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DOI: 10.1 002/cmdc.201300085

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Probing the Structural Properties of DNA/RNA Grooveswith Sterically Restricted Phosphonium Dyes: Screening ofDye Cytotoxicity and UptakeIvo Cmolatac,'" Lidija-Marija Turnir.!" Nedyalko Y. Lesev}bl Aleksey A. Vasllev,"Todor G. Deliqeorqlev'?' Katarina Mlkovl.'? Ljubica Glava-Obrovac,'?' Oliver vuqrek,'" andIvo Piantanida"!"

To explore in greater detail the recently reported rare kineticdifferentiation between homo-polymeric and alternating AT-DNA sequences by using sterically restricted phosphoniumdyes that form dirners within the DNA minor groove, new ana-logues were prepared in which the quinolone phosphoniummoiety was kept constant, while the size and hydrogen bond-ing properties of the rest of the molecule were varied. Struc-ture-activity relationship studies revealed that a slight increasein length by an additional methylene unit resuIts in loss of ki-netic AT selectivity, but yielded an AT-selective fluorescence re-

Introduction

The importance of DNA binding small molecules is under-scored by the many reviews that have been written on thistopic over the last 40 years!" Various noncovalent interactionmod es between small molecules and DNA have been ex-plained in detail, of which the main modes are: 1) electrostaticinteractions between polycationic compounds and the DNAouter surface (negatively charged phosphate backbone);2) binding in the DNA minor qroove.!':" or rarely in the majorqroove?' 3) various types of intercalation between adjacentbase pairs; and 4) metal coordination to the bases.'" Researchon DNA/RNA dyes has been focused primarily on the impacton DNA/RNA function or selective or specific DNA/RNA [abel-ing.[SIHowever, the enormous complexity of DNA-coded pro-cesses that depend not only on coding DNA base pair sequen-

[al Dr. I. Crnolatac. Dr. L.-M. Turnir, Dr. I. PiantanidaDivision of Organic Chemistry and BiochemistryRuer Bokovi Institute, PO Box 180, 10002 Zagreb (Croatia)E-mail: [email protected]

[b] N. Y. Lesev, Dr. A. A. Vasilev, Prof. T.G. DeligeorgievFaculty of Chemistry, University of SofiaJ. Bourchier Avenue, 1164 Sofia (8ulgaria)

[cl Dr. K. Mikovi, Prof. L. Glava-ObrovacDepartment of Medicinal Chemistry and BiochemistryClinicallnstitute of Nuclear Medicine and Radiation ProtectionSchool of Medicine, University Hospital Centre Osijek, 31000 Osijek (Croatia)

[dl Dr. O. VugrekDivision of Molecular MedicineRuer Bokovi Institute, PO Box 180, 10002 Zagreb (Croatia)

~ Supporting information for this article is available on the WWWunderhttp://dx.doi.org/1 0.1 002/cmdc.201300085.

sponse. These DNA/RNA-groove-bound dyes combine very lowcytotoxicity with efficient cellular uptake and intriguingly spe-cific fluorescent marking of mitochondria. In contrast to longeranalogues, a decrease in length (by methylene unit removal)and rearrangement of positive charge resulted in dyes thathad switched to the intercalative binding mode to GC DNA/dsRNA but that still form dimers in the minor groove of AT se-quences, consequently yielding a significantly different chiro-optical response. The latter dyes also revealed strongly selec-tive antiproliferative activity toward HeLa cancer cells.

ces, but that also involve epigene.tic mechanisms has only re-cently gathered attention.[6,71To address such complex systems,researchers within the last two decades have usually combinedtwo or more DNA/RNA binding modes in the design of novelsmall molecules, formulating specific 3D binding motifs to ach-ieve highly selective or specific interactions with slightly differ-ent DNA/RNA structures.l'Y" Fluorescence is the most popularmethod of choice for monitoring selective or specific interac-tions. Indeed, the latest developments in fluorescence-basedtechniques and applications have resulted in their widespreaduse as tools in reading DNA rnlcroarrays.!'?' gene expression ex-periments, automated DNA sequenclnq.l'" fluorescence-activat-ed cell sorting}121and fluorescence-based imaging,[13,14JFluores-cent probes are usually small organic molecules characterizedby a dramatic increase in fluorescence emission intensity uponassociation with biornacrornolecules.

The cyanine dyes have made a major impact in the field offluorescent probes for molecular bioloqy.'l" The most widelyknown are the yaya and TOTa dye families, showing sensitivi-ty in DNA detection on par with that of radioactive probes.l'"An attractive property of cyanines is that free dyes in solutionusually exhibit almost no intrinsic fluorescence, but show re-markable fluorescence enhancement upon DNA binding. Thor-ough research has shown that the DNA binding mode can bealtered by varying the substituents attached to the dye mole-cule or by using a different DNA sequence, thus offering an ex-cellent model to study structure-function relatlonshtps.'!"

Our previous resuIts are in line with observed substituent in-fluences on cyanine dyes; that is, long er and bulkier substitu-

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ents attached to the longer axis of the dye direct binding tothe DNA minor groove and also decrease biological activity.[18JTherefore, we prepared novel asymmetric monomethine cya-nine dyes based on benzothiazole-quinoline conjugates withnew types of sterically demanding, yet positively charged sub-stituents (tributylphosphonium, triphenylphosphonium; com-pou nds 4a and 4b in Figure 1). These asymmetric cyaninesform dimers in the DNA minor groove (the formation of largeraggregates was disfavored by large substituents protruding onboth sides of the dimer and hampering oligomer propagation,as mentioned earlier for asymmetric dyes)y7J revealing a rarekinetic differentiation between homo-polymeric and alternat-

taking into account the previously observed selectivity of4a,b.[19JBecause cyanine dyes have many applications in bio-medical research, we also carried out a preliminary screen ofdyes 4a-f for cytotoxicity and cellular uptake, with the aim ofbetter defining future research directions.

