 min to 90% B, 90% B for 10 min; gradient B: 22 °C, 0-2% B for 5 min, in 23 min to 20% B, in 7 min to 95% B, 95% B for 10 min). NMR spectra were recorded on a Bruker Avance 200 spectrometer. UV melting experiments were performed on a Varian Cary 100 Bio UV-Vis spectrophotometer measuring absorbance at 260 nm in 1-cm black wall semimicrocuvettes with sample volume 0.7 mL, 2 µM strand concentration in MOPS buffer (10 mM, pH 7), NaCl (50 or 150 mM), and with or without Zn2+ (4 µM). Cooling and heating rates were 0.5 °C/min. Melting points were averages of the extrema of the first derivative of the 61point smoothed curves from at least two cooling and two heating curves. Fluorescent spectra were acquired on a Varian Cary Eclipse fluorescence spectrometer. Synthesis of 3. Fmoc-Gly-OH was coupled to Wang resin (5 g, 4.5 mmol of free hydroxyl groups) using diisopropylcarbodiimide (16). Loading of Fmoc-group on the resin was found to be 0.7 mmol/g. Unreacted hydroxyl groups were capped with Ac2O/pyridine, and Fmoc-group was deprotected with piperidine/DMF. 1,4,5,8-Naphthalene-tetracarboxylic dianhydride (3.35 g, 12.5 mmol) was suspended in pyridine (65 mL) and added to the resin. The mixture was heated to 65 °C and DIEA (4.4 mL, 25 mmol) was added. The slurry obtained was refluxed for 8 h, then filtered hot, washed thoroughly with hot (90

Mokhir and Kraemer

°C) pyridine, with DMF (2 × 10 mL) and CH3CN (2 × 10 mL), and dried under vacuum (10-2 mbar). A solution of Ac2O (1.9 mL, 20 mmol) in pyridine (65 mL) was added to the resin. After 30 min the resin was filtered, washed with DMF (2 × 10 mL) and CH3CN (2 × 10 mL), and dried under vacuum (10-2 mbar). Trifluoroacetic acid in CH2Cl2 (20%, 60 mL) was added to the resin. After the sample was shaken for 3 h, the slurry was filtered and the resin was washed with CH2Cl2 (30 mL). The filtrates obtained were combined, volatiles were evaporated (70 mbar), and diethyl ether (200 mL) was added to the solid residue. The resulting suspension was cooled to 4 °C and kept at this temperature for 8 h. After this, the precipitate formed was filtered off, washed with diethyl ether, and dried under vacuum (10-2 mbar) to give yellow amorphous solid, 0.59 g, 54%. LDI-TOF MS (negative mode) m/z found 325.9, calcd. for C16H6NO7 [M•]- 325.2. 1 H NMR (200 MHz, DMSO-d6): δ 8.60 (s, 4H), 4.67 (s, 4H). Synthesis of 4. N-Allyloxycarbonylethylenediamine (0.27 g, 1.8 mmol), diisopropylethylamine (0.7 mL, 4.0 mmol), and 3 (0.59 g, 1.8 mmol) were mixed together in hot pyridine (80 mL, 65 °C), and the resulting suspension was stirred at this temperature for 8 h. Then volatiles were evaporated (10 mbar, 40 °C), aqueous HCl solution (1%, 50 mL) added, and the precipitate formed was filtered, washed with water (10 mL), and dried (0.01 mbar) to give the title product 0.59 g, 70%. LDI-TOF MS (positive mode) m/z found 451.6, calcd. for C22H17N3O8 [M•]+ 451.1. TLC in ethanol/AcOH/CH2Cl2 (5/2/97): Rf 0.6. 1 H NMR (200 MHz, DMSO-d6, at 60 °C): δ 8.61 (m, 4H), 6.97 (broad s, 1H), 5.66 (m, 1H), 5.08 (d, 3J ) 17 Hz, 1H), 4.97 (d, 3J ) 10 Hz, 1H), 4.68 (s, 2H), 4.27 (m, 2H), 4.11 (m, 2H), 3.28 (m, 2H). 13C NMR (50 MHz, DMSO-d6): δ 169.75, 163.50, 163.01, 156.88, 134.43, 131.66, 131.13, 127.71, 127.10, 126.79, 126.15, 117.34, 64.86, 42.26. UVVis: λmax (nm) 381, 361, 344 shoulder. Synthesis of PNA Conjugates. Synthesis of PNA Part of the Conjugates. The PNA part of the conjugates was synthesized on an Expedite 8909 PNA/DNA synthesizer according to the manufacturers’ recommendations for 2 µmolar scale synthesis. Coupling of Monoamines and Symmetric Diamines to Polymer Bound PNA. Bromoacetyl bromide (8.7 µL, 100 µM) and DIEA (19 µL, 110 µmol) in DMF (1 mL) are added to the resin bound PNA bearing free amino group. The resulting suspension is left shaking for 1 h, then filtered, the resin washed with DMF (2 × 1 mL), CH3CN (2 × 1 mL), and dried under vacuum (10-2 mbar). Amine (100 µM) and DIEA (19 µL, 110 µmol) in CH2Cl2 (1 mL) are added to the resin and the resulting mixture is left on a shaker for 8 h. Then, the resin is filtered, washed with CH2Cl2 (2 × 1 mL), DMF (2 × 1 mL), and CH3CN (2 × 1 mL), and dried under vacuum (10-2 mbar). Coupling of Carboxylic Acids to Polymer Bound PNA. Carboxylic acid (100 µmol), HBTU (34 mg, 90 µmol), and HOBT (14 mg, 100 µmol) are dissolved in DMF (1 mL) and DIEA (38 µL, 220 µmol) is added. This solution is vortexed and immediately added to H2N-PNAPG-RinkPS resin (2-4 µmol of free amino-groups). The slurry obtained is left under vigorous mixing for either 0.5 h for chiral amino acids or 1 h for the others. Then, the resin is filtered, washed with DMF (2 × 1 mL) and CH3CN (2 × 1 mL), and dried under vacuum (10-2 mbar). Removal of Fmoc Groups. Piperidine/DMF (1/4) mixture (1 mL) is added to the resin bound PNA containing Fmoc protected amino group(s) (2-4 µmol) and the reaction is allowed to proceed for 30 min. At the end of this time the resin is filtered, washed with DMF (2 × 1

