Synthesis and Bioconjugation of Diene-Modified Oligonucleotides

The preparation of diene-modified oligonucleotides as well as their properties and further derivatization are described. Self-complementary oligonucle...
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Bioconjugate Chem. 2005, 16, 837−842

837

Synthesis and Bioconjugation of Diene-Modified Oligonucleotides Rolf Tona and Robert Ha¨ner* Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern, Switzerland . Received February 1, 2005; Revised Manuscript Received June 14, 2005

The preparation of diene-modified oligonucleotides as well as their properties and further derivatization are described. Self-complementary oligonucleotides containing a diene moiety in the loop region form stable, hairpin-like secondary structures. These hairpin mimics can be further derivatized via the Diels-Alder reaction. Diene modification in the stem region leads, in contrast, to a marked destabilization of the hairpin structure. No further reduction in stability is observed, however, upon conjugation of the stem-modified derivatives via the Diels-Alder reaction with an N-substituted maleimide dienophile.

INTRODUCTION

The hairpin is one of the most common secondary structural motifs found in nucleic acids (1). In RNA, it is an essential element for the assembly of higher order structures (2-5). By enabling the proper folding, the hairpin contributes to the many functional properties of RNA. Extra stable hairpins containing four bases in the loop (tetraloops) have emerged as a distinct class of hairpins forming highly specific interactions with tetraloop receptor sites (1, 6, 7). The hairpin motif is also found in DNA, although to a much lesser extent due to the intrinsic double-stranded nature of DNA. The requirements for the formation of DNA hairpins (8) and cruciform structures (9) in palindromic DNA sequences have been investigated in detail, and the involvement of such structures in the regulation of gene expression has been discussed (10, 11). Furthermore, the hairpin loop also acts as a site of specific metal coordination (12-14). The central role of the hairpin as a structural and functional element has stimulated the design and synthesis of chemically modified hairpin analogues. Hairpin mimics can serve as tools for the investigation of structure and function of nucleic acids. In addition, they are increasingly gaining importance as building blocks for the construction of defined, nucleic acid-based molecular structures (15). Thus, the hairpin loop has been replaced with flexible oligoethylene glycol linkers (16-18) as well as with more rigid aromatic derivatives (19-23) and metal complexes (24-26). Furthermore, the construction of a stilbene-based hairpin mimic forming G-tetrades has been reported very recently (27). The Diels-Alder reaction has been used for the derivatization (28-30) and immobilization (31) of nucleic acids. It was shown that oligonucleotides bearing a 5′linked diene moiety react site-specifically with dienophiles without interference of the many functional groups present in RNA (28) and DNA (29, 31). Despite its established chemoselectivity, however, the Diels-Alder reaction has hitherto not found widespread use for the purpose of DNA modification. Recently, we communicated the successful derivatization of a hairpin mimic with various functional groups (32). We showed that a 1,3-butadiene building block can serve as an excellent * To whom correspondence should be addressed. Fax +41 31 631 8057; e-mail [email protected].

replacement of the hairpin loop. The so obtained dienemodified hairpin was amenable to derivatization via the Diels-Alder reaction with maleimide dienophiles carrying different pendant groups. We have subsequently extended these studies to the investigation of several aspects, such as the influence of changes in the 1,3-diene building blocks as well as the effect of placement of the 1,3-diene in the stem of the hairpin. Here, we report the synthesis and properties of these modified hairpins as well as their derivatization via the Diels-Alder reaction. EXPERIMENTAL PROCEDURES