Resuits and DiscussionSynthesis

The preparation of the target monomethine dyes requiresseveral building blocks. Tributyl-(3-iodopropyl)phosphonium

iodide 1a and (3-iodopropyl)tri-phenylphosphonium iodide 1 bwere synthesized by monoqua-ternization of tributylphosphineand triphenylphosphine, respec-tively, with 1,3-diiodopropane atroom temperature (Scheme 1).By using a modified reactionprocedureP" lepidine was qua-ternized by simple melting withthe appropriate intermediate 1aor 1 b to afford the new quater-nary salts 4-methyl-1-(3-(tributyl-phosphonio)propyl)quinolin-1-ium iodide (2 a) and 4-methyl-1-(3-(triphenylphosphonio)propyl)-quinolin-1-ium iodide (2 b) inhigh yields (Scheme 1). The in-termediates 1a, 1 b, 2 a, and 2 bare highly hygroscopic and wereused in the next step withoutfurther purification.

The target dyes 4a-f(Scheme 1) were prepared ac-

cording to the slightly modified method of Brooker et al.P1Jwhereby base-triethylamine was replaced by the more sterical-Iy hindered N-ethyldiisopropylamine (Hunig's base), in this wayavoiding the possible undesired side reaction of Hoffman elim-ination.

Figure 1. Structures of compounds 4a-f studied in this work.

ing AT DNA sequences.i'" Moreover, 4a and 4b were found tobe highly selective probes for induced circular dichroism (ICO)because of dimer formation in the minor groove of (dGdC)nhomo-polynucleotides and not in alternating (dGdC-dGdC) se-quences.

To explore in greater detail the fascinating seledivity poten-tial of cyanine phosphonium dyes (compounds 4a,b), new an-alogues 4 c-f were prepared, with the quinolone phosphoniumpart remaining unchanged. Specifically, in 4c,d an additionalmethyl group was attached to the pyridinium moiety (to intro-duce additional steric hindrance for dimer formation) and theoxazolo function was replaced by a thiazolo heterocycle toslightly alter the hydrogen bonding capacity with in DNA/RNAgrooves. In contrast to compounds 4a-d, benzothiazolium an-alogues 4e,f are significantly less sterically hindered (Iackingboth methyl substituents on the fused benzene ring), and theDNA/RNA binding ability of the thiazolo heterocycle was signif-icantly influenced by methylation of the endocyclic nitrogenatom. The interactions of novel compounds 4c-f with a rangeof dsDNA/RNA sequences representing various secondary heli-cal structures were studied by a set of methods, particularly

Physicochemical properties of studied compounds insolution

The studied compounds (Figure 1) are moderately soluble inredistilled water (c~l x10-4M). Aqueous solutions of 4a,b,eand 4 f showed no significant change in their UVNis spectraover a period of two months, whereas solutions of 4c and 4dshowed up to 10% degradation after several days. Compoundswere checked for their thermal stability upon heating at 9soC;UVNis spectra for all compounds studied did not change sig-nificantly, and minor changes 5%) were reversible after cool-ing back to room temperature. Aqueous solutions of 4a-fshowed linear correlation between absorption and concentra-tion in the experimental range (c= 1x 10-6-2 x 10-5 M, sodiumcacodylate buffer, 1=0.05 M, pH 7) and exhibited negligible

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2013, 8, 1093 - 1103 1094


cantly more potent stabilizer ofdsDNA than tributylphosphoni-um 4c; these findings are similarto those previously reported forcompounds 4a,b.(l9J In contra stto 4a-d, 4e and 4 f stabilizednot only dsDNA but also dsRNA.This latter finding strongly sug-gests a different RNA bindingmode in tha n that for com-pou nds 4a-d. Moreover, sub-stituent dependence was re-versed: stronger stabilizationwas observed with tributylphos-phonium than with triphenyl-phosphonium substituents.

I~I~1R1

I~P/+,

1- R1

1a: R1 = nBu1b: R1 = Ph

+

or

Scheme 1. Reagents and conditions: a) hexane, 24 h, Rl; 82% (1 a), 92% (1 b); b) melting at 120C for 1 h,76%(2a), 84% (2b); c) NEt(iPr)" acetic anhydride, 2 h, RT.

fluorescence upon excitation at respective absorption maxima(Table 1).

Study of interactions of 4 c-f with DNA and RNA

Thermal denaturation experiments

It is known that polynucleotide double helices dissociate intotwo single-stranded polynucleotides upon heating, at a well-defined and sequence-specific temperature (Tm value). Nonco-valent binding of small molecules to double-stranded (ds)polynucleotides usually has a certain stabilization effect onthermal denaturation, resulting in a change in Tm value. Thedifference between the Tm value of a free polynucleotide andthat in complex with a small molecule (L\Tm) is an importantparameter in the characterization of small-molecule/ds poly-nucleotide interactions. The effects of adding 4c, 4d, 4e, and4 f as stabilization against thermal denaturation of dsDNA anddsRNA are listed in Table 1.

Compounds 4c,d significantly stabilized dsDNA, but showedno impact on the thermal denaturation of dsRNA. Moreover,4d (triphenylphosphonium analogue) was found to be a signifi-

Table 1. Changes in melting temperature of studied polynucleotidesupon addition of various ratios (r) of 4c, 4d, 4e, and 4 f.

Compd st; [OC]['[poly-dAdT -poly-dAdT poly-A-poly-U

r:[b1 0.1 0.2 0.3 0.34a[191 2.3 3.6 5.9 O4b[191 4.4 8.2 11.5 O4c 1.3 2.3 3.9 O4d 8.4 15.5 18.4 O4e 7.1 12.0 15.1 11.04f 2.5 3.9 5.0 4.1

[al Error: 0.5 C; determined at pH 7.0 (sodium cacodylate buffer, 1=0.05 M). [b] r= [compound]/[polynucleotide].

Viscometry

Viscosity measurements on solu-tions of calf thymus (ct) DNA in-

cubated with 4d and 4e were carried out to further explorethe DNA binding mode. Ethidium bromide (EB) was used asreference compound, as it is known for its DNA intercalativebinding mode.[22JCompound 4e initiated an increase in theviscosity index, which indicates intercalative binding to dsDNA.However, relative to the viscosity index of ctDNA solution withEB,4e showed a somewhat lower value, which can be attribut-ed to either partial intercalation or kinking of the DNA helicalaxis due to the steric impact of the bulky tributylphosphoniumgroup. In contrast, 4d showed anegative viscosity index, im-plying a decrease in the solution viscosity upon addition of thecompound, which is not compatible with an intercalative bind-ing mode. The obtained viscosity indices (a) are listed inTable 2.