Conjugates of PNA with Naphthalene Diimide

Bioconjugate Chem., Vol. 14, No. 5, 2003 879

Table 1. Optimization of the Linker between Naphthalene Diimide and the N-Terminus of PNA no

duplex

linkera

Tm (°C)b

∆Tmc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

12:26 19a:26 16:26 14:26 20:26 19a:30 20:30 19f:32 25:32 19e:31 24:31 19b:27 21:27 19c:28 22:28 19d:29 23:29

-CH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH(CH2NH2)NHCH2C(O)-C(dO)CH(NH2)(CH2)4NHCH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH2NHCH2C(O)-C(dO)CH2NHCH2C(O)-

63.5 ( 1.1 68.7 ( 1.4 (57.6 ( 1.1) 69.9 ( 2.3 69.2 ( 1.0 61.0 ( 1.0 (52.9 ( 1.2) 55.1 ( 0.3 47.8 ( 0.9 68.5 ( 1.4 55.9 ( 0.7 22.0 ( 0.7 +7.0 0 +13.9 0 +12.3 0 +12.6 0

a Linker connecting naphthalene diimide with N-terminus of PNA. b Melting point of PNA-DNA at 10 mM MOPS, pH 7, 50 mM NaCl, [PNA] ) [DNA]) 2 µM. In brackets are melting points for 150 mM NaCl solutions. c Difference in melting points of corresponding modified and nonmodified PNA-DNA. Vertical lines in the table separate PNAs with different sequences.

