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Nucleic Acids Research, 2003, Vol. 31, No. 11 2725-2734
© 2003 Oxford University Press

Syntheses and structural studies of calix[4]arene–nucleoside and calix[4]arene–oligonucleotide hybrids

Su Jeong Kim and Byeang Hyean Kim

National Research Laboratory, Department of Chemistry, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja Dong, Pohang 790-784, Korea

*To whom correspondence should be addressed. Tel: +82 54 279 2115; Fax: +82 54 279 3399; Email: bhkim{at}postech.ac.kr

Received March 13, 2003; Revised and Accepted April 9, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
We have synthesized three types of calix[4]arene– nucleoside hybrid efficiently by amide bond formation between the amine functional groups of 1,3-diaminocalix[4]arene and the carboxyl groups of thymidine nucleoside derivatives. X-ray crystallography of a homocoupled calix[4]arene–nucleoside hybrid revealed an interesting hydrogen bonding pattern between thymine bases and the amide linkages. We designed the calix[4]arene–oligonucleotide hybrids (5'-AAAAGATATCAAXTTGATATCTTTT-3', 5'-T12-X-T12-3', and 5'-A12-X-T12-3') to be V-shaped oligodeoxyribonucleotides and synthesized them by using a calix[4]arene–nucleoside hybrid (X) as a key building block. Thermal denaturation experiments, monitored by UV spectroscopy at 260 and 284 nm, and circular dichroism spectra of the calix[4]arene–oligonucleotide hybrids suggest that the modified oligonucleotides indeed adopt V-shaped conformations, making them suitable for use as building blocks in the construction of programmed oligonucleotide nanostructures.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Oligodeoxyribonucleotides (ODNs) provide an attractive framework for the construction of self-assembling nanoscale architectures because of the fidelity of their hybridization and their well defined double-helical structure (1–3). The sequence specificity of DNA hybridization allows several strands to be linked in a predictable fashion, which can lead to complex, highly functional networks. This feature has resulted in the use of ODNs as programmable assemblers (4–6). To explore the use of ODNs for the formation of controllable nanoscale architectures through self-assembly, a key step is the synthesis of functional phosphoramidites and their incorporation into ODNs.

Calix[4]arene is a structurally well defined macrocyclic molecule that is readily available in large quantities and easily modified by chemical reactions (7). It is a promising host molecule because of the directional preorganization of its functional binding groups and its capacity to rapidly modify its guest-binding site by low-energy conformational changes (8–10). Suitably functionalized calix[4]arene derivatives have been used as building blocks for the construction of larger molecules and molecular assemblies, and have been used as building blocks for multifunctional enzyme models (11,12). Recently, calix[4]arenes have been coupled with sugars (13–15), amino acids (16–19), peptides (20), nucleobases (adenine, thymine, uracil) (21–23) and guanosine (24), to develop biologically active synthetic receptors and enzyme mimics.

Since calix[4]arene has various structural advantages and meets general criteria for the modification of ODNs (25–27), we have designed and synthesized the calix[4]arene– nucleoside hybrids (28) (calixnucleosides) 1–4 (Fig. 1) as structural scaffolds for preparing the calix[4]arene– oligonucleotide hybrids (calixoligonucleotides) 5'-AAA AGATATCAAXTTGATATCTTTT-3', 5'-T12-X-T12-3' and 5'-A12-X-T12-3' (where X denotes the calixnucleoside unit 1) as rigid V-shaped ODN derivatives (Fig. 2). In this paper we describe the details for the synthesis and structural studies of four calixnucleosides and three calixoligonucleotides (Oligo 1, Oligo 2 and Oligo 3).



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  		Figure 1. Structures of synthesized calix[4]nucleosides 1–4.

 


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  		Figure 2. Possible structures adopted by calixnucleotides. (a) Hairpin structure: intramolecular base pairing; (b) bulged duplex: intermolecular base pairing; (c) V-shaped aggregate: intermolecular base pairing.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Synthesis of calix[4]arene–nucleoside hybrids
The key step in the calixnucleoside synthesis was amide bond formation between the amine functional groups of calix[4]arene 7 and the carboxyl groups of thymidine nucleosides 6 and 8 (Scheme 1). To activate the 5'-carboxyl functionality of thymidine derivative (29) 6, various peptide coupling reagents were investigated, such as (COCl)2, EDC, 2,4,6-trichlorobenzoyl chloride and O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU). Only the use of TBTU provided the homocoupled reaction product 3, in 69% yield. In a similar fashion, the 3'-carboxyl-functionalized thymidine derivative (30) 8 was treated with 1,3-diaminocalix[4]arene (31) 7 to give homocoupled calixnucleoside 4 in 43% yield. To synthesize the heterocoupled calixnucleosides 1 and 2, we first prepared mono-coupled calixnucleoside 5 in 58% yield by peptide coupling of the 5'-carboxyl-functionalized thymidine derivative 6 (1.2 equivalents) with 1,3-diaminocalix[4]arene 7. Calixnucleoside 2 was obtained in 64% yield by a subsequent peptide coupling of the 3'-carboxyl-functionalized thymidine derivative 8 with mono-coupled calixnucleoside 5. Finally, calixnucleoside 1 was prepared in 83% yield by deprotection of 2. In these syntheses we have utilized the amide bond as the linking unit in the hybrids because it is a moiety often used in nucleotide backbone modification for antisense ODNs (32).



