g - quadruplexes ( g4 ) , secondary dna structures consisting of g - quartet planers in g - rich regions , play significant biological roles   for example , control of transcription and telomeric lengths [ 119 ] . one typical g4 forming dna sequence is a telomere , which exists at the ends of chromosomes consisting of ( ttaggg)n repeating single - stranded sequences [ 112 ] . telomeres protect chromosomes from end to end fusion events , which result in replication of the chromosome ( the hayflick limit ) . the telomere repeats are elongated by the reverse transcriptase telomerase , which is overexpressed in most tumor cells . since the activity of this enzyme is inhibited by the g4 structure of telomeres owing to steric hindrance , small molecules that selectively bind and stabilize the telomeric g4 should be potential anticancer agents .   as a result , a number of g4 ligands , inspired by artificial dna intercalators as well as natural products , have been developed during the past decade . telomestatin ( tms ) is a natural product isolated from streptomyces anulatus 3533-sv4 , which displays one of the most potent telomeric g4 binding activity ( figure 1 ) [ 2328 ] . interaction analysis has shown that two molecules of tms induce conversion of telomeric g4 into an antiparallel type by way of an end stacking mode [ 2528 ] . we have recently developed macrocyclic hexaoxazole compounds 6otd , containing a variety of side chain functional groups , that serve as a novel tms derivative [ 2932 ] . in addition , by considering the proposed binding mode of tms with telomeric g4 , we have carried out further structural development of dimeric 6otd derivatives ( figure 1 ) . the results of molecular dynamics calculations guided the selection of 6otd dimer 1 that contains an appropriate length of a linker between the monomeric units of 6otd . studies showed that dimer 1 binds to telomeric g4 more tightly than do other 6otd dimers with linkers of shorter or longer lengths .   one possible structural development strategy to enhance the stabilizing ability of 1 against the g4 would be to install cationic functional groups on the side chain . below , we describe synthesis of new 6otd dimers 2 and 3 that derivatize 1 but possess cationic amine and guanidine functional groups on their side chains .   in addition , the ability of these substances to stabilize telomeric g4 along with their interaction mode was investigated . flash chromatography was performed on silica gel 60 ( spherical , particle size 0.040  ~  0.100  m ; kanto ) . optical rotations were measured on a jasco p-2200 polarimeter , using the sodium d line.h andc nmr spectra were recorded on jeol jnm - ecx 300 , 400 , and 500 . the spectra are referenced internally according to residual solvent signals of cdcl3 ( h nmr ;  = 7.26  ppm , c nmr  = 77.0 ; ppm ) and dmso d  6 ( h nmr ;  = 2.50  ppm , c nmr ;  = 39.5  ppm ) . data for h nmr are recorded as follows : chemical shift (  , ppm ) , multiplicity ( s , singlet ; d , doublet ; m , multiplet ; br , broad ) , integration , and coupling constant ( hz ) .   data for c nmr are reported in terms of chemical shift (  , ppm ) . mass spectra were recorded on a jeol jms - t100x spectrometer with esi - mass mode using methanol as solvent . all oligonucleotides purified were obtained from sigma genosys and dissolved in double - distilled water to be used without further purification . fluorescence resonance energy transfer ( fret ) melting assay was made with an excitation wavelength of 470505  nm and a detection wavelength of 523543 nm using the dna engine opticon 2 real - time cycler pcr detection system ( biorad ) . cd spectra were recorded on a jasco-810 spectropolarimeter ( jasco , easton , md ) using a quartz cell of 1  mm optical path length and an instrument scanning speed of 500  nm / min with a response time of 1  s , and over a wavelength range of 220320  nm . .  
 synthesis of 5to a solution of trioxazole 4 ( 2.1  g , 3.6  mmol ) in meoh - thf ( 1  :  1 , 60  ml ) was added pd(oh)2/c ( 420  mg ) , and the reaction mixture was stirred at room temperature under hydrogen gas ( balloon ) .   after 3  h , the catalyst was removed by filtration through a pad of celite , and the filtrates were concentrated in vacuo to give amine 5 , which was used without further purification . 
  to a solution of trioxazole 4 ( 2.1  g , 3.6  mmol ) in meoh - thf ( 1  :  1 , 60  ml ) was added pd(oh)2/c ( 420  mg ) , and the reaction mixture was stirred at room temperature under hydrogen gas ( balloon ) .   after 3  h , the catalyst was removed by filtration through a pad of celite , and the filtrates were concentrated in vacuo to give amine 5 , which was used without further purification .  
 synthesis of 7to a solution of trioxazole 6 ( 2.1  g , 3.6  mmol ) in thf - h2o ( 3  :  1 , 80  ml ) was added lioh ( 230  mg , 5.4  mmol ) at 0c .   after stirring at room temperature for 1  h , to the resulting mixture was added 1  n hcl , to give carboxylic acid 7 , which was used without further purification . 