General. Reagents were purchased from Fluka, SigmaAldrich, or Acros and were used without further purification. Fluoresceine-derived maleimide 7 was purchased from Vector Laboratories, Inc. Reactions were monitored by thin-layer chromatography (TLC, Machery-Nagel precoated TLC plates SIL G-25 UV254) using mixtures of ethyl acetate/hexane or dichloromethane/methanol. Compounds were visualized with UV light and/or dipping in permanganese- or cerium(IV) reagent followed by heating. Common workup consisted of sequential washing of the organic extract with water and brine, followed by drying over anhydrous sodium sulfate and evaporation under reduced pressure. Column chromatography (CC) was performed on silica gel (sds, SILICE 60 A C.C 4063 µm). For tritylated compounds, TLC plates and silica gel columns were prepared with solvents containing 0.1% triethylamine. Melting points were recorded using a Bu¨chi 510 apparatus (open capillaries, uncorrected values). 1H (300 MHz), 13C (75 MHz), and 31P (122 MHz) NMR were recorded on a Bruker AC 300 and fitted with 1D WINNMR, and the chemical shifts (δ/ppm) were referenced to chloroform-d for 1H and 13C NMR and to phosphoric acid for 31P NMR. Mass spectra were recorded on a VG Platform single quadrupole ESI-MS, Micromass Instruments, Manchester, UK. Preparation of DNA Oligomers. The DNA oligomers were assembled on cpg solid supports (Transgenomic, Glasgow, UK) using an ABI 392 or an ABI 394 Nucleic Acid synthesizer from Applied Biosystems using the program for a 0.2 µmol synthesis except that the coupling time for modified phosphoramidites was extended to 5 min. Standard reagents and solvents for the phosphotriester method were used. Long chain aminoalkyl controlled pore glass (LCAA-CPG) solid supports with the

10.1021/bc050025t CCC: $30.25 © 2005 American Chemical Society Published on Web 06/29/2005

838 Bioconjugate Chem., Vol. 16, No. 4, 2005

Tona and Ha¨ner

Table 1. Summary of Molecular Weights of Modified Oligonucleotides oligomer

calcd [g/mol]a

found [g/mol]

∆ mass [g/mol]

6a 6b 6c T4 A4 8 9 10 11

3291 3319 3347 4243 4279 3817 7334 7916 8414

3292 3319 3348 4243 4279 3817 7334 7913 8414

1 0 1 0 0 0 0 -3 0

a

For molecular weight -H+.

4,4′-dimethoxytrityl protected 3′-end nucleotide from Transgenomic (Glasgow, UK) were used. Synthesis involved repetitive cycles, each comprising (i) deprotection of the 5′-hydroxyl group by removing the 4,4′-dimethoxytrityl group with trichloroacetic acid, (ii) tetrazole activation and coupling of the following phosphoramidite, (iii) capping of nonprotected 5′-OH with acetic anhydride/ 1-methylimidazole, and (iv) iodine oxidation to the phosphotriester. The DNA oligomers were cleaved from the solid support using 25% aqueous ammonia hydroxide and then deprotected in the same solution at 55 °C for 15 h. After filtration through a 0.45 µm nylon syringe filter, the crude materials were purified with RP-HPLC using a LiChroCART 250-4 column from Merck (A: 0.1 M triethylammonium acetate in water, B: acetonitrile) at 40 °C. The dried oligonucleotides were desalted with SepPak Cartridges (Waters) and analyzed with ESI-MS and denaturating polyacrylamide gel electrophoresis (PAGE) [Mini-PROTEAN 3 electrophoresis module (Bio-Rad Laboratories), 0.75-mm-thick 12% polyacrylamide, 1× TBE (Tris-borate-EDTA) and 10 M urea, 1× TBE as electrolyte]. Oligonucleotide concentrations were calculated from the absorbance at 260 nm with extinction coefficients calculated according to the program provided at http:// paris.chem.yale.edu/extinct.html. For diene-modified and bioconjugated oligonucleotides, extinction coefficients of the respective unmodified sequences were used. Pure oligonucleotides were lyophilized and stored at -30 °C. The molecular weights of the purified oligonucleotides were determined by electrospray mass spectroscopy (see Table 1). Bioconjugation Reactions. Diene-modified oligodeoxyribonucleotides were incubated in 10 mM sodium acetate buffer (pH 6.5) with 10 equiv of the maleimide derivatives at room temperature for 7 days. The absence of side reactions between maleimide and nucleic acid bases was monitored by MS analysis in control experiments. Fluoresceine-labeled oligodeoxyribonucleotides were purified by RP HPLC purification. The dried fractions were desalted via Sep-Pak Cartridges (Waters) and stored at -30 °C. The molecular weights of the purified conjugates were determined by electrospray mass spectroscopy (see Table 1). Thermal Denaturation Experiments. UV melting curves were recorded on a Cary 3E UV/VIS spectrophotometer (Varian) equipped with a Cary Temperature Controller, a Sample Transport Accessory and a Multi Cell Block. Aqueous solutions of 2.5 µM oligomer, 10 mM Tris-HCl (pH 7.5), and 100 mM NaCl were used. The strands were allowed to anneal by heating at 85 °C for 1-2 min, followed by slow cooling to room temperature. The samples were heated at a rate of 0.5 °C/min, and the absorbance at 260 nm was plotted every 1 min. The percent hyperchromicity at 260 nm was plotted as a function of temperature. Melting temperatures were

