small organic molecules with specific interactions with dna have become antitumor , antiviral , and antibiotic drugs [ 1 , 2 ] .
duplex dna - binding drugs interact in two main ways , through groove binding and through intercalation .
medicinal chemistry has made a considerable effort in searching for and testing of a large number of drugs with increased selectivity to a range of dna sequences or structures .
more recently , some of this interest has moved to the search of new ligands for g - quadruplexes .
this structure motif is formed by the planar association of four guanines in a cyclic hoogsteen hydrogen bonding tetrad .
guanine - rich sequences form g - quadruplex structures and have been found in telomeres   and in transcriptional regulatory regions of critical oncogenes such as c - myc and c - kit [ 5 , 6 ] .
ligands that selectively bind and stabilize these structures have become anticancer drugs of interest .  
the g - quadruplex stabilization occurs in most cases by - stacking and electrostatic interaction .
g - quadruplex ligands are normally planar aromatic molecules that are prone to stacking with g - tetrads . some of them are also positively charged or have hydrophilic groups to favor electrostatic interaction .
although there is a long way to go in the development of potent drugs that target g - quadruplexes , some promising lead compounds have been achieved .
several ligand structures have been studied , such as anthraquinones , cationic porphyrins , perylene derivatives , and a large number of compounds . among the acridine compounds , 3,6,9-trisubstituted acridines have inhibitory activity in the nanomolar range and they have entered preclinical studies [ 8 , 10 , 11 ] .   in previous studies we described the preparation of sequence specific oligomers of dna - intercalating drugs using protocols based on solid - phase synthesis in an attempt to facilitate the preparation of compounds with improved dna - binding selectivity [ 12 , 13 ] .
it has been proposed that bis- and tris - intercalating drugs show promising activity and selectivity [ 14 , 15 ] .
here we described solid - phase synthesis protocols for the preparation of several acridine oligomers linked through 2-aminoethylglycine units as well as their dna - binding properties .
although the acridine derivatives described in this study are not cytotoxic , they show a clear affinity for several dna g - quadruplex structures , especially those sequences found in the promoter regions of c - myc   and bcl-2 [ 17 , 18 ] oncogenes .
the phosphoramidites and ancillary   reagents used during oligonucleotide synthesis were obtained from applied biosystems ( usa ) and link technologies ltd .
the slide - a - lyzer mini dialysis units 3500  mwco were purchased from pierce .
2-(acridine-9-carboxamide)acetic acid was prepared by reaction of acridine-9-carboxylic acid with glycine methyl ester and subsequent saponification of the methyl ester as described .
boc-(2-aminoethyl)glycine(fmoc ) ( boc - aeg(fmoc)-oh ) was obtained from iris biotech and fmoc - glycine ( fmoc - gly - oh ) was obtained from bachem .
oligonucleotide sequences ( table 1 ) were prepared on an automatic applied biosystems 3400 dna synthesizer on 1  mol ( cpg resin ) scale using commercially available 2-cyanoethyl phosphoromidites .
after the assembly of the sequences , oligonucleotide - supports were deprotected using 32% aqueous ammonia at 55c for 16  h. ammonia solutions were concentrated to dryness and the residue was desalted by a nap-10 ( sephadex g-25 ) column .
acridine dimers and trimers ( 14 , figure 1 ) were prepared with the 2-aminoethylglycine scaffold , which allows the growth of a polyamide skeleton on solid - phase and the following incorporation of acridine unit .
the assembly of 2-aminoethylglycine derivatives was carried out on methylbenzhydrylamine ( mbha ) polystyrene-1%-divinylbenzene solid support applying an fmoc / boc hybrid strategy using boc - aeg(fmoc)-oh , fmoc - gly - oh , and acridine-9-carboxylic acid as building blocks ( figure 2 ) .
fmoc - sarcosine - oh ( 5  eq ) was coupled to the resin using standard coupling conditions ( 5  eq .
diea , 1  h ) , then the fmoc group was removed ( 20% of piperidine in dmf , 30  min ) , and boc-6-aminohexyl hemisuccinate ( boc  nh(ch2)6ococh2ch2cooh ) was coupled ( 5  eq r - cooh , 5  eq . pybop and 10  eq .
the residual unreacted amino groups were acetylated with 5  eq . of acetic anhydride and 5  eq .
next , the boc group was removed ( 40% trifluoroacetic acid in dichloromethane ) and the 2-aminoethylglycine skeleton was synthesized by repetitive couplings of boc - aeg(fmoc)-oh until reaching the desired dimer or trimer compound .