mL) and CH3CN (2 × 1 mL), and dried under vacuum (10-2 mbar). Removal of Alloc Groups. A mixture of tetrakis(triphenylphosphine)palladium(0) complex (5 mg, 4.3 µmol) and triphenylphosphine (1 mg, 3.8 mmol) is dissolved in CH2Cl2 (0.5 mL), and thoroughly purged with argon. Separately, diethylammonium hydrocarbonate (5 mg, 38 µmol) is suspended in CH2Cl2, purged with argon and added to the solution of the Pd complex. The resulting solution is added to the resin bound PNA containing Alloc protected amino group(s) (2-4 µmol) and the reaction is allowed to proceed for 2 h. At the end of this time, the resin is filtered, washed with CH2Cl2 (2 × 1 mL), DMF (2 × 1 mL) and CH3CN (2 × 1 mL), and dried under vacuum (10-2 mbar). PNA Cleavage, Deprotection, and Workup. The resin bound PNA conjugate (0.5-4 µmol) is treated with TFA/ m-cresol mixture (4/1 v/v, 120 µL) for 1.5 h. The resin is filtered off, the filtrate mixed with diethyl ether (2.5 mL), and the precipitate formed is filtered, washed with diethyl ether (2 × 1 mL), dried (0.01 mbar) and HPLC purified. Compound 10. HPLC gradient A: Rt ) 25.7 min. Yield 7.0%. MALDI-TOF MS for C146H197N66O35 [M+H]+: calcd 3434.6, found 3434.1. Compound 11. HPLC gradient A: Rt)23.6min.Yield8.2%.MALDI-TOFMSforC142H188N65O35 [M+H]+: calcd 3363.5, found 3363.5. Compound 12. HPLC gradient B: Rt ) 43.8 min. Yield 6.1%. MALDITOF MS for C132H170N61O33 [M+H]+: calcd 3137.3, found 3138.0. Compound 19a. HPLC gradient A: Rt ) 22.5 min. Yield 9.2%. MALDI-TOF MS for C134H173N62O34 [M+H]+: calcd 3194.4, found 3194.8. Compound 19b. HPLC gradient A: Rt ) 23.0 min. Yield 10.1%. MALDITOF MS for C133H172N63O33 [M+H]+: calcd 3179.4, found 3178.3. Compound 19c. HPLC gradient A: Rt ) 24.3 min. Yield 11.4%. MALDI-TOF MS for C134H172N65O32 [M+H]+: calcd 3203.4, found 3204.7. Compound 19d. HPLC gradient A: Rt ) 24.0 min. Yield 5.2%. MALDITOF MS for C134H172N65O33 [M+H]+: calcd 3219.4, found 3220.6. Compound 19e. HPLC gradient A: Rt ) 17.8 min. Yield 14.2%. MALDI-TOF MS for C92H120N41O22 [M+H]+: calcd 2150.9, found 2149.2. Compound 19f. HPLC gradient A: Rt ) 20.0 min. Yield 11.0%. MALDITOF MS for C114H146N55O28 [M+H]+: calcd 2733.2, found 2732.2. Compound 16. HPLC gradient B: Rt ) 43.9 min. Yield 5.1%. MALDI-TOF MS for C135H176N63O34 [M+H]+: calcd 3223.4, found 3222.0. Compound 14. HPLC

gradient B: Rt ) 44.3 min. Yield 3.7%. MALDI-TOF MS for C138H182N63O34 [M+H]+: calcd 3265.4, found 3265.2. Compound 18. HPLC gradient A: Rt ) 24.7 min. Yield 11.0%. MALDI-TOF MS for C128H186N63O30 [M+H]+: calcd 3085.5, found 3085.9. Compound 20. HPLC gradient A: Rt ) 20.7 min. Yield 9.5%. MALDI-TOF MS for C114H159N58O28 [M+H]+: calcd 2788.3, found 2786.0. Compound 21. HPLC gradient A: Rt ) 19.1 min. Yield 11.8%. MALDI-TOF MS for C113H158N59O27 [M+H]+: calcd 2773.3, found 2771.8. Compound 22. HPLC gradient A: Rt ) 19.6 min. Yield 12.1%. MALDI-TOF MS for C114H158N61O26 [M+H]+: calcd 2797.3, found 2796.8. Compound 23. HPLC gradient A: Rt ) 20.4 min. Yield 7.9%. MALDI-TOF MS for C114H158N61O27 [M+H]+: calcd 2813.3, found 2814.1. Compound 24. HPLC gradient A: Rt ) 14.0 min. Yield 12.3%. MALDI-TOF MS for C72H106N37O16 [M+H]+: calcd 1744.9, found 1743.1. Compound 25. HPLC gradient A: Rt ) 18.0 min. Yield 12.3%. MALDI-TOF MS for C94H131N51O22 [M+H]+: calcd 2326.1, found 2325.0. RESULTS

The design of N-terminal PNA modifications utilizes a linker component, a planar aromatic fragment and a DNA binding ligand. The linker component is varied to modulate π-stacking interactions between the duplex and the planar aromatic fragment. We have chosen polyamide linkers (Table 1), since their length and flexibility can be easily controlled by a variation of amino acid fragments using commercially available derivatives. Naphthalene diimide (NADI) was selected as a planar aromatic fragment because its geometric parameters are closely related to those of natural AT and GC pairs and it efficiently interacts with DNA duplexes and to some extent with triplexes by means of π-stacking (19-21). Moreover, it offers two imide groups available for modification, positioned on opposite sides, on the line dividing this molecule into two equal parts. One imide group was used for the conjugation with PNA and the other one for the attachment of DNA binding ligands. The latter were chosen to be macrocyclic ligands: L1 or L2 (Scheme 2). These ligands are able to bind negatively charged DNA due to electrostatic interactions (L1 and L2 are positively charged at pH 7), hydrogen bonding or, when they form metal complexes with, e.g., Zn2+, due to metal coordination (e.g., LZn- - -OP). Moreover, they increase PNA