determined from the maximum of the first derivative of the melting curve (A260 against temperature); each Tm is the average of three independent experiments; exp error: ( 0.5 °C. Molecular Modeling. The structures shown in Figure 1 were minimized using the amber force field (Hyperchem 7.0, Hypercube, Waterloo, Ontario). General Methods. (A) Under argon, the diol (10 equiv) in absolute THF (1 mL/mmol) was added dropwise to a suspension of sodium hydride (4 equiv) in absolute THF (1 mL/mmol). After the sample was stirred for 15 h at room temperature, the dibromide, dissolved in absolute THF (1 mL/mmol), was added at room temperature and the reaction mixture was stirred for 1 h at 80 °C. The reaction mixture was then poured onto brine and extracted with EtOAc. After the sample was concentrated under reduced pressure, excess diol was evaporated under high vacuum in a kugelrohr oven (Bu¨chi). (B) The diol was dissolved in absolute pyridine (20 mL/ mmol), and 4,4′-dimethoxytrityl chloride in absolute pyridine (0.8 equiv, 20 mL/mmol) was added dropwise over a period of about 2 h. After the sample was stirred at room temperature for 3 h, the reaction was quenched by the addition of water. The crude product was isolated by extraction with CH2Cl2 and concentrated using a rotary evaporator. To avoid decomposition of the product on silica gel, the column was prepared with eluent containing 0.5% triethylamine. Before addition of the crude product, the column was washed with pure eluent to remove surplus triethylamine. (C) To a suspension of N,N-diisopropylammonium tetrazolide (2 equiv) and 2-cyanoethyl-bis(N,N-diisopropyl)amino phosphite (2 equiv) in absolute CH2Cl2 (5 mL/ mmol), the alcohol (1 equiv) dissolved in absolute CH2Cl2 (10 mL/mmol) was added. After the sample was stirred at room temperature for 15 h, the reaction was quenched with saturated aqueous NaHCO3 and extracted with CH2Cl2. To avoid decomposition of the product on silica gel, the column was prepared with eluent containing 0.5% triethylamine. Before addition of the crude product, the column was washed with pure eluent to remove surplus triethylamine. 1,6-Dibromo-hexa-2t,4t-diene (2). A solution of hexa1,6-diene-3,4-diol (1.50 g, 13.1 mmol) in absolute diethyl ether (5 mL) was added dropwise to phosphorus tribromide (2.24 g, 9.0 mmol) in absolute diethyl ether (5 mL) at 0 °C. After the addition was complete, the mixture was allowed to warm to room temperature. After 3 h the reaction mixture was poured, slowly and under stirring, into ice-water. The mixture was carefully neutralized by the addition of saturated aqueous sodium carbonate solution. The mixture was extracted with diethyl ether and then purified by CC (EtOAc/MeOH, 19:1). The product is a strong lachrymator and should be handled in a fume hood (2.63 g, 11.4 mmol, 87%). 1H NMR (CDCl3) δ: 4.00 (d, 4 H, J ) 7.7 Hz), 5.84-5.99 (m, 2 H), 6.216.31 (m, 2 H). 13C NMR (CDCl3) δ: 32.50, 130.86, 133.25. mp: (EtOAc) 85-87 °C. 2-[6-(2-Hydroxy-ethoxy)-hexa-2t,4t-dienyloxy]-ethanol (3a). 3a was synthesized according to general method A from 2 and ethylene glycol. The crude material was purified by CC (EtOAc/MeOH, 19:1). Yield: 24%, as a yellowish oil.1H NMR (CDCl3) δ: 2.02 (s br, 2 H), 3.523.57 (m, 4 H), 3.73 (t, 4 H, J ) 4.4 Hz), 4.05 (d, 4 H, J ) 5.6 Hz), 5.71-5.80 (m, 2 H), 6.22-6.27 (m, 2 H). 13C NMR (CDCl3) δ: 61.92, 71.26, 77.24, 129.90, 132.03. 3-[6-(3-Hydroxy-propoxy)-hexa-2t,4t-dienyloxy]propan-1-ol (3b). 3b was synthesized according to general method A from 2 and 1,3-propandiol. The crude