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  		Scheme 1. Synthesis of calixnucleosides. (a) (i) 6 (2.4 equivalents), TBTU, HOBT, 4-methylmorpholine, CH2Cl2, room temperature, 30 min; (ii) 7 (1.0 equivalent), 4 h, 69%. (b) (i) 6 (1.2 equivalents), TBTU, HOBT, 4-methylmorpholine, CH2Cl2, room temperature, 30 min; (ii) 7 (1.0 equivalent), 1 h, 58%. (c) (i) 8 (2.4 equivalents), TBTU, HOBT, 4-methylmorpholine, CH2Cl2, room temperature, 30 min; (ii) 7 (1.0 equivalent), 1 h, 43%. (d) (i) 8 (1.2 equivalents), TBTU, HOBT, 4-methylmorpholine, CH2Cl2, room temperature, 30 min; (ii) 5 (1.0 equivalent), 3 h, 64%. (e) TBAF, THF, room temperature, 10 min, 83%.

 

Compound 3 (3'-O-TBDMS-thymidine–calix[4]arene–3'-O-TBDMS-thymidine). 5'-Carboxyl-functionalized thymidine derivative 6 (65 mg, 0.18 mmol) was added to a solution of HOBT (26 mg, 0.19 mmol), TBTU (59 mg, 0.18 mmol), 4-methylmorpholine (20 µl, 0.18 mmol) in CH3CN/CH2Cl2 (2:1, 6 ml). The solution was stirred at room temperature for 30 min and then charged with 1,3-diaminocalix[4]arene 7 (49 mg, 0.079 mmol). The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure and the residue partitioned between 5% aqueous NaHCO3 solution and CH2Cl2. The organic layer was dried over MgSO4 and the solvent was evaporated under reduced pressure to give an orange solid. Purification by flash chromatography (hexane/EtOAc, 2:1) provided the product as an orange solid (50 mg, 0.038 mmol, 48%). M.p. 212.3–214.2°C; MS (FAB): m/z 1327.3 [M + H]+; [{alpha}]24D = –9.42° (c 0.0046, CH2Cl2); IR (neat): {nu} = 3284, 3063, 2957, 2930, 2857, 1682, 1466 cm–1; 1H NMR (300 MHz, CDCl3): {delta} = 9.02 (s, 2H), 8.95 (s, 2H), 7.37 (br, 2H), 7.17 (br, 4H), 6.27 (s, 6H), 6.00 (dd, J = 4.9, 9.8 Hz, 2H), 4.72 (d, J = 4.5 Hz, 2H), 4.40 (s, 2H), 4.39 (d, J = 13.2 Hz, 4H), 3.91 (t, J = 7.8 Hz, 4H), 3.67 (t, J = 6.9 Hz, 4H), 3.12 (d, J = 13.4 Hz, 2H), 3.10 (d, J = 13.3 Hz, 2H), 2.79–2.72 (m, 2H), 2.04 (dd, J = 5.0, 12.7 Hz, 2H), 1.96–1.80 (m, 14H), 1.04 (t, J = 7.3 Hz, 6H), 0.92 (s, 18H), 0.88 (t, J = 7.5 Hz, 6H), 0.18 (s, 6H), 0.15 (s, 6H); 13C NMR (75.5 MHz, CDCl3): {delta} = 167.5, 163.8, 155.6, 154.8, 150.7, 138.9, 137.1, 137.1, 133.5, 133.5, 131.4, 127.8, 122.3, 120.5, 111.9, 110.8, 91.8, 87.8, 77.1, 75.8, 37.4, 31.2, 26.0, 23.6, 23.1, 18.2, 12.6, 10.9, 10.2, –4.5, –4.7; Anal. Calc. for C72H98N6O14Si2·3H2O: C, 62.58; H, 7.58; N, 6.08. Found: C, 62.92; H, 7.65; N, 5.84.

