Synthesis and structure investigation of stabilized aromatic oligoamides and their interaction with g quadruplex structures 5

Chapter Circular Aromatic Oligoamides as New G-quadruplex-Binding Ligands 5.1 Introduction Nucleic acid sequences that comprise tandem guanine (G) nucleotides are predisposed to forming four stranded structures called G-quadruplexes.1,2 G-quadruplexs can be folded either from a single G-rich sequence by intramolecular hydrogen bonding or two (dimeric) or four (tetrameric) separate strands by intermolecular association. Since these stands may have different directionalities, there exist topological variations for these strands.3 These strands can be parallel, alternating parallel or three parallel and one anti-parallel, resulting in distinct conformations named as parallel, anti-parallel and propeller quadruplexes, respectively. Intramolecular quadruplexes have been intensively investigated due to their biological relevance. For example, intramolecular quadruplex formation in the promoter region of some oncogenes seems to play a vital role in regulating transcription of the corresponding gene.4,5 Among all reported intramolecular quadruplexes, human telomere DNA is of particular interest.6 Telomere DNAs, located at the end of chromosomes, are known to contain repeating single-strand sequences of (TTAGGG)n folding into a quadruplex structure.7 The formation of 130 G-quadruplexes has been shown to inhibit the activity of telomerase (an enzyme overexpressed in 84-90% of cancer cells), which catalyses the addition of telomeric DNA repeats onto the single-stranded 3’ end, contributing to the immortalization of human tumor cells.8-10 The topologies of telomere G-quadruplex in different salts have been extensively studies. In Na+ solution, the 22nt sequence d[AGGG(TTAGGG)3] was proven to form a basket-type structure which has one diagonal and two lateral TTA loops in Na+ solution.11 In the presence of K+, the telomere DNA adopts a propeller-type parallel-stranded G-quadruplex structure that contains no loop structure by a solid state study.9,12 However, the topology of telomere DNA in K+ solution was controversial and many scientists have made efforts in elucidating it.13-16 It was generally suggested that telomere DNA adopts a mixed-parallel/antiparallel qudruplex stucture.17-20 Figure 5.1. Human telomeric DNA of different conformations:18 (A) Antiparallel basket-type quadruplex in a Na+ solution determined by NMR, (B) Parallel propeller-type quadruplex in K+ crystal determined by X-ray, (C) Mixed-parallel/antiparallel quadruplex in a K+ solution proposed based on CD and NMR. As mentioned, G-quadruplexs are stabilized with either potassium or sodium ions. Since Zahler and co-workers demonstrated in 1991 the telomerase inhibitory 131 behaviors of potassium-stabilized G-quadruplex structures, G-quadruplex DNA has emerged as an attractive target for designing telomerase inhibitors, leading to the development of new anticancer drugs.21-23 Recently, there is a growing interest in developing ligands that stabilize quadruplex DNA structures and so disrupt the interactions between quadruplex DNA and telomerase, causing telomerase to lose its function.41-43 a) d) b) c) e) f) Figure 5.2. Compounds reported to bind to G-quadruplex. (a) TMPyP4, (b) Se2SAP, (c) Telomestatin, (d) Telomestatin mimic oligoamide, (e) Oligoamide, (f) Oxazole-based oligoamide. Among all the reported G-quadruplex stabilizers, most of them are based on cyclic π-delocalized systems owing to the cyclic nature of G-tetrad, a repeating unit composed of four guanines that forms the G-quadruplex structure. Telomestain24 is the first natural telomerase inhibitor of high potency due to its ability to stabilize the G-qudruplex structure. It appears to interact preferably with basket-type 132 intramolecular G-quadruplexes rather than intermolecular G-qudruplexes. However, this macrocyclic compound tends to occupy the whole G-quartet with little selectivity over different intromolecular structures. Heterocyclic compounds such as porphyines,25,26 porphyrazines27 and cyclopyyroles28,29 have also been demonstrated to display high binding affinities for G-quadruplex. Among the most popular macrocyclic ligands are oligoamides which showed unique G-quadruplex selectivity.30,31 For example, oxazole-based peptide32 which contains three stereo amine side chains demonstrated high selectivity toward c-kit over human telomeric quadruplex. Recently, Masayuki Tera, et al, also developed the macrocyclic hexazole binders selective for telo23 DNA sequence.33 In the study of G-quadruplex structure, effective assays are required for studying the G-quadruplex formation and for determining the interaction between ligands and G-quadruplexes. Titration experiments analyzed by spectroscopic methods such as fluorescence,34 circular dichroism35 (CD) and ultraviolet (UV)36 can provide significant information on ligand-quadruplex interactions. CD was used mostly to identify G-quadruplex structures, especially to distinguish parallel from antiparallel structures. It also serves as an efficient method to measure the melting temperature of