880 Bioconjugate Chem., Vol. 14, No. 5, 2003 Scheme 1. Synthesis Building Blocka

of

Naphthalene

Mokhir and Kraemer Diimide

Scheme 2. Synthesis of PNA Conjugates 10 and 11a

a(a) Piperidine, DMF, (b) 1,4,5,8-naphthalene-tetracarboxylic dianhydride, DIEA, pyridine, (c) Ac2O, pyridine, (d) TFA, CH2Cl2, (e) N-(allyloxycarbonyl)ethylenediamine, DIEA, pyridine.

solubility in water in contrast to typically hydrophobic intercalators and groove binders. Modeling. Molecular modeling using AMBER force field energy minimization (Hyperchem 5.0.2) was conducted to probe whether the ligands attached to the imide group of the NADI∼PNA conjugate (conjugate 19a was used as example, -NH2 as the ligand) are well positioned for interaction with DNA. Geometry of the terminal basepair of the duplex was taken from a reported NMR structure (22), the NADI structure was energy-minimized using AMBER force field and initial parameters for amino acids were from Hyperchem template set. In the energy minimized structure (Figure 1C), the NADI and a basepair are positioned one over another with 3.4 Å distance between their mean planes, which indicates their π-stacking interaction. Distance between PNA and DNA termini in the duplex is ∼18.3 Å. In the conjugate modeled the -NH2 ligand is positioned on the side of the DNA strand in the PNA-DNA duplex and the distance between the point of the ligand connection and the DNA terminus is ∼8.7 Å. Thus, ligands conjugated to PNA via the NADI linker are positioned 9.6 Å closer to the DNA backbone than those attached directly to the PNA terminus. Synthesis. Unsymmetrically substituted intermediate 4 (Scheme 1) was required for synthesis of PNA conjugates 10-12, 14, 16, and 19a-f. This compound has a carboxylic group for coupling with the free amino group of PNA and an Alloc protected amino group, which serves for the conjugation of L1 and L2 (Scheme 2). Alloc group can be cleaved under mild neutral conditions, which do not affect acid (Bhoc, Boc) and base (Fmoc) sensitive groups employed in PNA protection. In known methods of synthesis of unsymmetrical naphthalene diimides (1921), two amines are reacted with 1,4,5,8-naphthalenetetracarboxylic dianhydride to produce a statistical product mixture, which is separated by column chromatography. Application of this methodology for synthesis of 4 has produced the desirable product with low yield, mainly because of low product solubility in organic solvents and

a(a) PNA synthesis, (b) (1) (Fmoc)-Gly-OH, HBTU, HOBT, DIEA, DMF, (2) piperidine, DMF, (c) (1) (Alloc)-NADI-OH (4), HBTU, HOBT, DIEA, DMF, (2) Ac2O, pyridine, (3) Pd(PPh3)4, PPh3, (NEt2H2)(HCO3), CH2Cl2, (d) bromoacetylbromide, DIEA, DMF, (e) (1) HL1(2), DIEA, CH2Cl2, (2) TFA, m-cresol. (*) PG) protecting groups: Bhoc for PNA nucleobases: A, C, and G and Boc for -NH2 of PNA lysines.