Compound 4 (5'-O-TBDPS-thymidine–calix[4]arene–5'-O-TBDPS-thymidine). 3'-Carboxyl-functionalized thymidine derivative 8 (652 mg, 1.25 mmol) was added to a solution of HOBT (84 mg, 0.62 mmol), TBTU (400 mg, 1.24 mmol), 4-methylmorpholine (137 µl, 1.25 mmol) in CH3CN/CH2Cl2 (2:1, 15 ml). The solution was stirred at room temperature for 30 min and then charged with 1,3-diaminocalix[4]arene 7 (311.43 mg, 0.5 mmol). The solution was stirred at room temperature for 1 h and then THF (6 ml) was added to dissolve the precipitate. The reaction mixture was heated at 45°C for 1 h. The solvent was evaporated under reduced pressure and the residue was partitioned between saturated aqueous NaHCO3 and CH2Cl2. The organic layer was separated, dried (MgSO4) and the solvent was evaporated under reduced pressure to give an orange solid. Purification by flash chromatography (CH2Cl2/EtOAc, 2:1) provided the product (282 mg, 0.216 mmol, 43%) as a white solid. The solid was dissolved in hot MeOH and X-ray diffraction-grade single crystals were obtained through slow evaporation of the solution. M.p. 222.4–223.7°C. MS (FAB): m/z 1630.5 [M+]; [{alpha}]24D = +9.27° (c 0.0035, CH2Cl2); IR (neat): {nu} = 3314, 3068, 2960, 2931, 2873, 1688, 1605, 1544, 1468 cm–1; 1H NMR [300 MHz; CDCl3/CD3OD, 15:1 (v/v)]: {delta} = 10.5 (br, 2H), 8.46 (br, 2H), 7.59 (s, 2H), 7.41 (s, 2H), 7.34–7.24 (m, 12H), 6.80 (s, 2H), 6.75 (s, 2H), 6.51 (d, J = 7.1 Hz, 4H), 6.42 (t, J = 7.1 Hz, 2H), 6.05 (t, J = 5.8 Hz, 2H), 4.36 (d, J = 13.2 Hz, 4H), 3.94 (d, J = 7.8 Hz, 2H), 3.75 (s, 10H), 3.30 (s, 1H), 3.17 (s, 1H), 3.05 (d, J = 13.3 Hz, 4H), 2.84 (br, 2H), 2.34–2.03 (m, 8H), 1.84 (q, J = 7.2 Hz, 8H), 1.49 (s, 6H), 1.01 (s, 18H), 0.91 (dd, J = 7.6, 16.2 Hz, 12H); 13C NMR [75.5 MHz; CDCl3/CD3OD, 15:1 (v/v)]: {delta} = 169.2, 164.5, 156.4, 153.7, 150.9, 135.7, 135.7, 135.4, 134.7, 133.3, 132.7, 131.6, 130.1, 130.0, 128.4, 128.1, 128.0, 128.0, 122.1, 120.6, 120.5, 111.2, 85.3, 84.6, 76.9, 64.5, 39.7, 38.2, 35.5, 31.1, 27.0, 23.3, 23.2, 19.5, 12.0, 10.4, 10.3; Anal. Calc. for C96H114N6O14Si2·2H2O: C, 69.12; H, 7.13; N, 5.04. Found: C, 69.19; H, 7.13; N, 4.81. Crystal data for compound 4 (C96H114N6O14Si2·4H2O): Mr = 1704.18, monoclinic, space group P2(1), a = 14.36670(10) Å, b = 19.87010(10) Å, c = 18.0957(2) Å, {alpha} = 90.0000°, ß = 103.9570(10), {gamma} = 90.0000°, V = 5013.23(7) Å3, Z = 2, {rho}calcd = 1.129 Mg m–3, MoK{alpha} radiation ({lambda} = 0.71073 Å), crystal dimensions 0.40 x 0.30 x 0.10 mm3. Of 20 498 reflections collected on a Siemens SMART diffractometer equipped with a CCD detector, 12 375 were observed (Rint = 0.0327) and used for all calculations (program SHELXL-97). After absorption correction (psi scans), the structure was solved by direct methods and refined anisotropically on F2. Final residuals R1 = 0.1033, wR2 = 0.2738 [I > 2{sigma}(I)]; R1 = 0.1334, wR2 = 0.3134 (all data), 1103 parameters. Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-145437. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 1223 336 033; Email: deposit{at}ccdc.cam.ac.uk).