oligonucleotides. Calorimetric techniques such as isothermal titration calorimetry (ITC) can also provide thermodynamics information on ligand-quadruplex interactions.37 However, those analytical methods always require a large amount of oligonucleotides and are time-consuming. Therefore, gel-shift assays have been extensively used, including polyacrylamide gel electrophoresis38 (PAGE) and 133 polymerase chain reaction (PCR) stop assays.39 PCR stop assays could selectively detect G-quadruplexes ligands by using only trace amounts (pmole) of oligonucleotides. Although the topologies of telomere G-quadruplexs have been recently elucidated in a good detail, few compounds have been reported as selective binders to telomere G-quadruplexes with different structures. In this study, we developed a new generation of aromatic oligoamides for stabilizing telomeric G-quadruplex structures. These stabilizers demonstrated better binding affinity to mixed-parallel/antiparallel over anti-parallel basket-type G-quadruplexes. PCR stop assay has been extensively adopted herein because of its high sensitivity and selectivity. We also confirmed the switching oligoamides from macrocylic to helical structures resulted in loss of G-quadruplex binding affinity and selectivity. 5.2 Result and discussion Table 5.1. Sequences of oligomers (primers) used in this study. oligomers ODN1 Sequence 5’-GGGTTAGGGTTAGGGTTAGGGT-3’ Description Telomeric DNA forming -parallel/antiparallel G-quadruplex in K ODN2 5’-TAGGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric DNA mixed + forming mixed -parallel/antiparallel G-quadruplex in K+ ODN3 5’- TTAGGGTTAGGGT-3’ Telomeric DNA forming a parallel G-quadruplex ODN4 5’-GAGTTAGAGTTAGAGTTAGAGT-3’ Mutated ODN1 that can’t form a G-quadruplex ODN5 5’-AGGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric DNA forming -parallel/antiparallel G-quadruplex in K Primer1 5’-TCTCTGTCACACCCTAAC-3’ mixed + Partial complementary sequence of ODN1,ODN2 and ODN3 in PCR stop assay Primer2 5’-TCTCTGTCACACTCTAAC-3’ Partial complementary sequence of ODN4 in PCR stop assay 134 Scheme 5.1. Cyclic pentamers recognizing G-quadruplexes O O O N H OMe HN OMe OMe MeO OMe HN O HN MeO OMe O MeO MeO HN MeO NH O O OMe O O Me HN OH 10a NH O N H O Me HN OH 9a NH O N H OH NH O O O O N H HO HN 10b OH O MeO HN MeO NH NH NH NH O O O O O O C8 H1 O O O N H OC8H1 OH 9b NH O OMe 11a NH O HN OH O NH C8 H1 7O O Me HN OH MeO MeO O N H OMe HN MeO HO HN O NH O O OC8H17 Scheme5. 2. Synthetic route for 9a, 10a, 10b, 11a O a + O 2N H2 N COOH N H OR COOMe OMe OR 5e R1 = Me 5f R1 = Bn NO 5g O O NO2 O O N H R 1O OR2 OR MeO b, c O HN NO2 MeO b, c 5h R1 = Me 5i R1 = Bn HN OR2 R 1O NH b, c HN COOMe OMe MeO O MeO 5j R1 = Me, R2 = Me 5k R1 = Bn, R2 = Bn 5l R1 = Bn, R2 = Me O 5m R1, R2, R3 = Me 5n R1, R2 = Bn, R3 = Me 5o R1, R3 = Bn, R2 = Me O O O HN OR R 1O NH OR3 NH HN NH O O O NH O MeO NO2 O HN H N O 5p 5q 5r 5s a R1, R2, R3 = Me, R4 = Bn R1, R2 = Bn, R3, R4 = Me R1, R3 = Bn, R2, R4 = Me R1, R2, R4=Bn, R3=Me 5u R1, R2, R3 = Me, R4 = Bn 5v R2 = Bn, R3, R4 = Me 5w R1, R3 = Bn, R2, R4 = Me 5x R1, R2, R4=Bn, R3=Me HN R 1O NH O OR MeO OR OR3 f R 1O OR3 MeO N H HN OR b, d, e OR O O O O HN OR MeO H N O 9a R1, R2, R3 = Me, R4 = H 10a R1, R2 = H, R3 R4 = Me 10b R1, R3 = H, R2, R4 = Me 11a R1, R2, R4 = H, R3 = Me a) ethyl carbonochloridate, 4-methylmorpholine, CH2Cl2, 64~71%; b) Fe, AcOH, EtOH, 93%; c) 5e (or 5f), SOCl2, DIEA, DCM, 57~80%; d) 1M KOH, MeOH, 95%; e) BOP, DIEA, CH2Cl2, 75~86%; f). H2, Pd/C, THF/MeOH, 50 oC, 57~90%. 135 Scheme 5.3. Synthetic route for 9b OC8 H 17 O2N COOH OMe H2N OBn O O 6e 5f OC 8H 17 O O MeO a 5f + 6e N H O NO2 Bn O O N H OMe b, c O OMe b, c O HN Bn O NO2 6g OC H17 6h MeO C8 H 17O R1 O O NH C 8H 17O O HN OMe Bn O O HN NH O OMe b, c HN R3 6j NO O R2 Bn O R4 OC 8H 17 O MeO C 8H 17 O O O O HN O f O NH O Bn O Bn O a C 8H 17O HN O O NH HN O N H O O NH O O NO2 C 8H 17O 6p OC 8H 17 O Bn O C 8H 17O b, d, e O HN 6m NH O MeO N H O O H 9b O H O O HN O NH OC8H17 O OC8 H17 a) SOCl2, reflux, 2h, DIEA, CH2Cl2, 86%; b) Fe, AcOH, EtOH, 95%; c) 5f, SOCl2, DIEA, DCM, 90%; d). 1M KOH, MeOH, 99%; e) BOP, DIEA, CH2Cl2, 57%; f) H2, Pd/C, THF/MeOH, 50 oC, 83%. Scheme 5.4. Primers were designed to be partially complementary to ODNs 136 5.2.1. Binding Selectivity of Oligoamides toward Telemeric G-quadruplexes The ligands’ ability to stabilize a quadruplex structure was primarily evaluated by PCR stop assay. The detection principle is based on the fact that the binding of ligands with G-quadruplex structures could inhibit the action of the DNA polymerase.41 The single-stranded DNA could be induced into a G-quadruplex structure that blocks hybridization with a complementary strand overlapping the last G repeat in the presence of aromatic oligoamides. As a result, the DNA extension with Taq polymerse is inhibited, causing a reduction on the final double-stranded PCR product. For all the Oligonucleotides (ODNs), PCR stop assay was performed in the presence of different concentrations of aromatic oligoamides. A typical PAGE for PCR stop assay on circular pentamer 10a is shown in Figure 5.3. The synthesis of double-stranded PCR product was