similar mobility of symmetrical and unsymmetrical products in silica gel. Therefore, a new method of synthesis was developed (Scheme 1). First, compound 1 was synthesized by coupling Fmoc-Gly-OH to Wang resin under standard conditions (18), then Fmoc group was cleaved and free amino group was acylated with an excess of 1,4,5,8-naphthalene-tetracarboxylic dianhydride. After acetylation of unreacted amino groups, cleavage from the solid support and precipitation using diethyl ether compound 3 was obtained (>90% purity). Finally, the reaction of 3 with N-(allyloxycarbonyl)ethylenediamine gave the desired product 4 (>90% purity). In contrast to known procedures, this method can be used for combinatorial synthesis of unsymmetrical naphthaline diimides, since it does not require laborious purification and a range of suitable starting materials (monoprotected diamines and Fmoc-protected amino acids) are commercially available. For the optimization of the interaction of naphthalene diimide with PNA-DNA basepairs product 4 was attached to the N-terminus of PNA via linkers of different length: directly (PNA 12), via a three-atom achiral linker (PNA 19a), three-atom chiral linker (PNA 16) and sevenatom linker (PNA 14). Synthesis of 19a and 16 was performed by first coupling corresponding Fmoc protected amino acid to resin bound PNA (6, Scheme 2) using HBTU, HOBT, DIEA activating mixture (7, Scheme 2, 15, Scheme 3) then Fmoc deprotection, acylation with 4, Alloc cleavage using Pd0 catalyst and finally cleavage from the solid support using TFA. MALDI-TOF mass spectrum of the crude product 19a (Figure 2, A) il-

Conjugates of PNA with Naphthalene Diimide Scheme 3. 18, 19a-fa

Bioconjugate Chem., Vol. 14, No. 5, 2003 881

Synthesis of PNA Conjugates 12, 14, 16,

Figure 2. MALDI-TOF mass spectra of crude 19a (A) and 10 (B). Scheme 4 Nonmodified PNA and DNA Used for Hybridization Experiments

a (a) (1) (Alloc)-NADI-OH (4), HBTU, HOBT, DIEA, DMF, (2) Ac2O, pyridine, (3) Pd(PPh3)4, PPh3, (NEt2H2)(HCO3), CH2Cl2, (4) TFA, m-cresol, (b) (Fmoc)Lys(-N-Alloc)-OH, HBTU, HOBT, DIEA, DMF, (c) (1) a3, (2-4) a1-3, (5) piperidine, DMF, (6) a4, (d) (1) (Fmoc)-Dpr(β-N-Boc)-OH, HBTU, HOBT, DIEA, DMF, (2) b5, (3-6) a1-4, (f) bromoacetyl bromide, DIEA, DMF, (g) (1) HL1, DIEA, CH2Cl2, (2) a4, h. a1-4.

lustrates high synthesis efficiency. Compound 14 was synthesized analogously, except that after coupling FmocLys(-Alloc)-OH, Alloc group was cleaved first, free amino group was acylated with the 4, and Alloc and Fmoc groups were consequently cleaved (Scheme 3). Intermediate 8 was used for the conjugation of amines (Scheme 2). Its bromoacetylation gave intermediate 9, which is suitable for the attachment of a variety of mono and symmetric polyamines. The desired conjugates 10, 11 were obtained by reacting 9 with an excess of HL1 or HL2. MALDI-TOF mass spectrum of crude 10 is shown in Figure 2B. Following TFA/m-cresol deprotection and PNA precipitation using diethyl ether the conjugates were purified in one (12, 14, 16, 18, 19a-f) or two (10, 11) HPLC runs. Nonmodified PNA and DNA used in this study are shown in Scheme 4. Hybridization of PNA with ssDNA. Naphthalene dimide stabilizes PNA-DNA duplexes best, when it is

attached to the N-terminus of the PNA via linkers consisting of five atoms (∆Tm ) +7.7-8.9 °C, entries 2 and 3, Table 1). If the linker is longer than five atoms, its size seems to be unimportant (entry 4). However, stabilization of the duplex is much reduced when a shorter linker is used (∆Tm ) 2.5 °C, entry 1). Therefore, in all further experiments the NADI has been conjugated with the N-terminus of the PNA via the -C(O)CH2NHCH2C(O)- linker. Analogues of 19a with other PNA sequences (19b-f) were synthesized to test generality of the effect observed. In all cases, PNA-DNA duplexes were more stable than corresponding nonmodified PNA-DNA duplexes (entries 6-17, Table 1). Moreover, duplexes of PNA 19a with long, 26 and short, 30 DNA were stabilized to the same extent (∆Tm ) +7.7 and +7.3 °C respectively, entries 2 and 6). UV-spectra of the NADI∼PNA show characteristic absorption bands for polynucleotides (λmax ) 260 nm) and for naphthalene diimides (λmax ) 381, 361, and 344 nm) (19-21) (Figure 3). Upon titration of PNA solutions with complementary DNA, 26, the intensity of the absorption bands of NADI

