synthesis, dna binding, and cleavage studies of novel pna binding cyclen complexes

Synthesis, DNA Binding, and Cleavage Studies of Novel PNA Binding CyclenComplexes

by Yu Zhanga), Ming-Qi Wanga), Ji Zhanga), Da-Wei Zhangb), Hong-Hui Lin*b), and Xiao-Qi Yu*a)

a) Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry,Sichuan University, Chengdu 610064, P. R. China

b) Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of LifeSciences, Sichuan University, Chengdu 610064, P. R. China

(fax: þ86-28-85415886; e-mail: [email protected])

A novel coumarin-appended PNA binding cyclen derivative ligand, C1, and its copper(II) complex,C2, have been synthesized and characterized. The interaction of these compounds with DNA wassystematically investigated by absorption, fluorescence, and viscometric titration, and DNA-melting andgel-electrophoresis experiments. DNA Melting and viscometric titration experiments indicate that thebinding mode of C1 is a groove binding, and C2 is a multiple binding mode that involves groove bindingand electrostatic binding. From the absorption-titration data, we can state that the primary interactionbetween CT DNA and the two compounds may be H-bonds between nucleobases. Fluorescence studiesindicate that the binding ability of C1 to d(A)9 is as twice or thrice as that of other oligodeoxynucleotides.Agarose gel-electrophoresis experiments demonstrate that C2 is an excellent chemical nuclease, whichcan cleave plasmid DNA completely within 24 h.

1. Introduction. – Peptide nucleic acids (PNAs), reported by Nielsen in 1991 [1], areartificial DNA mimics and have attracted considerable interests in their synthesis andapplications. PNAs offer important advantages including high binding affinity towardsDNA/RNA strands, high chemical stability, and resistance to nucleases [2– 5], makingthem useful in antisense and antigene therapies [6– 8]. Recently, many researchesfocused on the modification of PNAs in order to improve their solubility, cellpenetrability, and binding specificity [9 – 11]. One of the most interesting modificationsturns out to be the replacement of glycine by a-amino acids with hydrophobic,hydrophilic, or charged a-substituents [12] [13]. Howarth and Wakelin reported a-PNAs, in which the basic amino acid moieties are derived from homoserine, and inwhich the basic amino acids are interspaced with glycine [14]. However, as an idealDNA-binding material, PNA has hardly been reported as DNA cleavage reagent. Anexcellent chemical nuclease should have both strong binding affinity and cleavageability towards DNA.

As a well-known macrocyclic polyamine, 1,4,7,10-tetraazacyclododecane (cyclen)has been widely studied for its strong coordination ability towards a wide range ofcations. Many cyclen complexes were used as chemical nucleases in DNA recognitionand cleavage [15 – 17]. In our previous study, a chiral PNA monomer was synthesizedby condensation of uracil-containing l-cysteine with natural l-amino acids [18], and itsmetal complexes could catalyze the cleavage of DNA efficiently [19].

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 827

� 2011 Verlag Helvetica Chimica Acta AG, Z�rich

Due to the indistinct fluorescence quantum yield of native DNA and unmodifiedPNA [1] [20] [21], the development of fluorescent probes is gaining importance. Manyinvestigations on the interaction of fluorescent molecules with DNA have been carriedout [22 –24]. In this study, we introduced a coumarin group into PNA as a fluorescentprobe for better investigation. Experiments confirmed that the coumarin moiety showsno intercalation with DNA, and, therefore, it would not affect the binding mode of theligand towards DNA. Structures of the novel coumarin-appended PNA binding cyclenligand, C1, and its copper(II) complex, C2, were shown in Fig. 1. Their interactions withDNA were studied by absorption, fluorescence, and viscometric titration, and DNAmelting and gel-electrophoresis experiments. The results indicate that C1 and C2exhibited good interactions with DNA.