inhibited by oligoamides in a dose-dependent manner. IC50 was used to represent the concentration of oligoamides required to achieve 50% inhibition of the reaction. Oligoamide 10a was investigated at different concentrations ranging from 0.5 to 16 μM. With the increasing concentration, PCR product decreased correspondingly. PCR products can be hardly detected when the concentration of 10a reached μM or higher. This micro-molar inhibition capability of our circular pentamer was almost the same as TMPyP4 (1.9 μM) and telomestatin (3 μM) to Pu22mu with G-quadruplex structure39. a) b) + + + + + + + 10a (μM) Lane 16 4 0.5 K Na + + + + + + + 10a (μM) Lane 16 4 0.5 ds ds ss ss 137 c) Figure 5.3. Effect of 10a on the double-stranded PCR product using ODN-1 as the PCR template. (a) PAGE analysis of the inhibiting effect in 50 mM KCl, (b) PAGE analysis of inhibiting effect in 50 mM NaCl, (c) Comparison of inhibiting effect by 10a in 50 mM KCl, 50 mM NaCl, and without salts. A control experiment was carried out with an oligonuclotide (ODN-4) that contains one G to A mutation in every guanine tandem repeat (5’-GAGTTAGAGTTAGAGTTAGAGT-3’) and no inhibition was observed even at a very high concentration of 20 μM (Figure 5.4). 10a (μM) Lane 20 4 ds ss Figure 5.4. Effect of 10a on the double-stranded PCR product of ODN-4 in 50 mM KCl. Interestingly, pentamer 10a demonstrated distinct inhibitory behaviors in K+ solution compared to Na+ solution. At lower concentrations (< μM), the intensity of the PCR product decreased more slowly in Na+ solution than in K+ solution. The IC50 of 10a is found to be 2.68 μM in 50 mM K+ solution and 6μM in 50 mM Na+ solution, respectively. The difference may be attributed to the variation of G-quadruplex structures in different salt solutions. It can be deduced that pentamer 10a was a better 138 stabilizer for hybrid G-quadruplex in K+ solution than the basket structure in Na+ solution. The binding selectivity of pentamer 10a is similar to Se2SAP which was reported to prefer the mixed parallel/antiparallel hybrid structure.1 The result suggests that our circular pentamer 10a may use the same binding mechanism as Se2SAP that requires external loops for ionic interactions with the phosphate contained in these loops.38 5.2.2 Stability of Telemeric G-quadruplex It was found that, the inhibition still occurred in PCR stop assay without adding any salts. This may be due to the presence of potassium chloride (KCl) in Taq polymerase buffer. Some interesting results could be obtained by comparing the IC50 of 10a with and without 50 mM KCl or sodium chloride (NaCl). The IC50 of 10a was 2.98 μM without adding additional salts, slightly higher than 2.68 μM in 50 mM KCl. This result suggests that higher KCl concentration causes a decrease in IC50 value. Moreover, compared with that in 50 mM NaCl solution, IC50 of 10a without additional salts decreased from μM to 2.98 μM, indicating that the addition of sodium salt into the buffer produces more PCR product. This may be attributed to the fact that the anti-parallel G-qudruplex stabilized by Na+ solution is less stable than the mixed-parallel/antiparallel G-quadruplex in K+. To support this assumption, the variation of the Taq polymerase activity in different salts was tested by performing PCR stop assay in 50 mM KCl or NaCl solution without 10a. It was found that the quantity of PCR products were equal in 50 mM KCl and 50 mM NaCl, indicating that 139 was added acetate acid (2.5 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. Evaporation of the solvent gave the amine product used for the next step reaction without purification. A solution of 5f (1.12 g, 4.2 mmol) in SOCl2 (2.0 mL) was heated at reflux for h. After removal the SOCl2, the amine product (1.7 g, 2.1 mmol) and DIEA (0.8 mL, 5.0 mmol) in dry CH2Cl2 (30 mL) were added to the residue. The reaction was allowed to proceed for h. After washing with HCl solution, aqueous sat. NaHCO3 and Brine, the organic layer was dried over Na2SO4. Purification by column chromatography on silica gel (40:1 v/v CH2Cl2: acetone) yielded the pure product 5s as a light brown solid. Yield: 1.49 g, 72%. 1H NMR (300 MHz, CDCl3): δ 9.82 (s, 1H), 9.76 (s, 1H), 9.39 (s, 1H), 9.01 (s, 1H), 8.76 (m, 4H), 8.33 (dd, 1H, J = 1.8 Hz, J = 7.9 Hz), 8.04 (dd, 1H, J = 1.8 Hz, J = 8.1 Hz), 7.86 (dd, 1H, J = 1.6 Hz, J = 7.9 Hz), 7.73 (m, 3H), 7.64 (dd, 1H, J = 1.7 Hz, J = 7.9 Hz), 7.43 (m, 5H), 7.20 (m, 4H), 7.07 (m, 7H), 6.90 (m, 3H), 5.10 (s, 2H), 4.88 (s, 2H), 4.85 (s, 2H), 3.89 (s, 3H), 3.37 (s, 3H), 3.29 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 170.42, 167.18, 165.33, 163.44, 162.50, 162.38, 161.51, 148.96, 147.64, 146.97, 146.63, 145.36, 144.45, 135.82, 135.52, 134.16, 133.69, 132.97, 132.37, 132.05, 131.62, 131.19, 130.61, 130.47, 129.05, 128.82, 128.70, 128.39, 127.87, 127.67, 126.34, 126.10, 125.77, 125.65, 125.14, 124.94, 124.79, 124.51, 124.18, 123.97, 123.44, 79.72, 79.52, 78.46, 77.21, 67.64, 62.12, 61.97, 59.82, 51.77, 38.31, 29.96, 29.22, 28.49, 23.36, 22.54, 20.51, 13.78, 13.64, 10.57. MS-ESI: calculated for [M-H]- (C59H48N5O13): m/z 1034.3, found: m/z 1034.0. 