882 Bioconjugate Chem., Vol. 14, No. 5, 2003

Mokhir and Kraemer

Figure 3. UV-visible spectra of 10 (solid line) and 10:26 (dotted line) recorded in MOPS buffer 10 mM pH 7, NaCl 50 mM, [PNA] ) [DNA] ) 2 µM. Table 2. Melting Points of PNA-DNA Duplexes no

duplex

Tm (°C)a

1 2 3 4 5 6

10:26 10:26/Zn2+ 11:26 11:26/Zn2+ 18:26 18:26/Zn2+

[NaCl] 50 mM 72.6 ( 0.7 72.0 ( 1.0 70.3 ( 1.6 70.3 ( 0.7 60.8 ( 1.8 60.5 ( 1.9

∆Tm (°C)b [NaCl] 150 mM 61.3 ( 1.0 60.6 ( 1.7 60.1 ( 1.1 60.2 ( 1.2 53.1 ( 2.1 52.5 ( 1.5

11.6 (8.3) 11.0 (7.7) 9.3 (7.2) 9.3 (7.3) -0.2 (+0.2) -0.5 (-0.4)

a Melting point of PNA-DNA at 10 mM MOPS, pH 7, [PNA] ) [DNA] ) 2 µM and with 4 µM Zn2+ or without. For melting experiments 2 equiv of Zn2+ were used (4 µM), since at lower concentrations dissociation and association curves were considerably shifted from each other (hysteresis effect). b Difference in melting points of PNA-DNA duplexes and the control 20:26 duplex at 50 mM NaCl, and in brackets at 150 mM NaCl.

decreases, which signals an intercalation of NADI in the PNA-DNA duplex (19). Moreover, melting transition of 19a:26 duplex monitored at 260 and 361 nm occurs at the same temperature, which indicates cooperative dissociation of both PNA-DNA basepairs and NADI. Importantly, the N-terminal modification of PNA with NADI does not significantly alter PNA sequence specificity toward DNA (Supporting Information). The duplex of DNA 26 with conjugate 10, which consists of PNA, NADI, and macrocycle L1 melts at a temperature 3.9 °C higher than that of 19a:26 and 11.6 °C higher than nonmodified 20:26 (entries 1 and 3, Table 2). Interestingly, DNA affinity of L1-NH-Gly-PNA conjugate (18), which does not contain the NADI, is practically the same as that of the nonmodified PNA 20 (entry 5, Table 2). Due to the high affinity of L1 and L2 toward Zn2+, this cation is expected to be fully bound by conjugates 10, 11 at micromolar concentrations. In agreement with this, fluorescent Zn2+ indicator Newport Green does not detect Zn2+ ions in 10(11)/Zn2+ solutions (1:1, 2 µM). However, stability of 10:26 and 11:26 duplexes is not affected by Zn2+ (4 µM). DISCUSSION

In the duplexes of DNA with NADI∼PNA conjugates, NADI is π-stacking with the duplex basepairs, as it is evident from a substantial increase in thermal stability of the duplexes of PNA 19a-f with corresponding complementary DNA (∆Tm) 7.7-13.9 °C, Table 1) and an apparent hypochromicity of the absorption bands of the NADI chromophore in duplexes. The absence of terminal mismatch discrimination in NADI∼PNA-DNA duplexes is in agreement with intercalative binding, since alternative stacking on the terminal basepair is expected to increase terminal mismatch discrimination (23). Basestacking interaction between the NADI and the duplex is further indicated in that melting profiles obtained at