2. Results and Discussion. – 2.1. Preparation of the Complex. The synthetic route ofligand C1 and its CuII complex C2 is depicted in the Scheme. First, compounds 1 and 2were coupled in the presence of N,N’-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt). Subsequent deprotection gave product 4. On the otherhand, S-thyminyl-l-cysteine hydrochloride 5 was esterified with MeOH, and the

Fig. 1. The structures of coumarin-appended PNA binding cyclen ligand, C1, and its CuII complex C2

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)828


Scheme. Synthetic Route of the PNA Binding Cyclen Ligand, C1, and its CuII Complex, C2

product was then coupled with N-[(tert-butoxy)carbonyl]glycine in the presence of 4-methylmorpholine (NMM) and i-C4H9OCOCl to give compound 6. After saponifica-tion, the coupling between compound 7 and 4 was carried out to afford the product 8.The deprotected product 9 and compound 12, which was derived from compound 6,could be coupled under similar conditions as those for the preparation of 8 to givecompound 13. The deprotection of 13 and subsequent treatment with CuCl2 · 2 H2O inEtOH yielded the target ligand C1 and its CuII complex C2, respectively. All newcompounds were characterized by 1H-NMR, IR, and ESI-MS or HR-MS analyses.

2.2. DNA Melting Experiments. DNA Melting experiments were carried out toinvestigate the DNA binding ability of C1 and C2 by thermal denaturation studies usingCT DNA [25]. The melting curves of CT DNA in the absence and presence of PNAbinding cyclen complexes are given in Fig. 2. The Tm value of CT DNA alone is 67.78.After addition of compound C1 or C2, the DTm values of 3.68 and 4.88 respectively, wereobserved. Melting studies show that both of the compounds can stabilize the CT DNAduplex while being heated. As the result of electrostatic interaction caused by the CuII

cation of C2, there is a little difference in the DTm value between the two complexes(C2>C1 for both values).

2.3. Viscometric Titration. Viscometric titration is a useful method to determine thebinding modes of small molecules towards DNA [26] [27]. In the three binding modes(intercalation, groove binding, and outside binding), only intercalation causes asignificant increase of viscosity of the DNA solution. This is due to the unwinding of theDNA duplex that leads to the receiving the intercalator into the base pairs and theincrease of DNA length. Groove binding and outside binding do not need to unwindthe DNA duplex and, therefore, keep the DNA length, and, as a result, the viscosity ofDNA solution does not show any significant change.

The titration of the ligand was performed by the addition of small volumes ofconcentrated stock solutions of the DNA sample into the viscometer. Relative

Fig. 2. Plots for the thermal denaturation of CT DNA upon addition of C1 and C2 (errors inmeasurements ca. �0.18)


viscosities for DNA in either the presence or absence of the complex were calculatedfrom the equation:

h¼ (t� t0)/t0

where t is the observed flow time of the DNA-containing solution, and t0 is the flowtime of buffer alone. Viscosity data was plotted as (h/h0)1/3 vs. the binding ratio r,according to the theory of Cohen and Eisenberg [28].

The results of viscometric titration of CT DNA with C1 and C2 are shown in Fig. 3.The viscosity of the DNA slightly decreased with the addition of C1. This decreasemight be explained by the reduction of the effective length of DNA caused by bends orkinks [29]. Based on the above data, we assumed that C1 could bind to DNA throughthe groove binding. On the other hand, a very little increase was found in theviscometric experiments involving C2 and DNA. According to the theory according towhich the slope of the curve for classical intercalation should be close to 1.0, weconcluded that the CuII complex C2 binds to DNA by the groove and electrostaticbinding mode.

2.4. Absorption Titration. To further investigate the interaction of the twocompounds, C1 and C2, with DNA, we performed titration monitored by absorptionspectra. Fig. 4 shows the change in the absorption spectra of C1 with an increase in thepeak intensity at ca. 265 nm, which was attributed to the absorption of nucleobase.Meanwhile, little difference in the peak intensity caused by coumarin [30] at ca. 320 nmwas observed. The fluorescence titration experiments by using C2 gave similar results(inset in Fig. 4). These indicate that the coumarin group has no intercalation with CTDNA. According to these results, together with those of the viscometric titration, theprimary interaction between CT DNA and the two compounds might be the H-bondsbetween the nucleobase moieties and DNA.

Fig. 3. Result of viscometric titration in the presence of C1 and C2


2.5. Fluorescence Titration. The binding ability of C1 towards four differentoligodeoxynucleotides was studied by fluorescence spectroscopy. The results werequite consistent with the classical Stern�Volmer equation [31].