  to a solution of trioxazole 6 ( 2.1  g , 3.6  mmol ) in thf - h2o ( 3  :  1 , 80  ml ) was added lioh ( 230  mg , 5.4  mmol ) at 0c .   after stirring at room temperature for 1  h , to the resulting mixture was added 1  n hcl , to give carboxylic acid 7 , which was used without further purification . synthesis of 8to a solution of amine 5 ( abovementioned ) in thf - h2o ( 1  :  1 ) was added the carboxylic acid 7 ( abovementioned ) , n - methylmorpholine  ( 1.2  ml , 11  mmol ) , and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride ( dmt - mm ) ( 3.0  g , 11  mmol ) , and the mixture was stirred at room temperature for 15  h.   to the reaction mixture was added h2o and precipitate was formed . this precipitate was collected with filtration using filter paper , to give 8 as a white solid ( 3.3  g , 3.2  mmol 89% 2 steps , mp = 200203c ) . spectral data for 8 : [ ]d = 2.7 ( c 1.1 , chcl3meoh ( 1  :  1 ) ) ; h nmr ( 300  mhz , cdcl3 )  8.43 ( s , 1h ) , 8.358.27 ( m , 5h ) , 7.53 ( d , j = 8.9  hz , 1h ) , 7.34 ( m , 5h ) , 5.985.81 ( m , 1h ) , 5.575.44 ( m , 2h ) , 5.30 ( d , j = 18  hz , 1h ) , 5.21 ( d , j = 11  hz , 1h ) , 5.154.97 ( m , 2h ) , 4.82 ( br , 1h ) , 4.58 ( d , j = 5.5 hz , 3h ) , 3.95 ( s , 3h ) , 3.303.01 ( m , 4h ) , 2.251.80 ( m , 4h ) , 1.701.30 ( m , 17h ) ; c nmr ( 125  mhz , dmso d  6 )  165.7 , 165.2 , 160.9 , 159.8 , 156.1 , 155.8 , 155.7 , 155.6 , 155.0 , 154.4 , 145.7 , 142.8 , 141.0 , 140.9 , 140.7 , 137.3 , 136.6 , 133.4 , 133.3 , 130.1 , 130.0 , 128.9 , 128.8 , 128.3 , 127.7 , 117.3 , 77.3 , 65.1 , 64.9 , 64.7 , 52.0 , 48.9 , 46.8 , 31.8 , 31.3 , 29.2 , 28.9 , 28.2 , 28.1 , 22.8 , 22.6 ; hrms ( esi , m + na ) calcd for c48h52n10o15na 1031.3511 , found 1031.3479 . 
  to a solution of amine 5 ( abovementioned ) in thf - h2o ( 1  :  1 ) was added the carboxylic acid 7 ( abovementioned ) , n - methylmorpholine  ( 1.2  ml , 11  mmol ) , and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride ( dmt - mm ) ( 3.0  g , 11  mmol ) , and the mixture was stirred at room temperature for 15  h.   to the reaction mixture was added h2o and precipitate was formed . this precipitate was collected with filtration using filter paper , to give 8 as a white solid ( 3.3  g , 3.2  mmol 89% 2 steps , mp = 200203c ) . spectral data for 8 : [ ]d = 2.7 ( c 1.1 , chcl3meoh ( 1  :  1 ) ) ; h nmr ( 300  mhz , cdcl3 )  8.43 ( s , 1h ) , 8.358.27 ( m , 5h ) , 7.53 ( d , j = 8.9  hz , 1h ) , 7.34 ( m , 5h ) , 5.985.81 ( m , 1h ) , 5.575.44 ( m , 2h ) , 5.30 ( d , j = 18  hz , 1h ) , 5.21 ( d , j = 11  hz , 1h ) , 5.154.97 ( m , 2h ) , 4.82 ( br , 1h ) , 4.58 ( d , j = 5.5 hz , 3h ) , 3.95 ( s , 3h ) , 3.303.01 ( m , 4h ) , 2.251.80 ( m , 4h ) , 1.701.30 ( m , 17h ) ; c nmr ( 125  mhz , dmso d  6 )  165.7 , 165.2 , 160.9 , 159.8 , 156.1 , 155.8 , 155.7 , 155.6 , 155.0 , 154.4 , 145.7 , 142.8 , 141.0 , 140.9 , 140.7 , 137.3 , 136.6 , 133.4 , 133.3 , 130.1 , 130.0 , 128.9 , 128.8 , 128.3 , 127.7 , 117.3 , 77.3 , 65.1 , 64.9 , 64.7 , 52.0 , 48.9 , 46.8 , 31.8 , 31.3 , 29.2 , 28.9 , 28.2 , 28.1 , 22.8 , 22.6 ; hrms ( esi , m + na ) calcd for c48h52n10o15na 1031.3511 , found 1031.3479 . 