361 nm for modified PNA-DNA duplexes exhibited cooperative increase in hyperchromicity with a midpoint in the transition that corresponded to that obtained at 260 nm (19). π-Stacking of naphthalene diimide with DNA duplexes and triplexes is well documented (17-19), while for PNA-DNA duplexes it is observed for the first time. This is an interesting result, because due to considerable structural differences of PNA-DNA and DNA-DNA duplexes and the lower negative charge density of the former, usual intercalators and groovebinders of double stranded DNA are often poor ligands for PNA-DNA duplexes. In contrast to naphthalene diimide, N-terminally conjugated naphthalene imide was found to stabilize the PNA-DNA duplex insignificantly (∆Tm ) 0.5 °C) (24). Surprisingly, NADI equally stabilizes the fully matched PNA-DNA duplexes and those with a single mismatch in the terminal and penultimate positions (Supporting Information). This might indicate that the naphthalene diimide is positioned between the second and the third basepairs of the PNA-DNA duplexes. However, more detailed structural studies are required for unambiguous conclusions about the structure of these duplexes. The five atom linker, -C(O)CH2NHCH2C(O), which is as well used for the attachment of nucleobases in PNA, positions NADI well for π-stacking in the duplex (∆Tm ) +7.7 °C, entries 2 and 5, Table 1). The use of a shorter linker allows obtaining a PNA of lower DNA affinity (∆Tm ) + 2.5 °C, entries 1,5, Table 1), while the positively charged linker with the same number of atoms as well as the longer one give the PNAs of slightly higher DNA affinity (∆Tm ) +8.9 and +8.2 °C, respectively, entries 3-5, Table 1). Since the stabilization trend for the five atom linker is reproduced for six PNA sequences and for two DNA targets, we believe that these effects have a general character. The results presented in this study outline an approach for increasing efficiency of ligand-DNA interaction by a combination of proper ligand positioning and ligand motion constraining. Since the intercalator is connected with the N-terminus of PNA at one end and its orientation is fixed by π-π interaction with the terminal and the penultimate basepairs of PNA-DNA, the other end is positioned at the negatively charged DNA backbone (Figure 1C). If a DNA binding ligand is attached, its orientation in relation to the DNA will be predefined by the NADI position. Such constraining of the ligand position should facilitate ligand-DNA interaction due to proximity effects, reduced possibilities for internal motions of the ligand and environment effects (duplex interior is hydrophobic, therefore polar interactions will be stronger than in water). This is illustrated by stabilization of 10:26 in comparison with 19a:26 (∆Tm ) +3.9 °C). As expected, when L1 is attached directly to the PNA, no stabilization is observed (entry 5, Table 2). At higher salt concentration, 10:26 melts at a temperature 3.7 °C higher than 19a:26. This salt independent stabilization cannot be rationalized in terms of electrostatic interactions between positively charged L1 and negatively charged DNA backbone, since it would be reduced in the presence of higher salt concentration. Hydrogen bonding (with, e.g., deprotonated phopsodiester groups of the DNA backbone) is a feasible possibility, since it is a salt independent interaction, and it is expected to be considerably stronger in the hydrophobic duplex interior than in water solution. The method of synthesis of 10, 11 can be easily adapted to other amine-containing ligands interacting with or modifying DNA, e.g. intercalators, groove binders, metal

Conjugates of PNA with Naphthalene Diimide

binding ligands and alkylating agents. Due to the factors mentioned above for L1, these ligands, when positioned by the NADI in close proximity to the DNA, should have a stronger effect on the DNA than the ligands directly attached to the PNA. Finally, the N-terminal PNA modifications increasing thermal stability of PNA-DNA duplexes by 2.5, 7.7, 8.2, 8.9, 9.3, and 11.0 °C found in this study can be potentially used for the design of PNA probes, whose DNA binding affinity is sequence independent. ACKNOWLEDGMENT

We thank Ruprecht-Karls-Universitaet Heidelberg, Fonds der Chemischen Industrie and Ministerium fu¨r Wissenschaft, Forschung und Kunst Baden-Wu¨rttemberg for financial support. A.M. thanks Prof. C. Richert for his help on early stages of the development of this project. Supporting Information Available: Melting points of duplexes of conjugate 19a and nonmodified PNA 20 with complementary and mismatch DNAs. MALDI-TOF mass spectra of all new compounds reported in this paper. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Nielsen, P. E., and Egholm, M. (1999) An Introduction to PNA. Peptide Nucleic Acids Protocols and Applications (Nielsen, P. E., and Egholm, M., Eds.) pp 1-19, Horizon Scientific Press, England. (2) Seitz, O., and Koehler, O. (2001) Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase. Chem. Eur. J. 7, 3911-3925. (3) Verheijen, J. C., van der Marel, G. A., van Boom, J. H., and Metzler-Nolte, N. (2000) Transition metal derivatives of peptide nucleic acid (PNA) oligomers- synthesis, characterization, and DNA binding. Bioconjugate Chem. 11, 741-743. (4) Hess, A., and Metzler-Nolte, N. (1999) Labeling of [Leu5]enkephalin with organometallic Mo complexes by solid-phase synthesis. Chem. Commun. 11, 885-886. (5) Okamoto, A., Tanabe, K., and Saito, I. (2001) Synthesis and properties of peptide nucleic acids containing psoralen unit. Org. Lett. 3, 925-927. (6) Uhlmann, E., Peyman, A., Breipohl, G., and Will, D. W. (1998) PNA: synthetic polyamide nucleic acids with unusual binding properties. Angew. Chem. Int. Ed. 37, 2796-2823. (7) Ratilainen. T., Holmen, A., Tuite, E., Nielsen, P. E., and Norden, B. (2000) Thermodynamics of sequence-specific binding of PNA to DNA. Biochemistry 39, 7781-7791. (8) Nielsen, P. E., and Christensen, L. (1996) Strand displacement binding of a duplex-forming homopurine PNA to a homopyrimidine duplex DNA target. J. Am. Chem. Soc. 118, 2287-2288. (9) Demidov, V. V., Protozanova, E., Izvolsky, K. I., Price, C., Nielsen, P. E., and Frank-Kamenetskii, M. D. (2002) Kinetics and mechanism of the DNA double helix invasion by