Compound 5 (mono-3'-O-TBDMS-thymidine–calix[4]arene). 5'-Carboxyl-functionalized thymidine derivative 6 (346 mg, 0.935 mmol) was added to a solution of HOBT (126 mg, 0.935 mmol), TBTU (450 mg, 1.40 mmol), 4-methylmorpholine (154 µl, 1.40 mmol) in CH3CN/CH2Cl2 (2:1, 30 ml). The solution was stirred at room temperature for 30 min and then charged with 1,3-diaminocalix[4]arene 7 (490 mg, 0.748 mmol). The reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure and the residue partitioned between 5% aqueous NaHCO3 solution and CH2Cl2. The organic layer was dried over MgSO4 and the solvent was evaporated under reduced pressure to give an orange solid. Purification by flash chromatography (hexane/EtOAc, 2:1) provided the product as an orange solid (426 mg, 0.438 mmol, 58%). M.p. 159.4–161.2°C. MS (FAB): m/z 975.4 [M + H]+; [{alpha}]23D = +29.2° (c 0.0095, CH2Cl2); IR (neat): {nu} = 3188.3, 2960.1, 2930.8, 2876.3, 1695.0, 1681.9, 1606.3, 1470.4, 1278.2 cm–1; 1H NMR (300 MHz; CDCl3): {delta} = 9.52 (s, 1H), 7.93 (s, 1H), 7.06 (d, 4H, J = 6.5 Hz), 6.90 (br, 2H), 6.45 (t, 1H, J = 6.7 Hz), 6.21 (s, 1H), 6.17 (s, 1H), 5.17 (s, 2H), 4.63 (s, 1H), 4.55 (d, 2H, J = 14.1 Hz), 4.50 (d, 2H, J = 14.0 Hz), 4.27 (s, 1H), 4.03 (br, 5H), 3.82 (t, 3H, J = 6.4 Hz), 3.74 (t, 3H, J = 6.4 Hz), 3.26 (d, 2H, J = 13.8 Hz), 3.14 (d, 2H, J = 13.5 Hz) 2.40 (br, 1H), 2.26 (br, 1H), 1.99 (s, 11H), 1.17 (dd, J = 6.79, 12.6 Hz, 6H), 1.02 (s, 15H), 0.02 (s, 6H); 13C NMR (75.5 MHz; CDCl3): {delta} = 169.67, 164.69, 158.24, 158.18, 154.84, 151.23, 150.28, 139.40, 136.97, 136.92, 136.81, 135.53, 135.46, 135.26, 135.12, 130.94, 129.28, 129.22, 129.14, 125.04, 124.79, 122.39, 122.28, 116.04, 115.87, 111.77, 87.51, 87.07, 77.24, 76.95, 75.84, 60.82, 40.10, 31.49, 26.29, 23.83, 23.47, 18.48, 13.21, 11.20, 10.39, –4.24, –4.28.

Compound 2 (5'-O-TBDPS-thymidine–calix[4]arene–3'-O-TBDMS-thymidine). 3'-Carboxyl-functionalized thymidine derivative 8 (129 mg, 0.246 mmol) was added to a solution of HOBT (17 mg, 0.12 mmol), TBTU (79 mg, 0.25 mmol), and 4-methylmorpholine (27 µl, 0.25 mmol) in CH3CN/CH2Cl2 (2:1, 15 ml). The solution was stirred at room temperature for 30 min and then charged with compound 5 (110 mg, 0.113 mmol). The reaction mixture was then stirred at room temperature for 3 h. The solvent was evaporated under reduced pressure and the residue was partitioned between 5% aqueous NaHCO3 solution and CH2Cl2. The organic layer was dried (MgSO4) and the solvents were evaporated under reduced pressure to yield an orange solid. Purification by flash chromatography (CH2Cl2/EtOAc, 3:1) provided the product as an orange solid (107.5 mg, 0.072 mmol, 64%). M.p. 176.4–178.2°C; MS (FAB): m/z 1479.3 [M + H]+; [{alpha}]23D = +16.8° (c 0.0079, CH2Cl2); IR(neat): {nu} = 3306, 3066, 2958, 2931, 2958, 1694, 1552, 1468 cm–1; 1H NMR (300 MHz, acetone-d6): {delta} = 10.23 (s, 1H), 9.95 (s, 1H), 9.46 (s, 1H), 8.97 (s, 1H), 7.84 (s, 1H), 7.79–7.75 (m, 4H), 7.54 (s, 1H), 7.48–7.37 (m, 8H), 7.29 (s, 1H), 7.28 (s, 1H), 6.40–6.33 (m, 6H), 6.27 (dd, J = 5.3, 9.3 Hz, 1H), 6.20 (dd, J = 4.9, 7.0 Hz, 1H), 4.79 (d, J = 5.0 Hz, 1H), 4.48 (d, J = 13.1 Hz, 2H), 4.46 (d, J = 13.1 Hz, 2H), 4.38 (d, J = 1.3 Hz, 1H), 4.09 (dd, J = 7.7, 9.0 Hz, 1H), 4.00–3.95 (m, 6H), 3.76 (t, J = 7.0 Hz, 4H), 3.14, 3.12 (2 x d, J = 13.1 Hz, 4H), 3.01 (q, J = 7.7 Hz, 1H), 2.63–2.48 (m, 2H), 2.42–2.18 (m, 3H), 1.96 (m, 8H), 1.87 (s, 3H), 1.56 (d, J = 0.9 Hz, 3H), 1.13–1.06 (m, 15H), 0.99–0.95 (m, 15H), 0.20 (s, 3H), 0.19 (s, 3H); 13C NMR (75.5 MHz, acetone-d6): {delta} = 170.0, 168.9, 164.6, 157.0, 155.1, 154.7, 152.4, 151.7, 139.9, 137.6, 137.5, 136.9, 136.8, 136.7, 135.0, 134.9, 134.5, 133.8, 131.2, 129.2, 129.0, 123.3, 121.5, 121.3, 111.8, 111.2, 90.9, 88.7, 86.6, 85.4, 78.1, 78.0, 77.3, 65.8, 40.7, 39.4, 39.0, 36.5, 32.2, 27.9, 26.7, 24.6, 24.3, 20.5, 19.1, 13.0, 12.9, 11.5, 10.9, –4.1, –4.2; Anal. Calc. for C84H106N6O14Si2: C, 68.17; H, 7.21; N, 5.67. Found: C, 67.79; H, 7.53; N, 5.85.