161 Compound 5u: To a solution of 5p (442 mg, 0.50 mmol) and iron (112 mg, 2.0 mmol) in EtOH (5.0 mL) was added acetate acid (1.0 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was not purified, but used directly in the next step. To the solution of resiude in hot methanol (1.0 mL) was added 1M KOH (1.0 mL). The reaction was heated under reflux for hours. After quenching with water (15 ml), the aqueous layer was neutralized by addition 1M HCl (1.0 mL). The mixture was extracted with CHCl3 (3x15 mL). The organic extracts were dried over NaSO4 and concentrated under reduced pressure. To the solution of the residue in dry CH2Cl2 (10 mL) was added BOP (0.66 g, 1.5 mmol) and DIEA (0.35 mL). The reaction was stirred in room temperature for hours. The reaction was washed with HCl solution, aqueous sat. NaHCO3 and Brine. After removal of the solvent, the residue was purified by flash column chromatography on silica gel using CH2Cl2/Ethyl Acetate (8:1 v/v) as the eluent to give the product 5s, three-step total yield: 177 mg, 43%. Light yellow solid. 1H NMR (500 MHz, CDCl3) δ 10.84 (s, 1H), 10.83 (s, 1H), 10.71 (s, 1H), 10.50 (s, 1H), 10.40 (s, 1H), 9.01-8.97 (m, 3H), 8.92 (dd, 1H, J = 8.2, 1.3 Hz), 8.84 (dd, 1H, J = 8.2, 1.3 Hz), 8.05-7.98 (m, 3H), 7.92-7.89 (m, 2H), 7.47-7.38 (m, 5H), 7.13 (d, 2H, J = 7.0 Hz), 7.02-6.94 (m, 3H), 5.11 (d, 2H, J = 5.0 Hz), 4.04 (s, 1H), 4.01 (s, 1H), 4.00 (s, 1H), 3.98 (s, 1H). 13 C NMR (125 MHz, CDCl3) δ 162.7, 162.4, 162.3, 162.2, 162.1, 146.6, 146.5, 146.4, 146.3, 145.0, 133.4, 133.3, 132.8, 162 132.8, 132.8, 132.5, 129.5, 129.5, 128.6, 127.3, 126.5, 126.4, 126.4, 126.2, 126.2, 126.1, 126.1, 126.0, 125.9, 125.7. 125.6, 125.6, 124.4, 124.3, 124.2, 123.8, 80.0, 63.5, 63.5, 63.0, 62.9, 60.4, 14.2. HRMS-ESI: calculated for [M+Na]+(C46H39O10N5+Na): m/z 844.2589, found: m/z 844.2628. Compound 5v: To a solution of 5q (465 mg, 0.50 mmol) and iron (112 mg, 2.0 mmol) in EtOH (5 mL) was added acetate acid (1.0 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was not purified, but used directly in the next step. To the solution of resiude in hot methanol (1.5 mL) was added 1M KOH (1.0 mL). The reaction was heated under reflux for hours. After quenching with water (15 ml), the aqueous layer was neutralized by addition 1M HCl (1.5 mL). The mixture was extracted with CHCl3 (3x15 mL). The organic extracts were dried over NaSO4 and concentrated under reduced pressure. To the solution of the residue in dry CH2Cl2 (10 mL) was added BOP (0.55 g, 2.5 mmol) and DIEA (0.25 mL). The reaction was stirred in room temperature for hours. The reaction was washed with HCl solution, aqueous sat. NaHCO3 and Brine. After removal of the solvent, the residue was purified by flash column chromatography on silica gel using CH2Cl2/Ethyl Acetate (20:1 v/v) as the eluent to give the product 5t, three-step total yield: 210 mg, 47%.1H NMR (300 MHz, CDCl3) δ 10.76 (s, 1H), 10.64 (s, 1H), 10.49 (s, 1H), 10.22 (s, 1H), 10.03 (s, 1H), 8.93-8.86 (m, 4H), 8.71 (d, J = 8.1 Hz, 1H), 8.00 (dd, 2H, J = 14.0, 8.0 Hz), 7.93-7.84 163 (m, 2H), 7.78 (d, 1H, J = 7.8 Hz), 7.47-7.34 (m, 5H), 7.11 (m, 4H), 7.03-6.86 (m, 6H), 5.12 -4.93 (m, 4H), 3.95 (s, 3H), 3.88 (s, 6H); 13 C NMR (75 MHz, CDCl3) δ 162.9, 162.5, 162.4, 162.2, 146.5, 146.4, 144.9, 133.7, 133.0, 132.9, 132.7, 132.6, 132.4, 129.3, 129.2, 129.1, 128.5, 127.4, 127.1, 126.4, 126.2, 126.2, 126.0, 125.8, 125.7, 124.2, 124.1, 124.0, 123.6, 79.7, 79.6, 63.6, 63.2, 63.1. HRMS-ESI: calculated for [M+Na]+(C52H43O10N5+Na): m/z 920.2902, found: m/z 920.2879. Compound 5w: To a solution of 5r (465 mg, 0.50 mmol) and iron (112 mg, 2.0 mmol) in EtOH (5 mL) was added acetate acid (1.0 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was not purified, but used directly in the next step. To the solution of resiude in hot methanol (1.5 mL) was added 1M KOH (1.0 mL). The reaction was heated under reflux for hours. After quenching with water (15 ml), the aqueous layer was neutralized by addition 1M HCl (1.5 mL). The mixture was extracted with CHCl3 (3x15 mL). The organic extracts were dried over NaSO4 and concentrated under reduced pressure. To the solution of the residue in dry CH2Cl2 (10 mL) was added BOP (0.55 g, 2.5 mmol) and DIEA (0.25 mL). The reaction was stirred in room temperature for hours. The reaction was washed with HCl solution, aqueous sat. NaHCO3 and Brine. After removal of the solvent, the residue was purified by flash column chromatography on silica gel using CH2Cl2/Ethyl Acetate (20:1 v/v) as the eluent to give the product 5w, three-step total yield: 228 mg, 51%.1H NMR (300 MHz, 164 CDCl3) δ 10.86, 10.68, 10.65, 10.52, 10.40, 10.23, 10.18, 9.99, 8.98, 8.96, 8.93, 8.91, 8.88, 8.84, 8.03, 8.00, 7.92, 7.88, 7.85, 7.45, 7.42, 7.40, 7.26, 7.15, 7.09, 7.00, 6.96, 5.29, 5.21, 5.17, 5.10, 5.06, 4.05, 3.99, 3.98, 3.93, 3.77; 13C NMR (75 MHz, CDCl3) δ 163.0, 162.7, 162.5, 162.3, 162.0, 146.4, 146.3, 146.1, 145.1, 144.6, 144.4, 133.7, 133.4, 133.2, 133.2, 133.0, 132.8, 132.6, 132.3, 129.8, 129.6, 129.5, 129.4, 129.1, 128.5, 127.7, 127.6, 127.4, 126.9, 126.3, 126.0, 125.7, 125.6, 125.4, 124.1, 124.0, 123.8, 123.6, 123.6, 123.5, 80.0, 79.9, 79.6, 63.4, 63.2, 62.7, 62.6, 