 synthesis of 9   to a solution of bis - trioxazole 8 ( 510  mg , 0.50  mmol ) in dmf - thf ( 1 : 5 , 30  ml ) was added morpholine ( 440  l , 5.0  mmol ) and pd(pph3)4 ( 29  mg , 0.025  mmol ) , and the mixture was stirred at room temperature for 1  h.   to the reaction mixture was added ether and precipitate was formed . the solid was purified by column chromatography on silica gel ( chcl3-meoh = 9 : 1 ) to give 9 ( 460  mg , 0.50  mmol 99% ) . spectral data for 9 : [ ]d = 28 ( c 1.0 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 300  mhz , cdcl3 )  8.43 ( s , 1h ) , 8.358.25 ( m , 5h ) , 7.53 ( d , j = 8.9  hz , 1h ) , 7.33 ( m , 5h ) , 5.565.43 ( m , 1h ) , 5.08 ( br , 2h ) , 4.80 ( br , 1h ) , 4.56 ( br , 1h ) , 4.144.05 ( m , 1h ) , 3.95 ( s , 3h ) , 3.263.04 ( m , 4h ) , 2.251.75 ( m , 4h ) , 1.731.30 ( m , 17h ) ; c nmr ( 125  mhz , dmso d  6 )  169.3 , 165.2 , 160.9 , 159.8 , 156.1 , 155.9 , 155.7 , 155.6 , 154.9 , 154.4 , 145.7 , 142.7 , 141.0 , 140.9 , 140.6 , 140.5 , 137.3 , 136.6 , 133.3 , 130.0 , 129.9 , 128.8 , 128.5 , 128.3 , 127.7 , 77.3 , 65.1 , 52.0 , 49.4 , 46.7 , 35.3 , 31.3 , 29.2 , 28.2 , 28.1 , 22.8 , 22.6 ; hrms ( esi , m + na ) calcd for c44h48n10o13na 947.3300 , found 947.3308 . 
    to a solution of bis - trioxazole 8 ( 510  mg , 0.50  mmol ) in dmf - thf ( 1 : 5 , 30  ml ) was added morpholine ( 440  l , 5.0  mmol ) and pd(pph3)4 ( 29  mg , 0.025  mmol ) , and the mixture was stirred at room temperature for 1  h.   to the reaction mixture was added ether and precipitate was formed . the solid was purified by column chromatography on silica gel ( chcl3-meoh = 9 : 1 ) to give 9 ( 460  mg , 0.50  mmol 99% ) . spectral data for 9 : [ ]d = 28 ( c 1.0 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 300  mhz , cdcl3 )  8.43 ( s , 1h ) , 8.358.25 ( m , 5h ) , 7.53 ( d , j = 8.9  hz , 1h ) , 7.33 ( m , 5h ) , 5.565.43 ( m , 1h ) , 5.08 ( br , 2h ) , 4.80 ( br , 1h ) , 4.56 ( br , 1h ) , 4.144.05 ( m , 1h ) , 3.95 ( s , 3h ) , 3.263.04 ( m , 4h ) , 2.251.75 ( m , 4h ) , 1.731.30 ( m , 17h ) ; c nmr ( 125  mhz , dmso d  6 )  169.3 , 165.2 , 160.9 , 159.8 , 156.1 , 155.9 , 155.7 , 155.6 , 154.9 , 154.4 , 145.7 , 142.7 , 141.0 , 140.9 , 140.6 , 140.5 , 137.3 , 136.6 , 133.3 , 130.0 , 129.9 , 128.8 , 128.5 , 128.3 , 127.7 , 77.3 , 65.1 , 52.0 , 49.4 , 46.7 , 35.3 , 31.3 , 29.2 , 28.2 , 28.1 , 22.8 , 22.6 ; hrms ( esi , m + na ) calcd for c44h48n10o13na 947.3300 , found 947.3308 . synthesis of 10to a solution of bis - trioxazole 9 ( 2.2  g , 2.4  mmol ) in thf - h2o ( 3 : 1 , 200  ml ) was added lithium hydroxide ( 300  mg , 7.2  mmol ) , and the mixture was stirred at room temperature for 2  h.   to the reaction mixture was added 1n hcl , and the resulting mixture was concentrated in vacuo .   to the residual solution in dmf - ch2cl2 ( 1 : 2 , 800  ml ) was added dmap ( 1.5  g , 12  mmol ) , diisopropylethylamine ( 2.0  ml , 12  mmol ) , and dppa ( 2.6  ml , 12  mmol ) , and the resulting mixture was stirred for 22  h at 90c .   to the reaction mixture was added h2o and the organic layer was extracted with ethyl acetate . the extracts were washed with brine , dried over mgso4 , filtered , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( ethyl acetate 100% ) to give 10 as a white solid ( 1.7  g , 1.9  mmol 79% , mp = 220225c dec ) . spectral data for 10 : [ ]d = 12 ( c 0.4 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 400 mhz , cdcl3 )  8.54 ( d , j = 7.3  hz , 2h ) , 8.258.16 ( m , 6h ) , 7.367.27 ( m , 5h ) , 5.475.37 ( m , 2h ) , 5.05 ( br , 2h ) , 4.89 ( br , 1h ) , 4.59 ( br , 1h ) , 3.222.98 ( m , 4h ) , 2.301.89 ( m , 4h ) , 1.621.18 ( m , 17h ) ; c nmr ( 100  mhz , cdcl3 )  164.8 , 164.7 , 161.2 , 159.9 , 159.8 , 156.3 , 156.0 , 155.9 , 154.6 , 141.0 , 140.9 , 139.1 , 138.4 , 136.9 , 136.8 , 136.6 , 130.9 , 129.6 , 128.4 , 128.1 , 128.0 , 79.0 , 66.5 , 47.8 , 47.7 , 40.7 , 40.3 , 34.6 , 29.5 , 29.2 , 28.4 , 21.9 , 21.8 ; hrms ( esi , m + na ) calcd for c43h44n10o12na 915.3038 , found 915.2999 . 