Table 2. Viscosity indices for 4d, 4e, and ethidium bromide (ES).!'1

Compd a 5D4d -1.67 0.074e 1.29 0.04ES 1.71 0.02

[al Values are the mean standard deviation from n=3 experiments per-formed in triplicate.

Spectrophotometric titrations

Addition of any ds polynucleotide resulted in pronouncedbathochromic and hypochromic shifts in the UVNis spectra ofcompounds 4c-f (Figure 2; see also Supporting Information).The titrations mostly reveal a systematic deviation from theisosbestic points, which supports the formation of several dif-ferent types of complex. Closer inspection of the titration datarevealed that one type of complex is dominant if polynucleo-tide is present at large excess over dye, whereas if dye is inexcess, mixed binding modes appear. For cyanine dyes, such

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0.5

0.4

0.3


the dsDNA/RNAs studied, highlighting the impact of the addi-tional methyl group attached to the pyridinium moiety ofcompounds 4c and 4d.

Circular dichroism experiments

Up to this point the described spectroscopic experiments werebased on monitoring the spectroscopic properties of the stud-ied compounds upon addition of polynucleotides. To get infor-mation about the changes induced by the small molecule onthe spectroscopic properties of polynucleotides, the CD spec-troscopy was applied as a highly sensitive method to evaluateconformational changes in the secondary structure of polynuc-leottdes.P" Additionally, the induced CD spectrum, which canappear upon binding of achiral small molecules to polynucleo-tides, could give useful information about modes of interac-tion.[26]Notably, the studied compounds are achiral and there-fore do not possess intrinsic CD spectra. Previously studiedcompounds 4a,b revealed an ICD pattern specific for each ofthe studied polynucleotides, among which the most interest-ing was slow kinetics of binding exclusively to poly-dA-poly-dl, which was attributed to dye-dimer formation changing thepolynucleotide secondary structure into a B-form helix.[19] Incontrast to 4a,b, the close analogues 4c-f did not reveal slowkinetics of binding to any of the studied dsDNA/RNAs.

Addition of 4 c-f to studied polynucleotides resulted in de-creased intensity of the DNA/RNA CD bands below 300 nmdue to the partial unwinding of the DNA/RNA helix and conse-quent loss of helical chirality. Simultaneously, ICD bands wereobserved > 300 nm, whereby ICD band shape and sign wasstrongly dependent on a [compoundl/[polynucleotidel ratio (r),again indicating mixed binding mode (as observed in UVNis ti-trations, Figure 2; see also Supporting Information). At excessof DNA/RNA over dye (r < 0.2), ICD bands were rather weakand of positive sign (Supporting Information), whereas at r=0.2-0.5, dual-sign ICD bands appeared, originating from the di-merization of dye molecules along the polynucleotide (Fig-ures 4 and 5; see also Supporting lnforrnationl.l'" Intriguingly,the intensity and shape of dual-sign ICD bands were stronglydependent on a dye/polynucleotide combination, stressing thehigh potential of dye dimers in fine sensing of DNA/RNA sec-ondary structure, with respect to the steric and bond ing pa-rameters of minor and/er major grooves of a double helix(Table 4).

For the 4d, the ICD signal patterns can be correlated to theavailable space for the dimer formation within the DNA/RNAgrooves (Table 4). Namely, the width of a poly-dA-poly-dTminor groove (3.3 A) corresponds to the width of poly-A-poly-U major groove (3.8 Al. and also to the half width of the poly-dG-poly-dC minor groove (9.5/2=4.2 A; the guanine half of itsminor groove is occupied by guanine amino groups, thus leav-ing only the cytosine side of the minor groove for a dyedimer). All three polynucleotides yielded an ICD pattern char-acterized by anegative Cotton effect at 450-500 nm coupledwith apositive Cotton effect at 510-580 nm. However, the op-posite sign combination (positive Cotton effect at 450-520 nmcoupled with negative Cotton effect at 520-600 nm) was ob-

Table 4. Groove widths and depths for selected nucleic acid conforma-tlons.?"

Width [A] Depth [A]Major Minor Major Minor

poly-dAdT -poly-dAdT('( 11.2 6.3 8.5 7.5

poly-da-poly-dF'" 11.4 3.3 7.5 7.9poly---poly-U'" 3.8 10.9 13.5 2.8poly-dGdC -poly-dGdC(') 13.5 9.5 10.0 7.2

[al B-helical structure (i.e., B-form DNA); [b] C-helical structure (i.e., C-formDNA). [cl A-helical structure (i.e., A-form DNA).

served for alternating AT DNA characterized by a significantlybroader minor groove (6.3 A). Finally, poly-dGdC-poly-dGdC,with guanine amino groups protruding alternately from bothsides of the minor groove, thereby heavily impairing approachto the minor groove, yielded no dual-sign ICD bands (Support-ing Information), thus excluding dimer formation (Figure 4).

~!poly(dAdT),-2-4

1 f poly-dA-poly-dT ~o ----......... / ""'"

~-1 ~E

o 21o 1 poly-dG - poJy-dC ~Or-------~~~~-----T--~=-----~-1 ~

0.5

jJ AJU

::: ~y- -=s:z:=:400 425 450 475 500 525

)./ nm550 575 600

Figure 4. CD titration of the indicated polynucleotides (c= 3.0x 10-5 M) with4d at molar ratio r [4d]![polynucleotide] =0.5 (pH 7.0, sodium cacodylatebuffer, 1=0.05 M).