the last boc group of the sequence was removed and the resulting amino group was acetylated ( 5  eq .
once the aminoethylglycine backbone was built , the fmoc - protecting groups of the side chains were removed ( 20% of piperidine in dmf , 30  min ) , and fmoc - gly - oh followed by 9-acridine carboxyl acid was coupled to the support .
the progress of the coupling reactions was followed by ninhydrine test and by uv monitoring of the 9-methylene-9h - fluorene released during deprotection , which allowed optimization of the coupling conditions .  
the acridine dimer 1 and trimer 3 were obtained by treatment of the appropriate solid supports with hf anhydrous at 0c .
finally the acridine dimer 2 and trimer 4 were obtained by treatment of dimer 1 and trimer 3 respectively with 32% aqueous ammonia ( 1  h , 55c ) .   
good yields and purities were obtained for the products ( around 85% for 1 and 2 , 75% for 3 and 4 ) .
hplc and maldi - tof spectra are shown in supplementary material available onlie at doi : 10.4061/2010/489060 .
the compounds were analyzed by maldi - tof , 1 [ m+na ] = 1080.3 ( expected 1054.2 ) , 2 [ m ] = 885.2 ( expected 884.0 ) , 3 [ m ] = 1418.9 ( expected 1416.5 ) , and 4 [ m ] = 1247.4 ( expected 1246.4 ) .
maldi - tof spectra were obtained using a perseptive voyager detmrp mass spectrometer , equipped with nitrogen laser at 337 nm using a 3  ns pulse .
the matrix used contained 2,5-dihydroxybenzoic acid ( dhb , 10  mg / ml in water ) .
analytical hplc was performed using xbridge ost c18 ( waters ) , 2.5  m , 4.6  50  mm column using a 10-minute linear gradient from 9% to 45% b , flow rate 1  ml / min ; solution a was 5% acn in 0,1  m   aqueous teaa , and b 70% acn in 0.1 m aqueous teaa .
fluorescence spectra were recorded using a jasco fp-6200 spectrofluorometer equipped with a peltier temperature controller , em  =  435  nm ( exc = 252  nm ) .
uv spectra were recorded using a jasco spectrophotometer v-650 , max   = 252  nm , 360  nm .
the in vitro cytotoxicity of the compounds ( figure 1 ) was evaluated by colorimetric assays with tetrazole salts ( mtt ) on jurkat clone e6 - 1 ( human leukemia ) , glc-4 clone ( human lung carcinoma ) cell lines , and one mouse fibroblast cell line ( niht-3t3 ) .
glc4 and jurkat cell lines were cultured in rpmi and nih3t3 in dmem and supplemented with 10% fetal calf serum , 10000  u / ml penicillin , 10  g / ml streptomycin and 200  mm l - glutamine .
cells were grown in a humidified atmosphere of air containing 5% co2 at 37. cells were plated in triplicate wells ( 1.510 cells well ) in 100  l of growth medium in 96-well plates and proliferate for 24  h and then treated with increasing concentrations of acridine oligomers .
after 72  h of incubation , 10  m of mtt ( 5  mg / ml in phosphate buffer saline 10% ) was added for an additional 4  h. the absorbance at 570  nm was measured on a multiwell plate reader after addition of 100  l of isopropanol  :  1n hcl ( 24  :  1 ) .
cell viability was expressed as a percentage of control and ic50 was determined as the concentration of drug that produced a 50% reduction of absorbance at 570  nm .
100  l of a 50  m oligonucleotide ( table 1 ) in potassium phosphate buffer ( 185  mm nacl , 185  mm kcl , 2  mm nah2po4 , 1  mm na2edta , 6  mm na2hpo4 at ph 7 ) was introduced into a separated dialysis unit .
all 12 dialysis units were then placed in the beaker containing the 1  m solution of the appropriate acridine derivative .
sds is usually added to denature the dna sample and release the acridine oligomer , but in our case the presence of k ions induced the formation of a white precipitate , which interfered with the measurement of the fluorescence spectra of the samples .   in order to measure the compound retained in the dialysis unit ,
samples were treated with snake - venom phosphodiesterase to degrade the dna and release the acridine oligomer .