  to a solution of bis - trioxazole 9 ( 2.2  g , 2.4  mmol ) in thf - h2o ( 3 : 1 , 200  ml ) was added lithium hydroxide ( 300  mg , 7.2  mmol ) , and the mixture was stirred at room temperature for 2  h.   to the reaction mixture was added 1n hcl , and the resulting mixture was concentrated in vacuo .   to the residual solution in dmf - ch2cl2 ( 1 : 2 , 800  ml ) was added dmap ( 1.5  g , 12  mmol ) , diisopropylethylamine ( 2.0  ml , 12  mmol ) , and dppa ( 2.6  ml , 12  mmol ) , and the resulting mixture was stirred for 22  h at 90c .   to the reaction mixture the extracts were washed with brine , dried over mgso4 , filtered , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( ethyl acetate 100% ) to give 10 as a white solid ( 1.7  g , 1.9  mmol 79% , mp = 220225c dec ) . spectral data for 10 : [ ]d = 12 ( c 0.4 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 400 mhz , cdcl3 )  8.54 ( d , j = 7.3  hz , 2h ) , 8.258.16 ( m , 6h ) , 7.367.27 ( m , 5h ) , 5.475.37 ( m , 2h ) , 5.05 ( br , 2h ) , 4.89 ( br , 1h ) , 4.59 ( br , 1h ) , 3.222.98 ( m , 4h ) , 2.301.89 ( m , 4h ) , 1.621.18 ( m , 17h ) ; c nmr ( 100  mhz , cdcl3 )  164.8 , 164.7 , 161.2 , 159.9 , 159.8 , 156.3 , 156.0 , 155.9 , 154.6 , 141.0 , 140.9 , 139.1 , 138.4 , 136.9 , 136.8 , 136.6 , 130.9 , 129.6 , 128.4 , 128.1 , 128.0 , 79.0 , 66.5 , 47.8 , 47.7 , 40.7 , 40.3 , 34.6 , 29.5 , 29.2 , 28.4 , 21.9 , 21.8 ; hrms ( esi , m + na ) calcd for c43h44n10o12na 915.3038 , found 915.2999 . 
 synthesis of 11to a solution of macrocyclic bis - amide 10 ( 200  mg , 220  mol ) in meoh ( 50  ml ) was added pd(oh)2/c ( 80  mg ) and the reaction mixture was stirred at room temperature under hydrogen ( balloon ) .   after 5  h , the reaction mixture was filtered through a pad of celite , and the filtrates were concentrated in vacuo .   to the residual solution in dmf - mecn ( 1  :  1 , 4.0  ml ) was added diisopropylethylamine ( 190  l , 1.1 mmol ) and adipoyl chloride ( 16  l , 110  mol ) , and the mixture was stirred at room temperature for 11  h.   the reaction mixture was concentrated in vacuo , and the residue was acidified with 0.1 n hcl and extracted with chcl3 . the organic layer was dried over mgso4 , filtrated , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( chcl3acoet  meoh = 3  :  2  :  1 ) to give 11 ( 51  mg , 31  mol , 28% ) . spectral data for 11 : [ ]d = 11 ( c 0.95 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 500  mhz , cdcl3 )  8.678.48 ( m , 4h ) , 8.278.15 ( m , 12h ) , 6.38 ( br , 2h ) , 5.475.38 ( m , 4h ) , 4.84 ( br , 2h ) , 3.302.98 ( m , 8h ) , 2.151.90 ( m , 12h ) , 1.651.10 ( m , 38h ) ; c nmr ( 125  mhz , cdcl3 )  173.0 , 164.9 , 164.8 , 159.8 , 159.7 , 156.2 , 156.1 , 156.0 , 154.7 , 154.6 , 141.0 , 140.9 , 139.5 , 139.3 , 138.7 , 138.6 , 136.9 , 136.8 , 130.8 , 129.5 , 129.3 , 78.9 , 47.8 , 47.7 , 40.3 , 38.9 , 36.0 , 34.5 , 34.3 , 29.7 , 29.5 , 28.8 , 28.4 , 25.1 , 21.9 , 21.7 ; hrms ( esi , m + na ) calcd for c76h82n20o22na 1649.5810 , found 1649.5811 . to a solution of macrocyclic bis - amide 10 ( 200  mg , 220  mol ) in meoh ( 50  ml ) was added pd(oh)2/c ( 80  mg ) and the reaction mixture was stirred at room temperature under hydrogen ( balloon ) .   