Compounds 4e,f are the shortest among the studied com-pou nds, as they lack both methyl groups on the dye's fusedbenzene ring, thus facilitating accommodation of- the dimerwith in the DNA/RNA grooves. Consequently, among the stud-ied dyes, only 4 e.f revealed exclusively positive ICD for dsRNAabove 475 nm. Furthermore, 4 e has the smallest overall struc-ture, in both the cyanine port ion as well as the flexible aliphat-ic phosphonium tail, allowing 4e dimer accommodationwithin the minor grooves of dsDNAs of varying steric proper-ties (Table 4); this yields similar ICD patterns for all DNA/RNA(Figure 5 al. However, the sterically more demanding triphenyl-phosphonium analogue 4 f revealed a distinctly different ICDpattern for dsDNAs (Figure 5 b) from that of 4e. The observedchang es in CD spectra (particularly ICD bands >400 nm) of4 f-GC DNA complexes were quite similar to those observedwith 4d, leading to the same conclusions: a 4 f dimer formedonly with a dGdC homo-polynucleotide, but not within thesterically hindered alternating dGdC-dGdC minor groove. Most

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a) 2b /"--..polv(o ~~__ -:-.p_O""Iy_(d:;:;::dC;:;::l;::;,=:;::::;=::::::::====::::::-2

~b~_Po'y_dC

g> -~~-4O ~ poly(dAdT), ," ", - - - poly-dA - poly-I


Table 5. Antiproliferative activity of 4(, 4d, 4e, and 4 f against a panel of normal and solid tumor celllines.

Compd IC50 [IlM]]']Normal lines Solid tumor lines

BJ MDCK AGS MIAPaCa2 CaCo-2 HEp-2 HeLa NCI-H358

4a >100 100 >100 >100 >100 >100 >100 >1004b >100 >100 >100 >100 >100 >100 >100 >1004c 240.1 >100 390.7 21 0.2 48 1.3 790.3 >100 >1004d 230.1 >100 41 0.1 320.1 >100 220.5 260.9 430.14e 520.1 >100 840.2 460.1 >100 51 0.1 >100 >1004f 600.1 790.2 37 0.7 13 0.3 4114.7 430.9 60.5 88 0.7DOX]b] 0.50.1 0.30.1 0.20.1 0.40.1 0.70.1 0.40.1 40.3 0.80.1

[al Drug concentration required to inhibit cell growth by 50%; data represent the mean SD of three independent experiments. Cells in exponentialgrowth phase were treated with substances for 72 h, and cytotoxicity was analyzed by MIT cell survival assay. [b] Doxorubicin.

Biological testing

Cytotoxicity assays

The six dyes 4a-f were tested in vitro for their cytotoxicity po-tential against normal and tumor cells (Table5). Apart fromcompounds 4a and 4b, which did not show activity againstany of the cell lines used in the study and are therefore exclud-ed from the discussion, the other studied compounds demon-strated unexpected variation in inhibitory activity, with ICsovalues ranging from 6x 10-6 to > 1x 10-4 M toward humansolid tumor cells and human foreskin fibroblasts (BJ cells).Under the same experimental conditions Madine-Oarby caninekidney (MOCK)cells were more resistant.

More detailed analysis of the resuIts revealed that the profileof growth inhibitory activity of all tested compounds againstMOCK cells is very similar, while for the human BJ fibroblasts,dyes 4c and 4d exhibited antiproliferative effects at concentra-tions twofold lower than for 4 e and 4 f. Generally, 4 f (benzo-thiazole quinolone with a triphenylphosphonium side chain)showed the strongest inhibitory potency on tumor cell growthamong all other tested compounds. The most sensitive to 4 fwere HeLa cells (lCso= 6x 10-6 M), showing potency similar tothat of the chemotherapeutic doxorubicin, used as reference.Intriguingly, 4 e, differing from 4 f only by amore flexible ali-phatic tributylphosphonium side chain, showed significantlylower cytotoxic potential against both normal and tumor cells.According to their antiproliferative potential, studied com-

pounds 4a-f can be divided in two categories of further re-search interest:

1) Compounds 4e,f revealed interesting antiproliferative ef-fects, as well as selectivity with respect to cell type, influ-enced by small modification of the phosphonium sidechain, strongly supporting more detailed studies into theirmechanism of cellular action (outside the scope of thiswork).

2) Compounds 4a,b were relatively inert toward all cell lines.Considering their intriguing spectrophotometric response,controlled by DNA/RNA secondary structure, 4a,b couldfind application as biologically safe biochemical markers.Thus, to gauge the possibility of 4a,b for use in monitoringcellular processes,we studied the cellular uptake and intra-

cellular distribution of these compounds, taking advantageof their strong fluorescence increase in visible the range(A-em: 540-620 nm) upon binding to biomacromolecular tar-gets.

Cellular uptake of compounds 4 a,b

Preliminary experiments performed by epifluorescence micros-copy revealed that 4a,b (e= 1.0x 1O-s M) efficiently enter HeLacells after somewhat longer period s of time (120 min) relativeto the commercially available DNA marker Hoechst 33258(30 min; data not shown). However, the distribution of 4a,bwith in the cell was different from that of Hoechst 33258,which accumulated within the cell nucleus. Fluorescence fromcompounds 4a,b was observed within the cytoplasm, mostlylocated in particles. Under visible light microscopy, no signifi-cant morphological changes of treated cells were observed,again underscoring the biological inertness of compounds4a,b.

To gain further insight regarding their intracellular targets,4a and 4 b were investigated in a co-Iocalization study usingconfocal microscopy. Live HeLa cells expressing mitochondria-targeted protein cyan fluorescence protein (CFP) with bluefluorescence emission (complementary to the green fluores-cence of 4a,b) were prepared'i'' and treated with 4a,b. Thefluorescence of 4a,b suggested dye accumulation in mitochon-dria (Figure 6a; see also Supporting lnforrnatlon), which wassupported by measuring CFP fluorescence in the same cells(Figure 6b and Supporting Information) and superimposingthe resulting micrographs (Figure 6c and Supporting Informa-tion). This revealed excellent co-Iocalization of CFP and 4a,b,indicating that after 120 min 4 a and 4 b (e= 1.0x 1O-s M) enterthe cells, pass the mitochondrial membrane, and accumulatewith high selectivity into the mitochondrial DNA and/or RNAexpressing stable green fluorescence, without a considerablephotobleaching effect.