F0/F¼1þKsv[Q]

where F0 and F are the fluorescence intensities in the absence and presence of thecomplex, respectively, Ksv is a linear Stern�Volmer quenching constant, [Q] is theconcentration of complex. According to the quenching curve (insets of Fig. 5), the Ksv

values for C1 binding with d(A)9, d(T)9, d(C)9, and d(G)9 were estimated as 6.09�104,3.14�104, 2.62�104, and 2.05�104

m�1, respectively. It was observed that C1 binds to

d(A)9 most strongly, probably as the result of base complementation.2.6. Cleavage of Plasmid DNA. The cleavage activities of C1 and C2 towards pUC

19 supercoiled DNA were studied under physiological conditions. Copper complex C2catalyzed the conversion of supercoiled plasmid DNA (Form I) to nicked form (FormII) and linear form (Form III). Agarose gel electrophoresis was used to monitor theconversion.

First, we compared the DNA-cleavage abilities of several complexes under pH 7.4at 378, and the results are shown in Fig. 6. Lines 2 – 5 represent the DNA cleavagecatalyzed by CuCl2, cyclen-CuII, C1, and C2, respectively. Electrophoresis anddensitometry indicated that the catalytic cleavage efficiency was in the order ofC2�CuCl2>C1>cyclen.

The cleavage conditions for C2 catalysis were then optimized. Effects of theconcentration and reaction time on the cleavage process catalyzed by C2 were studied(Figs. 7 and 8). The yield of Form II and Form III increased with increasing catalystconcentration or reaction time. Almost 100% total yield of Form II and Form III couldbe achieved with 429 mm of C2 in 24 h. In other words, C2 shows better catalytic activitythan the CuII complex of PNA monomer reported earlier [19].

Copper complexes can cleave DNA via hydrolytic and/or oxidative pathways[32] [33]. In the oxidative pathway, they have been shown to react with molecular


Fig. 4. Result of absorption titration in the presence of C1 and C2 (inset)


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oxygen or H2O2 to produce a variety of reactive oxidative species (ROS). The cleavagemechanism was studied by using a series of scavengers that could inhibit production ofthe ROSs. For example, DMSO and t-BuOH can be used as scavenger of HO. radical,while NaN3 can be used as singlet-oxygen scavenger. Plasmid pUC19 DNA wasincubated with C2 in the presence of DMSO, t-BuOH, and NaN3, respectively, and theresults are shown in Fig. 9. DMSO, t-BuOH, and NaN3 can all inhibit the process. Thismeans that C2 might cleave plasmid DNA via oxidative pathway.

3. Conclusions. – In summary, we have successfully synthesized a novel PNAbinding cyclen ligand, C1, and its CuII complex, C2, and investigated their DNA-binding affinities as well as cleavage abilities towards DNA. Fluorescence studiesindicated that C1 could bind with d(A)9 more strongly than the other threeoligodeoxynucleotides, which might be attributed to base complementation. DNAMelting experiments showed that both C1 and C2 can stabilize the CT DNA duplexwhile being heated, and DTm of C2 was higher than that of C1. Agarose gelelectrophoresis demonstrated that C2 could also catalyze the cleavage of plasmid DNAefficiently. Almost 100% yield of nicked DNA could be achieved in the cleavagereaction catalyzed by 429 mm of C2 within 24 h. These results suggest that C2 may act asa promising chemical nuclease due to its excellent DNA binding affinity and cleavageability.


Fig. 6. Cleavage of pUC 19 DNA (14 mg/ml) catalyzed by different complexes in Tris · HCl buffer (50 mm,pH 7.4) at 378 for 12 h. a) Agarose gel-electrophoresis diagram: Lane 1: DNA control; Lane 2 : CuCl2;Lane 3 : cyclen-CuII; Lane 4 : C1; Lane 5 : C2. b) Quantitation of % plasmid relaxation relative to plasmid

DNA per lane.


Fig. 7. Effect of concentration of complex C2 on the cleavage reaction of pUC 19 DNA (14 mg/ml) in Tris ·HCl buffer (50 mm, pH 7.4) at 378 for 12 h. a) Agarose gel-electrophoresis diagram: Lane 1: DNAcontrol; Lanes 2–6 : [C2]¼36, 71, 143, 357, and 429 mm, resp. b) Quantitation of % plasmid relaxation

relative to plasmid DNA per lane.