after 5  h , the reaction mixture was filtered through a pad of celite , and the filtrates were concentrated in vacuo .   to the residual solution in dmf - mecn ( 1  :  1 , 4.0  ml ) was added diisopropylethylamine ( 190  l , 1.1 mmol ) and adipoyl chloride ( 16  l , 110  mol ) , and the mixture was stirred at room temperature for 11  h.   the reaction mixture was concentrated in vacuo , and the residue was acidified with 0.1 n hcl and extracted with chcl3 . the organic layer was dried over mgso4 , filtrated , and concentrated in vacuo . the residue was purified by column chromatography on silica gel ( chcl3acoet  meoh = 3  :  2  :  1 ) to give 11 ( 51  mg , 31  mol , 28% ) . spectral data for 11 : [ ]d = 11 ( c 0.95 , chcl3-meoh ( 1 : 1 ) ) ; h nmr ( 500  mhz , cdcl3 )  8.678.48 ( m , 4h ) , 8.278.15 ( m , 12h ) , 6.38 ( br , 2h ) , 5.475.38 ( m , 4h ) , 4.84 ( br , 2h ) , 3.302.98 ( m , 8h ) , 2.151.90 ( m , 12h ) , 1.651.10 ( m , 38h ) ; c nmr ( 125  mhz , cdcl3 )  173.0 , 164.9 , 164.8 , 159.8 , 159.7 , 156.2 , 156.1 , 156.0 , 154.7 , 154.6 , 141.0 , 140.9 , 139.5 , 139.3 , 138.7 , 138.6 , 136.9 , 136.8 , 130.8 , 129.5 , 129.3 , 78.9 , 47.8 , 47.7 , 40.3 , 38.9 , 36.0 , 34.5 , 34.3 , 29.7 , 29.5 , 28.8 , 28.4 , 25.1 , 21.9 , 21.7 ; hrms ( esi , m + na ) calcd for c76h82n20o22na 1649.5810 , found 1649.5811 . 
 synthesis of 2a solution of 11 ( 50  mg , 31  mol ) in ch2cl2-tfa ( 95 : 5 , 25  ml ) was stirred at room temperature for 2  h.   the reaction mixture was concentrated in vacuo to give 2 as a white solid ( 50  mg , 30  mol , 98% , mp = 225230c dec ) .   spectral data for 2 : [ ]d = 72 ( c 0.3 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 500 mhz , dmso d  6 )  9.159.08 ( m , 8h ) , 8.958.89 ( m , 4h ) , 8.38 ( d , j = 6.9 hz , 2h ) , 8.30 ( d , j = 6.9  hz , 1h ) , 7.807.53 ( m , 6h ) , 5.505.35 ( m , 4h ) , 2.982.89 ( m , 4h ) , 2.782.89 ( m , 4h ) , 2.151.85 ( m , 12h ) , 1.551.00 ( m , 20h ) ; c nmr ( 125 mhz , dmso d  6 )  171.7 , 164.5 , 164.3 , 158.8 , 158.7 , 155.6 , 154.5 , 142.5 , 141.9 , 141.8 , 141.1 , 136.0 , 129.8 , 129.7 , 128.5 , 128.4 , 47.4 , 47.3 , 38.6 , 38.1 , 35.1 , 33.1 , 28.7 , 26.7 , 24.9 , 21.3 , 20.8 ; hrms ( esi , m + h ) calcd for c66h67n20o18 1427.4942 , found 1427.4961 . 
  a solution of 11 ( 50  mg , 31  mol ) in ch2cl2-tfa ( 95 : 5 , 25  ml ) was stirred at room temperature for 2  h.   the reaction mixture was concentrated in vacuo to give 2 as a white solid ( 50  mg , 30  mol , 98% , mp = 225230c dec ) . spectral data for 2 : [ ]d = 72 ( c 0.3 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 500 mhz , dmso d  6 )  9.159.08 ( m , 8h ) , 8.958.89 ( m , 4h ) , 8.38 ( d , j = 6.9 hz , 2h ) , 8.30 ( d , j = 6.9  hz , 1h ) , 7.807.53 ( m , 6h ) , 5.505.35 ( m , 4h ) , 2.982.89 ( m , 4h ) , 2.782.89 ( m , 4h ) , 2.151.85 ( m , 12h ) , 1.551.00 ( m , 20h ) ; c nmr ( 125 mhz , dmso d  6 )  171.7 , 164.5 , 164.3 , 158.8 , 158.7 , 155.6 , 154.5 , 142.5 , 141.9 , 141.8 , 141.1 , 136.0 , 129.8 , 129.7 , 128.5 , 128.4 , 47.4 , 47.3 , 38.6 , 38.1 , 35.1 , 33.1 , 28.7 , 26.7 , 24.9 , 21.3 , 20.8 ; hrms ( esi , m + h ) calcd for c66h67n20o18 1427.4942 , found 1427.4961 . 