Compound 1 (5'-OH-thymidine–calix[4]arene–3'-OH-thymidine). TBAF (1 M in THF, 2 ml) was added to a solution of compound 2 (604.5 mg, 0.408 mmol) in THF. The reaction mixture was stirred at room temperature for 10 min, and then distilled water and CH2Cl2 were added. The organic layer was separated, dried over MgSO4, and concentrated under reduced pressure. Purification by flash chromatography (CH2Cl2/MeOH, 12:1) provided the product as an orange solid (383.4 mg, 0.340 mmol, 83.2%). M.p. > 212.5°C (decomp.); MS (FAB) m/z: 1127.6 [M + H]+; [{alpha}]20D = +6.70° (c 0.0094, CHCl3); IR (neat): {nu} = 3309, 3065, 2961, 2931, 2875, 1682, 1465 cm–1; 1H NMR [300 MHz; acetone-d6/CDCl3, 1/1 (v/v)]: {delta} = 10.05 (s, 1H), 9.88 (s, 1H), 9.02 (s, 1H), 8.66 (s, 1H), 7.94 (s, 1H), 7.92 (s, 1H), 7.66 (s, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.79 (s, 1H), 6.65–6.45 (m, 7H), 6.31 (br, 1H), 6.01 (dd, J = 5.3, 1.4 Hz, 1H), 5.39 (s, 1H), 4.85 (br, 1H), 4.57 (br, 1H), 4.38 (d, J = 13.1 Hz, 4H), 4.34 (s, 1H), 4.22 (br, 1H), 3.87 (br, 1H), 3.82–3.70 (m, 9H), 3.09 (d, J = 13.8 Hz, 2H), 3.05 (d, J = 13.6 Hz, 2H), 2.81–2.75 (m, 1H), 2.51–2.45 (m, 2H), 2.34–2.30 (m, 2H), 2.24–2.08 (m, 2H), 1.88 (br, 11H), 1.80 (s, 3H), 0.94 (q, J = 3.6 Hz, 12H); 13C NMR [75.5 MHz; acetone-d6/CDCl3, 1/1 (v/v)]: {delta} = 169.9, 168.6, 164.5, 164.2, 156.8, 153.8, 153.5, 151.4, 150.9, 135.6, 135.5, 135.2, 132.5, 132.1, 128.5, 122.4, 120.8, 111.2, 109.7, 88.7, 87.1, 86.7, 85.1, 77.1, 77.0, 75.0, 61.6, 39.5, 38.5, 38.1, 33.9, 31.3, 23.5, 12.6, 10.6, 10.5; Anal. Calc. for C62H74N6O14: C, 66.06; H, 6.62; N, 7.45. Found: C, 65.75; H, 6.97; N, 7.73.

Synthesis of calix[4]arene–oligonucleotide hybrids
With calixnucleoside 1 in hand, we prepared the DMTr-protected 2-cyanoethyl phosphoramidite building block of 1 and directly applied it to solid-phase oligonucleotide synthesis protocols (33) with an automated DNA synthesizer (PerSeptive Biosystems 8909 ExpediteTM Nucleic Acid Synthesis System). For comparison, the unmodified ODNs were also obtained. The synthesized oligonucleotides were cleaved from the solid support by treatment with 30% aqueous NH4OH (1.0 ml) for 10 h at 55°C. The crude products from the automated ODN synthesis were lyophilized and diluted with distilled water (1 ml). The ODNs were purified by HPLC (Merck LichoCART C18 column, 10 x 250 mm, 10 µm, 100 Å pore size). The HPLC mobile phase was held isocratically for 10 min with 5% acetonitrile/0.1 M triethylammonium acetate (TEAA) (pH 7.0) at a flow rate of 3 ml/min. The gradient was then increased linearly over 10 min from 5% acetonitrile/ 0.1 M TEAA to 50% acetonitrile/0.1 M TEAA at the same flow rate. The fractions containing the purified ODN were pooled and lyophilized. Aqueous AcOH (80%) was added to the ODN. After 30 min at ambient temperature, the AcOH was evaporated under reduced pressure. The residue was diluted with water (1 ml), and the solution was purified by HPLC using the same condition as described above. The ODNs were analyzed by HPLC (Hewlett–Packard, ODS Hypersil, 4.6 x 200 mm, 5 µm, 79916OD-574) with almost the same eluent system, but with a different flow rate (1 ml/min). For characterization, matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometric data of the calix[4]oligonucleotides were collected in a PE Biosystems Voyager System 4095 instrument in positive-ion mode using a 1:1 mixture of 3-hydroxypicolinic acid (0.35 M) and ammonium citrate (0.1 M) as the matrix and with an accelerating voltage of 25 kV.