Fig. 8. Effect of reaction time on the cleavage reaction of pUC 19 DNA (14 mg/ml) with complex C2(429 mm) in Tris · HCl buffer (50 mm, pH 7.4) at 378. a) Agarose gel-electrophoresis diagram: Lane 1:DNA control, 24 h; Lane 2 : DNA control, 0 h; Lanes 3–6 : 3, 6, 12, and 24 h, resp. b) Quantitation of %

plasmid relaxation relative to plasmid DNA per lane.

Experimental Part

1. General. Anh. MeCN was dried and purified under N2 according to standard methods and distilledimmediately before use. All other chemicals and reagents were obtained commercially and used withoutfurther purification. Electrophoresis-grade agarose and plasmid DNA (pUC 19) were purchased fromTakara Biotechnology Company. CT DNA purchased from Sigma was directly dissolved in H2O at aconcentration of 1 mg/ml and stored at 48. Coumarin-3-acetic acid (¼ (2-oxo-2H-chromen-3-yl)aceticacid ; 1), ethyl N-(2-Boc-aminoethyl)glycinate (¼ethyl N-(2-{[(tert-butoxy)carbonyl]amino}ethyl)glyci-nate ; 2), S-thyminyl-l-cysteine hydrochloride (¼ S-[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]-l-cysteine hydrochloride ; 5) and 2-{4,7,10-tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetic acid (11), were prepared according to the literature [18] [34–36]. Absorption spectra wererecorded in phosphate buffer soln. on Hitachi U1900 spectrophotometer with a Polyscience temp.controller system (�0.18). Fluorescence spectra: at r.t. in air, Horiba Jobin Yvon Fluoromax-4spectrofluorometer; corrected for the system response. 1H-NMR Spectra: Bruker AV II-400 MHzspectrometer; d in ppm referenced to residual solvent peaks or internal TMS. HR-MS: Bruker DaltonicsBio TOF mass spectrometer. Electrophoresis: Biomeans stack II-electrophoresis system, PPSV-010 ;bands visualized with UV light and photographed using a gel documentation system by the estimation ofthe intensity of the DNA bands, recorded on an Olympus Grab-IT 2.0 annotating image computersystem.

2. Preparation of Ethyl N-(2-{[(tert-Butoxy)carbonyl]amino}ethyl)-N-[(2-oxo-2H-chromen-3-yl)-acetyl]glycinate (3) . To a soln. of 1 (2.04 g, 10 mmol) and 2 (2.46 g, 10 mmol) dissolved in dry CH2Cl2, 1-hydroxybenzotriazole (HOBt; 1.8 g, 11 mmol) was added, and then N,N’-dicyclohexylcarbodiimide(DCC; 2.48 g, 12 mmol) dissolved in CH2Cl2 was added dropwise at 08. The mixture was stirred for 6 h atr.t. The mixture was evaporated to dryness in vacuo. The residue was dissolved in AcOEt, and theprecipitated dicyclohexylurea (DCU) was removed by filtration. The org. phase was washed with sat.NaHCO3 twice, the solvent was removed under vacuum, and the residue was purified by column


Fig. 9. Inhibition studies on cleavage of pUC 19 DNA (14 mg/ml) catalyzed by C2 (429 mm). Reactions arecarried out in Tris ·HCl buffer (50 mm, pH 7.4) at 378 for 24 h. a) Agarose gel-electrophoresis diagram:Lane 1: DNAþC2þDMSO; Lane 2 : DNAþC2þ t-BuOH; Lane 3 : DNAþC2þNaN3; Lane 4 : DNAþC2 ; Lane 5 : DNA control. b) Quantitation of % plasmid relaxation relative to plasmid DNA per lane.

chromatography (CC; silica gel (SiO2); petroleum ether (PE)/AcOEt 2 :1 (v/v)) to afford 3 (62%).White solid. 1H-NMR (400 Hz, CDCl3): 7.67 (s, H�C(4) of coumarin); 7.37 –7.41 (m, H�C(5) ofcoumarin, H�C(7) of coumarin); 7.14–7.21 (m, H�C(6) of coumarin, H�C(8) of coumarin); 4.10–4.17(m, MeCH2); 4.00 (s, NCH2CO); 3.45–3.60 (m, CH2CO of coumarin, NCH2); 3.21–3.29 (m, NHCH2 ofBoc); 1.34 (s, t-Bu); 1.21 (t, J¼4.4, MeCH2). ESI-MS: 403.4 ([M�Et]þ ).