Synthesis of Diene-Modified Oligonucleotides

material was purified by CC (EtOAc/MeOH, 19:1). Yield: 24%, as transparent crystals.1H NMR (CDCl3) δ: 1.78-1.86 (m, 4 H), 2.34 (s br, 2 H), 3.59 (t, 4 H, J ) 56.0 Hz), 3.75 (t, 4 H, 6.0 Hz), 3.99 (d, 4 H, J ) 6.0 Hz), 5.69-5.77 (m, 2 H), 6.16-6.26 (m, 2 H). 13C NMR (CDCl3) δ: 32.06, 61.95, 69.41, 71.24, 129.85, 131.88. 4-[6-(4-Hydroxy-butoxy)-hexa-2t,4t-dienyloxy]-butan-1-ol (3c). 3c was synthesized according to general method A from 2 and 1,4-butanediol. The crude material was purified by CC (EtOAc/MeOH, 19:1). Yield: 17%, as a yellowish oil. 1H NMR (CDCl3) δ: 1.60-1.67 (m, 8 H), 2.26 (s br, 2 H), 3.42 (t, 4 H, J ) 5.9 Hz), 3.60 (t, 4 H, 3.0 Hz), 3.97 (d, 4 H, J ) 5.6 Hz), 5.67-5.76 (m, 2 H), 6.156.25 (m, 2 H). 13C NMR (CDCl3) δ: 26.77, 30.11, 62.52, 70.24, 70.98, 129.83, 131.95. 2-(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxy]ethoxy}-hexa-2t,4t-dienyloxy)-ethanol (4a). 4a was synthesized according to general method B from 3a and 4,4′-dimethoxytrityl chloride. The crude product was purified by CC (EtOAc). Yield: 30%, as a transparent, viscous material. 1H NMR (CDCl3) δ: 1.80-1.87 (m, 4 H), 3.53-3.58 (m, 4 H), 3.76 (s, 6 H), 3.95-4.01 (m, 4 H), 4.07-4.14 (m, 2 H), 5.68-5.75 (m, 2 H), 6.18-6.23 (m, 2 H), 6.77-6.82 (m, 4 H), 7.18-7.31 (m, 7 H), 7.41 (d, 2 H, J ) 7.0 Hz).13C NMR (CDCl3) δ: 52.45, 54.18, 60.80, 68.70, 70.25, 84.96, 112.05, 125.64, 126.73, 127.22, 129.07, 130.25, 131.32, 135.35, 144.08, 157.38. 3-(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxy]propoxy}-hexa-2t,4t-dienyloxy)-propan-1-ol (4b). 4b was synthesized according to general method B from 3b and 4,4′-dimethoxytrityl chloride. The crude material was purified by CC (EtOAc/Hex, 2:3). Yield: 59%, as a transparent oil. 1H NMR (CDCl3) δ: 1.61-1.84 (m, 4 H), 1.97 (s br, 2 H), 3.08 (t, 4 H, J ) 6.0 Hz), 3.48-3.56 (m, 4 H), 3.71 (s, 6 H), 3.90-3.95 (m, 4 H), 5.61-5.71 (m, 2 H), 6.10-6.20 (m, 2 H),6.72-6.77 (m, 4 H), 7.10-7.26 (m, 7 H), 7.35 (d, 2 H, J ) 12 Hz). 