 synthesis of 12to a solution of 2 ( 50  mg , 30  mol ) in meoh ( 5.0  ml ) was added amberlyst a-26(oh ) ion - exchange resin , and the mixture was stirred for 30  minutes . the resulting mixture was filtered through a cotton with meoh , and the filtrates were concentrated in vacuo .   to a residual solution of 2 in dmf ( 5.0  ml ) was added diisopropylethylamine ( 52 l , 0.31  mmol ) , hgcl2 ( 50  mg , 0.18  mmol ) , and 1,3-bis(tertbutoxycarbonyl)-2-methyl-2-thiopseudourea ( 66  mg , 0.18  mmol ) , and the mixture was stirred for 1  h at room temperature .   to the reaction mixture was added h2o , and the organic layer was extracted with ethyl acetate . the residue was chromatographed on silica gel ( chcl3-ethyl acetate - meoh = 3  :  2  :  1 ) to give 12 as a white solid ( 30  mg , 16  mol , 52% ) . spectral data for 12 : [ ]d = 3.8 ( c 1.4 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 500  mhz , cdcl3 )  11.5 ( br , 2h ) , 8.578.48 ( m , 4h ) , 8.308.17 ( m , 14h ) , 6.17 ( br , 2h ) , 5.455.36 ( m , 4h ) , 3.413.32 ( m 4h ) , 3.283.10 ( m , 4h ) , 2.201.88 ( m , 12h ) , 1.651.20 ( m , 56h ) ; c nmr ( 125  mhz , cdcl3 )  172.9 , 164.8 , 164.7 , 163.5 , 159.9 , 159.7 , 156.1 , 156.0 , 154.7 , 154.6 , 153.2 , 141.0 , 140.9 , 139.3 , 139.2 , 138.6 , 138.5 , 136.9 , 136.8 , 130.8 , 129.6 , 129.5 , 82.9 , 79.1 , 47.7 , 47.6 , 40.5 , 39.1 , 36.0 , 34.7 , 28.7 , 28.6 , 28.2 , 28.0 , 25.0 , 22.1 , 22.0 ; hrms ( esi , m + na ) calcd for c88h102n24o26na 1933.7295 , found 1933.7332 . 
  to a solution of 2 ( 50  mg , 30  mol ) in meoh ( 5.0  ml ) was added amberlyst a-26(oh ) ion - exchange resin , and the mixture was stirred for 30  minutes . the resulting mixture was filtered through a cotton with meoh , and the filtrates were concentrated in vacuo .   to a residual solution of 2 in dmf ( 5.0  ml ) was added diisopropylethylamine ( 52 l , 0.31  mmol ) , hgcl2 ( 50  mg , 0.18  mmol ) , and 1,3-bis(tertbutoxycarbonyl)-2-methyl-2-thiopseudourea ( 66  mg , 0.18  mmol ) , and the mixture was stirred for 1  h at room temperature .   to the reaction mixture was added h2o , and the organic layer was extracted with ethyl acetate . the residue was chromatographed on silica gel ( chcl3-ethyl acetate - meoh = 3  :  2  :  1 ) to give 12 as a white solid ( 30  mg , 16  mol , 52% ) . spectral data for 12 : [ ]d = 3.8 ( c 1.4 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 500  mhz , cdcl3 )  11.5 ( br , 2h ) , 8.578.48 ( m , 4h ) , 8.308.17 ( m , 14h ) , 6.17 ( br , 2h ) , 5.455.36 ( m , 4h ) , 3.413.32 ( m 4h ) , 3.283.10 ( m , 4h ) , 2.201.88 ( m , 12h ) , 1.651.20 ( m , 56h ) ; c nmr ( 125  mhz , cdcl3 )  172.9 , 164.8 , 164.7 , 163.5 , 159.9 , 159.7 , 156.1 , 156.0 , 154.7 , 154.6 , 153.2 , 141.0 , 140.9 , 139.3 , 139.2 , 138.6 , 138.5 , 136.9 , 136.8 , 130.8 , 129.6 , 129.5 , 82.9 , 79.1 , 47.7 , 47.6 , 40.5 , 39.1 , 36.0 , 34.7 , 28.7 , 28.6 , 28.2 , 28.0 , 25.0 , 22.1 , 22.0 ; hrms ( esi , m + na ) calcd for c88h102n24o26na 1933.7295 , found 1933.7332 . 