3. Preparation of Ethyl N-(2-Aminoethyl)-N-[(2-oxo-2H-chromen-3-yl)acetyl]glycinate Hydrochlor-ide (4) , Glycyl-N-(2-{(2-ethoxy-2-oxoethyl)[(2-oxo-2H-chromen-3-yl)acetyl]amino}ethyl)-S-[(1,2,3,4-tet-rahydro-2,4-dioxopyrimidin-5-yl)methyl]-l-cysteinamide Hydrochloride (9) , and Methyl Glycyl-S-[(1,2,3,4-tetrahydro-2,4-dioxopyrimidin-5-yl)methyl]-l-cysteinate Hydrochloride (10) . Typical Procedurefor the Synthesis of 4. An excess amount of HCl/EtOH soln. was added to 3 (2.68 g, 6.2 mmol). Then, thesoln. was stirred for 2 h, and the reaction was monitored by TLC. After completion of the reaction, thesolvent was removed under reduced pressure to afford the title compound as a white solid. Yield: 95%.

4. Preparation of Methyl N-[(tert-Butoxy)carbonyl]glycyl-S-[(2,4-dioxo-1,2,3,4-tetrahydropyrimi-din-5-yl)methyl]-l-cysteinate (6). SOCl2 (6 g, 0.27 mmol) was added dropwise to MeOH (300 ml) at 08.After being kept at this temp. for 0.5 h with an ice bath, 5 (4 g, 14.2 mmol) was added. The mixture wasstirred for another 0.5 h at r.t., and then it was refluxed for 4 h. After being cooled to r.t., the mixture wasfiltered, evaporated, and recrystallized from Et2O. S-Thyminyl-l-cysteine methyl ether hydrochloridewas obtained as a white solid.

To a soln. of N-[(tert-butoxy)carbonyl]glycine (1.75 g, 10 mmol) in DMF (20 ml), N-methylmorpho-line (NMM; 1.21 ml, 11 mmol) and i-C4H9OCOCl (1.30 ml, 10 mmol) were added at ca. �158. After10 min, a soln. of S-thyminyl-l-cysteine methyl ether hydrochloride (3.25 g, 11 mmol) and NMM(1.33 ml, 12.1 mmol) in DMF (20 ml) was poured into the mixture. The mixture was stirred for another0.5 h at �158 and left overnight at r.t. After evaporation of the solvent under reduced pressure, the solidresidue was dissolved in AcOEt and washed with brine. The org. layer was dried (Na2SO4). Afterremoval of the solvent, the residue was purified by CC (SiO2; AcOEt/MeOH 10 : 1 (v/v)) to afford 6(74%). White solid. 1H-NMR (400 MHz, DMSO): 11.16 (s, H�N(1) of uracil); 10.83 (s, H�N(3) ofuracil); 8.30 (d, J ¼ 8.0, CH2CONH); 7.40 (d, J ¼ 5.6, H�C(6) of uracil); 6.95 (t, BocNHCH2); 4.48–4.53(m, H�CCOO); 3.64 (s, MeOOC); 2.83–2.94 (m, CH2S�uracil); 2.67–2.77 (m, CH2S); 1.38 (s, Me3C).ESI-MS: 439.0 ([MþNa]þ ).

5. Preparation of N-[(tert-Butoxy)carbonyl]glycyl-S-[(1,2,3,4-tetrahydro-2,4-dioxopyrimidin-5-yl)-methyl]-l-cysteine (7) . To the soln. of 6 (0.84 g, 2 mmol) in MeOH was added aq. NaOH soln. (2 mol/l,10 ml) at 08. The mixture was stirred for 2 h, and the reaction was monitored by TLC. MeOH wasevaporated, and the pH was adjusted to 6. After evaporation of H2O, MeOH was added, and most ofNaCl was filtered. The product from the deprotection of methyl ester group was obtained as a white solid.