ConclusionsTo explore in greater detail the fascinating selectivity potentialof cyanine phosphonium dyes (compounds 4a,b)}191 new ana-

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Figure 6. Fluorescence confocal micrographs of HeLa cells expressing mito-chondria-targeted CFP after incubation with compound 4a (e=1.0 x 10-5 Mlfor 120 min. al Fluorescence emitted by the dye 4a, A.ex: 514 nm, A.em: 540-620 nm. b) Fluorescence emitted by CFP, A.ex: 458 nm, A.em: 470-510 nm.cl Overlay of micrographs from panels al and b),

logues were prepared, the quinolone phosphonium portionwas unchanged, while the size and hydrogen bond ing proper-ties of the rest of molecule were varied. In comparison with4a,b, the pyridinium analogues 4c,d are extended by an addi-tional methylene unit, whereas the benzothiazolium analogues4eJ are sterically shortened; in addition, heterocyclic atomsare varied by position and positive charge. Comparison of theinteractions with dsDNA/RNA revealed that all three pairs(4a,b versus 4 c,d versus 4 e,f) bind in different ways to varioustypes of double-helical structures, yielding corresponding spe-cific spectrophotometric signals. It should be stressed that only4a,b revealed highly interesting and rare slow kinetics in struc-tural interchange from homo-polymeric to alternating-line ATsequence.l'"

The antiproliferative activity screening of studied com-pounds showed that 4a,b are nontoxic under conditions usedfor fluorescence and confocal microscopy. They accumulate ex-clusively in the mitochondria and show no photobleachingeffect, which makes them promising lead structures for the de-velopment of biochemical markers for cell biology.In contrast to 4a,b, the sterically less hindered compounds

4e,f revealed much more pronounced cytotoxicity, which canbeattributed to the intercalation capacity of these analoguesin GCDNA and dsRNA. Moreover, the cytotoxicity is stronglydependenton cell type as well as the nature of the phosphoni-umside chain, the latter property also having been observedin different interactions of 4 e versus 4 f with various dsDNAIRNA(Figure4). Such pronounced cytotoxic selectivity is un-commonfor DNA-targeting small molecules, which usually dis-

play similar toxicities toward all cell lines. This strongly sup-ports more detailed studies into the mechanism of biologicalaction for compounds 4e,f, which is outside the scope of thiswork. The convenient fluorimetric properties of 4e,f will be ofgreat assistance in this regard.

Our future research will also focus on the AT-targeting cya-nine dye dimers (both covalently and noncovalently bound).These are intriguing for many reasons, such as the importantrole played by AT sequences in dynamic protein-DNA recogni-tion events,'?' ar small molecules that bind to alternating ATsequences related to antiparasitic activity.[331 Moreover, thefinely tuned selectivity in spectrometric responses presentedherein offers a new approach to tools for the study of interac-tions between the nuclear genome and mitochondrial DNA,which was very recently shown to be essential for proper cellu-lar function.P"Thus, in our future research a new series of analogues is

foreseen based on the systematic modification of cationic sidechains similar to the phosphonium tails discussed herein: spe-cifically, the preparation of positively charged centers connect-ed to quinolone by propyl linkers, with positive charges steri-cally masked by aliphatic or aromatic substituents.

Experimental SectionMelting points were determined on a Kofler apparatus and are un-corrected. 1HNMR spectra were obtained on a Bruker Avance DRX600 MHz spectrometer in [D6lDMSOas solvent. Elemental analyseswere performed on a Vario 3 instrument. Absorption spectra werescanned on a Cecil Aurius 3021 spectrophotometer (1x 10-5M inCH30H). Intermediates 3a-e were synthesized by known proce-dures.[3S.36)All of the solvents were obtained commercially fromSigma-Aldrich and were used without further purification.

Synthesis

Tributyl-(3-iodopropyl)phosphonium iodide 1a and (3-iodopro-pyl)triphenylphosphonium iodide 1b: 1,3-Diiodopropane(0.01mol) and tributylphosphine or triphenylphosphine (0.009mol)were dissolved in dry hexane (50 ml). The reaction mixture wasflushed with argon and stirred at room temperature for 24 h. Thewhite precipitate was suction filtered and dried in a vacuum desic-cator. The target compounds were highly hygroscopic and wereused without further purification. Yield for 1a: 82%, for 1b: 92%.

4-Methyl-1-(3-(tributylphosphonio)propyl)quinolin-1-ium iodide2 a and 4-methyl-1-(3-(triphenylphosphonio)propyl)quinoliniumiodide 2b: The appropriate intermediate 1a or 1b (0.001mol) andlepidine (0.001mol) were melted together at 120C for 1 hin a 50-ml round-bottom flask equipped with a thermometer, reflux con-denser, and an electromagnetic stirrer. After cool ing to room tem-perature, dry acetone (10 ml) and dry Et20 (20 ml) were added.The formed precipitate was suction filtered, washed with Et20, andair dried. Intermediates 2 a and 2 b are highly hygroscopic andwere used directly in the next step. Yield for 2a: 76%, for 2b:84%.

Oyes 4a-f: Compounds 2a or 2b (0.001mol) and compounds 3a-e (0.001mol) were finely powdered together and suspended in8 ml EtOH (for dyes 4c-f) or 5 ml acetic anhydride (for dyes 4aand 4b) in areaction vessei equipped with an electromagnetic stir-

2013 Wiley-VCHVerlagGmbH&Co. KGaA,Weinheim ChemMedChem 2013, 8, 1093-11 03 1100

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org~~~~~==========~========~===rer. A twofold molar excess of N-ethyldiisopropylamine was addeddropwise for -1 min, and then the reaction mixture was stirred atroom temperature for 2 h. The resulting precipitate was suction-fil-tered, washed with CH30H, and dried in desiccator. For compounds4a and 4b when acetic anhydride was used as reaction media,Et20 was added to the reaction mixture, and the precipitated dyewas filtered off and dried in desiccator. The target dyes 4a-f werepurified by multiple recrystallizations from CHpH.