62.2; HRMS-ESI: calculated for [M+Na]+(C52H43O10N5+Na): m/z 920.2902, found: m/z 920.2905. Compound 5x: To a solution of 5s (517 mg, 0.50 mmol) and iron (112 mg, 2.0 mmol) in EtOH (5 mL) was added acetate acid (1.0 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was not purified, but used directly in the next step. To the solution of resiude in hot methanol (1.5 mL) was added 1M KOH (1.0 mL). The reaction was heated under reflux for hours. After quenching with water (15 ml), the aqueous layer was neutralized by addition 1M HCl (1.5 mL). The mixture was extracted with CHCl3 (3x15 mL). The organic extracts were dried over NaSO4 and concentrated under reduced pressure. To the solution of the residue in dry CH2Cl2 (10 mL) was added BOP (0.55 g, 2.5 mmol) and DIEA (0.25 mL). The reaction was stirred in room temperature for hours. The reaction was washed with HCl solution, aqueous sat. NaHCO3 and Brine. After removal of the solvent, the residue was purified by flash 165 column chromatography on silica gel using 60:1 v/v CH2Cl2: acetone as the eluent to give the product 5x. Yield: 0.14 g, 30%. isomers were present in a : 0.7 ratio. The peaks due to both isomers overlapped seriously and cannot be differentiated from one another. Only the peaks corresponding to the amide protons were distinct. 1H NMR (300 MHz, CDCl3): Isomer 1: δ 10.75, 10.51, 10.29, 9.91, 9.73. Isomer 2: δ 10.65, 10.37, 10.36, 10.04, 9.80. 13 C NMR (75 MHz, CDCl3): δ 163.25, 162.79, 162.71, 162.56, 162.47, 162.12, 146.42, 146.31, 146.12, 145.10, 144.82, 144.44, 144.28, 134.07, 133.64, 133.58, 133.35, 133.27, 133.11, 133.08, 132.98, 132.79, 132.75, 132.67, 132.64, 132.39, 132.35, 129.98, 129.64, 120.60, 129.53, 129.40, 129.35, 129.28, 129.13, 129.03, 128.79, 128.68, 128.55, 128.49, 128.43, 128.41, 128.19, 128.11, 127.83, 127.54, 127.50, 126.73, 126.32, 126.18, 126.05, 125.89, 125.78, 125.73, 125.66, 125.52, 125.42, 125.21, 124.41, 124.31, 124.02, 123.93, 123.78, 123.71, 123.54, 123.41, 123.30, 79.82, 79.67, 79.34, 65.76, 63.53, 63.37, 62.47, 62.38, 30.83, 30.43, 29.61, 28.88. MS-ESI: calculated for [M-H]- (C58H46N5O10): m/z 972.3, found: m/z 972.3. Compound 9a: Compound 5s (82.0 mg, 0.10 mmol) was reduced by catalytic hydrogenation in THF (20 mL) at 50 oC, using 10% Pd-C (12.0 mg, 15%) as the catalyst for h. The reaction mixture was then filtered and the solvent removed in vacuo and washed with ether to give the green solid 2a. Yield: 61.0 mg, 84%. 1H NMR (500 MHz, d6-DMSO) δ = 11.77 (b, 1H), 11.2 (s, 1H), 11.05 (s, 1H), 11.04 (s, 1H), 11.00 (s, 1H), 8.89-8.84 (m, 4H), 8.74 (d, 1H, J = 7.7 Hz), 7.88-7.82 (m, 5H), 7.49-7.43 (m, 4H), 7.26 (s, 1H), 166 4.12 (s, 6H), 4.11 (s, 3H), 4.06 (s, 3H). 13 C NMR (125 MHz, d6-DMSO) δ = 162.9, 161.6, 161.5, 147.0, 147.0, 147.0, 146.8, 133.2, 133.0, 132.9, 132.9, 132.1, 126.0, 125.7, 125.7, 125.6, 125.6, 125.3.125.2, 125.1, 125.0, 124.8, 124.7, 123.4, 123.2, 123.3, 123.0, 122.9, 63.6, 63.5, 63.4, 63.3, 54.9. HRMS-ESI: calculated for [M-H](C39H32N5O10)：m/z 730.2149, found: m/z 730.2189. Compound 10a: Compound 5t (89.7 mg, 0.10 mmol) was reduced by catalytic hydrogenation in THF (20 mL) at 50 oC, using 10% Pd-C (18.0 mg, 20%) as the catalyst for h. The reaction mixture was then filtered and the solvent removed in vacuo and washed with ether to give the green solid 3a. Yield: 58 mg, 81%. 1H NMR (500 MHz, d6-DMSO) δ 11.72 (s, 1H), 11.18 (s, 1H), 11.05 (s, 1H), 10.91 (s, 1H), 10.19 (s, 1H), 8.93-8.79 (m, 3H), 8.70 (dd, 1H, J = 8.0, 1.4 Hz), 8.61 (d, 1H, J = 7.9 Hz), 7.95-7.86 (m, 3H), 7.81-7.80 (m, 2H), 7.60 (d, 1H, J = 6.9 Hz), 7.38-7.28 (m, 3H), 7.16 (dd, 2H, J = 17.7, 8.0 Hz), 4.08 (s, 6H), 4.03 (s, 3H); 13C NMR (125 MHz, d6-DMSO) δ 163.9, 163.5, 162.3, 162.2, 162.1, 147.2, 146.9, 146.9, 133.4, 133.2, 133.0, 132.1, 131.9, 131.8, 126.3, 125.9, 125.8, 125.7, 125.6, 125.6, 125.5, 125.3, 125.3, 124.9, 124.5, 124.5, 124.2, 123.9, 123.4, 123.2, 122.6, 122.5, 63.3, 63.1; HRMS-ESI: calculated for [M-H]- (C38H31N5O10-H)：m/z 716.1998, found: m/z 716.2019. Compound 10b: Compound 5u (89.7 mg, 0.10 mmol) was reduced by catalytic hydrogenation in THF (20 mL) at 50 oC, using 10% Pd-C (18.0 mg, 20%) as the catalyst for h. The reaction mixture was then filtered and the solvent removed in vacuo and washed with 167 ether to give the green solid 3a. Yield: 62 mg, 86%.1H NMR (500 MHz, d6-DMSO) δ 11.64 (s, 1H), 11.53 (s, 1H), 11.03 (s, 2H), 10.88 (s, 1H), 10.31 (s, 1H), 8.87 – 8.73 (m, 3H), 8.68 (s, 2H), 7.86-7.79 (m, 6H), 7.34 – 7.22 (m, 3H), 7.13 (s, 2H), 4.04 (s, 3H), 4.00 (s, 3H), 3.98 (s, 3H); 13C NMR (75 MHz, d6-DMSO) δ 159.3, 157.7, 147.1, 142.8, 142.6, 142.5, 139.8, 132.5, 128.8, 128.6, 128.2, 128.0, 123.6, 121.7, 121.5, 121.2, 121.2, 121.1, 121.0, 121.0, 120.8, 120.6, 120.1, 120.0, 119.7, 119.3, 119.0, 118.9, 118.3, 58.8, 58.6, 58.4; HRMS-ESI: calculated for [M-H]- (C38H31N5O10-H)： m/z 716.1998, found: m/z 716.1987. Compound 11a: Compound 5x (0.07 g, 0.10 mmol) was reduced by catalytic hydrogenation in solvent mixture (4 mL) ethanol and THF at 50 oC using Pd-C (0.03 g, 45%) as the catalyst for h. The reaction mixture was then filtered and the solvent removed in vacuo Purification by flash column chromatography on silica gel (6:1 v/v CH2Cl2 : acetone) yielded the pure product as a green solid. Yield: 0.05 g, 75%. 