pH-Induced changes in electronic absorption and fluorescence spectra of phenazine derivatives

Spectrochimica Acta Part A 66 (2007) 849–859

pH-Induced changes in electronic absorption and fluorescence spectra of phenazine derivatives O.A. Ryazanova a,∗ , I.M. Voloshin a , V.L. Makitruk b , V.N. Zozulya a , V.A. Karachevtsev a a

B. Verkin Institute for Low Temperature Physics & Engineering of NAS of Ukraine, 47 Lenin ave., 61103 Kharkov, Ukraine b Institute of Molecular Biology & Genetics of NAS of Ukraine, 150 Zabolotnogo str., 03143 Kyiv, Ukraine Received 12 January 2006; received in revised form 26 April 2006; accepted 26 April 2006

Abstract The visible electronic absorption and fluorescence spectra as well as fluorescence polarization degrees of imidazo-[4,5-d]-phenazine (F1), 2-methylimidazo-[4,5-d]-phenazine (F2), 2-trifluoridemethylimidazo-[4,5-d]-phenazine (F3), 1,2,3-triazole-[4,5-d]-phenazine (F4) and their glycosides, imidazo-[4,5-d]-phenazine-N1-␤-d-ribofuranoside (F1rib), 1,2,3-triazole-[4,5-d]-phenazine-N1-␤-d-glucopyranoside (F4gl), were investigated in aqueous buffered solutions over the pH range of 0–12, where the spectral transformations were found to be reversible. The effects of protonation and deprotonation on spectral properties of these dyes were studied. We have determined the ranges of pH, where individual ionic species are predominant. In aqueous buffered solutions the fluorescence was found only for neutral species of F1, F1rib, F2, and F4gl dyes, whereas for the ionic forms of these dyes, as well as for F3 and F4 ones, the fluorescence has not been detected. The concentrational deprotonation pKa values were evaluated from experimental data. It was shown that donor–acceptor properties of the substituent group in the second position of the pentagonal ring substantially affect the values of the deprotonation constants and the character of protonation for chromophore. The substitution of a hydrogen atom in the NH-group by the sugar residue blocks the formation of the anionic species, and results in enhancement of the dye emission intensity. The steep emission dependence for F1 and F1rib over pH range of 0–7 with intensities ratio of IpH 7 /IpH 1 = 60 allows us to propose them as possible indicator dyes in luminescence based pH sensors for investigation of processes accompanied by acidification, e.g. as gastric pH-sensors. A comparative analysis of the studied dyes has shown that F4gl is the most promising compound to be used as a fluorescent probe for investigation of molecular hybridization of nucleic acids. © 2006 Elsevier B.V. All rights reserved. Keywords: Phenazine dyes; Spectroscopy; Absorption; Fluorescence; pH; Acid–base equilibrium; Sensor

1. Introduction In the past decade a considerable interest and research efforts in the life science have been given to development of miniaturized sensor devices for monitoring of pH, concentration of glucose and lactose, carbon dioxide, cholesterol and others, which can be used in molecular biology, chemistry and medicine [1–5]. In optical pH sensors the organic dyes are used as indicators, which properties depend on environment characteristics. Therefore, a permanent search takes place for the most suitable dyes for wide range of applications. Phenazines are heteroaromatic dyes, which are widely employed in biology and medicine for staining of submolecular

∗

Corresponding author. Tel.: +380 57 3308558; fax: +380 57 3450593. E-mail address: [email protected] (O.A. Ryazanova).

1386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2006.04.027

particles and tissues [6], as well as the active antituberculosis drugs [7]. Phenazine derivatives are redox-active antibiotics, which promote a microbial mineral reduction [8]. Besides, they can be used in the solar cells based on dyes [9]. Molecules of phenazine derivatives may be also employed to trace molecular and physiological events in living cells. In particular, they have been applied as electron transfer mediators in development of amperometric biosensor for hydrogen peroxide [10,11], glucose and lactose [11,12]. The planar structure of phenazine derivatives allows them to intercalate between bases of nucleic acids [13]. In this process their absorption and fluorescence vary substantially, depending on biopolymer secondary structure and a type of nucleotides in a binding site. The glycoside derivative of uncharged imidazo-[4,5-d]-phenazine (F1) (Fig. 1) was successfully used for the nucleic acids complexes stabilization. Also, it appeared to be the efficient fluorescent probe in the study of structure and dynamics of nucleic acids [14,15]. A number

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Fig. 1. Molecular structure of phenazine derivatives: phenazine (F), phenazine ethosulfate (FES), imidazo-(4,5-d)-phenazine (F1), imidazo-(4,5-d)-phenazineN1-␤-d-ribofuranoside (F1rib), 2-methylimidazo-(4,5-d)-phenazine (F2), 2-trifluoridemethylimidazo-(4,5-d)-phenazine (F3), 1,2,3-triazole-(4,5-d)-phenazine (F4), 1,2,3-triazole-[4,5-d]-phenazine-N1-␤-d-glucopyranoside (F4gl). The numeration of atoms included in aromatic system is given accordingly to the case F1.

of tetracyclic uncharged phenazine derivatives, which are distinguished by presence of the planar tetracyclic chromophore, were also synthesized [16,17] to choose the most promising fluorescent probe. There are found to be the 2-methylimidazo-[4,5-d]phenazine (F2), 2-trifluoridemethylimidazo-[4,5-d]-phenazine (F3) and 1,2,3-triazole-[4,5-d]-phenazine (F4) (Fig. 1). Their spectroscopic properties in organic solvents of different polarities and proton-donating abilities were investigated by the methods of absorption and polarized fluorescence spectroscopy [18,19]. This allowed us to model some features of these dyes behavior in intercalation complexes with nucleic acids, which formation is accompanied by changing of chromophore molecular environment. It was shown [18], that F3 and F4 fluorescence polarization degrees are sufficiently larger than corresponding F1 and F2 values, and this enables the former derivatives to be used as promising fluorescent probes for the polarization method application. Also it was shown that dye absorption and emission spectral shifts, as well as the fluorescence quantum yield, depend on physico-chemical properties of the environment, in

particular, on proton-donating and hydrogen bonding abilities [18]. Therefore, considering these compounds as potential fluorescent probes, it is necessary to take into account the effect of medium pH on dye spectral characteristics [20–22]. It was shown earlier, that the cationic form of an intercalating dye is more preferred for binding than the neutral one, and the anionic form is non-binding due to the Coulomb repulsion between negative charged backbone of nucleic acid and, respectively, charged dye molecules [23]. In the present work we have investigated the pHinduced changes in the spectra of F1, F2, F3, F4, as well as their glycosides, the imidazo-[4,5-d]-phenazine-N1-␤-dribofuranoside (F1rib) and the 1,2,3-triazole-[4,5-d]-phenazineN1-␤-d-glucopyranoside (F4gl), in aqueous buffered solutions over the pH range of 0–12 by using the absorption and fluorescence spectroscopy. We have determined the pH range where the predominance of individual prototropic forms is observed. The possibility to employ such phenazine derivatives as indicator dyes in optical sensor for pH monitoring is discussed.

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2. Experimental 2.1. Reagents The molecular structures of the investigated dyes are shown in Fig. 1. The phenazine derivatives were synthesized and purified according to the procedure reported in [17]. Buffered aqueous solutions for investigation of pH effects were prepared with high quality pure water and analytical grade chemicals, such as sodium hydroxide, sodium tetraborate, potassium dihydrogenphosphate, potassium hydrogen phtalate, oxalic acid dihydrate and hydrochloric acid from Sigma–Aldrich. In the pH range of 1.68–12 the ionic strength changed within the range of 0.025–0.06. At lower and higher pH values the ionic strength of solutions corresponded to hydrochloric acid and sodium hydroxide concentrations, respectively. Since the water solubility of the phenazine derivatives F1–F4 is very low, at the first stage we prepared the basic solutions of high concentration by dissolving of pre-weighted amount of dye powder in a fixed volume of methanol. To obtain a dye solution of required pH, it was diluted about 1:100 with the corresponding buffered solution, so that the final dye concentration was approximately 10−5 M/dm3 . Hence, the methanol influence on spectroscopic properties of the sample is expected to be negligibly small. 2.2. Apparatus and techniques The pH values of solution were determined by potentiometric method with the laboratory pH-meter—millivoltmeter pH-340 (produced by SOYUZANALYTDEVICE of Gomel plant of measuring instruments) using the glass electrode ESL-43-07. The calibration of pH meter and preparation of glass electrode prior to work were carried out before each measurement in accordance with a manual by using standard buffered solutions prepared with the “for pH-metry” MRTU-6-09-1289-64 (Russia) reagents, issued in the form of fixanals. All electronic absorption spectra were measured in quartz cells of two types, 2 cm or 5 cm optical path length, on SPECORD UV/VIS (VEB Carl Zeiss, Jena) spectrophotometer. Spectral data were collected and analysed by means of a personal computer. The absorption spectra of dye were measured with the pH step of 0.5. In the region of co-existence of different ionic forms the step was reduced to 0.2. The intensity and polarization of fluorescence were measured in 1 cm2 quartz cell with a laboratory spectrofluorimeter by the method of photon counting. The apparatus and procedure of measurements were described earlier [14]. The halogen lamp was used as a light source, λex = 463 nm. The emission was observed at 90◦ from the excitation beam, and obtained fluorescence spectra were corrected for spectral sensitivity of the spectrofluorimeter. The polarization degree of fluorescence (P) has been defined from the equation [24]: III − I⊥ P= III + I⊥

(1)

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where III and I⊥ are intensities of the emitted light, which are polarized parallel and perpendicular to the polarization direction of exciting light, respectively. The experiments were carried out at the room temperature (18 ◦ C). 3. Results 3.1. Effect of pH on absorption spectra of phenazine derivatives 3.1.1. Imidazo-[4,5-d]-phenazine Fig. 2 shows a comparison of absorption spectra in the visible region for distinct forms of F1. As can be seen, the absorption spectrum of neutral species consists of the unstructured intense shortwave band with a maximum at 385 nm (extinction ε ≈ 16,300) and the longwave broad shoulder extending to 490 nm, so that its maximum lies approximately at 440 nm. The neutral form was observed over the pH range 5–8. For pH >8 the shape and position of absorption spectra are changed. In particular, the hyperchromism, the appearance of intense absorption band at 397.5 nm, and also the widening and the large red shift of longwave shoulder, approximately by 60–70 nm, have been observed. We suppose that such spectral transformations are caused by deprotonation of the dye molecule. In the pH range of 8–12 there are two isosbestic points, at 413.5 nm and 469 nm, in which absorption of neutral and anionic forms of F1 are equal. For pH above 12 the dye exists in the anionic form only. This was confirmed by absorption measurements at pH 13, which revealed the spectrum identical to that at pH 12. The reduction of pH from 5 to 3 induces a rise of the band at 377.5 nm with a simultaneous decrease of that at 385 nm, as well as a blue shift of the longwave shoulder, about 30 nm, and the overall shape of spectrum becomes more structured. In the pH range between 3 and 2 no notable spectral changes were observed. Obviously, the spectrum in this range belongs to a distinct cationic species, named “cationic form 1”. The further

Fig. 2. Absorption spectra of imidazo-(4,5-d)-phenazine (F1) in different ionic forms: anionic (- - -) at pH 12, neutral (—) at pH 7, cationic1 (--) at pH 2, cationic2 (. . .) at pH 0, measured in buffered aqueous solution at room temperature. Dye concentration is 10−5 mol/l, optical path length is 2 cm.

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decrease of pH results in appearance of a band with a maximum at 395 nm, and additional pH reduction below 1 induces a strong rise of this band, so that at pH 0 its intensity is in one and a half times larger than that for the band at 377.5 nm. Also, under the reduction of pH from 1 to 0 a longwave shoulder is broadened strongly and red shifted approximately by 60 nm. The existence of isosbestic point at 407 nm has allowed us to assume the presence of transition to another cationic form, denoted as “cationic form 2”. The decrease of pH from 0 to (−1) is accompanied by further absorption hyperchromism and a red shift of the longwave band. This indicates that at pH 0 the transition is not completed. We have also carried out the similar investigation for the imidazophenazine derivatives F1rib, F2, F2rib, F3, and the triazolophenazine ones, F4 and F4gl. The values of absorption band maximum wavelength, typical for distinct forms of dyes, along with the ranges of pH, where corresponding form is dominant, are summarized in the Table 1. Table 1 Absorption spectral properties of the phenazine derivatives in aqueous buffered solutions of different pH Compound

F1

pH

Species

Absorption maxima λ1 (nm)a

λ2 (nm)b

λ3 (nm)c

510 440 404 435 465

570 490 465

430 402 433 465

490 465

580 540 540 505 460

12 5–8 2–3

Anionic Neutral Cationic 1

397.5 385 377.5

0

Cationic 2

395

5–12 2–3

Neutral Cationic 1

384.5 379

0

Cationic 2

395

14 12

Anionic Anionic + neutral

6.5–9.18 3

Neutral Cationic 1

402 388 400.5 388 379

0

Cationic 2

398

501 –d –d 438 410 430 462

F3

9–12 3–6.0 0–1

Anionic Neutral Cationic

394 384.5 401

495 430 478

560 500 560

F4

11–12 3–6.5 0

Anionic Neutral Cationic

390 383 401

508 458 515

580 520 590

F4gl

3–12 0

Neutral Cationic

382 400

458 515

520 590

F

12 6.5 1

Neutral Neutral Cationic

370.5 370.5 383

400 400 430

425 425 475

0–12

Cationic

387.5

440

490

F1rib

F2

FES

520

520

b

3.1.3. 2-Methylimidazo-[4,5-d]-phenazine Fig. 3 shows the effect of pH on the absorption spectrum of F2. As can be seen from the figure, the spectral transformations induced by the pH changes in solution are analogous to those of F1. Also, the existence of neutral, anionic and two cationic forms of the dye was found. Their absorption spectra shapes are similar to those of F1, however, the spectrum maxima are red shifted approximately by 3 nm. For the neutral form the value of extinction coefficient in absorption maximum is equal to 18,700 and appeared to be higher than that for F1. As is seen in Table 1, the regions of neutral and, especially, anionic forms lie at higher pH values. Even at pH 12 no complete deprotonation was observed. The additional absorption measurements show that the transition to anionic species is completely finished only at pH 14 (see Fig. 3). The transition of the dye from neutral species to cationic 1 and cationic 2 ones occurs approximately within the same pH region as for F1 (Table 1). Regarding to the position of absorption bands for neutral species, it can be noted, that absorption spectra typical for anionic forms of F1 and F2 are red shifted more strongly than those for cationic ones. 3.1.4. 2-Trifluoridemethylimidazo-[4,5-d]-phenazine Fig. 4 presents a comparison of absorption spectra in the visible range for anionic (pH 12), neutral (pH 5.5) and cationic (pH 0) species of F3. The data relevant to other pH values were analyzed, and results are also summarized in Table 1. The neutral form of the dye is observed in the pH range from 3 to 6. The shape of absorption spectrum for this species is similar to those of F1 and F2, but it lies in somewhat shorter wavelength

520

λ1 is a position of the maximum of shortwave absorption band. λ2 is a position of the maximum of longwave absorption band (shoulder). c λ is a position of the red edge of longwave absorption band (shoulder). 3 d The wavelength is not indicated because at pH 12 the mixture of neutral and anionic forms is observed for F2 derivative. a

3.1.2. Iimidazo-[4,5-d]-phenazine-N1-β-d-ribofuranoside For F1rib we have observed only the neutral and two cationic forms. The shape and position of absorption bands are very similar to those of F1 (see Table 1). However, the value of the extinction measured in the maximum of absorption band, ε ≈ 20,100, appeared to be higher than that for F1.

Fig. 3. Absorption spectra of F2 in different ionic forms: anionic (– – –) at pH 14, mixture of anionic and neutral ones (- - -) at pH 12, neutral (—) at pH 7, cationic 1 (--) at pH 3, cationic 2 (. . .) at pH 0, in buffered aqueous solution at room temperature. Dye concentration is 9.1 × 10−6 mol/l, optical path length is 2 cm.

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Fig. 4. Absorption spectra of F3 in different ionic forms: anionic (- - -) at pH 12, neutral (—) at pH 6, cationic (. . .) at pH 0, in buffered aqueous solution at room temperature. Dye concentration is 8.8 × 10−6 mol/l, optical path length is 5 cm.

region. It consists of one intense shortwave band with a maximum at 384.5 nm (ε ≈ 10,300) and the longwave broad shoulder extending to 500 nm. For the pH increasing from 6 to 9 we have observed few spectral changes, which can be attributed to the molecule deprotonation. There are, specifically, the appearance of a new band at 394 nm, the rise of absorption intensity, and the strong batochromic shift of the longwave absorption band (≈60 nm), resulted in transformation of the shoulder to a separate unstructured band. We have also observed the isosbestic point at 407 nm. These spectral transformations are accompanied by changes of solution color from yellow for the neutral species to purple red for the anionic one. At the pH range from 9 to 12 the only insignificant hyperchromism of absorption spectrum takes place. In comparison with F1, the ranges where distinct neutral and anionic species exist are shifted to lower pH values. The spectral changes, caused by acidification, are very similar to those at high pH values. Unlike to F1 and F2, we have observed only one cationic type of F3. The decrease of solution pH from 3 to 1 leads to the absorption hyperchromism, accompanied by a rise of intensive band at 401 nm and by a red shift of the longvave shoulder. In the pH range between 1 and 0 we have observed only the absorption hyperchromism without any changes of the spectrum shape. Therefore, the protonation and deprotonation of the molecule result in similar absorption spectral changes, namely, the appearance of new absorption bands, the hyperchromism and the red shifts of absorption spectra, in comparison with the neutral form. The differences in the absorption intensity of F3 anionic and cationic species, in comparison with the neutral one, are substantially higher than those for F1 and F2. 3.1.5. 1,2,3-Triazole-[4,5-d]-phenazine In Fig. 5 we present a comparison of absorpion spectra related to F4 neutral and different ionic species. The spectral changes induced by pH alteration are similar to those for F3. Their detailed description will be given in Section 3.2.

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Fig. 5. Absorption spectra of F4 in different ionic forms: anionic (- - -) at pH 12, neutral (—) at pH 5, cationic (. . .) at pH 0, in buffered aqueous solution at room temperature. Dye concentration is 1.4 × 10−5 mol/l, optical path length is 5 cm. In the inset the pH dependence of electronic absorption spectra of F4 in the neutral/basic pH range is shown. The pH values are given in order of decreasing of absorbance at 515 nm: 12; 11; 10; 9.5; 9; 8.5; 8; 7.75; 7.5; 7.25; 7; 6.5; 6; 5.

3.1.6. 1,2,3-Triazole-[4,5-d]-phenazine-N1-β-d-glucopyranoside For F4gl derivative the absorption spectra at neutral and acidic pH are almost identical to those of F4. However, in comparison with the case of neutral form, at basic pH values no spectral changes were observed. For F4gl neutral species the value of the extinction measured at maximum of absorption band, ε ≈ 12,200, appeared to be significantly lower than that for F4 (ε ≈ 17,950). It is easy to see that in neutral solutions the absorption maxima of both F3 and F4 are blue shifted in comparison with F1. However, for cationic species of these derivatives the values of red shifts observed for corresponding maxima, and especially for longwave shoulder, are stronger than those of F1 and F2. As it was shown earlier [18], the spectra of F3 and F4 derivatives are more sensitive to changes in solvent polarity and hydrogen bonding ability. We want to emphasize that for all F1–F4 dyes a strong deviation from neutral pH values results in reversible changes of solution color from yellow, for neutral species, to purple, for anionic and cationic ones, due to a shift of the longwave absorption band. By the series of measurements with repeated changing of a solution pH from minimum (pH 0) to maximum (pH 12), we have confirmed the reversibility of this process. At the same time, the solubility increases strongly for their cationic and anionic forms. A comparison of the normalized to 1 absorption spectra of F1–F4 dye solutions, measured at pH 0 (cationic form), pH 6 (neutral form), pH 12 (anionic form for F1, F3, F4) and pH 14 (anionic form for F2), has revealed a relative shift for both the shortwave and longwave bands. The results of this comparison are presented in Table 2, where we have shown a comparative disposition of absorption bands for distinct ionic species of the same phenazine derivatives. The symbols in Table 2 denote the relative positions of the maxima for the dye shortwave absorp-

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Table 2 Relative disposition of absorption bands for distinct ionic species of the phenazine derivatives in aqueous buffered solutions Compound

Shortwave absorption banda

Longwave absorption band (shoulder)b

F1 F2 F3 F4

λa ≥ λc > λn λa ≥ λc > λn λc > λa > λn λc > λa > λn

λa > λc > λn λa > λc > λn λa > λc > λn λc > λa > λn

λa is a position of the maximum of dye absorption band in anionic form (at pH 12 for F1, F3, F4 and at pH 14 for F2). λn is a position of the maximum of dye absorption band in neutral form (at pH 6). λc is a position of the maximum of dye absorption band in cationic form (at pH 0). a The wavelengths correspond to λ values from Table 1. 1 b The wavelengths correspond to λ values from Table 1. 2

tion band (left column) and the longwave one (right column) in anionic, neutral and cationic forms. The corresponding values of the shortwave and longwave absorption maxima wavelengths are indicated in Table 1 as λ1 and λ2 , respectively. In such a way, the inequalities in Table 2 demonstrate a trend in the absorption spectra transformation at transition from one ionic species to another. Fig. 6 presents a comparison of the absorption spectra for F1, F2, F3 and F4 derivatives in a neutral form, as well as the fluorescence spectrum of F1 resulting from excitation at 463 nm. As can be seen from this figure, the F4 has the most structurized absorption spectrum among considered derivatives. Its absorpion maximum lies at the farthest shortwave position (383 nm), and for the longwave shoulder we have observed a red shift by ∼15 nm in comparison with those of F1–F3. In order to explain the spectral behavior of F1–F4 in acidic range the absorption spectra of ordinary phenazine, F, and its cationic derivative, phenazine ethosulfate (FES), were measured in the wide range of pH.

Fig. 6. Normalized absorption spectra (left) of F1 (—), F2 (- - -), F3 (-+-), F4 (--) and normalized fluorescence spectra (right) of F1 (—) in a neutral form, measured in buffered aqueous solution at 18 ◦ C. Dyes concentration are 10−5 mol/l. Wavelength of fluorescence excitation (463 nm) is indicated by arrow.

3.1.7. Phenazine For phenazine, F, the absorption spectra are found to be identical at neutral and basic pH values. As pH decreases to acidic values, few spectral changes, presumably caused by protonation, were observed, namely, the appearance of a new band at longwave side of absorption band, the rise of intensity, widening and red shift of the longwave shoulder (see Table 1). 3.1.8. Phenazine ethosulfate For FES the absorption spectra (Fig. 7) in acidic, neutral and basic solutions appeared to be invariable over the whole range of pH (Table 1). Their shapes are similar to that of the protonated species of F1 (cationic form 2), however, the maximum of FES absorption is blue shifted by approximately 7.5 nm in comparison with the corresponding value for F1. 3.2. Determination of the ionisation constants pKa The F1–F4 phenazine derivatives have a single possible site of deprotonation. It is NH-group in the pentagonal ring. In the alkaline solution (B) the dye (FH) containing a mobile proton is transformed to the anionic species (F− ): FH + B ↔ F− + HB+

(2)

This process is accompanied by absorption spectral transformations being similar for all abovementioned derivatives. It can be illustrated by the example of F4 (Fig. 5). The inset in Fig. 5 shows changes in the electronic absorption spectra of F4 over the pH range of 5–12. As can be seen, the increase of pH from 6.5 to 12 is supplemented by the hyperchromism of absorption, the appearance of the intense band with the maximum at 390 nm, and, finally, by transformation of the longwave shoulder in a separate wide unstructured band, resulting in the red shift of absorption spectrum. Also, two isosbestic points were observed at 406 nm and 482 nm, in which the absorption coefficients for the neutral and anionic forms are equal. Since absorption spectra of neutral and anionic species are essentially different, we applied the spectrophotometric method to estimate a value of its

Fig. 7. Absorption spectra of phenazine ethosulfate (FES) at pH 12 (- - -), pH 7 (—), pH 1 (. . .) measured in buffered aqueous solution at room temperature, dye concentration is 5.5 × 10−5 mol/l, optical path length is 2 cm.

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ground state deprotonation equilibrium constant, pKa . For this purpose, a titration curve was plotted as the absorbance changes at 515.5 nm versus pH (not shown here), and the inflection point of this curve gives pKa = 7.3 ± 0.1. The alternative way to estimate the deprotonation constant pKa is to employ the equation from Ref. [25]:  pKa = pH + log

Dmax − D D − Dmin

 (3)

where Dmax is the absorption of the anionic form of dyes (at pH 12) at the maximum of the longwave absorption band at 515.5 nm, Dmin is the absorption of the neutral form of F4 (pH 5), D is the absorption of F4 solution for intermediate pH values (5< pH <12) at the same wavelength. The corresponding pKa values found by both ways appeared to be very close. For F3 the value of pKa was found by the first way by considering the absorption changes at 397 nm. Its estimated value, about 7.1 ± 0.1, is somewhat lower than that for F4. In distinction to F3 and F4, the full deprotonation of F1 and F2 occurs at higher pH values, correspondingly at pH 12 and pH 14. By plotting a titration curve of F1 as absorbance changes at 505 nm versus pH, we have similarly found its pKa = 10.9 ± 0.1. Due to difficulties of measuring high pH values (above 12), we could not find exactly the deprotonation constant of F2. From analysis of spectral changes of this dye we have estimated this pKa value as close to 12. Based upon molecular structure of F1–F4 dyes, one can expect the existence of several possible sites of protonation. Due to the presence of the lone pair of electrons at the nitrogen atoms of phenazole, imidazole or triazole rings of the dye, a protonation would occur in an acidic medium accompanied by formation of different cationic species. The absorption spectral changes, which are induced by acidification, show that a competitive protonation providing to a mixture of distinct cationic species take place. Therefore, no evident isosbestic points were observed. Obviously, the values of protonation equilibrium constants are close for different cationic species. From consideration of spectral changes in strongly acidic buffered solutions of F1–F4 (pH ranging between 3 and 0), it is estimated that the pKa values for transition to the corresponding cationic form are located in the range from 0.5 to 1.3. In a similar way the values of ground state protonation constants pKa were found from the pH dependence of emission intensity (see Section 3.3).