4-Methyl-2-{[1-(3-(tributylphosphonio)propyl)quinolin-4(1 H)-ylidene]methyl}oxazolo[4,5-b]pyridinium diiodide (4a): Pink solid(0.52 g, 67%): Rr=O (CH2CI/CH30H 10:1); mp: l34-l36C; IH NMR(600 MHz, [06]OMSO): 0=0.89 (t,)= 7.2 Hz, 9H, CH3), 1.36-1.41 (m,6 H, CH2), 1.42-1.44 (m, 6 H, CH2). 2.05-2.08 (m, 2 H, CH2), 2.19-2.23(rn, 6 H, P+CH2), 2.35-2.40 (m, 2 H, P+CH2), 4.24 (5, 3 H, N+CH3), 4.60(t, )=7.1 Hz, 2H, WCH2), 6.57 (5, 1 H, CH), 7.39 (t, )=7.2 Hz, 1H,ArH), 7.65 (t, )=7.4 Hz, 1 H, ArH), 7.92 (t, )=8.2 Hz, 1 H, ArHJ. 8.07(d, )=8.8 Hz, 1H, ArH), 8.18 (d, )=7.6 Hz, 1H, ArH), 8.33-8.36 (rn,3 H, ArH), 8.59 ppm (d, ) = 8.3 Hz, 1 H, ArH); 13CNMR (600 MHz,[OJOMSO): 0= l3.7 CH3, 15.6 CH2, 17.7 CH2< 17.9 CH2, 20.2 CH2,23.0 CH2, 23.8 CH2, 23.9 CH3, 57.1 CH, 119.7-159.5 CH; UVNis(CH30H): Amax (s) = 521 nm (108000); Anal. calcd for C32H46N30P212:C 49.69, H 5.99, N 5.43, O 2.07, found: C 49.73, H 5.97, N 5.45, O2.10.

4-Methyl- 2{[ 1-(3-(triphenylphosphon io )propyl)qu inoli n-4( 1H)-ylidene]methyl}oxazolo[4,5-b]pyridinium diiodide (4b): Pink solid(0.33 g, 40%): Rr=O (CH2CI/CHPH 10:1); mp: 266-269C; IH NMR(600 MHz, [06l0MSO): 0=2.09-2.12 (m, 2H, CH2), 3.74-3.78 (m, 2H,P+CH2), 4.2 (5, 3H, NCH3), 4.63 (t, )=7.2 Hz, 2H, WCH2), 6.53 (5,1 H, CH), 7.36 (t, )=6.6 Hz, 1H, ArH), 7.59 (t, )=7.81 Hz, 1 H, ArH),7.72-7.79 (m, l3H, ArH), 7.84 (t, )=8.1 Hz, 1H, ArH), 7.88-7.90 (m,4H, ArH), 8.15 (d, )=7.7 Hz, 1 H, ArH), 8.26 (d, )=7.3 Hz, 1H, ArH),8.30 (d, ) = 6.6 Hz, 1 H, ArH), 8.53 ppm (d, ) = 7.9 Hz, 1H, ArH);13CNMR (600 MHz, [OJOMSO): 0=18.5 CH2, 20.1 CH2, 22.5 CH2,29.7 CH3, 59.3 CH, l30.5-215.4 CH; UVNis (CH30H): Amax (10)=521 nm (204000); Anal. calcd for C3sH34N30P12:C 56.27, H 3.37, N4.24, found: C 56.31, H 3.42, N 4.19.

4,6-0imethyl- 2-{[ 1-(3-(tributylphos phonio )propyl)qu inolin-4( 1H)-ylidene]methyl}thiazolo[4,5-b]pyridinium dihexafluorophos-phate (4c): Oark-purple solid (0.81 g, 96%): Rr=O (CH2CI/CH30H10:1); mp: l38-140C; IHNMR (600 MHz, [OJOMSO): 0=0.89 (t,)=7.1 Hz, 9H, CH3), 1.39-1.45 (rn, 12H, CH2), 2.17-2.20 (m, 2H,CH2), 2.40 (5, 3 H, CH3), 2.58-2.60 (m, 6 H, P+CH2), 3.72-3.77 (m, 2 H,P+CH2), 4.03 (5, 3H, NCH3), 4.75 (t, )=9.1 Hz, 2H, WCH2), 6.53 (5,1 H, CH), 7.01 (d, )=7.2 Hz, 1H, ArH), 7.51 (d, )=7.2 Hz, 1 H, ArH),7.83 (t, )=6.1 Hz, 1H, ArH), 8.06 (t, )=7.7 Hz, 1H, ArH), 8.255 (1 H,ArH), 8.32 (5, 1H, ArH), 8.42 (5, 1 H, ArH), 8.70 (d, )=7.3 Hz, 1H,ArH); 8.86 ppm (d, ) = 7.6 Hz, 1 H, ArH); 13CNMR (600 MHz,[OJOMSO): 0= l3.7 CH3, 17.5 CH2, 17.8 CH2, 20.2 CH2, 22.9 CH2,23.0 CH2, 23.8 CH2, 23.9 CH3, 129.5 CH, l30.2-161.8 CH; UVNis(CH30H): Amax (10)=549 nm (l31 000); Anal. calcd for C33H4sF12N3P3S:C 47.20, H 5.76, N 5.00, found: C 47.22, H 5.79, N 5.03.

4,6-0i methyl- 2-{[ 1-(3-(triphenyl ph osphon io) propyl)q uinolin-4(1 H)-ylidene]methyl}thiazolo[4,5-b]pyridinium diiodide (4 dl:Oark-purple solid (0.55 g, 64%): Rr=O (CH2CI/CH30H 10:1); mp:266-269C; IH NMR (600 MHz, [OJOMSO): 0=2.08-2.10 (rn, 2H,CH2), 2.37 (5, 3 H, CH3), 3.70-3.82 (rn, 2 H, P+CH2), 4.26 (5, 3 H, WCH3), 4.58 (t, )=7.4 Hz, 2H, NCH2), 6.99 (5, 1 H, CH). 7.44 (d, )=7.1 Hz, 1 H, ArH), 7.54 (t, )=7.2 Hz, 1H, ArH), 7.72-7.96 (rn, 15H,ArH), 8.16 (d, ) = 7.5 Hz, 1H, ArH), 8.38 (5, 1H, ArH), 8.52 (5, 1H,ArH), 8.71 (d, )=7.1 Hz, 1 H, ArHJ. 8.82 ppm (d, )=8.0 Hz, 1H, ArH);13CNMR (600 MHz, [OJOMSO): 0=19.8 CH2, 21.1 CH2, 30.3 CH3,

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2013, 8, 1093 -11 03 1101

32.5 CH2, 58.9 CH, l35.5-199.5 CH; UVNis (CH30H): Amax (e)=549 nm (150000); Anal. calcd for C39H3612N3PS:C 54.24, H 4.87, N4.20, S 3.71, found: C 54.21, H 4.91, N 4.19, S 3.71.