UV melting curves
UV melting curves were measured on a SHIMADZU UV2501PC spectrophotometer equipped with a circulating bath (PolyScience digital temperature controller 9110). A temperature gradient of 1.0°C/min was applied. At temperatures below 15°C, the cell compartment was flushed with dry air (ZANDER Ecodry-air dryer system) to prevent condensation of water on the cuvettes. Sample solutions were covered with a thin layer of dimethylpolysiloxane (Sigma) to prevent evaporation of water. Values of Tm were defined as the maxima of the first-order derivatives of the melting curves, and they corresponded to within ±1°C to the values determined at half of the maximal hyperchromicity after baseline correction (34).

Circular dichroism (CD) spectra
The mixture of ODNs was equilibrated by cooling to 5°C and after 30 min the CD spectra was recorded on a Jasco J-715 CD spectropolarimeter. The temperature was controlled by a Jasco PTC-348WI temperature controller.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
X-ray crystal structure of compound 4
Single crystals suitable for X-ray diffraction were grown by slow evaporation of an MeOH solution of calixnucleoside 4. The resulting X-ray crystallographic data offers a wealth of structural information and molecular interactions. Calix nucleoside 4 has C2 symmetry and the two thymine bases have an anti-parallel orientation. The two amide linkages, through which the nucleosides and calix[4]arene are joined together, also have an anti-parallel orientation. Dihedral angles between the amide linkages and the thymine bases are nearly 90° (Fig. 3). The calix[4]arene moiety adopts a pinched-cone conformation. The distances between the upper position of the substituted and unsubstituted benzene rings of the calixarene unit are 3.9 and 9.9 Å, respectively. The preference for the pinched-cone conformation results from the fact that each amide linkage is hydrogen bonded to a thymine base of an adjacent calixnucleoside. Each molecule of calixnucleoside 4 is linked to others in a two-dimensional network through eight intermolecular [N–H···O] hydrogen bonds between thymine bases and amide linkages (Fig. 4). This network can be viewed as four layers that resemble a bilayer-type structure (Fig. 5). Layers 1 and 4 contain hydrophobic residues (calix[4]arene moieties) and layers 2 and 3 contain hydrophilic residues (thymidine units and amide linkages). We believe that similar aggregation phenomena exist in solution also. The signals obtained in the 1H NMR spectra (300 MHz) of this sample recorded in CDCl3 were too broad to assign to specific protons. The addition of a small amount of CD3OD, however, sharpened the signals and made them assignable (figures not shown). This dramatic change suggests to us that compound 4 does not remain as a monomeric species in CDCl3 solution, but is aggregated through hydrogen bonding in a manner similar to that observed in the crystal structure.



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  		Figure 3. X-ray crystal structure (Chem 3D rendering) of 4. Disorder around the propyl ether units is not shown.

 


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  		Figure 4. Plan view of the hydrogen bonding patterns in the packing of 4 (Chem 3D rendering). Four calixnucleoside units (A, B, C and D) are displayed. The calix[4]arene moieties and protecting groups have been omitted for clarity.

 


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  		Figure 5. Side view of the packing of 4. Four calixnucleoside units (A, B, C and D) are depicted. The propyl and protecting groups have been omitted for clarity. Calixnucleoside molecules B and D are eclipsed.

 
Design and synthesis of calixoligonucleotides
To investigate the effect of incorporating calixnucleosides into ODNs, the calixnucleoside 1 was introduced, with high coupling efficiencies, into the middle of ODN sequences using an automated DNA synthesizer. We confirmed the successful synthesis of these calixoligonucleotides with DMTr monitoring. These calixoligonucleotides were characterized by MALDI-TOF mass spectra. The sequences and mass spectral data are summarized in Table 1. Inverted repeat sequence in ODNs (palindromers) capable of forming hairpin, as well as cruciform, structures frequently occur in regions known to have peculiarities, such as regulation and promotion sites (35–38). Oligo 1 and Oligo 3 are palindromers for the determination of the structural properties of the calixoligonucleotides, namely either hairpin mimics or simple bent V-shaped ODNs. Oligo 2 was designed for comparison with Oligo 3 and natural hairpin ODNs.


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  		Table 1. Designed sequences of calixnucleosides and their selected MALDI-TOF mass spectral data
 
HPLC data
Reverse-phase HPLC (RP-HPLC) was the most efficient method for the purification of large amounts of synthesized ODNs. The general chromatographic conditions that were useful for purification and characterization of ODNs were also useful for checking their purity before analytical experiments (CD, Tm, PAGE). Figure 6 shows that not only are the synthetic ODNs pure enough to be used in other experiments, but also that the calixoligonucleotides have longer retention times than regular ODNs. The retention time of a calixoligonucleotide is similar to that of a DMTr-protected natural ODN. This feature suggests that the structure of a calixoligonucleotide is similar to that of an ODN bearing a non-polor moiety at one of its ends. That is to say, the calix[4]arene moiety is not just buried in the long ODN, but is exposed at the end of calixoligonucleotide chains and has a V-shaped structure in solution.



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  		Figure 6. HPLC data for Oligo 1 and Oligo 4. The experimental conditions are described in Material and Methods for the synthesis of calix[4]arene– oligonucleotide hybrids.

 
UV melting experiments of ODNs
We analyzed the binding affinities of calixoligonucleotides by UV melting curves, with melting transitions monitored at both 260 and 284 nm. The UV absorbance of A–T Watson–Crick duplexes do not change when their melting transitions are monitored at 284 nm, since that wavelength is an isosbestic point of double helix structures with A–T-rich sequences (39). Thus, any change of absorbance at 284 nm is induced by structural transitions other than simple duplex formation/destruction, such as triplex or more complex aggregations. Figure 7 shows the relative absorbance of curves used for the determination of the Tm value. The melting temperatures are summarized in Table 2. Entries 1 and 2 suggest that Oligo 1 and Oligo 4 could have double-stranded hairpin structures. Oligo 1 has a Tm value of 40°C when detected at 284 nm and two Tm values of 40 and 70°C when measured at 260nm. The hetero-sequence-containing calixoligonucleotide (Oligo 1) may be a mixture of two different structures (hairpin structure and intermolecular aggregates). Oligo 2, which has a unit of compound 1 incorporated into the middle of dT24, cannot adopt a double-stranded secondary structure (entry 3). Thus, the structure of Oligo 2 must be random coils, which is confirmed by the lack of measurable Tm values at both 260 and 284 nm. Oligo 3, which incorporates a unit of compound 1 into the middle of dT12A12, appears to adopt a double-stranded secondary structure (entry 4). Oligo 5, which incorporates a C4 unit (a natural hairpin domain) into the middle of the dT12A12 sequence, almost certainly adopts a secondary structure (hairpin structure, entry 5) and has a high Tm value (note that the Tm value at 284 nm is induced by the C4 units, since 284 nm is not an isosbestic point for the cytidine base). The mixture of Oligo 2 and Oligo 6 (entry 7) has two Tm values. One dT12 sequence of one Oligo 2 forms a double helix with added dA12 and then the dT12 sequence of another Oligo 2 forms a triplex (Fig. 8a). The mixture of Oligo 3 and Oligo 7 (entry 8) also has two Tm values, possibly because the dT12 and dA12 sequences of two Oligo 3 units form a double helix through intermolecular base pairing and then the added dT12 sequence could form a triplex with it (Fig. 8b). The mixture of Oligo 5 and Oligo 7 (entry 9) has two Tm values, but the pattern of base pairing is different from those of entries 7 and 8. The dT12 and dA12 sequences of Oligo 5 assemble into a double helix through intramolecular base pairing (hairpin structure, Tm = 70°C) and then the added dT12 sequence forms a triplex (Fig. 8c). Thus, while in principle Oligo 2, Oligo 3 and Oligo 5 could form similar double- and triple-stranded assemblies when third strands are added, the structures of the triplexes are all different from each other. Finally, a mixture of Oligo 2 and Oligo 8 (entry 10) has a similar Tm value (36°C) to that of the mixture of Oligo 2 and Oligo 6 (entry 7). If Oligo 2 had a linear structure, it could form a long double helix with dA28 and so would have a higher Tm value than does the mixture of Oligo 2 and Oligo 6. Since it does not, we infer that the structure of the calixoligonucleotide Oligo 2 is not linear, but instead is bent.



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  		Figure 7. Thermal denaturation curves of synthetic ODNs. The experimental conditions are described in Table 2. (a) Oligo 2; (b) a mixture of Oligo 2 and Oligo 6; (c) Oligo 3; (d) a mixture of Oligo 3 and Oligo 7; (e) Oligo 5; (f) a mixture of Oligo 5 and Oligo 7. All absorbance data are normalized (At, absorbance at temperature t; A0, absorbance at initial temperature; Af, absorbance at final temperature).

 

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  		Table 2. Melting temperatures of synthetic ODNs
 


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  		Figure 8. Possible structures adopted by calixnucleotides. (a) Hydrogen bonding pattern between Oligo 2 and Oligo 6. (b) Hydrogen bonding pattern between Oligo 3 and Oligo 7. (c) Hydrogen bonding pattern between Oligo 5 and Oligo 7.

 
Polyacrylamide gel electrophoresis (PAGE)
With denaturing PAGE (7 M urea, 20%, acrylamide/bisacrylamide, 19:1) at two different temperatures (50 and 5°C), the structures can be estimated as adopting either duplexes with intramolecular base pairing (see Fig. 2a) or random coils. At a high temperature, Oligo 1 has two bands (Fig. 9, lane 3). The upper band has the same mobility as do Oligo 2 and Oligo 3 (lanes 1 and 2). At a low temperature, Oligo 1 has two bands also (lane 6). In this case, the lower band has the same mobility as Oligo 3. From these observations, we can suggest the structures of the calixoligonucleotides. The Oligo 2 has an unfolded structure, because its sequence does not allow for base pairing through hydrogen bonding. At a high temperature, Oligo 1 (upper band) and Oligo 3 have structures similar to that of Oligo 2 (i.e. an unfolded structure). At a low temperature, Oligo 1 and Oligo 3 have similar folded structures with intramolecular base pairing. The upper and lower bands of Oligo 1 were separated by PAGE at room temperature. The purified ODNs were incubated at 5°C for annealing and then the PAGE experiment was repeated and gave the same results (figure not shown).