 synthesis of 3a solution of 12 ( 29  mg , 31  mol ) in ch2cl2-tfa ( 3  :  1 , 2.0 ml ) was stirred at room temperature for 2  h.   to the reaction mixture was added ether and precipitate was formed . this precipitate was collected with filtration using filter paper , to give 3 as a white solid ( 20  mg , 12  mol , 80% , mp = 220225c dec ) . spectral data for 3 : [ ]d = 18 ( c 0.75 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 400  mhz , dmso d  6 )  9.149.08 ( m , 8h ) , 8.948.90 ( m , 4h ) , 8.37 ( d , j = 7.3  hz , 1h ) , 8.32 ( d , j = 7.3  hz , 1h ) , 7.757.69 ( m , 2h ) , 7.517.45 ( m , 2h ) , 5.485.37 ( m , 4h ) , 3.082.90 ( m , 8h ) , 2.151.84 ( m , 12h ) , 1.501.00 ( m , 20h ) ; c nmr ( 125 mhz , dmso d  6 ) 171.7 , 164.5 , 164.4 , 158.8 , 158.7 , 156.7 , 155.7 , 155.6 , 154.5 , 142.5 , 141.8 , 141.1 , 136.0 , 129.8 , 129.7 , 128.5 , 128.4 , 47.4 , 40.5 , 38.1 , 35.1 , 33.4 , 33.3 , 28.7 , 28.2 , 24.9 , 21.3 , 21.0 ; hrms ( esi , m + h ) calcd for c68h71n24o18 1511.5378 , found 1511.5368 . 
  a solution of 12 ( 29  mg , 31  mol ) in ch2cl2-tfa ( 3  :  1 , 2.0 ml ) was stirred at room temperature for 2  h.   to the reaction mixture was added ether and precipitate was formed . this precipitate was collected with filtration using filter paper , to give 3 as a white solid ( 20  mg , 12  mol , 80% , mp = 220225c dec ) . spectral data for 3 : [ ]d = 18 ( c 0.75 , chcl3-meoh ( 1  :  1 ) ) ; h nmr ( 400  mhz , dmso d  6 )  9.149.08 ( m , 8h ) , 8.948.90 ( m , 4h ) , 8.37 ( d , j = 7.3  hz , 1h ) , 8.32 ( d , j = 7.3  hz , 1h ) , 7.757.69 ( m , 2h ) , 7.517.45 ( m , 2h ) , 5.485.37 ( m , 4h ) , 3.082.90 ( m , 8h ) , 2.151.84 ( m , 12h ) , 1.501.00 ( m , 20h ) ; c nmr ( 125 mhz , dmso d  6 ) 171.7 , 164.5 , 164.4 , 158.8 , 158.7 , 156.7 , 155.7 , 155.6 , 154.5 , 142.5 , 141.8 , 141.1 , 136.0 , 129.8 , 129.7 , 128.5 , 128.4 , 47.4 , 40.5 , 38.1 , 35.1 , 33.4 , 33.3 , 28.7 , 28.2 , 24.9 , 21.3 , 21.0 ; hrms ( esi , m + h ) calcd for c68h71n24o18 1511.5378 , found 1511.5368 . the dual fluorescently labeled oligonucleotides flu - telo21 5-fam-[ggg(ttaggg)3]-tamra-3 and flu - ds26 5-fam-[(ta)2gc(ta)2t6(ta)2gc(ta)2]-tamra-3 were used in this protocol . the donor fluorophore was 6-carboxyfluorescein , fam , and the acceptor fluorophore was 6-carboxytetramethylrhodamine , tamra . the oligonucleotides were initially dissolved as a 100  m stock solution in milliq water ; further dilutions were carried out in 60  mm potassium cacodylate buffer ( ph 7.4 ) . dual - labeled dna was annealed at a concentration of 400  nm by heating at 94c for 5  minutes   followed by cooling to room temperature . we added the different concentrations of ligands into different samples , using a total reaction volume of 40  l , with 200  nm of labelled oligonucleotide . following experiments should keep the temperature procedure in real - time pcr and procedure was finished as following : 25c for 5  minutes , then a stepwise increase of 1c every minute from 25c to reach 99c .   during the procedures , we measured the fam after each stepwise . the 10  m oligonucleotide of telo24 : ( [ ttaggg]4 ) was dissolved in tris - hcl buffer ( 50  mm , ph 7.0 ) and the solution was heated to 90c for 5  minutes , then slowly cooled to 25c . g4 ligands were diluted from 10  mm stock solutions to give a concentration of 1  mm with water and added into the oligonucleotide samples at 50  m ( the 10  mm stock solutions of 2 and 3 were made in dmso ) . esi - mass spectra were recorded in a negative - ion mode with jeol jms - t100x spectrometer . the direct - infusion flow rate was   5.0  l  min  . all experiments were performed in 20  mm nh4oac containing 10  m of telo24 and 40  m of 2 and 3 . the 6otd dimers 2 and 3 were synthesized by using the sequences as shown in scheme 1 . trioxazoles 4 and 6 were synthesized starting with l - serine and l - lysine , respectively by using the previously reported procedure [ 29 , 30 , 3638 ] . the cbz group of 4 was removed by treatment with hydrogen in the presence of pd(oh)2/c to give amine 5 . hydrolysis of the ester group in 6 with lithium hydroxide followed by coupling of the resulting acid with amine 5 using dmt - mm   gave the bis - trioxazole amide 8 . cleavage of the allyloxycarbonyl group in 8 and hydrolysis of