3-Methyl-2-{[1-(3-(tributylphosphonio)propyl)quinolin-4(1 H)-yli-dene]methyl}benzo[d]thiazolium diiodide (4 el: Orange solid(0.49g, 63%): Rr=O (CH2C1/CH30H 10:1); mp: 116-118C; IH NMR(600 MHz, [OJOMSO): 0=1.21 (t,)= 7.2 Hz, 9 H, CH3), 1.22-1.31 (m,6H, CH2), 1.36-1.38 (m, 6H, CH2), 2.16-2.21 (m, 6H, P+CH2), 4.04 (5,3 H, WCH3), 4.68 (t, ) = 7.4 Hz, 2 H, NCH2), 6.96 (5, 1 H, CH), 7.39 (d,)=7.2 Hz, 1 H, ArH), 7.62-7.63 (m, 1 H, ArH), 7.64-7.65 (m, 1H, ArH),7.80-7.81 (m, 1H, ArH), 7.82 (d, )=8.4 Hz, 1H, ArH), 7.98-8.01 (m,1H, ArH), 8.08 (d, )=7.9 Hz, 1 H, ArH), 8.20 (d, )=8.8 Hz, 1H, ArH),8.63 (d, )=7.2 Hz, 1H, ArH), 8.83 ppm (d, )=8.3 Hz, 1H, ArH);13CNMR (600 MHz, [OJOMSO): 0=13.8 CH3, 17.6 CH2, 17.9 CH2,23.1 CH2, 23.1 CH2, 23.7 CH2, 23.9 CH2, 34.5 CH3, 88.9 CH, 108.2-168.5 CH; UVNis (CH30H): Amax (10)=508 nm (67 800); Anal. calcdfor C33H4712N2PS:C 50.26, H 6.01, N 3.55, found: C 50.31, H 5.98, N3.55.

3-Methyl- 2-( (1-(3-(tri phenylphos phonio )propyl)qu inoli n-4(1 H)-ylidene)methyl)benzo[d]thiazolium diiodide (4 f): Orange solid(0.48 g, 57%): Rr=O (CH2CI/CH30H 10:1); mp: 265-268C; IH NMR(600 MHz, [OJOMSO): 0=2.16-2.18 (rn, 2H, CH2), 3.79-3.84 (m, 2H,P+CH2), 4.05 (5, 3H, N+CH3), 4.77 (t, )=7.5 Hz, 2H, NCH2), 6.95 (5,1H, CH), 7.38 (d, )=7.2 Hz, 1 H, ArH), 7.45 (t, )=7.4 Hz, 1H, ArH),7.51 (t, )=7.4 Hz, 1 H, ArH), 7.64 (t, )=7.6 Hz, 1 H, ArH), 7.70 (t,)=7.8 Hz, 1 H, ArH), 7.78-7.97 (rn, 15H, ArH), 8.04 (d, )=8.7 Hz, 1H,ArHJ. 8.08 (d, )=7.8 Hz, 1H, ArH), 8.23 (d, )=7.8 Hz, 1 H, ArH), 8.60(d, )=7.3 Hz, 1 H, ArH), 8.81 ppm (d, )=8.3 Hz, 1H, ArH); 13CNMR(600 MHz, [06l0MSO): 0=29.4 CH2, 29.7 CH2, 30.2 CH2, 31.5 CH3,82.1 CH, l30.7-197.5 CH; UVNis (CH30H): Amax (10)=508 nm (76500); Anal, calcd for C39H3SI2N2PS:C 55.20, H 4.16, N 3.30, found: C55.25, H 4.15, N 3.29.

Study of DNA/RNA interactions

UVNis, CD, and fluorescence spectroscopy: UVNis spectra wererecorded on a Varian Cary 100 Bio spectrophotometer, fluorescencespectra on a Varian Cary Eclipse spectrofluorimeter, and CD spectrawere collected with a Jasco J-810 spectropolarimeter at 25C usingquartz cuvettes of 1 cm path length. The polynucleotides: poly-dGdC-poly-dGdC, poly-dAdT -poly-dAdT, poly-dG-poly-dC, poly-dA-poly-dT, poly-A-poly-U and calf thymus (ct) DNA (Sigma-AI-drich, St. Louis, MO, USA) were dissolved in sodium cacodylateb uffe r, 1= 0.05 M, pH 7; ctONA was additionally sonicated and fil-tered through a 0.45 um filter. Aqueous solutions of compoundswere buffered to pH 7 (sodium cacodylate b uffe r, 1=0.05 M). Thepolynucleotide concentration was determined spectroscopically asthe concentration of phosphates.P" Spectrophotometric titrationswere performed at pH 7 (sodium cacodylate buffer, 1= 0.05 M) byadding portions of polynucleotide solution into the solution of thestudied compound. CD experiments were performed by adding ali-quots of the aqueous solutions of compounds into the solution ofpolynucleotide. In fluorimetric experiments the excitation wave-length above 500 nm (A.x> 500 nm) was used to avoid potentialinner-filter effects caused by increasing absorbance of the polynuc-leotide. The emission spectra were collected in the range A.m: 510-650 nm. The titration data were processed using the Scatchardequation.P" and the resulting values n (ratio between the concen-tration of dye-polynucleotide complex and the concentration ofthe polynucleotide) were in the range of n=0.1-0.5; however, forthe purpose of comparison all Ks values, binding constants, were

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org============-===============================-re-calculated at a fixed value of n=0.2. Values for KS' listed inTable 2, have acceptable correlation coefficients (> 0.99).