13C NMR (CDCl3) δ: 30.60, 32.26, 55.33, 60.36, 62.07, 67.74, 69.45, 71.05, 71.43, 85.90, 113.12, 126.71, 127.82, 128.33, 129.62, 130.16, 130.62, 131.52, 132.25, 136.74, 145.46, 158.47. 4-(6-{4-[Bis-(4-methoxy-phenyl)-phenyl-methoxy]butoxy}-hexa-2t,4t-dienyloxy)-butan-1-ol (4c). 4c was synthesized according to general method B from 3c and 4,4′-dimethoxytrityl chloride. The crude material was purified by CC (EtOAc). Yield: 37%, as a beige solid. 1H NMR (CDCl3) δ: 1.64 (s, 8 H), 3.04 (t, 2 H, J ) 5.8 Hz), 3.37 (t, 2 H, J ) 6.0 Hz), 3.45 (t, 2 H, J ) 5.7 Hz), 3.603.77 (m, 2 H), 3.77 (s, 6 H), 3.98 (dd, 4 H, J ) 5.9 and 15.5 Hz), 5.68-5.77 (m, 2 H), 6.15-6.26 (m, 2 H), 6.786.82 (m, 4 H), 7.15-7.32 (m, 7 H), 7.41 (d, 2 H, J ) 7.1 Hz). 13C NMR (CDCl3) δ: 26.71, 26.80, 30.25, 55.19, 62.78, 63.05, 70.22, 70.29, 70.89, 71.08, 112.95, 126.55, 127.68, 128.20, 129.54, 130.01, 130.45, 131.54, 132.15, 136.67, 145.36, 158.28. Diisopropyl-phosphoramidous Acid 2-(6-{2-[Bis(4-methoxy-phenyl)-phenyl-methoxy]-ethoxy}-hexa2t,4t-dienyloxy)-ethyl Ester 2-Cyano-ethyl Ester (5a). 5a was synthesized according to general method C from 4a and 2-cyanoethyl-bis(N,N-diisopropyl)amino phosphite. The crude material was purified by CC (EtOAc). Yield: 98%, as a transparent oil. 1H NMR (CDCl3) δ: 1.15-1.18 (m, 12 H), 1.54 (s, 2 H), 2.61 (t, 2 H, J ) 6.4 Hz), 3.20 (t, 2 H, J ) 5.1 Hz), 3.55-3.63 (m, 6 H), 3.77 (s, 6 H), 3.73-3.87 (m, 2 H), 4.06 (t, 4 H, J ) 6.0 Hz), 5.71-5.78 (m, 2 H), 6.24-6.28 (m, 2 H), 6.77-6.82 (m, 4 H), 7.17-7.35 (m, 7 H), 7.45 (d, 2 H, J ) 7.2 Hz). 13C NMR (CDCl3) δ: 20.27, 24.52, 24.59, 24.62, 24.68, 42.98, 43.14, 55.21, 58.38, 58.63, 62.53, 62.78, 63.24, 69.68, 70.00, 70.09, 71.24, 71.32, 77.44, 113.03, 126.64, 127.74,

Bioconjugate Chem., Vol. 16, No. 4, 2005 839

128.21, 129.72, 130.07, 130.30, 131.44, 132.03, 136.33, 145.07, 158.37. 31P NMR (CDCl3) δ: 148.78. HRMS (ESI+): C44H60N2NaO7P: calc.: 727.3488 g/mol, found: 727.3497 g/mol. Diisopropyl-phosphoramidous Acid 3-(6-{3-[Bis(4-methoxy-phenyl)-phenyl-methoxy]-propoxy}-hexa2t,4t-dienyloxy)-propyl Ester 2-Cyano-ethyl Ester (5b). 5b was synthesized according to general method C from 4b and 2-cyanoethyl-bis(N,N-diisopropyl)amino phosphite. The crude material was purified by CC (EtOAc). Yield: 99%, as a transparent, glassy material.1H NMR (CDCl3) δ: 1.09-1.19 (m, 16 H), 1.81-1.89 (m, 4 H), 2.58-2.63 (m, 2 H), 3.13 (t, 2 H, J ) 6.2 Hz), 3.48-3.61 (m, 6 H), 3.71-3.85 (m, 2 H), 3.76 (s, 6 H), 3.97 (t, 2 H, J ) 5.5 Hz), 5.67-5.76 (m, 2 H), 6.16-6.26 (m, 2 H), 6.77-6.83 (m, 4 H), 7.14-7.32 (m, 7 H), 7.41 (d, 2 H, J ) 7.1 Hz). 13C NMR (CDCl3) δ: 14.06, 20.31, 20.40, 22.99, 24.52, 24.61, 24.70, 30.44, 31.47, 31.57, 42.91, 43.08, 55.19, 58.19, 58.45, 60.22, 60.43, 60.66, 66.88, 67.60, 70.98, 71.05, 85.73, 112.97, 126.58, 127.69, 128.17, 130.01, 130.15, 131.60, 131.75, 136.57, 145.32, 158.31. 31 P NMR (CDCl3) δ: 147.72. HRMS (ESI+): C42H58N2O7P: calc.: 733.3981 g/mol, found: 733.3971 g/mol. Diisopropyl-phosphoramidous acid 4-(6-{4-[bis(4-methoxy-phenyl)-phenyl-methoxy]-butoxy}-hexa2t,4t-dienyloxy)-butyl ester 2-cyano-ethyl ester (5c). 5c was synthesized according to general method C from 4c and 2-cyanoethyl-bis(N,N-diisopropyl)amino phosphite. The crude material was purified by CC (EtOAc/ Hex, 1:1). Yield: 97%, as a transparent oil.1H NMR (CDCl3) δ: 1.14-1.17 (m, 14 H), 1.65 (t, 8 H, J ) 3.0 Hz), 2.61 (t, 2 H, J ) 6.4 Hz), 3.04 (t, 2 H, J ) 5.8 Hz), 3.353.44 (m, 4 H), 3.51-3.74 (m, 4 H), 3.77 (s, 6 H), 3.96 (t, 4 H, J ) 6.8 Hz), 5.68-5.77 (m, 2 H), 6.15-6.26 (m, 2H), 6.77-6.82 (m, 4 H), 7.15-7.32 (m, 7 H), 7.41 (d, 2 H, J ) 12.2 Hz). 13C NMR (CDCl3) δ: 20.32, 20.41, 24.52, 24.60, 24.70, 26.31, 26.72, 28.02, 42.90, 43.07, 55.19, 58.17, 58.42, 63.05, 69.95, 70.26, 70.94, 77.21, 112.95, 126.55, 127.67, 128.19, 130.01, 130.09, 130.15, 131.69, 136.66, 158.29. 31P NMR (CDCl3) δ: 147.45. HRMS (ESI+): C44H62N2O7P: calc.: 761.4294 g/mol, found: 761.4283 g/mol. RESULTS AND DISCUSSION

Synthesis of the different conjugated diene building blocks was accomplished as shown in Scheme 1. Thus, diol 1 was converted into the dibromide 2, according to a literature procedure (33). Reaction with the corresponding R,ω-diols afforded the symmetrical diols 3a-c, which were further transformed into the DMT-protected derivatives 4a-c. Standard phosphitylation finally gave the phosphoramidites 5a-c. The different phosphoramidite building blocks were subsequently used for the synthesis of the hairpin mimics 6a-c, shown in Scheme 2, via automated oligonucleotide synthesis. All oligomers were purified by reverse phase HPLC and characterized by electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. For comparison, the two hairpins T4 and A4, containing either four thymidine or deoxyadenosine nucleotides in the hairpin loops, were synthesized. The stabilities of the different hairpin mimics were analyzed by thermal denaturation experiments. The denaturation curves showed a single, cooperative transition for all oligomers (data not shown). Furthermore, melting temperature (Tm) values were independent of the oligomer concentration, which indicates a unimolecular process, i.e., formation and melting of the hairpin structures. The different Tm values are summarized in Table

840 Bioconjugate Chem., Vol. 16, No. 4, 2005

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Scheme 1. Synthesis of Different Diene-Derived Phosphoramidite Building Blocksa

a Conditions: (a) PBr , rt (87%); (b) NaO(CH ) OH, THF, 50 3 2 n °C (30-43%); (c) 4,4′-dimethoxytrityl chloride, pyridine, rt (46%); (d) bis(N,N-di-iso-propylamino)-2-cyanoethylphosphoramidite, di-iso-propylammonium tetrazolide, CH2Cl2, rt (98%). PAM: 2-cyanoethyl-di-iso-propylaminophosphinoyl;DMT: 4,4′-dimethoxytrityl.

Scheme 2. Different Hairpin Mimics 6a-c Containing Diene Building Blocks as Loop Replacements and Their Comparative Analogues with a T4 or A4 Loop

Table 2. Tm Values of Different Hairpins and Hairpin Mimics hairpin Tm [°C]a ∆Tm [°C]b

T4 57.8

A4 56.8 -1.0

6a 63.5 5.7

6b 66.5 8.7

6c 65.5 7.7

8 67.1c 9.3

a Conditions: oligomer concentration 2.5 µM, 10 mM Tris-HCl, 100 mM NaCl, pH 7.5; temp gradient: 0.5 °C/min. b Difference in Tm relative to T4. c Value taken from ref 32.

2. All diene-modified oligomers form more stable secondary structures than the control oligonucleotides T4 or A4. An increase in the Tm’s of 6-9 °C was observed compared to the analogous hairpin containing a T4 loop. Somewhat surprisingly, the size of the linkers connecting the diene moiety to the hairpin stem had a relatively small influence on the structural stability. The hairpin mimics 6a and 6c containing building blocks with two or four methylene units in the linker arms are only 3 and 1 °C less stable than 6b, which contains three methylene units. Since the loop region of the hairpin is crucial for intraand intermolecular interactions, introduction of substituents at this site is desirable. We previously showed (32) that introduction of substituents can be readily achieved via the Diels-Alder reaction with maleimide-derived dienophiles, such as 7 (Scheme 3). The obtained conjugate