350  l of potassium phosphate buffer ( without edta ) , buffer at ph 8.5 , 50  l of 100  mm mgcl2 , and 1  m of snake venom phosphodiesterase solution were added for an additional overnight incubation at 37c .
finally , the fluorescence of each sample was measured ( ex and em   were set to 252 and 435  nm , resp . ) .
the study of the interaction equilibrium of acridine derivatives and oligonucleotides consists of recording the fluorescence spectra of a 0.2  m solution of the acridine derivative after the addition of increasing amounts of oligonucleotide ( from 0 to 10  m ) in potassium phosphate buffer ( 185  mm nacl , 185  mm kcl , sodium phosphate , 1  mm edta , ph 7 ) .
these experiments were carried out by adding small volumes of an oligonucleotide stock solution to the 0.2  m solution of the acridine derivative .
after 24  h the emission spectra of the resulting solutions were recorded from 300 to 500  nm at 252  nm excitation wavelength at 25c .  
the macroscopic binding constant ( k ) corresponding to the reaction 
 ( 1)dna+ligand    interaction  complex 
							 was calculated from the multivariate analysis of fluorescence data recorded in the range 300390  nm using the hard - modeling program equispec .
this program performs a nonlinear least squares optimization of k and of the pure fluorescence spectra corresponding to each of the species considered ( dna , ligand , and interaction complex )
. a 1  :  1 stoichiometry dna  :  ligand for the interaction complex was assumed .
the logarithms of the binding constants calculated are given as their weighted means with twice their standard errors ( units of the least significant digit ) .
an increasing amount of 1 ( from 0 to 8  m ) in potassium phosphate buffer ( 185  mm nacl , 185  mm kcl , sodium phosphate , 1  mm edta , ph 7 ) was added to a 1  m solution of the oligonucleotide . the cd spectra were recorded after 24  h on a jasco j-810 spectropolarimeter attached to a julabo f/25hd circulating water bath in 1  cm path - length quartz cylindrical cells , using a 50 nm / min scan rate , a spectral band width of 1  nm , and a time constant of 4  s. all the spectra were corrected with the buffer blank , normalized to facilitate comparisons and noise - reduced using matlab software .
the synthesis of acridine dimer and trimers has been described previously   using a boc-(2-aminoethyl)glycine derivative carrying the 2-(acridine-9-carboxamide)acetyl residue .
an alternative method developed is to first assemble the boc-(2-aminoethyl)glycine backbone on solid - phase , and then the intercalating agent is added .
this strategy is more convenient for rapid synthesis , as it is unnecessary to construct each intercalating monomer .  
thus , acridine oligomers were assembled using the methylbenzhydrylamine ( mbha ) resin by applying an fmoc / boc hybrid strategy and using boc-(2-aminoethyl)-(fmoc)glycine [ boc - aeg(fmoc)-oh ] as building block ( figure 2 ) .
the boc group was used to protect the aminoethyl group of each unit , which thus facilitated elongation of the backbone .
fmoc was the semipermanent protecting group for the amino group of glycine through which the intercalating compound was introduced .
it has been described that an intramolecular side reaction can lead to premature loss of the oligomers during the base treatment used to remove the fmoc group .
thus , n - methylglycine ( sarcosine ) was incorporated between the amino - support and the succinyl linker .
6-aminohexanol was used to connect the succinyl linker and the oligomer backbone , as described for the synthesis of peptide nucleic acid ( pna ) oligomers . the ( 2-aminoethyl)glycine backbone was assembled by consecutive additions of boc - aeg(fmoc)-oh to obtain the dimer and trimer sequence .
acetylation of the n - terminal position was carried out using acetic anhydride and a base .   
next , the removal of the fmoc group allowed the addition of a fmoc - glycine unit as a spacer , which was followed by the addition of acridine-9-carboxylic acid .
the desired oligomers were not released from the support even after prolonged time and high temperatures .
we therefore treated the supports with anhydrous hf to yield the acridine oligomers 1 and 3 , which contain the sarcosyl succinyl linker . at this point
hplc spectra showed a major peak that had the expected molecular weight for the oligomers 1 and 3 carrying the sarcosyl succinyl linker ( see supplementary data ) .
ammonia treatment of acridines 1 and 3 in solution now yielded the desired acridine dimer 2 and trimer 4 in excellent yields and purity ( see supplementary data ) .
the pka of the acridine ring of acridine-9-carboxylic acid and 2-(acridine-9-carboxamide)acetic acid was measured by uv titration .
we therefore estimated that the acridine rings of compounds 14 are mainly unprotonated at ph 7.0 .
the in vitro cytotoxicity of the compounds was evaluated by colorimetric assays with tetrazole salts ( mtt ) .
this assay is based on the capacity of living cells to incorporate and reduce mtt .
this reaction can be followed by the change of absorbance of the reduced and oxidized forms .