1H NMR (300 MHz, DMSO-d6/CDCl3): δ 14.80 (s, 1H), 13.82 (s, 1H), 12.72 (s, 1H), 11.24 (s, 1H), 11.03 (s, 1H), 8.77 (m, 2H), 8.52 (m, 1H), 8.38 (m, 2H), 7.61 (m, 4H), 7.18 (m, 3H), 6.41 (m, 2H), 6.29 (m, 1H), 4.00 (s, 6H). 13 C NMR (125 MHz, DMSO-d6/CDCl3): δ 167.30, 167.10, 165.86, 161.92, 161.45, 160.80, 161.84, 147.70, 147.22, 138.99, 136.20, 134.84, 132.96, 131.85, 128.25, 127.98, 127.48, 125.27, 215.08, 124.93, 124.59, 124.45, 124.28, 124.15, 123.74, 123.51, 123.49, 121.66, 120.49, 120.08, 119.83, 111.61, 109.79, 97.67, 63.38, 62.33, 34.69. MS-ESI: calculated for [M-H](C37H28N5O10-H): m/z 702.2, found: m/z 702.3. 168 Compound 6g: A solution of 2-(benzyloxy)-3-nitrobenzoic acid 5f (8.00 g, 29.3 mmol) in SOCl2 (22.0 mL) was heated at reflux for h. After removal the SOCl2, methyl 3-amino-2-methoxy-5-(octyloxy)benzoate 6e (7.60 g, 24.4 mmol) and DIEA (N,N-Diisopropylethylamine, 6.10 mL, 34.2 mmol) in dry CH2Cl2 (120 mL) were added to the residue. The reaction was allowed to proceed for h. After washing with HCl solution, aqueous sat. NaHCO3 and Brine, the organic layer was dried over Na2SO4. The residue was recrystallized from methanol to give the pure product 6c as a white solid. Yield: 11.80 g, 86%. mp 58-59 °C. 1H NMR (500 MHz, CDCl3) δ 9.79 (s, 1H), 8.36 (d, 1H, J = 3.2 Hz), 8.26 (dd, 1H, J = 7.6, 1.3 Hz), 7.98 (dd, 1H, J = 8.2, 1.9 Hz), 7.39 (t, 1H, J = 8.2 Hz), 7.24-7.21 (m, 4H), 7.10 (d, 1H, J = 3.2 Hz), 4.00 (t, 2H, J = 6.3 Hz), 3.91 (s, 3H), 3.53 (s, 3H), 1.81-1.78 (m, 2H), 1.51-1.45 (m, 2H), 1.35-1.26 (m, 8H), 0.90 (t, 3H, J = 6.9 Hz). 13 C NMR (125 MHz, CDCl3) δ 165.8, 161.8, 154.9, 149.3, 144.9, 143.1, 135.9, 133.8, 133.0, 131.2, 129.5, 129.2, 128.6, 128.4, 125.0, 123.6, 111.6, 111.1, 79.8, 68.7, 62.3, 52.3, 31.8, 29.3, 29.2, 26.0, 22.7, 14.1. HRMS-ESI: calculated for [M+Na]+ (C31H36O8N2+Na): m/z 587.2388, found: m/z 587.2364. Compound 6h: To a solution of 6g (5.52 g, 10.0 mmol) and iron (2.24 g, 40.0 mmol) in EtOH (100 mL) was added acetate acid (10 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. Evaporation 169 of the solvent gave the amine product used for the next step reaction without purification. A solution of 2-methoxy-3-nitro-5-(octyloxy)benzoic acid 6f (3.90 g, 12.0 mmol) in SOCl2 (20.0 mL) was heated at reflux for h. After removal the SOCl2, the amine product (2.61 g, 5.0 mmol) and DIEA (2.5 mL, 14 mmol) in dry CH2Cl2 (60 mL) were added to the residue. The reaction was allowed to proceed for h. After washing with HCl solution, aqueous sat. NaHCO3 and Brine, the organic layer was dried over Na2SO4. The residue was recrystallized from methanol to give the pure product 6h as a white solid. Yield: 7.70 g, 90%. mp 83-84 °C. 1H NMR (500 MHz, CDCl3) δ 9.99 (s, 1H), 9.81 (s, 1H), 8.70 (dd, 1H, J = 8.2, 1.3 Hz), 8.47 (d, 1H, J = 3.2 Hz), 7.80 (dd, 1H, J = 7.6, 1.3 Hz), 7.77 (d, 1H, J = 3.2 Hz), 7.47 (d, 1H, J = 3.2 Hz), 7.34 (t, 1H, J = 8.2 Hz), 7.19-7.04 (m, 6H), 5.01(s, 1H), 4.05-4.00 (m, 4H), 3.93 (s, 3H), 3.87 (s, 3H), 3.73 (s, 3H), 1.84-1.79 (m, 4H), 1.49-1.45 (m, 4H), 1.359-1.21 (m, 16H), 0.91-0.88 (m, 6H). 13 C NMR (125 MHz, CDCl3) δ 165.8, 163.4, 161.1, 155.2, 155.1, 145.8, 144.8, 144.2, 142.8, 134.1, 133.4, 132.6, 129.7, 129.5, 129.1, 128.5, 128.0, 126.3, 125.6, 124.2, 123.7, 121.0, 114.6, 111.4, 110.9, 79.4, 69.3, 68.2, 64.2, 62.4, 52.3, 31.8, 31.8, 29.3, 29.2, 29.2, 28.9, 26.0, 25.9, 22.6, 22.6, 14.1, 14.0. HRMS-ESI: calculated for [M+Na]+ (C47H59O11N3+Na): m/z 864.4034, found: m/z 864.4042. Compound 6j: To a solution of 6h (7.60 g, 9.20 mmol) and iron (2.04 g, 36.8 mmol) in EtOH (100 mL) was added acetate acid (9.2 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and 170 washed with water and Brine. The organic layer was dried over Na2SO4. Evaporation of the solvent gave the amine product used for the next step reaction without purification. A solution of 6f (2.11 g, 6.50 mmol) in SOCl2 (12.0 mL) was heated at reflux for h. After removal the SOCl2, the amine product (3.99 g, 5.0 mmol) and DIEA (1.3 mL, 7.0 mmol) in dry CH2Cl2 (40 mL) were added to the residue. The reaction was allowed to proceed for h. After washing with HCl solution, aqueous sat. NaHCO3 and brine, the organic layer was dried over Na2SO4. The residue was recrystallized from methanol to give the pure product 6j as a white solid. Yield: 4.60 g, 84%. mp 105-106 °C 1H NMR (500 MHz, CDCl3) δ 10.08 (s, 1H), 9.90 (s, 1H), 9.69 (s, 1H), 8.77 (dd, 1H, J = 8.2, 1.3 Hz), 8.44 (d, 1H, J = 3.2 Hz), 8.41 (d, 1H, J = 3.2 Hz), 7.95 (d, 1H, J = 3.2 Hz), 7.54 (d, 1H, J = 3.2 Hz), 7.35 (t, 1H, J = 7.6 Hz), 7.31 (d, 1H, J = 2.6 Hz), 7.20-7.18 (m, 2H), 7.12-7.05 (m, 4H), 5.00 (s, 2H), 4.08-4.00 (m, 6H), 4.00 (s, 3H), 3.99 (s, 3H), 3.78 (s, 3H), 3.69 (s, 3H), 1.85-1.78 (m, 6H), 1.50-1.45 (m, 6H), 1.35-1.26 (m, 24H), 0.91-0.88 (m, 9H). 13 C NMR (125 MHz, CDCl3) δ 165.8, 163.6, 162.8, 156.2, 155.4, 155.1, 145.7, 144.7, 144.4, 142.8, 140.7, 134.4, 133.4, 132.8, 132.5, 129.7, 129.0, 129.0, 128.5, 128.1, 126.8, 125.8, 125.6, 124.3, 123.6, 121.2, 115.1, 111.5, 111.4, 111.1, 111.0, 79.0, 69.4, 68.7, 68.6, 64.5, 63.1, 62.3, 52.3, 31.8, 31.8, 29.3, 29.2, 29.2, 29.1, 28.9, 26.0, 25.9, 22.6, 22.6, 14.1. HRMS-ESI: calculated for [M+Na]+ (C63H82O14N4+Na): m/z 1141.5720, found: m/z 1141.5712. Compound 6m: To a solution of 6j (1.82 g, 1.65 mmol) and iron (0.37 g, 6.60 mmol) in EtOH (20 mL) 171 was added acetate acid (1.65 mL). The reaction was refluxed for hours. After cooling, the solvent was evaporated and the residue was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. Evaporation of the solvent gave the amine product used for the next step reaction without purification. A solution of 6f (0.75 g, 2.30 mmol) in SOCl2 (10 mL) was heated at reflux for h. After removal the SOCl2, the amine product (1.77 g, 1.65 mmol) and DIEA (0.47 mL, 2.60 mmol) in dry CH2Cl2 (20 mL) were added to the residue. The reaction was allowed to proceed for h. After washing with HCl solution, aqueous sat. NaHCO3 and Brine, the organic layer was dried over Na2SO4. The residue was recrystallized from methanol to give the pure product 6m as light yellow solid. Yield: 2.00 g, 88%. mp 96-97 °C. 1H NMR (500 MHz, CDCl3) δ 10.27 (s, 1H), 9.85 (s, 1H), 9.85 (s, 1H), 9.79 (s, 1H), 9.74 (s, 1H), 8.73 (dd, 1H, J = 8.2, 1.3 Hz), 8.46-8.43 (m, 3H), 7.95 (d, 1H, J = 3.2 Hz), 7.78 (dd, 1H, J = 8.2, 1.3 Hz), 7.51 (d, 1H, J = 3.2 Hz), 7.41 (d, 1H, J = 3.2 Hz), 7.35 (t, 1H, J = 8.2 Hz), 7.24 (d, 1H, J = 3.2 Hz), 7.15-7.13 (m, 2H), 7.11 (d, 1H, J = 3.2 Hz), 6.99-6.98 (m, 3H), 4.98 (s, 2H), 4.09-3.99 (m, 8H), 4.01 (s, 3H), 3.87 (s, 3H), 3.86 (s, 3H), 3.81 (s, 3H), 3.70 (s, 3H), 1.82-1.78 (m, 8H), 1.49-1.47 (m, 8H), 1.36-1.25 (m, 32H), 0.90-0.87 (m, 12H). 13 C NMR (125 MHz, CDCl3) δ 165.6, 163.5, 163.0, 162.9, 161.4, 156.4, 156.3, 155.4, 155.1, 145.9, 144.6, 144.5, 142.9, 140.8, 140.6, 134.4, 133.5, 132.8, 132.7, 132.6, 129.6, 129.2, 128.9, 128.4, 127.9, 127.0, 126.9, 125.9, 125.6, 124.6, 123.5, 121.2, 115.2, 111.7, 111.5, 111.2, 111.0, 111.0, 110.8, 78.9, 69.3, 68.7, 68.7, 68.6, 64.5, 63.3, 63.0, 62.2, 52.1, 31.8, 31.7, 29.3, 29.2, 29.2, 28.9, 26.0, 25.9, 22.6, 22.6, 14.1, 14.0. HRMS-ESI: 172 calculated for [M+Na]+ (C79H105O17N5+Na): m/z 1418.7398, found: m/z 1418.7408 Compound 6p: To a solution of 6m (1.38 g, 1.0 mmol) and iron (220 mg, 4.0 mmol) in EtOH (20 mL) was added acetate acid (2.0 mL). The reaction was refluxed for hours. The reaction was dissolved with CH2Cl2 and washed with water and Brine. The organic layer was dried over Na2SO4. After removal of the solvent, the residue was not purified, but used directly in the next step. To the solution of resiude in hot methanol (1.0 mL) was added 1M KOH (2.0 mL). The reaction was heated under reflux for hours. After quenching with water (30 ml), the aqueous layer was neutralized by addition 1M HCl (2.0 mL). The mixture was extracted with CHCl3. The organic extracts were dried over NaSO4 and concentrated under reduced pressure. To the solution of the residue in dry CH2Cl2 (20 mL) was added BOP (1.33 g, 3.00 mmol) and DIEA (0.70 mL). The reaction was stirred in room temperature for hours. The reaction was washed with HCl solution, aqueous sat. NaHCO3 and Brine. After removal of the solvent, the residue was purified by flash column chromatography on silica gel using hexane/CH2Cl2 (1:1 v/v) as the eluent to give the product 6p, three-step total yield: 711 mg, 57%. Light grey solid, mp 171-172 oC. 1H NMR (500 MHz, CDCl3) δ 10.91 (s, 1H), 10.89 (s, 1H), 10.76 (s, 1H), 10.61(s, 1H), 10.37 (s, 1H), 8.90 (dd, 1H, J = 7.6, 1.3 Hz), 8.60-8.58 (m, 3H), 8.45 (d, 1H, J = 4.7 Hz), 7.88 (dd, 1H, J = 7.6, 1.3 Hz), 7.50-7.38 (m, 5H), 7.12 (d, 2H, J = 7.0 Hz), 7.04-6.96 (m, 3H), 5.10 (d, 2H, J = 2.6 Hz), 4.09-4.05 (m, 8H), 3.96 (s, 3H), 3.93 (s, 6H), 3.91 (s, 3H), 1.84-1.81 (m, 8H), 1.51-1.48 (m, 8H), 1.36-1.31 (m, 32H), 0.92-0.89 (m, 12H). 13 C NMR (125 MHz, 173 CDCl3) δ 162.7, 162.4, 162.2, 162.1, 156.9, 156.8, 156.8, 144.9, 140.4, 140.3, 140.2, 140.1, 133.4, 133.4, 133.4, 133.3, 133.1, 129.6, 129.4, 128.5, 127.3, 126.3, 126.0, 125.9, 125.8, 125.8, 123.7, 111.1, 111.1, 111.0, 111.0, 110.6, 110.5, 110.3, 79.9, 68.7, 63.6, 63.5, 63.1, 31.8, 29.3, 29.2, 29.2, 26.0, 22.7., 14.1. MS-FAB: calculated for [M]+ (C71H96N5O14)：m/z 1242.7, found: m/z 1242.5. Compound 9b: Compound 6p (462.0 mg, 0.35 mmol) was reduced by catalytic hydrogenation in THF/MeOH (4:1 v/v, 50 mL) at 50 oC, using 10% Pd-C (70 mg, 15%) as the catalyst for hours. The reaction mixture was then filtered and the solvent removed in vacuo and washed with ether to give the green solid 9b. Yield: 358 mg, 83%. Light green solid, mp > 250 oC (decomposed). 1H NMR (500 MHz, CDCl3) δ 11.16 (s, 1H), 11.15 (s, 1H), 11.11 (s, 1H), 11.01 (s, 1H), 10.61 (s, 1H), 8.64 (d, 2H, J = 3.0 Hz), 8.53 (d, 1H, J = 3.0 Hz), 8.37 (d, 1H, J = 7.0 Hz), 8.29 (s, 1H), 8.04 (s, 1H), 7.54 (d, 1H, J = 3.0 Hz), 7.51 (d, 1H, J = 3.0 Hz), 7.48-7.46 (m, 1H), 7.37 (dd, 2H, J = 7.5, 2.5 Hz), 6,94 (t, 1H, J = 8.0 Hz), 4.20 (s, 3H), 4.14 (s, 3H), 4.11 (s, 3H), 4.10-4.08 (m, 4H), 4.08 (s, 3H), 4.02 (t, 2H, J = 6.5 Hz), 3.93 (t, 2H, J = 6.5 Hz), 1.88-1.81 (m, 6H), 1.76-1.72 (m, 2H), 1.51-1.47 (m, 8H), 1.38-1.21 (m, 32H), 0.93-0.87 (m, 12H). 13 C NMR (125 MHz, CDCl3) δ 165.9, 162.6, 162.3, 162.2, 162.01, 156.8, 156.5, 156.1, 140.1, 140.4, 140.4, 133.6, 133.5, 132.2, 125.7, 125.4, 125.3, 124.7, 124.1, 111.9, 111.1, 111.0, 110.6, 111.5, 110.3, 110.1, 111.1, 108.8, 108.8, 68.7, 63.2, 63.2, 62.7, 31.8, 29.3, 29.3, 29.2, 29.2, 29.0, 26.0, 26.0, 25.9, 25.8, 22.7, 14.1. 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USA. 2005, 2, 10864. 176 [...]... difference in K+ and Na+ solution Circular dichroism (CD) spectroscopy (Figure 5. 4) was used to examine the stability of G- quadruplex with different structures The CD spectrum of the d[AGGG(TTAGGG)3] (ODN-1) in the presence of 100 mM Na+ had a 2 95 nm positive band and a 265nm negative band (Figure 5. 4a, red), indicative of a typical anti-parallel structure In contrast, the CD spectrum of ODN-1 in 100... 2 4 1 5 0 .5 6 9b (μM) Lane 1 8 2 4 3 2 4 1 5 0 .5 6 ds ss c) Figure 5. 12 Comparison of the effect of 9b and 9a on the double-stranded PCR product by using ODN-1 as the PCR template: (a) PAGE analysis of inhibitory effect of 9b in 50 mM KCl, (b) PAGE analysis of inhibitory effect of 9a in 50 mM KCl and (c) comparison of the effect of 9b and 9a on the double-stranded PCR product 5. 2 .5 Poor Binding Affinity... NaCl, (b) PAGE analysis of inhibitory effect of 10a in 50 mM KCl, (c) PAGE analysis of inhibitory effect of 10a in 50 mM NaCl, (d) PAGE analysis of inhibitory effect of 11a in 50 mM KCl and (e) PAGE analysis of inhibitory effect of 11a in 50 mM NaCl (f) Comparison of inhibitory effect of 10a in KCl and NaCl (g) Comparison of inhibitory effect of 11a in KCl and NaCl, 148 5. 3 Conclusion The interactions... CD Spectra of ODN-1 in the presence of 100 mM KCl, 100 mM NaCl and 50 mM KCl /50 mM NaCl mixture, (b) melting points of ODN-1 G- quadruplex in 100 mM KCl and 100 mM NaCl, respectively 2 .5 Blank 3b 2.0 Absorbance 1 .5 1.0 0 .5 0.0 -0 .5 -1.0 10 20 30 40 50 60 70 80 90 100 0 Temperature ( C) Figure 5. 6 Melting study of G- quadruplex ODN -5 in the phosphate buffer (pH = 8.0, 10 mM) in the presence of 100 mM KCl... concentrations The IC50 of 11a in K+ solution was estimated to be 0. 75 μM The above results suggested that by replacing more interior methoxy groups with hydroxyl groups, the binding affinity toward DNA G- quadruplex structures was greatly improved On the other hand, the quantity of PCR product of 11a in Na+ solution also decreased sharply compared to 10a, indicating the decrease of the ligands’ binding selectivity... binding affinity or selectivity to different G- quadruplexs could be varied However, the solubility of these aromatic oligoamides is still a limiting factor that constrains the research scope Further research can be focus on introducing positively charged substituents which have been used very often in quadruplex- targeting ligands to enable the ligand to efficiently interact with the grooves and loops of. .. intramolecular H-bonding forces The PCR stop assay implies that good planarity found in macrocycles is crucial for stabilizing G- qudruplexes a) b) 2c (μM) Lane 1 8 2 2 4 4 3 1 5 0 .5 6 3f (μM) Lane 1 8 2 4 3 2 4 1 5 0 .5 6 ds ds ss ss c) 5d (μM) Lane 1 8 2 4 3 2 4 1 5 0 .5 6 ds ss Figure 5. 7 Effect of oligoamides on the PCR product using ODN-1 as the PCR template in 50 mM KCl: (a) 2c, (b) 3f and (c) 5d 5. 2.4 The Tunable... series of circular pentamers (9b, 9a, 10b, 11a, 1) were designed by either adding the exterior side chains or replacing the interior methoxyl groups with hydroxyl groups The binding affinities of these aromatic oligomers have also been studied For example, Figure 5. 8 showed the effect of 10b on the formation of double-stranded DNA by PCR a) b) Figure 5. 8 Effect of 10b and 9a on the double-stranded PCR... 1 25. 8, 1 25. 6, 1 25. 6, 1 25. 5, 1 25. 1, 124.6, 124.6, 124.4, 124.3, 124.1, 123.3, 78.7, 78.7, 64.4, 62.9, 62.1, 52 .1; MS-ESI: calculated for [M-H]- (C53H45N5O13-H): m/z 958 .3, found m/z 958 .2 Compound 5r: 159 To a solution of 5n (3.4 g, 4.0 mmol) and iron (0.94 g, 16.8 mmol) in EtOH (20 mL) was added acetate acid (5 mL) The reaction was refluxed for 2 hours After cooling, the solvent was evaporated and the... 126.34, 126.10, 1 25. 77, 1 25. 65, 1 25. 14, 124.94, 124.79, 124 .51 , 124.18, 123.97, 123.44, 79.72, 79 .52 , 78.46, 77.21, 67.64, 62.12, 61.97, 59 .82, 51 .77, 38.31, 29.96, 29.22, 28.49, 23.36, 22 .54 , 20 .51 , 13.78, 13.64, 10 .57 MS-ESI: calculated for [M-H]- (C59H48N5O13): m/z 1034.3, found: m/z 1034.0 161 Compound 5u: To a solution of 5p (442 mg, 0 .50 mmol) and iron (112 mg, 2.0 mmol) in EtOH (5. 0 mL) was added . this study. oligomers Sequence Description ODN1 5 -GGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric DNA forming mixed -parallel/antiparallel G- quadruplex in K + ODN2 5 -TAGGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric. Telomeric DNA forming mixed -parallel/antiparallel G- quadruplex in K + ODN3 5 - TTAGGGTTAGGGT-3’ Telomeric DNA forming a parallel G- quadruplex ODN4 5 -GAGTTAGAGTTAGAGTTAGAGT-3’ Mutated ODN1. a G- quadruplex ODN5 5 -AGGGTTAGGGTTAGGGTTAGGGT-3’ Telomeric DNA forming mixed -parallel/antiparallel G- quadruplex in K + Primer1 5 -TCTCTGTCACACCCTAAC-3’ Partial complementary sequence of