Thermal denaturation experiments: Thermal melting curves fordsDNA, dsRNA, and their complexes with studied compounds weredetermined as previously described by following the change in theabsorption at 260 nm as a function of temperarure.P" The absorb-ance of the liga nds was subtracted from each curve, and the ab-sorbance scale was normalized. The Tm values are the midpoints ofthe transition curves, determined from the maximum of the firstderivative and checked graphically by the tangent method. LiTmvalues were calculated by subtracting the Tm of the complex fromthe Tm of the free nucleic acid. The reported LiTm values (with in-strumental error of O.5C) are the average of at least duplicateindependent measurements.

Viscometry: Viscometry measurements were conducted with anObbelohde viscometer system AVS 350. The temperature wasmaintained at 25 0.1 "C. Aliquots of dye stock solutions wereadded to 3 ml of 5 x 10-4 M ctDNA solution in sodium cacodylate

. b uffe r, 1=0.05 M, pH 7. The ratios r, between the concentrations ofctDNA and the studied compounds, were 0.075, 0.1, and 0.15. Dilu-tion never exceeded 4% and was corrected in the calculations. Theflow times were measured at least five times optically by light bar-rier with a deviation of 0.02 s. The viscosity index a was obtainedfrom the flow times at varying r according to the following equa-tion :[29.38]

for which to. tDNA, and r, denote the flow times of buffer, free DNA,and DNA complex at reagent/phosphate ratio r, respectively; LlLo isthe relative DNA lengthening. The plot of LlLo as a function of rwas fitted to a straight line that gave slope a; the error in a is0.1.

Biological experiments

Cell culture: The normal human foreskin fibroblast (BJ) and Madine-Darby canine kidney (MDCK) cell lines, as well as pancreatic carci-noma (MiaPaCa2), colon adenocarcinoma (CaCo-2), larynx carcino-ma (HEp-2), cervix adenocarcinoma (Hela), stomach epithelial gas-tric adenocarcinoma (AGS), and bronchoalveolar carcinoma (NCI-H358) tumor cell lines were cultured as monolayers in tissue cul-ture flasks (25 and 75 crrr'). BJ cells were used between 27-29 pas-sages, while MDCK cells were used between 24-26 passages. Cellswere cultured in Dulbecco's modified Eagle's medium (DMEM,Gibco, EU) supplemented with 10% heat-inactivated fetal bovineserum (FBS, Gibco, EU), 2 mM glutamine, and 100 U/O.1 mg penicil-lin/streptomycin. For cultivation of the AGS and NCI-H358 celllines, RPMI 1640 medium (Gibco, EU) supplemented with 10%heat-inactivated FBS (Gibco, EU), 2 mM glutamine, 1 mM sodiumpyruvate, 10 mM HEPES, and 100 U/O.l mg penicillin/streptomycinwas used. For detachment from the flask surface, cells were treatedwith 0.25 % trypsin/EDTA solution. Cells were cultured in a humidi-fied atmosphere at 3rC, 5% CO2 (Shell lab incubator, SheldonManufacturing, USA). The trypan blue dye exclusion method wasused to assess cell viability.

Cytotoxicity evaluation by MIT assay:[39] Tested compounds wereprepared as stock solutions (1 x 10-2 M) in DMSO. Doxorubicin wasdissolved in highly pure water as a stock solution at 1 x 10-2 M.Working solutions (10-3-10-7 M) were prepared in water of highestpurity prior to testing. The effect of solvent (DMSO) on cell growth

(1 )

was also tested by adjusting the concentration range to be thesame as in working concentrations of tested compounds. Cytotox-icity effects were determined by MTI (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brom ide) assay. Cells were seeded in 96-micro-well flat-bottorn plates (Greiner, Austria) at a concentrationof 2xl04 cells ml, " and incubated overnight to allow attachmentto the bottom of the wells. After 72 h incubation with test com-pou nds, the growth medium was discarded, and MTI solution(50 fll-, 5 mqrnl,"') was added. After 4 h incubation at 37(, water-insoluble MTI formazan crystals were dissolved in DMSO. Plateswere shaken at high speed to ensure total dissolution of crystal s,which is required for absorbance measurements. Absorbance wasmeasured at 570 nm on an ELISA micro plate reader (Stat fax 2100,Pharmacia Biotech, Uppsala, Sweden). Control cells untreated withcompound solution were grown under the same conditions. All ex-periments were performed at least three times in triplicate. ICsovalues, defined as the compound concentration leading to 50%decrease in cell viability, were calculated and used as a comparisonparameter. Doxorubicin was used as a reference compound.

Confocal microscopy: Transfected Hela cells (5 x 105) stably express-ing mitochondria-targeted cyan fluorescence protein (CFP)[31lwereseeded on glass-bottom Petri dishes (MatTek Corp., USA). The ex-pression plasmid carrying the mitoCFP gene was a generous giftfrom Dr. Yaron Shav-Tal, Bar-Ilan University, Ramat-Gan, Israel). After24 h cultivation at 37C in a humidified atmosphere with 5% CO2,compounds 4a and 4b were added (c= 10-5 M). Cells were furtherincubated at 37C for 120 min and analyzed by confocal microsco-py (leica TCS SP2 AOBS), with A. 458 and 514 nm excitation for CFPand 4a or 4 b, respectively; emission was detected at A. 470-510 nm for CFP and at A. 540-620 nm for 4a or 4b.

Acknowledgements

I.P., O.v., and L.G.-O. thank the Ministry of Science, Educationand Sport of the Repub/ic of Croatia for financiai support of this

study (grants 098-0982914-2918, 219-0982914-2176, and 098-

0000000-2463). This work was supported in port by the Bulgarian

Scientific Research Fund (grant DO 02-82/2008, project "Union".

We also thank Maja Herak Bosnar and Robert Belui for theirhelp in performing mitochondria-related experiments.

Keywords: cellular uptake circular dichroism DNA

recognition fluorescence . mitochondria RNA recognition

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Received: February 22, 2013Revised: April 24, 2013Published online on May 31, 2013

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