this reaction is done by the action of the mitochondrial enzyme succinatehydrogenase , which is active only in living cells . the intensity of color is directly correlated with the number of living cells in the sample .
no cytotoxicity activity was observed in compounds 14 at concentrations up to 50  m . in order to evaluate the selectivity of the compounds for dna structures
, a competitive dialysis experiment was performed using 11 oligonucleotides ( table 1 ) representing several nucleic acid structures .
the more acridine accumulated in the dialysis unit indicates a higher binding affinity to the oligonucleotide present in the dialysis unit . as model for single stranded structures we used t20 and the c - rich complementary strand of bcl-2 ( 24bclc )
this last oligonucleotide folds in an i - form quadruplex structure at acidic ph but has no structure in the conditions used in the dialysis ( ph 7 ) . as duplexes we used the self - complementary sequences dickerson - drew dodecamer ( dickerson ) and a 26 mer ( ds26 ) . a parallel triplex ( tc triplex ) and an antiparallel triplex ( ga triplex ) were also prepared by mixing a hairpin watson - crick sequence and the corresponding triplex - forming sequence .
finally , the following g - quadruplex sequences were prepared : the tetramolecular parallel g - quadruplex tg4 t , the antiparallel thrombin - binding aptamer ( tba ) , the human telomere sequence ( ht24 ) , and the promoter sequences of c - myc ( cmyc )   and bcl-2 ( 24bcl ) [ 17 , 18 ] .  
the buffer solution was similar to that described in   but k was included ( 185  mm ) to ensure the formation of the most stable g - quadruplex structures . at the end of the dialysis experiment , the amount of acridine derivative bound to the dna was analyzed by fluorescence measurement of the acridine compound .
after dialysis , we observed that the spectra of the acridine derivatives of some samples ( especially those from g - quadruplexes ) differed greatly from the fluorescence spectra of the initial compounds .
this difference is attributed to the interaction of the acridine derivatives with the g - quadruplex . in order to release the acridine oligomer ,
the presence of k ions resulted in the formation of a white precipitate with sds which did not allow the measurements of the fluorescence spectra . in order to solve this problem ,
the oligonucleotide was digested with snake venom phosphodiesterase at the end of the dialysis experiment .
thus , the fluorescence spectra of the acridine derivatives were recorded with high accuracy without the interference of dna and without the use of sds .    
we measured the binding preferences of compounds 14 and the acridine monomers acridine-9-carboxylic acid and 2-(acridine-9-carboxamide)acetic acid ( figure 1 ) .
unexpectedly , the addition of the glycine residue , used as spacer , induced a change in the affinity .
2-(acridine-9-carboxamide)acetic acid showed the highest affinity for the g - quadruplex sequences cmyc and 24bcl and less affinity for duplex ds26 .  
dimer 1 ( dimer with the sarcosylsuccinyl linker ) has a similar profile as 2-(acridine-9-carboxamide)acetic acid . in this case , the g - quadruplex 24bcl is preferred to the cmyc sequence .
surprisingly , dimer 2 ( without the sarcosylsuccinyl linker ) lost most of the selectivity although some residual higher affinity for g - quadruplex 24bcl was observed .  
in contrast , trimer 3 ( trimer with the sarcosylsuccinyl linker ) presented lower binding affinity than the other compounds .
however , trimer 4 ( without the sarcosylsuccinyl linker ) recovered most of the affinity for 24bcl and cmyc showing a similar profile to that observed for dimer 1 .
dialysis experiments suggest that some of the acridine derivatives prepared have special affinity for 24bcl and cmyc g - quadruplexes . in order to confirm this observation ,
hence , increasing amounts of oligonucleotides 24bcl , ht24 , cmyc , and dickerson were added to a solution with a fixed concentration of the acridine derivatives , and the fluorescence spectra were recorded at excitation wavelengths 252 and 360  nm . at both wavelengths , changes in the fluorescence spectra upon the addition of oligonucleotides were observed .
figure 4 shows the changes in the fluorescence spectra of acridine dimer 1 at excitation wavelengths 252  nm when oligonucleotides 24bcl , ht24 , cmyc , and dickerson were added . a dramatic increase in fluorescence intensity
was observed around 360  nm upon addition of g - quadruplex dna sequences ( 24bcl , ht24 , and cmyc ) .
similar results were found with compounds 2 , 3 , and 4 as well as 2-(acridine-9-carboxamide)acetic acid ( see supplementary data ) .
interestingly , when the dickerson dodecamer was added , no changes in the fluorescence spectra were detected .
the progressive modification of the fluorescence spectrum of these compounds reflects their interaction with the g - quadruplex .  