6. Preparation of N-[(tert-Butoxy)carbonyl]glycyl-S-[(1,2,3,4-tetrahydro-2,4-dioxopyrimidin-5-yl)-methyl]-N-(2-{(2-ethoxy-2-oxoethyl)[(2-oxo-2H-chromen-3-yl)acetyl]amino}ethyl)-l-cysteinamide (8) .To a soln. of 7 (0.80 g, 2 mmol) in dry CH2Cl2, NMM (0.24 ml, 2.2 mmol) and i-C4H9OCOCl (0.26 ml,2 mmol) were added at ca. �158. After 10 min, a soln. of 4 (0.74 g, 2 mmol) and NMM (0.24 ml,2.2 mmol) in dry CH2Cl2 was poured into the mixture. The mixture was stirred for another 0.5 h at �158and then for 6 h at r.t. After evaporation of the solvent under reduced pressure, the solid residue wasdissolved in AcOEt, and washed with H2O and brine, and then the org. layer was dried (Na2SO4). Afterremoval of the solvent, the residue was purified by CC (SiO2; CH2Cl2/MeOH 10 :1 (v/v)) to afford 8(42%). Yellow solid. 1H-NMR (400 MHz, DMSO): 11.15 (s, H�N(1) of uracil); 10.82 (s, H�N(3) ofuracil); 8.00–8.25 (m, 3 CONH); 7.85–7.94 (m, H�C(4) of coumarin); 7.67 –7.69 (m, H�C(7) ofcoumarin); 7.57 –7.59 (m, H�C(5) of coumarin); 7.34–7.43 (m, H�C(6) of coumarin, H�C(8) ofcoumarin, H�C(6) of uracil); 6.94 (d, J¼5.6, H�N�Boc); 4.46 (s, SCH2CHCO); 4.10 (q, J¼6.8,CH2Me); 3.48–3.69 (m, NCH2, coumarin�CH2CO); 3.25 (t, J¼5.8, Boc�NH�CH2); 1.34 (s, t-Bu), 1.20(t, J¼7.6, MeCH2). ESI-MS: 715.2 ([M�H]� ).

7. Preparation of N-({4,7,10-Tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetyl)-glycyl-S-[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]-l-cysteine (12) . Compound 10 (0.85 g,2.4 mmol) and Et3N (0.29 g, 2.88 mmol) were dissolved in DMF, and 11 (1.06 g, 2 mmol) was added.The soln. was cooled to 08, then HOBt (0.36 g, 2.2 mmol) and DCC (0.49 g, 2.4 mmol) were added. Themixture was stirred overnight at r.t. The solvent was removed under vacuum, and the residue was


dissolved in AcOEt. Then, the precipitated DCU was removed by filtration. The soln. was washed withH2O twice and with brine once. The org. phase was dried (Na2SO4). After removal of the solvent undervacuum, the residue was purified by CC (SiO2; AcOEt/MeOH 4 : 1 (v/v)) to afford a white solid. Yield:70%. 1H-NMR (400 MHz, CDCl3): 10.13 (s, H�N(1) of uracil); 9.81 (s, H�N(3) of uracil); 7.51 (d, J ¼ 4.8,2 CONH); 7.38 (d, J ¼ 4.4, H�C(6) of uracil); 4.70 (s, CHCOO); 3.91 (s, cyclen�CH2CO); 3.72 (s,COOMe); 3.10–3.60 (m, 8 CH2 of cyclen); 2.87–2.95 (m, SCH2�uracil); 2.78–2.85 (m, SCH2); 1.44 (s,3 t-Bu): ESI-MS: 851.4 ([MþNa]þ ).

To the soln. of the white solid obtained at the previous step (1.18 g, 1.4 mmol) in MeOH was addedaq. NaOH (2n, 20 ml). The mixture was stirred for 2 h, and the reaction was monitored by TLC. MostMeOH was evaporated, and the pH was adjusted to 6. After the extraction of the soln. with AcOEt (3�100 ml), the org. layer was dried (Na2SO4) and evaporated to yield 12 (93%). White solid. 1H-NMR(400 MHz, DMSO): 11.12 (s, H�N(1) of uracil); 10.89 (s, H�N(3) of uracil); 7.45 (s, H�C(6) of uracil);4.81–4.30 (m, NHCHCOOH); 3.75 (s, cyclen�CH2CO); 3.20–3.50 (m, 8 CH2 of cyclen); 2.85–2.87 (m,SCH2�uracil); 2.71–2.75 (m, SCH2); 1.40 (s, 3 t-Bu). ESI-MS: 837.4 ([MþNa]þ ).

8. Preparation of N-({4,7,10-Tris[(tert-butoxy)carbonyl]-1,4,7,10-tetraazacyclododecan-1-yl}acetyl)-glycyl-S-[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]-l-cysteinylglycyl-S-[(2,4-dioxo-1,2,3,4-tet-rahydropyrimidin-5-yl)methyl]-N-(2-{(2-ethoxy-2-oxoethyl)[(2-oxo-2H-chromen-3-yl)acetyl]amino}eth-yl)-l-cysteinamide (13) . To a soln. of 12 (0.94 g, 1.2 mmol) in dry CH2Cl2, NMM (0.14 ml, 1.25 mmol) andi-C4H9OCOCl (0.15 ml, 1.2 mmol) were added at ca. �158. After 10 min, a soln. of 9 (0.75 g, 1.2 mmol)and NMM (0.14 ml, 1.25 mmol) in dry CH2Cl2 was poured into the mixture. The mixture was stirred foranother 0.5 h at �158 and then for 4 h at r.t. After completion of the reaction, the mixture was washedwith H2O and brine, and the org. layer was dried (Na2SO4). After removal of the solvent, the residue waspurified by CC (SiO2; CH2Cl2/MeOH 8 : 1 (v/v)) to afford 13 (29%). Yellow solid. 1H-NMR (400 MHz,DMSO): 11.15 (s, 2 H�N(1) of uracil); 10.81 (s, 2 H�N(3) of uracil); 8.10–8.45 (m, 5 CONH); 7.92 (s,H�C(4) of coumarin); 7.66 –7.68 (m, H�C(7) of coumarin); 7.56–7.59 (m, H�C(5) of coumarin); 7.34 –7.42 (m, H�C(6) of coumarin, H�C(8) of coumarin, 2 H�C(6) of uracil); 4.51 (s, SCH2CHCO); 4.08 (q,J¼7.2, MeCH2); 3.63–3.83 (m, NCH2, coumarin�CH2CO); 3.45–3.20 (m, 8 CH2 of cyclen); 1.37 (s, 3 t-Bu); 1.22 (t, J¼6.8, MeCH2). ESI-MS: 1435.6 ([MþNa]þ ).

9. Preparation of N-(1,4,7,10-Tetraazacyclododecan-1-ylacetyl)glycyl-S-[(2,4-dioxo-1,2,3,4-tetrahy-dropyrimidin-5-yl)methyl]-l-cysteinylglycyl-S-[(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl]-N-(2-{(2-ethoxy-2-oxoethyl)[(2-oxo-2H-chromen-3-yl)acetyl]amino}ethyl)-l-cysteinamide (C1) . An excessamount of HCl/EtOH soln. was added to 13 (280 mg, 0.2 mmol), the mixture was stirred for 2 h, and thereaction monitored by TLC. After completion of the reaction, the solvent was removed under vacuum toafford a white solid, which was dissolved in twice dist. H2O, and a suitable amount of strongly alkalinestyrene anion exchange resin (20 1�7) was added to the soln., that was stirred until it turned to be basic.The soln. was filtered, evaporated, and dried (P2O5) to afford C1 (70%). White solid. 1H-NMR(400 MHz, DMSO): 8.10–8.55 (m, 5 CONH); 7.94 (s, H�C(4) of coumarin); 7.67–7.70 (m, H�C(7) ofcoumarin); 7.50 –7.59 (m, H�C(5) of coumarin); 7.36–7.46 (m, H�C(6) of coumarin, H�C(8) ofcoumarin, 2 H�C(6) of uracil); 4.46 (s, SCH2CHCO); 4.09 (q, J¼7.2, MeCH2); 3.75–3.86 (m, NCH2,coumarin�CH2CO); 3.00–2.70 (m, 8 CH2 of cyclen); 1.22 (t, J¼7.6, MeCH2). HR-ESI-MS: 1113.4237([MþH]þ , C47H65N14O14Sþ2 ; calc. 1113.4246).

10. Preparation of Compound C2. Excess CuCl2 · 2 H2O (5.2 mg, 0.0306 mmol) in 5 ml of EtOH wasadded dropwise to the EtOH soln. (15 ml) of C1 (33.4 mg, 0.03 mmol). The mixture was stirred overnightat r.t., and the precipitate was separated by centrifugation. Then, it was washed with EtOH (5�2.5 ml) toyield C2 (52%). Blue solid. IR (KBr): 2979, 2912, 1705, 1658, 1535, 1435, 1214, 1076, 1025, 819, 765, 692.HR-ESI-MS: 587.6731 ([M�2 Cl]2þ , C47H64CuN14O14Sþ2 ; calc. 587.6732).

11. DNA Melting Experiments. DNA Melting experiments were performed on a Hitachi U1900spectrophotometer with a Polyscience temp. controller system (�0.18) in a soln. consisting of 2.5 ml ofNaH2PO4/Na2HPO4 (5 mm, pH 7.4), 0.135 mm CT DNA, and 0.045 mm complex. Using the thermalmelting program, the temp. of the cell containing the cuvette increased from 25 to 958, and theabsorbance at 260 nm was measured every 1–28. The Tm value was calculated by plotting temp. vs.relative DA/DT.


12. Viscometric Titration. Viscometric titrations were performed at 258 with a Ubblehode viscometerin phosphate buffer (10.0 mm, pH 7.4). CT DNA (0.2 mm bp) was used. The concentrations of C1 werevaried, and the different flow times were measured by stopwatch. The plot of (h/h0)1/3 vs. r was obtainedwith the data, where h and h0 are the flow time of the compound bound DNA and DNA, resp., and r is[compound]/[DNA].

13. Absorption Titration. Phosphate buffer (5.0 mm, pH 7.4) with 50.0 mm NaCl was used for UVspectrophotometric titrations. CT DNA purchased from Sigma was used without further purification.The DNA concentration per nucleotide was determined by absorption spectroscopy, using the molarextinction coefficient of 6600 m

�1 cm�1 at 260 nm. In absorption titrations, the concentrations of C1 or C2were maintained, and different amounts of DNA were added. All experiments were performed at r.t.

14. Fluorescence Titration. The steady-state fluorescence spectroscopic experiments were carried outon a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer with a 1-cm pathlength cuvette. In fluorescencetitration experiments, the soln. of the ligands was diluted to 10 mm by 5.0 mm phosphate buffer containing2.0 mm NaCl at 258, and varying the concentrations of oligodeoxynucleotides. The fluorescence spectrawere recorded using excitation wavelength at 310 nm, and the emission range was set between 370 and550 nm.

15. Cleavage of Plasmid DNA. Plasmid DNA (pUC 19) cleavage activity of complexes wasmonitored by using agarose gel electrophoresis. In a typical experiment, supercoiled pUC19 DNA (5 ml,50 mg/ml) in Tris · HCl (50 mm, pH 7.4) was treated with C2 of different concentrations, followed bydilution with the Tris · HCl buffer to a total volume of 17.5 ml. The samples were then incubated at 378 fordifferent times, and loaded on a 1% agarose gel containing 1.0 mg/ml ethidium bromide (EB).Electrophoresis was carried out at 40 V for 30 min in Tris-acetate-EDTA (TAE) buffer. Bands werevisualized by UV light and photographed, followed by the estimation of the intensity of the DNA bandsusing a Gel Documentation System.

This work was financially supported by the National Science Foundation of China (Nos. 20725206and 20732004), the Specialized Research Fund for the Doctoral Program of Higher Education, and theScientific Fund of Sichuan Province for Outstanding Young Scientists. The authors also acknowledge theAnalytical and Testing Center of Sichuan University for NMR analysis.

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Received March 28, 2010


synthesis, dna binding, and cleavage studies of novel pna binding cyclen complexes

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