the ester group produced an amino acid , which was subjected to macrocyclization under high dilution conditions ( 5  mm ) to give 6otd 10 . the cbz group in 10 was removed with hydrogen in the presence of pd(oh)2/c to give corresponding amine . the procedure for synthesis of dimer 11 involved coupling of the amine with adipoyl chloride . bis - amine 2 was obtained by removal of the boc group of 11 with tfa . preparation of the guanidine derivative 3 was carried out by guanidination of the amine moiety in 2 by using 1,3-bis(tert - butoxycarbonyl)-2-methyl-2-thiopseudourea followed by deprotection of boc group with tfa . with the desired 6otd dimers 2 and 3 in hand , their mode of interaction with the telomeric dna ( telo24 ) firstly , conformational changes of telo24 , induced by these substances were evaluated using circular dichroism ( cd ) spectroscopy .   upon treatment of telo24 with 6otd dimers 2 and 3 ( 50  m ) in the presence of potassium chloride ( 100  mm ) , the mixed - type structure induced by potassium cation ( solid line in figure 2 ) is transformed to a typical antiparallel - type g4 structure ( dashed line in figure 2 ) [ 28 , 40 ] . the binding stoichiometries of the complexes formed between the telo24 and ligands   and 3 ( molar ratio = 1  :  4 ) were determined by using esi - mass spectrometric analysis [ 41 , 42 ] .   in both cases , only mass peaks that correspond to 1  :  1 complexes of both 2 and 3 with telo24 were observed ( at the 7- , 6- or in the 5-charge states ) . since these interaction modes are the same as that of 6otd dimer 1 , the newly synthesized 6otd dimers 2 and 3 appear to interact with telo24 through an end stacking mode using two 6otd moieties in a manner similar to that of tms and/or 6otd dimer 1 ( figures 4 and 6 ) [ 33 , ]    the ability to stabilize the g4 structure of telo24 by 6otd dimers 2 and 3 was evaluated by employing a fluorescence resonance energy transfer -(fret- ) based assay [ 34 , 35 ] . the tm values of labeled oligonucleotide flu - telo21 with 2 and 3 at a concentration of 1  m , which corresponds to 5 equivalents , are 10 and 14c , respectively ( figure 5(a ) and table 1 ) . since under the same conditions the tm value for 1 was 12c , among the substances explored to date guanidine 3 is a potent stabilizer of the g4 structure 
								 . this stabilization effect is likely caused by the attractive interaction between positively charged guanidinium residue and anionic phosphates backbone of the telomeric g4 . interactions of the ligands with the duplex form of flu - ds26 were also investigated by using the same protocol ( figure 5(b ) and table 1 ) . the observation that no differences in the tm values exist in the presence or absence of dimers 2 and   suggests that these ligands are selective for the telomere dna sequence . in summary , the efforts described above have led to the design and syntheses of 2 and 3 , two novel macrocyclic hexaoxazole dimeric derivatives of 6otd that have amine and guanidine groups in their respective side chains . these compounds , together with 6otd dimer acetate 1 , were found to induce a change of the telomeric dna sequence of telo24 into an antiparallel structure through the formation of 1  :  1 complexes with the dna .   the guanidine functionalized 6otd dimer 3 was determined to have the greatest ability to stabilize the telomere dna sequence . also , both dimers selectively bind to the telomeric dna sequence and not double - stranded dna . further studies , aimed at the structural development of 6otd dimers with different linkers , are currently underway .
structure - activity relationship studies were carried out on macrocyclic hexaoxazole ( 6otd ) dimers , whose core structure stabilizes telomeric g - quadruplexes ( g4 ) . 
 two new 6otd dimers having side chain amine and guanidine functional groups were synthesized and evaluated for their stabilizing ability against a telomeric g4 dna sequence . 
 the results show that the 6otd dimers interact with the dna to form 1:1 complexes and stabilize the antiparallel g4 structure of dna in the presence of potassium cation . 
 the guanidine functionalized dimer displays a potent stabilizing ability of the g4 structure , as determined by using a fret melting assay ( tm = 14c ) .