fluorescence data obtained at an excitation wavelength of 252  nm were analyzed with the hard - modelling equispec program in order to calculate the corresponding binding constants ( table 2 ) .
the values of the logarithm of the binding constant ( log  k ) obtained lie in the range 46 , suggesting a weak interaction with dna .
of all the compounds , dimer 1 showed the highest binding constants , thereby suggesting a stronger interaction with dna than the other compounds studied .
however these values were slightly lower than the binding constants calculated for other similar ligands , such as the acridine monomers braco-19 ( log  k = 7.4 ) and bsu6048 ( log  k = 6.5 )   or a hemicyanine - peptide ligand ( log  k = 7.1 ) , when interacting with human telomere quadruplex .  
changes in the fluorescence spectra at an excitation wavelength of 360  nm were also recorded . fluorescent emission at this wavelength 360  nm was much lower than that recorded at 252 nm ; so the fluorescent signal was low .
upon addition of the oligonucleotide to a solution of compounds 14 , the formation of a new maximum at 442  nm was observed ( see supplementary data ) .
although the fluorescent intensity was low , we could estimate the binding constant of the stronger interactions of compound 1 with 24bcl ( 7.2  0.4 ) and cmyc ( 5.5  0.2 ) sequences ( see supplementary data ) .
these values are in agreement with those recorded at an excitation wavelength of 252  nm ( table 1 ) .   
finally , cd spectra of the dna  :  ligand mixtures showed no significant differences in relation to those of dna ( see supplementary data ) .
this observation suggests that the dna g - quadruplex structure is not altered significantly upon binding of the acridine derivatives .
in summary , here we have described a new optimized protocol for the synthesis of acridine oligomers with a ( 2-aminoethyl)glycine backbone . in this method ,
the boc-(2-aminoethyl)glycine backbone is first assembled on solid - phase , and then the intercalating agent is assembled on the backbone .
this strategy is faster and more efficient than the one described previously   and yields the desired oligomers with good yields .
the succinyl linker attached to sarcosine was unexpectedly too stable and oligomers could not be directly released from the support by a single ammonia treatment .
instead a two - step protocol was used obtaining the desired compounds and an intermediate oligomer carrying a long succinyl sarcosine chain at the c - terminal position .    
competitive dialysis experiments have shown differences on the affinity of acridine oligomers to g - quadruplexes .
higher affinities are found in g - quadruplex sequences present on the promoter regions of c - myc and bcl-2 oncogenes .
this affinity is modulated by the number of acridines and the presence of the succinyl sarcosine chain at the c - terminal position , dimer 1 and trimer 4 being the more relevant compounds for g - quadruplex binding .
the monomer 2-(acridine-9-carboxamide)acetic acid also shows binding properties of interest and it is the simplest compound to be prepared .
unfortunately , the compounds synthesized in this study did not have antiproliferative activity in spite of their affinity to quadruplex .
this observation contrasts with other reported quadruplex - binding acridine derivatives , such as braco-19 [ 11 , 30 ] which shows anticancer activity .
the lower affinity to telomere g - quadruplex sequence and the larger size of the acridine derivatives described in the present study may hinder cellular uptake and may explain the absence of antiproliferative activity .
depending on the substituents , the acridine nitrogen can be charged when bound to dna , and with the ring stacked on a g - tetrad , the charge will occupy a position similar to that of the potassium cation that stabilizes the g - quadruplex .
the introduction of protonable side chains on the acridine ligand enhances binding by electrostatic interactions . in our case
the acridines had no protonable substituents and the acridine nitrogen was not charged when bound to dna .
for this reason , the affinity of oligomeric acridines to g - quadruplexes is due to the multimeric nature of the compounds as well as the addition of a glycine to acridine-9-carboxylic acid .
an interesting possibility for future development is the introduction of protonable sites at the oligomeric acridines , which may increase solubility in water , affinity to target , and cellular uptake .
the method described here will contribute to accelerating the preparation of potential active oligomeric compounds .
 the synthesis of oligomers containing two or three acridine units linked through 2-aminoethylglycine using solid - phase methodology is described . 
 subsequent studies on cell viability showed that these compounds are not cytotoxic . 
 binding to several dna structures was studied by competitive dialysis , which showed a clear affinity for dna sequences that form g - quadruplexes and parallel triplexes . 
 the fluorescence spectra of acridine oligomers were affected strongly upon binding to dna . 
 these spectral changes were used to calculate the binding constants ( k ) . 
 log k were found to be in the order of 46 . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions