Synthesis of 5‐Alkyl‐ and 5‐Phenylamino‐Substituted Azothiazole Dyes with Solvatochromic and DNA‐Binding Properties

Abstract A series of new 5‐mono‐ and 5,5′‐bisamino‐substituted azothiazole derivatives was synthesized from the readily available diethyl azothiazole‐4,4′‐dicarboxylate. This reaction most likely comprises an initial Michael‐type addition by the respective primary alkyl and aromatic amines at the carbon atom C5 of the substrate. Subsequently, the resulting intermediates are readily oxidized by molecular oxygen to afford the amino‐substituted azothiazole derivatives. The latter exhibit remarkably red‐shifted absorption bands (λ abs=507–661 nm) with high molar extinction coefficients and show a strong positive solvatochromism. As revealed by spectrometric titrations and circular and linear dichroism studies, the water‐soluble, bis‐(dimethylaminopropylamino)‐substituted azo dye associates with duplex DNA by formation of aggregates along the phosphate backbone at high ligand–DNA ratios (LDR) and by intercalation at low LDR, which also leads to a significant increase of the otherwise low emission intensity at 671 nm.


Introduction
Aromatic azo compounds have been extensively investigated with regard to their photophysical and photochromic properties since they are commonly utilized in severald ifferent fields. [1] For example, they are used in solar cells [2] and solar thermalf uels, [3] sensors, [4] photopharmacology, [5] and biomedical applications. [6] Furthermore, they have been incorporated as molecular switchesi nto polymers [7] and carbohydrates [8] as well as into biological systems [9] such as oligonucleotides, [10] peptides, andp roteins. [11] And azo derivatives have also been used in the design of (photoswitchable) ligands for duplex [12] and quadruplex DNA. [13] Most importantly,a zo dyes represent the largestg roup of colorants with respect to their number and production volume in the chemical industry,m ainly because they generally exhibit high molare xtinction coefficients and colorfastness. [14] Moreover,t he most commonly employed synthesis of azo dyes by diazotization and azo coupling is not just straightforwardb ut offersahuge structural diversity. [14,15] It is well known that the introductiono fd ifferent electron-donating and electron-withdrawings ubstituents into the structure of azobenzene enables as ignificant variation of color. [14] In this regard,t he replacement of one or even both phenyls ubstitu-ents by ah eterocyclic unit, for example, pyridines, indoles, purines, thiophenes or various azoles, has recently gained great attention in order to furtherf ine-tune the optical and photochromic properties for different applications. [16] Notably,t he development of disperse azo dyes that are synthesized from heteroaromatic diazonium or heteroaromatic coupling components has already startedo ver 30 years ago [17] and led, for example,t ot he replacement of red andb lue anthraquinone dyes. [18] Thiazoles in particular have often been employed for the design of hetaryl-substituteda zo derivatives, and the photophysical properties and colorfastnesso ft hose azo dyes have been studied frequently. [19] Specifically,t he integration of a thiazoleu nit may lead to biologically activea zo compounds, as thiazoles possess great biological relevance. For example, one of the most important compounds comprising at hiazole subunit is thiamine (vitaminB 1 ). [20] Thiazoles also occur in several other naturalp roducts such as cyclopeptides, whichh ave shown cytotoxica ctivity [21] or antibiotic properties. [22] It was found, too,t hat thiazolep olyamides, such as thiazotropsinA (1a)a nd thiazotropsin B( 1b)a sw ell as their analogues (e.g. 1c), bind to the minor groove of DNA ( Figure 1). [23] Furthermore, some thiazole-based derivatives exhibit antiproliferative activity against selected human cancer cell lines [24] and antibacterial activity. [25] In general,t hiazoles are synthesized by the Hantzsch reaction, which is the condensation of an a-halocarbonyl with a primary thioamide. [26] By replacingt he thioamide with bisthiourea hydrazothiazoles are available, which may be further oxidizedb yn itrous or nitric acid to afford the symmetric azothiazolederivatives 2a-h ( Figure 2). [27] In our search for novel, potentially photoswitchable DNAbinding ligands, [12,13] we identified the ester-substituted azothiazolederivatives 2e-h as promising platform for the synthesis of polyamides with DNA-binding properties. These substrates are easily accessible and, even more importantly,t he two identicalt hiazole substituents would allow as imultaneous modification of the ester groups on both sides of the azo unit. Remarkably,t he corresponding azothiazole carboxylic acids, which would serve as useful intermediates for furtherd erivatization, have not been reported, so far.I nf act, attempts of their synthesis by saponificationo f2e-h failed. [27c,d] Based on these reports, we tried to perform ad irect amidation of the ester groups by the reaction with amines;h owever,i no ur attempt to functionalize the 4,4'-substituted derivative 2g,w es urprisingly discovered that the reaction of these substrates led to the formationo fs trongly colored 5-alkylamino-or 5-phenylamino-substituted azothiazolesi nstead.A st his appearedt ob e an efficient access to novel amino-substituted azothiazole derivatives with ap ronounced red-shifted absorption from readily available substrates, we investigated this reactioni nm ore detail. And herein, we present the scope and limits of this synthetic approach, and we demonstrate that the resulting 5alkyl-and 5-phenylamino-substituted azothiazolesh ave favorable absorption and DNA-binding properties.

Synthesis
The knowna zothiazole diethyl ester 2g was synthesized by condensation of 2,5-dithiobiurea (3)w ith ethyl 3-bromopyruvate followed by oxidation of the intermediate hydrazothiazole 4 according to literature procedure (see Supporting Information). [27d] The addition of n-butylamine( 5a)t oas uspension of 1 and MgCl 2 as Lewis acid in THF (Scheme 1) [28] resulted in an immediate color change of the suspension from orange to magenta indicating ad rastic bathochromic shift of the absorption maximum caused by significant structural changes in the conjugated aromatic system.T he startingm aterial 2g slowly dissolved and-as indicated by TLC control-was consumed after 3h of stirring. Furthermore, an ew intense magenta-and a faint purple-colored spot were detected duringT LC analysis (SiO 2 , n-hexane/EtOAc 7:3, R f = 0. 31 and 0.44). The magentacolored main product was isolated by columnchromatography (Table 1, entry 1). The NMR-spectroscopic and the mass-spectrometrica nalysis revealed that the ester functionalities were intact and that the amide 6 was not formed. Instead, the azothiazole 2g underwent formally as ubstitution in 5-position resulting in the formation of the 5-butylamino-substituted azothiazole 7a.T his unexpected finding led us to investigate the scope of the reactionf urther. We already suspected that the purple-colored spot corresponded to the bis-substituted product 8a and, in fact, prolongation of the reactiont ime and the use of five molar equivalents (equiv.) of n-butylamine (5a)r esulted in the formation of 8a along with merely trace amounts of 7a (Table 1, entry 3).
Similarly,t he reactiono f2g with N,N'-dimethyl-1,3-propanediamine (5b), that was chosen because ad imethylamino group potentially increases water solubility,g ave the respec-  tive bis-substituted azothiazole 8b (entry 6). In order to allow furtherf unctionalization of the addition product we also wanted to introduce an alkyl chain bearing at erminal amino group. To avoid possible intermoleculars ide reactions, 2g was treated with the mono-Boc-protected propane-1,3-diamine (5c). However, even with relatively long reaction times (11d ) and 6m olar equivalents of the amine (entry 8), the conversion to the bis-substituted product 8c was very slow.A nd it turned out to be tedious work to separatet he mono-a nd bis-substituted products 7c and 8c by column chromatography with conventional solvent systems (DR f < 0.1). Hence, in order to simplifyp urification and increasey ields, the reaction conditions were optimized such that ideally either the respective mono-o rt he bis-substituted azothiazole are formed exclusively.F irstly,t he solventw as changed from THF to MeCN. Depending on the reactiont ime, the mono-substituted product 7c and the bis-substituted 8c were isolated after 5a nd 19 hi n 12 and 5% yield, respectively (entries 9, 10). Remarkably,t he addition of 2equivalents of 1,4-diazabicyclo[2.2.2]octane (DABCO) as an additional base led to as ignificant increase in the yield of 8c to 35 %i namuch shorter reactiont ime (entry 11). In CHCl 3 as the solvent, the bis-substituted azothiazole 8c was not formed even after 120 do fs tirring, and the monosubstituted dye 7c wasi solated as the only addition product in 16 %y ield (entry 7). Likewise, the reactionw ith the primary amines 5a and 5b in CHCl 3 gave the mono-substituted derivatives 7a and 7b as the only isolated addition products (entries 2, 5). Interestingly,i nt he case of 7b,t he yield wass lightly lower in the presenceo fo ne equivalent of DABCO (entry 4). The reactiono fs ubstrate 2g with the aromatic amines p-toluidine (5d)a nd p-(dimethylamino)aniline (5e)w as also attempted to obtain the respective 5-phenylamino-substituted derivatives. While the reactionw ith 5d under the optimized reaction conditions in MeCN with DABCO as base gave the products 7d and 8d in 18 and 3% yield, respectively,t he derivatives 7e and 8e were hardly available by this method ( entries 12, 13). Even with an excesso f5e (8 molar equivalents), we were only able to isolate the bis-substituted product 8e in ay ield of 1%.

Absorption properties
The absorption properties of the azothiazole derivatives 2g, 7a--d, 7f,a nd 8a--f were investigated in representative nonpolar (n-hexane, CHCl 3 ), polar aprotic (THF,a cetone, MeCN, DMSO)a nd polar protic solvents (H 2 O, MeOH, EtOH) ( Table 2, Supporting Information Table S1). The solubility of the parent azothiazole 2g is somewhatl imited,a nd the shift of itsl ongwavelength absorption maximum is essentially independent from the solventa nd just ranges from 397 nm in MeCN to 401 nm in THF (e max = 16 300-20 000 m À1 cm À1 )( Figure S2 A). In contrast, the monoalkylamino-substituted derivatives 7a-c are sufficiently soluble in the investigated solvents. Furthermore, these strongly magenta-colored dyes show broad absorption maximat hat are significantly red-shifted in comparison to the parenta zothiazole 2g (l abs = 507-577 nm, e max = 33 800-48 000 m À1 cm À1 )( Table 2, Figure S2 B-D). As ag eneral trend, the absorption bands of 7a--c are red-shifted going from a nonpolars olvent( n-hexane for 7a and 7b,C HCl 3 for 7c)t o DMSO.R emarkably,t he absorption maxima of the derivative 7b with the terminal dimethylamino group are slightly redshifted (4-9 nm) in comparison to 7a or 7c in all solvents except DMSO (31-36 nm). Thea forementioned trends also apply to the respective bisalkylamino-substituted azothiazoles 8a-c,b ut the additional amino functionalityg enerally causes a more pronounced bathochromic shift of the long-wavelength Scheme2.Synthesis of the 5-ammoniumpropylamino-substituted azothiazole derivatives 7fand 8f. Table 2. Absorption Properties of the Azothiazole Derivatives 2g, 7a-d and 8a-e. absorption band (l abs = 556-600 nm) and higher extinctionc oefficients (e max = 48 700-71 700 m À1 cm À1 ), so that these dyes are strongly purple-coloredi ns olution (Table 2, Figure S3 A-C, Figure 3A). The monophenylamino-substituted azothiazole 7d exhibitst he strongest positive solvatochromism;n amely,t he absorption band is red-shifted by 74 nm from n-hexane( l abs = 526 nm) to DMSO (l abs = 600 nm) ( Figure 3B,S 2E). The spectra of the bisphenylamino-substituted dyes 8d and 8e show absorptionb ands that already lie in the red region of the visible spectrum( l abs = 600-619nmf or 8d and l abs = 626-661 nm for 8e)( Ta ble2,F igure S3 D-E). Unfortunately,t heir absorption properties in polar protic solvents and n-hexane could not be determined due to the very low solubility in these solvents.

DNA-binding properties
The changes of the absorbance upon addition of doublestranded calf thymus (ct) DNA to the azothiazoles 7b, 8b,a nd 8f were followed by spectrophotometric titrations (Figure 4, Figure S6, Ta ble 3). The addition of ct DNA to as olution of 7b caused only as light decrease of the absorbance and as mall red shift (Dl = 3nm) ( Figure 4A1), whereas the addition of DNA to asolution of 8b also resulted in the formation of adistinct additional blue-shifted band with am aximum at 499 nm along with the decrease of the initial absorbance and formation of an isosbestic point at 515 nm. At as maller ligand-DNA ratio (LDR) of < 1.4 the blue-shifted peak disappeared and the absorbance increased, so that eventually ar ed shift of Dl = 9nmo ft he maximum was observed( Figure4A2). The titration  of ct DNA to 8f essentially resulted in the same course of the titration,t hat is, the formation of ab lue-shifted band located at 504 nm at LDR > 0.8 and as ubsequent increasei na bsorbance with ar ed shift of Dl = 8nm( Figure S6).
Additionally,t he interactions of the dimethylaminopropylamino-substituted derivatives 7b and 8b with the quadruplexforming oligonucleotide d[A(GGGTAA) 3 GGG] (22AG)a sw ell as with polystyrene sulfonate (PSS) were studied. While the titration of 22AG to as olution of 7b only led to av ery small decrease of the absorbance ( Figure 4B1), the addition to 8b also resultedi napronouncedr ed shift of the absorption maximum (Dl = 12 nm) ( Figure 4B2). The results of the photometric DNA titrations were analyzed according to the established protocol, [29] but the fitting of the binding isotherms to the theoretical model was only possible for titration of 8b with 22AG (K 22AG = 2.4 10 4 , n = 2.1) (Inset in Figure C2). On titration of PSS to 7b and 8b,t he absorbance at 528 nm (7b)a nd 576 nm (8b)d ecreased until ligand-PSS ratios of > 60 (7b)a nd > 115 (8b)w ere reached (Figure 4C1a nd C2). This hypochromic effect was accompanied by ah ypsochromics hift of the absorptionb and to 508 nm (7b)a nd 513 nm (8b). In both cases, the absorption increased at lower ligand-PSS ratios, and when saturation was reached the absorptionm aximaw ere eventually red-shifted by 7nma nd 13 nm, respectively,i nc omparison to the pure ligand solutions.
Upon excitation at l ex = 515 nm the derivative 7b is essentially non-fluorescent both in the absence or presence of DNA or PSS. The azothiazole 8b,h owever,e xhibits ab road emission band with very low fluorescencei ntensity (F fl = 0.002 relative to cresyl violet [30] )a t6 71 nm. The addition of ct DNA to 8b led initially to af urther decrease of the fluorescencei ntensity with increasing DNA concentration until an LDR of > 3w as reached ( Figure 5A). At lower LDR values the development of an ew blue-shifted emission band was observed (I/I 0 = 39, F fl = 0.029) ( Table 3). The addition of 22AG to 8b resultedi nt he formation of essentially the same blue-shifted emission band ( Figure 5B) with as maller light-up effect (I/I 0 = 9). On titration of PSS to 8b the increaseo ft he fluorescence intensity at low ligand-PSS ratios of < 8w as, however,s lightly more pronounced( I/I 0 = 50) ( Figure 5C)a sc ompared to addition of ct DNA. Remarkably, a solution of 8b in ah ighly viscous mediums uch as glycerol also exhibitedabroad fluorescenceb and at l fl = 655 nm (F fl = 0.10) ( Figure S1).
In order to examine the binding mode of 8b with DNA, circular dichroism (CD) and linear dichroism (LD) studies with ct DNA were performed ( Figure 6). At small LDR values of 0.2 and 0.5 no apparent induced circulard ichroism (ICD) band was detected ( Figure 6A), but ab road negative LD signal at around 585 nm appeared ( Figure 6B). At an LDR > 0.5 an intense ICD band at 410-590 nm with ab isignate shape and an isoelliptic point at 500 nm as well as an egative LD signala t5 00 nm developed,w hile the latter signal decreased in intensity from LDR = 1t o2 .N otably,t he intensity of the negative LD signal of the DNA at 258 nm decreased and eventually vanished at LDR = 2.

Synthesis
Our attempts to perform an amidation of the azothiazole diester 2g led to the discovery of novel 5-alkylamino-and 5-phenylamino-substituted azothiazole derivatives, that resulted from the initial nucleophilic addition of the respective amines at the C5 carbon atom. Usually,t hiazoles readily react in elec- Table 3. Absorption and Emission Properties of 7b and 8b in the Presence of ct DNA, 22AG and PSS.
[b] Molar extinction coefficient. [c] Shift of the long-wavelengtha bsorptionm aximum between free and bound ligand( in nm).
[ e] Shiftof the long-wavelength emission maximum between free and bound ligand (in nm).
[f] Not determined. trophilica romatic substitution reactions, if the thiazole ring is substituted with electron-donating groups. [31] For example, 2aminothiazole reacts with electrophiles such as bromine or diazonium salts at C5 to afford the respective 5-bromo-and 5arylazo-substituted thiazoles. [19d,e, 26, 32] The ester functionality of azothiazole 2g at C4,h owever,s ignificantly reduces the electron density of the thiazole ring. Therefore, it stands to reason that in the first step of the reaction mechanism, the thiazole undergoes aMichael-type addition at C5,which is in b-position to the ester (Scheme 3A). This proposedm echanism is consistent with the observation that the reaction with ar ather weak nucleophile such as p-toluidine (5d)g ave lower yields of the addition products 7d and 8d (Table1,e ntry 12) as compared to the reactions with the primary alkyl amines 5a--c,e ven thoughl onger reactiont imes were employed. Thev ery low yield of 8e (Table 1, entry 13) is probablyc aused by slow oxidation of p-(dimethylamino)aniline (5e)u nder the employed aerobic conditions. During the optimization of the reaction conditions, we found that the addition of DABCO led to as ignificantlyi ncreased yield of derivative 8c (Table 1, entries 10 and 11). However,i ts hould be notedt hat the substrate 2g also slowly decomposes in the presence of ab ase such as DABCO and therefore, the reaction has to be monitored carefully in order to avoid the formation of unidentified side prod-ucts. Nevertheless, the presenceo fD ABCO in the reaction mixture appearstobeadvantageous. In fact, it has been showna lready that the oxidation of thiazoline 4-carboxylate to thiazole 4-carboxylates by molecular oxygen is supported by bases, presumably as the latter promotes the formation of the enolate A (Scheme 3), which is ap rerequisite for the subsequent oxidation to B. [33,34] Hence, we propose that the a-hydroxy thiazoline C is formeda sa ni ntermediate that finally undergoes elimination, supposedly also assisted by DABCO, to achieve aromatization [20] and to form the monoamino-substituted thiazole 7.T he electron-donating 5-amino-substituent, however,r educes the reactivity of the mono-substituted azothiazoles so that these intermediates are less susceptible towards as econd nucleophilic addition at C5',a ss hown by the respective resonance structure of 7 (Scheme 3). This particular electron distribution resultsi nalower reactionr ate and therefore longer reactiontimes are required to obtain the bis-substituted products (Table 1, entries 3a nd 6). Remarkably,t he monoalkylamino-substituted azothiazoles 7a--c were isolated as the only addition products (Table 1, entries 2, 4, 5, 7), when the reactions of azothiazole 2g with the alkylamines 5a-c were carriedo ut in CHCl 3 ,w hile the use of an aprotic, polar solvents uch as THF or MeCN also afforded the respective bissubstituted derivatives. We assume that in contrast to the nonpolar CHCl 3 the latter solvents have as tabilizing effect on the charged intermediates that are formed duringt he second nucleophilic addition. In this regard, the use of two different solvents,t hat is, CHCl 3 and MeCN, in separates ubsequentr eaction steps may even allow the introduction of two different alkyl-or phenylamino substituents in order to further tune the absorption properties.

Absorption properties
The introduction of amino substituents as stronge lectron-donating groups into the azothiazole structure generally enhances the delocalization of the conjugated system and therefore causesasignificant red shifto ft he long-wavelength absorption maximum in comparison to the parent compound 2g.A ccordingly, the most pronounced red shift was observed for azothiazole 8f (l abs = 626-661 nm) bearing two p-(dimethylaminophenyl)amino substituents as the strongest electron Scheme3.Proposed mechanism for the formationo f8 (cf. ref. [33], [34]). donors. As ag eneral trend, the derivatives 7a-d and 8a-c exhibit positive solvatochromism with the longest wavelength absorption band in DMSO and the shortest in an onpolars olvent (n-hexane or CHCl 3 ). This characteristic behavior may originate either from the stabilization of the Franck-Condon excited state or from the destabilization of the ground state with increasing solvent polarity. However,d onor-acceptor-substituted azo dyes have ah igher dipole moment in the excited state than in the ground state, and therefore, the excited state is better stabilized by solvation in ap olar solvent. [35] With a strong donor substituent on just one side of the azo unit, the 5-(p-toluidylamino)-substituted derivative 7d has the highest dipole momenta nd therefore exhibits the strongest solvatochromism of all derivatives ( Figure 3B). To analyze the solvent-dependent shifto ft he absorption maximum several commons olvatochromic empirical parameters describing nonspecific or specific solute-solventi nteractions were employed. [35,36] Amongt hose parameters are the widely used Kamlet-Taft parameters, namely the hydrogenbond-donating ability (a), the hydrogen-bond-accepting ability (b)a nd the dipolarity/polarizability polarity( p*), [37] and the more recently developed Catalµnp arameters, namely the solvent acidity (SA), the solvent basicity (SB), the solventp olarizability (SP) and the solventd ipolarity (SdP). [38] Thereby,t he latter empirical scales by Catalµne tal. are advantageous in so far as they are based on defined reference processes and provide an independent polarizabilitya nd dipolarity scale. Overall, we found that the solvatochromism of the azothiazoles 7a-c is mainly affected by the dipolarity of the solvent. Specifically, the plots of the absorption maximac orrelate well with the SdP scale (7a: r 2 = 0.98, 7b: r 2 = 0.99, 7c: r 2 = 0.92), at least if DMSO and H 2 Oa re excluded as solvents (Figure 7, S4 A). This analysis indicates that an increasing dipolarity of the solvent leads to a more pronounceds tabilization of the excited molecule thus leadingtoar ed-shifted absorption.
The effect that the absorption spectra in DMSO are shifted to lower wavenumbers to am uch greater extent wasp reviously attributed to the high propensity of DMSO to act as hydrogen-bond acceptorr esulting in the formationo fh ydrogenbondingc omplexes that cause an even more pronounceds tabilizationo ft he excited state. [35c,d] In contrast to the alkylamino-substituted azothiazoles 7a--c,t he absorption maxima of 7d and 8a--c do not correlatew ellw ith the dipolarity (Figure S4 B-E) or any other particular solventproperty.Apparently, severalsolventproperties contribute simultaneously, buttodifferent extentt ot he stabilization of the excited state of these derivatives whichi nt urn influences the shift of the absorption bands.
The longest wavelengtha bsorption maximum of the bisphenylamino-substituted derivatives 8d und 8e was observed in DMSO,w hichi ndicates as trong stabilization of theses dyes in the excited state by this solvent, too ( Figure S3 D, E). Unfortunately,t he solvatochromism could not be investigated in more detail because of the limited solubility of these derivatives, especially in protic solvents.

DNA-binding properties
The water-soluble derivatives 7b, 8b,a nd 8f were also investigated regarding their DNA-bindingp roperties. Firstly,t he course of the spectrophotometric titrations revealed that all three azothiazoles associate with DNA;h owever, the addition of ct DNA or the quadruplex-forming oligonucleotide 22AG to the monoamino-substituted azothiazole 7b only causedm inor changes in the absorption spectra indicating aw eak binding affinity to both forms of DNA ( Figure 4A1, B1). In contrast, the titrations of DNA to the bis-substituteda zothiazoles 8b and 8f revealed that these dyes have at least as trong binding affinity to ct DNA with differentb inding modes at varying LDR ( Figure 4B 2, S6). Specifically,t he development of ad istinct blue-shifted band at higher LDR, that is, at high dye loading at the DNA, implied that these dyes at first form aggregates along the phosphate backbone of the DNA ("outside edge binding")d ue to the lacko fa vailable binding sites. This binding mode was confirmed by the additional CD-spectroscopic studies performed with derivative 8b since the bisignateshape of the ICD bands at around 410-590 nm as wella st he zero transition of this band at the absorption maximum of the bound dye clearly indicatee xciton coupling between the stackedc hromophores ( Figure 6A). [39] The respective negative LD signal at the same wavelength points towards the conclusion that these aggregates are orientedw ith the aromatic plane of the dyes perpendicular to the DNA helix ( Figure 6B). Remarkably,t he negative LD signal of the DNA is successively diminished at LDR > 0.5, which implies that the DNA is significantly bent or compacted due to the formation of the dyea ggregates and therefore becomes less oriented by the shear flow. [40] With an increasing number of binding sites available at lower LDR, the azothiazoles 8b and 8f have am ore specific binding to the DNA which is supported by the increase in absorptiona ccompanied by as ignificant red shift. [41] Thereby,t he negative LD signal at 585 nm, coinciding with the absorption maximum of the red-shifted band of the ligand-DNA complex, shows that 8b intercalates into the DNA at this LDR, [42] which is in agreement with previous DNA bindings tudies of the guanidinium-substituted azobenzene derivative 9a. [12a] The corresponding reduced linear dichroism (LD r )s pectra provide additional information about the averageo rientation of the ligand  Figure S7). At an LDR = 0.2, the LD r values at around 585 nm are smaller than at 260 nm, thus indicating that the transition momento ft he dye 8b is tilted relative to the plane of the DNA base pairs by approximately 248.M oreover,t he terminal, positivelyc harged protonated amino substituents of 8b and 8f may associate with the minor groove causing an additional stabilization of the ligand-DNA complex. [43] The spectrophotometric titration of 22AG to 8b ( Figure 4B2) revealed that this derivative also binds to quadruplex DNA, however with low binding affinity (K 22AG = 2.4 10 4 ). The continuousd ecrease of the absorption accompanied by asmall red shift upon addition of 22AG indicates that 8b does not form aggregates in the presenceo f22AG andb inds mostly with one binding mode to quadruplexD NA, namely by terminal p-stacking as was shown for the structurally similara zobenzene derivatives 9b-e ( Figure 8). [13] The aggregation behavior of the azothiazole derivatives 7b and 8b was investigatedf urther by spectrometric titrations of PSS-asarepresentative, anionic polyelectrolyte-to aqueous solutionso f7b and 8b ( Figure 4C1, C2). The initial decrease in absorption and the hypsochromic shift of the respective absorptionb ands indicated the formation of H-aggregates at low PSS concentrations. [44] Similarly to the titration with DNA, af urther increase of the PSSc oncentration again led to ab athochromic shift of the absorption band due to electrostatic interactions of the azothiazoles 7b and 8b with PSS on well-separated binding sites. [45] In the absence of DNA or PSS, the derivative 8b is only weakly fluorescent in aqueous solution because of radiationless deactivation of the excited state, presumably by E-Z isomerization of the azo double bond which is in turn followed by av ery fast thermal Z-E isomerization. [1a, 16a,g] At low ct DNA or PSS concentrations, the formation of aggregates( see above) initially leads to self-quenching, andafurtherd ecrease of the already low fluorescencei ntensity was detected (Figure 5A,C). [45,46] Accordingly,t his behavior was not observed upon addition of 22AG ( Figure 5B )b ecause 8b does not form aggregates in the presenceo fq uadruplex DNA. When binding in am ore specific manner to ct DNA or PSS at ah igherc oncentration of the respective host system, 8b experiences as ignificanti ncrease in fluorescencei ntensity,w hich can also be seen by the nakede ye under UV light (Inset in Figure 5A). Considering that this light-up effect takes place only as soon as ap articularl igand-DNA ratio is adjusted ( Figure 5A), the limiting value for this fluorimetric detectioni sc DNA = 7 mm. Since 8b exhibits the same emission in ah ighly viscous solvent such as glycerol ( Figure S1), it may be concluded that the conformational flexibility within the bindings ite is reduced leadingt os uppression of the radiationless deactivation pathways of the excited state. [47] Similarf luorescencel ight-up effects in media with restricted free volume, that is, in the presence of DNA or in glycerol, were also observed for amino-and aminostyryl-substituted quinolizinium derivatives. [48] The fact that the fluorescencel ight-up effect in the presence of 22AG is comparatively less pronouncedl eads to the conclusion that binding of 8b to the quadruplexD NA by terminal p-stacking does not reduce its conformational flexibility as much as in the intercalation binding site in duplex DNA.

Conclusions
The readily availabled iethyl azothiazole-4,4'-dicarboxylate (2g) unexpectedly undergoes aM ichael-type addition with primary alkyl and aromatic amines at C5, andt he intermediates are readily oxidized by molecular oxygen under aerobic conditions to afford novel mono-and bis-substituted 5-alkyl-and 5-phenylamino azothiazole derivatives. While their synthesis admittedly requires further optimizationt oi ncrease the yields, the aminopropylamino-substituted azothiazoles 7f and 8f even offer the opportunity for furtherf unctionalization. In general, all azothiazoles exhibit not only high color strength, but also remarkably red-shifted absorption bands in comparison to the parentc ompound 2g.I nt his regard, they also show positive solvatochromism with derivative 7d featuring the strongest bathochromic shift when changing from an onpolart oap olar solvent. As revealed by spectrometric titrations, the water-soluble dyes 7b and 8b associate with DNA, whereas the bis(dimethylaminopropylamino)-substituted azothiazole 8b has a higher binding affinity to ct DNA and quadruplexD NA in comparisont ot he mono-substituted derivative 7b.T he additional CD and LD studies revealed that 8b forms chiral aggregates in the presence of ct DNA at high dye loading and intercalates into the DNA with ample availability of binding sites at lower dye loading. Thus, the derivative 8b represents one of the rare representatives of azo dye-based DNA intercalators.M ostn otably,t he association of 8b to DNA also leads to as ignificant increase of the low emissioni ntensity at 671 nm, that is, in an advantageous range for biological applications,s ot hat these novel azothiazoles may be considered as promising starting point for the development of DNA-sensitivefluorescent dyes.

Experimental Section
Equipment NMR spectra were recorded with aB ruker Avance4 00 ( 1 H: 400 MHz, 13   given in ppm. Absorption spectra were recorded with aC ary 100 Bio or with an Analytik Jena Specord S6 00 spectrophotometer in Hellma quartz cells 110-QS or 114B-QS (10 mm) with baseline correction at 20 8C. Emission spectra were collected with aC ary Eclipse spectrophotometer in Hellma quartz cells 114F-QS (10 mm 4mm) at 20 8C. Circular dichroism (CD) and linear dichroism (LD) spectra were measured with an Applied Photophysics Chirascan spectropolarimeter.F or LD spectra the CD spectrometer was equipped with aH igh Shear Couette Cell Accessory.T he LD samples were recorded in ar otating couette with as hear gradient of 1200 s À1 .M ass spectra (ESI) were recorded on aF innigan LCQ Deca (U = 6kV; working gas:A rgon;a uxiliary gas:N itrogen;t emperature of the capillary:2 00 8C). High-resolution mass spectra were acquired with aT hermo Fisher Scientific Exactive mass spectrometer with Orbitrap mass analyzer and the exact masses of the analyte ions were measured. For analyte ionization ah eated electrospray ionization source (HESI-II) in positive-ion detection mode (U = 4.5 kV) was used. Samples were introduced into the HESI-II-MS system via flow injection by an Agilent 1200 HPLC instrument (analyte concentration:1 0o r1 00 mm,i njected sample volume:1 0mL, flow rate:0 .2 mL min À1 ,m obile phase:M eOH with 0.1 %f ormic acid). The melting points were measured with aB ÜCHI 545 (BÜCHI, Flawil, CH) and are uncorrected.

Methods
Solutions were prepared for each measurement from stock solutions in as uitable solvent (CHCl 3 for 2g, 7a-d, 8a-e;M eOH for 7f and 8f; c = 1.0 mm). For experiments in different solvents, aliquots of the stock solution were evaporated under as tream of nitrogen and redissolved in the respective solvent. In general, absorption spectra were determined in ar ange between 200 and 850 nm (260-850 nm for DMSO, 330-850 nm for acetone, 240-850 nm for CHCl 3 ,a nd 220-850 nm for THF) and subsequently smoothed in the Origin software with the function "adjacent-averaging" (factor of 10). For the detection of emission spectra the excitation and emission slits were adjusted to 5nm, the detection speed was 120 nm min À1 ,a nd the detector voltage was adjusted between 550 and 700 Vd epending on the fluorescence intensity.T he emission spectra were smoothed with the implemented moving-average function by af actor of 5. Emission spectra in the range between 600 and 850 nm were corrected using an instrument specific correction curve. The fluorescence quantum yields of derivative 8b were determined relative to cresyl violet (F fl = 0.54 in MeOH) [30a] according to the established procedures. [30b, c] The spectrometric titrations were performed according to published protocols, [48b] and the binding constants were determined by fitting the binding isotherms to the established theoretical model according to the independent-site model. [29] For the CD and LD experiments six samples were prepared with a fixed ct DNA concentration (c DNA = 10 or 20 mm). In five of the samples different amounts of ligand 8b were added to obtain ligand-DNA ratios of 0.2, 0.5, 1.0, 1.5, 2.0. All samples contained 5% v/v DMSO. CD and LD spectra were recorded in ar ange between 240 and 600 nm with ab and width of 1nm, ar ecording speed of 1nms À1 and at ime per data point of 0.5 s. The reduced linear dichroism, LD r ,a nd the angle a between the electric dipole moment of the ligand and the DNA helix axis were determined according to the established procedures. [42,49]