Bioconjugate Chem., Vol. 14, No. 5, 2003 883 pseudocomplementary peptide nucleic acids. Proc. Natl. Acad. Sci. U.S.A. 99, 5953-5958. (10) Lohse, J., Dahl, O., and Nielsen, P. E. (1999) Double duplex invasion by peptide nucleic acid: A general principle for sequence-specific targeting of double-stranded DNA. Proc. Natl. Acad. Sci. U.S.A. 96, 11804-11808. (11) Graves, D. E., and Velea, L. M. (2000) Intercalative binding of small molecules to nucleic acids. Curr. Org. Chem. 4, 915929. (12) Wilson, W. D. (1999) DNA intercalators. Comprehensive Natural Products Chemistry, Vol. 7 DNA and Aspects of Molecular Biology (Kool, E. T., Ed.) pp 427-476, Elsevier Science B. V., Amsterdam, Netherlands. (13) Erkkila, K. E., Odom, D. T., and Barton, J. K. (1999) Recognition and Reaction of Metallointercalators with DNA. Chem. Rev. 99, 2777-2795. (14) Wittung, P., Kim, S. K., Buchardt, O., Nielsen, P., and Norden, B. (1994) Interactions of DNA binding ligands with PNA-DNA hybrids. Nucleic Acids Res. 22(24), 5371-5377. (15) Eldrup, A. B., Christensen, C., Haaima, G., and Nielsen, P. E. (2002) Substituted 1,8-naphthyridin-2(1H)-ones are superior to thymine in the recognition of adenine in duplex as well as triplex structures. J. Am. Chem. Soc. 124, 32543262. (16) Armitage, B., Koch, T., Frydenlund, H., Ørum, H., and Schuster, G. B. (1998) Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone. Nucleic Acids Res. 26(3), 715-720. (17) Cama, L. D., Ratcliffe, R. W., Wilkening, R. R., Wildonger, K. J., and Sun, W. (1999) Synthesis of naphthosultamylcarbapenems as antibacterial compounds for treatment of methicillin resistant Staphylococcus. PCT Int. Appl. AN 1999: 282219, 153 p. (18) Advanced ChemTech, Inc. (1998) Advanced Chemtech Handbook of Combinatorial & Solid-Phase Organic Chemistry. A Guide to Principles, Products & Protocols (Bennett, W. D., Christensen, J. W., Hamaker, L. K., Peterson, M. L., Rhodes, M. R., and Saneii, H. H., Eds.) Advanced ChemTech, Inc., Louisville, Kentucky. (19) Bevers, S., Schutte, S., and McLaughlin, L. W. (2000) Naphthalene- and perylene-based linkers for the stabilization of hairpin triplexes. J. Am. Chem. Soc. 122, 5905-5915. (20) Lokey, R. S., Kwok, Y., Guelev, V., Pursell, C. J., Hurley, L. H., and Iverson, B. L. (1997) A new class of polyintercalating molecules. J. Am. Chem. Soc. 119, 7202-7210. (21) Yen, S.-F., Gabbay, E. J., and Wilson, W. D. (1982) Interaction of aromatic imides with DNA. 1. Spectrophotometric and viscometric studies. Biochemistry 21, 2070-2076. (22) Eriksson, M., and Nielsen, P. E. (1996) Solution structure of a peptide nucleic acid-DNA duplex. Nat. Struct. Biol. 3, 410-413. (23) Bleczinski, C. F., and Richert, C. (1999) Steroid-DNA interactions increasing stability, sequence-selectivity, DNA/ RNA discrimination, and hypochromicity of oligonucleotide duplexes. J. Am. Chem. Soc. 121, 10889-10894. (24) Ikeda, H., Nakamura, Y., and Saito, I. (2002) Synthesis and characterization of naphthaimide-containing peptide nucleic acids. Tetrahedron Lett. 43, 5525-5528.

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