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  		Figure 9. PAGE of calixoligonucleotides (acrylamide/bisacrylamide, 19:1). Lanes 1 and 4, Oligo 3; lanes 2 and 5, Oligo 2; lanes 3 and 6, Oligo 1.

 
CD spectroscopic studies
CD spectroscopy has been a useful method for distinguishing the structures of ODNs (40). We applied this technique to study the conformational changes arising from the modification of the ODNs. In Figure 10a, we have superimposed CD spectra of the single strands and duplexes. The CD spectrum of Oligo 2 (solid line) has weak positive CD bands at 218 and 281 nm and a weak negative CD band at 248 nm. This pattern is almost the same as that of Oligo 7 (dT12). The CD spectrum of Oligo 3 (dashed line) has positive CD bands at 218 and 283 nm and a negative CD band at 249 nm. This CD spectrum is almost similar to those of Oligo 5 and the mixture of Oligo 6 and Oligo 7, which indicates B-form duplexes (40). In Figure 10b are superimposed the CD spectra that represent the formation of triplexes. The CD spectrum of Oligo 2:Oligo 6 (dashed line) has strong positive CD bands at 217 and 281 nm and negative CD bands at 207 and 248 nm. The CD spectrum of Oligo 3:Oligo 7 (solid line) has strong positive CD bands at 219 and 281 nm and strong negative CD bands at 209 and 248 nm. The CD spectrum of Oligo 5:Oligo 7 (dotted line) has strong positive CD bands at 217 and 283 nm and strong negative CD bands at 209 and 248 nm. Because the shapes of these spectra are similar to each other, these three samples probably have similar secondary structures, namely triplexes. The CD spectrum of Oligo 2 alone reflects its random coil structure. The CD spectrum of Oligo 2 in the presence of Oligo 6, however, is changed dramatically to have almost the same pattern as the spectrum of the mixture between Oligo 5 and Oligo 7. This observation suggests that the mixture of Oligo 2 and Oligo 6 adopts a triplex structure similar to the one formed by Oligo 5 and Oligo 7.



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  		Figure 10. CD spectra of synthesized ODNs. (a) Oligo 2 (solid line); Oligo 3 (dotted line); Oligo 5 (large dashed line); Oligo 6 (dash-dot-dashed line); Oligo 7 (dash-double dot-dashed line); a mixture of Oligo 6 and Oligo 7 (dashed line). (b) A mixture of Oligo 3 and Oligo 7 (solid line); a mixture of Oligo 2 and Oligo 6 (dashed line); a mixture of Oligo 5 and Oligo 7 (dotted line). Conditions: pH 7.0, 10 mM Tris–HCl buffer, [NaCl] = 0.1 M, [MgCl2] = 20 mM, 10°C.

 

   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Calix[4]arenes are emerging as a new class of synthetic hosts that have attracted interest in several areas of bioorganic and biomimetic chemistry. For this reason, we designed and synthesized calix[4]arene–nucleoside hybrids as structural scaffolds and host molecules. These hybrids were synthesized by simple amide bond formation and characterized fully by 1H NMR, 13C NMR and IR spectroscopy, mass spectrometry, and elemental analyses. One unit of a calixnucleoside was introduced into ODNs (to form calixoligonucleotides) and separated by RP-HPLC. These calixoligonucleotides were characterized by MALDI-TOF mass spectra. The calixoligonucleotides could have two different structures resulting from either intra- or inter-molecular base pairings. The secondary structures were confirmed by determining Tm values and by comparing analyses made by HPLC, PAGE and CD spectroscopy. These analyses revealed that the calixoligonucleotides have a V-shaped structure. We confirmed by HPLC that the structures of calixoligonucleotides were different from linear ODNs. By PAGE, we confirmed that calixoligonucleotides can adopt hairpin structures with double-helix formation through intramolecular hydrogen bonding between complementary sequences of ODNs, which mimic natural DNA hairpin structures. By measuring Tm values, it is apparent that intermolecular base pairings are more favorable than intramolecular ones. The CD spectra exhibit the distinct characteristics of B-form DNA and indicate that the calixoligonucleotides can act in a manner similar to that of natural ODNs. The calixoligonucleotides aggregate in solution, with the driving forces of intermolecular base pairing of the ODN units and hydrophobic interactions of the hydrophobic residues of the calix[4]arene moieties. In the preliminary microscopy experiments, the calixoligonucleotides might be forming double helices with intermolecular base pairing (see Fig. 2c) rather than making linear double helices through intermolecular base pairing (see Fig. 2b). In conclusion, we have shown that calixoligonucleotides can be good V-shaped building blocks for constructing architectures with DNA, as well as being efficient turning points in long ODN sequences.


   ACKNOWLEDGEMENTS
 
We are grateful to KISTEP for the financial support through the NRL (Laboratory for Modified Nucleic Acid Systems) program. We also thank the Korea Health 21 R&D project and the BK21 program for partial support.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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