Scheme 3. Diels-Alder Reaction 1,3-Butadiene-Based Hairpin Mimic 6b Maleimide 7

of with

forms a stable secondary structure. Thus, a Tm value of 67.1 °C was obtained for 8 (see Table 2) (32). Despite its relative bulkiness, the fluoresceine substituent is well tolerated. The circular dichroism (CD) spectra of 6b and 8 were consistent with B-form DNA structures (data not shown). On the basis of all the information obtained, we derived models of 6b and of the fluoresceine bioconjugate 8 (Figure 1). According to amber force field calculations (Hyperchem) (34), both possible diene conformations of 6b (i.e., s-cis and s-trans) form a stable hairpin-like secondary structure. Since the s-cis isomer represents the relevant conformation for the subsequent Diels-Alder reaction, the hairpin mimic is presented in this orientation (Figure 1, middle). The model shows the nonnucleotidic linker on top of the helix, bridging the two nucleic acid strands. The plane of the diene is oriented parallel to the adjacent base pair, providing the proper arrangement for reaction with dienophiles. Thermal Diels-Alder reactions proceed through a concerted syn addition, leading to a cis geometry in the cycloadduct (35). Furthermore, the endo pathway is usually favored by far owing to a lowering of its transition state’s energy as the result of a secondary π overlap (35). We can, thus, assume that the formation of the endo products is preferred in our experiments. Finally, the two faces of the dienophiles used in this study are homotopic. This leaves two unique ways in which the two partners can approach each other. The two faces of the diene are rendered diastereofacial by the chiral nucleic acid. Therefore, the two diastereomeric products shown in Figure 1 can be formed. In one diastereomer, the fluoresceine substituent (green) points into the minor groove (left), and in the other diastereomer, it is oriented toward the major groove. Thus far, we could not find any evidence that one of the diastereomers is formed preferentially. Analysis and purification of the products by HPLC did not result in the resolution or separation of the possible diastereomers. Furthermore, thermal denaturation experiments with the obtained product 8 showed no evidence of two separate transitions as a result of the presence of two diastereomeric hairpins of different structural stability. The results demonstrate that the hairpin mimics containing diene modifications in the loop region, such as 6b, are amenable to derivatization through the DielsAlder reaction with maleimide-derived dienophiles. At-

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Synthesis of Diene-Modified Oligonucleotides

Figure 1. Optimized structures (HyperChem 7.0, amber force field) of the diene-modified hairpin mimic 6b (middle) and the two possible diastereomeric products 8 arising from an endo Diels-Alder reaction with maleimide 7. The two diastereomeric products have the fluoresceine group (highlighted in green) pointing either into the minor groove (left) or into the major groove (right). Scheme 4. Hairpins 10 and 11, Containing a Diene Modification or a Diels-Alder Adduct, Respectively, in the Stema

Table 3. Tm Values of Hairpins Modified in the Stem Region hairpin Tm [°C]a ∆Tm [°C]b

9 69.0

10 55.0 -14.0

11 57.5 -11.5

a Conditions: oligomer concentration 2.5 µM, 10 mM Tris-HCl, 200 mM NaCl, pH 7.5; temp gradient: 0.5 °C/min. b Difference in Tm relative to 9.

a For the detailed structure of the fluoresceine derivative see Scheme 3.

tachment of functional or reporter groups might, however, also be desirable in the stem part of a hairpin. Therefore, we investigated whether the approach of bioconjugation via the Diels-Alder reaction might be extended to the stem region of hairpin analogues. For this purpose, a hairpin analogue containing a diene building block in the stem was prepared. Again, the diene modification with three methylene units in the linkers was selected for incorporated into the hairpin oligonucleotide, this time, however, into the stem region. The unmodified hairpin 9 and the diene-containing analogue 10 are shown in Scheme 4. Treatment of 10 with the fluoresceine-substituted maleimide 7 under the same conditions as described above resulted in the formation of the Diels-Alder adduct 11. The thermal denaturation data are summarized in Table 3. As can be seen, addition of a diene-building block into the stem region causes a strong destabilization of 14 °C. Subsequent Diels-Alder reaction of 11, however, does not result in a further reduction of stability. In fact, the relatively bulky fluoresceine even reduces the destabilization to some extent (+2.5 °C). Thus, bioconjugation of hairpin analogues containing a diene modification in the stem can also be achieved via the Diels-Alder reaction. The resulting product is markedly destabilized. The destabilization is, however, a consequence of the insertion of an additional,

nonnucleosidic building block rather than of the conjugation of the dienophile. In conclusion, hairpin analogues containing diene modifications in the loop or in the stem regions have been synthesized. The hairpin mimics containing the diene modification in the loop region form stable secondary structures, which are relatively insensitive to the length of the synthetic linkers. The diene moiety is amenable to derivatization with an N-substituted maleimide, and the resulting conjugate adopts a remarkably stable secondary structure. On the other hand, derivatization with a diene in the stem region leads to a substantial reduction in stability. Subsequent Diels-Alder reaction with an N-substituted maleimide, however, occurs smoothly and does not result in any further destabilization. ACKNOWLEDGMENT

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