Fig. 8. Effect of pH on the fluorescence intensity of F1 (×) and F1rib (䊉) at maximum of band, λfl = 562 nm, in buffered aqueous solution at room temperature. The data are normalized to the intensity at pH 7. Wavelength of excitation is λex = 463 nm. The solid line represents the fitting according to the Eq. (3), where pKa = 3.6.

3.3. Fluorescence spectra of phenazine derivatives The emission spectra of the phenazine derivatives as well as their fluorescence polarization degrees were measured in aqueous buffered solutions of different pH under the excitation in the longwave absorption band (λexc = 463 nm). 3.3.1. F1 emission For F1 in the neutral solution of pH 7, the fluorescence spectrum represents a broad unstructured band with a maximum at 562 nm (see Fig. 6). The fluorescence polarisation degree, P, measured at the emission band maximum, was found to be around 0.015 (see Table 3). The position and shape of the emission band are relatively independent on pH, but the fluorescence quenching is observed at low and high pH values. The emissions of F1 cationic and anionic species were not found. Fig. 8 shows the titration curve obtained by plotting the F1 fluorescence changes at 562 nm normalized to 1.0 versus pH. As can be seen, the most intensive emission is observed at pH 7, gradually reducing at acidic and base pH values. In the diluted dye solutions the emission intensity is proportional to concentration of the neutral form. Therefore, fluorescence changes can be used for estimation of the ground state equilibrium constant pKa . In Fig. 8 the plot of fluorescence intensity within the range from

Table 3 Spectral properties of the phenazine derivatives in neutral form measured in aqueous buffered solutions Compound

pH

λmax abs , (nm)

ε, (M−1 cm−1 )

λex , (nm)

λmax flu , (nm)

P

F1 F1rib F2 F3 F4 F4gl

6 6 6 6 6 6

385 384.5 388 384.5 383 382

16,300 20,100 18,700 10,300 17,950 12,200

463 463 463 463 463 463

562 555 566 – – 522

0.015 0.017 0.023 – – 0.060

λmax abs is the wavelength of absorption band maximum. ε is the extinction measured in maximum of absorption band. λex is the fluorescence excitation wavelength. λmax abs is the wavelength of fluorescence band maximum. P is the fluorescence polarization degree.

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pH 6 to pH 2 shows a clear inflection point at pH 3.6, giving the value of protonation constant, pKa . 3.3.2. F1rib emission Additionally, we have investigated the fluorescence spectra of F1rib. It is necessary to note that substitution of hydrogen near N1 by ribose moiety does not influence a shape of the dye emission band, but it induces a small blue shift of the fluorescence maximum by 7 nm (λmax flu = 555 nm, P = 0.017) and increases the emission intensity. From Fig. 8 it can be seen that in the pH range of 0–7 both the F1 and F1rib exhibit essentially identical pH-dependent spectra with pKa value of 3.6. However, the pH region in which the F1rib emission intensity is invariable is somewhat wider than that for F1. 3.3.3. F2 emission The shape of F2 emission spectrum is similar to the F1’s one, and we have observed only fluorescence of the neutral form. The maximum of this band lies at 566 nm, and its polarisation degree was found to be around 0.023. The corresponding fluorescence intensity is somewhat below than that for F1, and their dependencies on pH are similar. However, for F2 the most intensive emission is detected at pH 9.18. From the pH dependence of F2 emission intensity the value of ground state protonation constant, pKa , is estimated to be around 4.5. 3.3.4. F2rib emission For the glycoside derivative F2rib the fluorescence intensity increases strongly, and its maximum is blue shifted to 557 nm. 3.3.5. F3 In contrast to the organic solvent [18], in the aqueous buffered solution a fluorescence of F3 has not been revealed under excitation of 463 nm. For molecular ionic forms with red shifted longwave bands we attempted to excite the fluorescence by light with the wavelength of 524 nm. However, the fluorescence has not been observed either. 3.3.6. F4 emission For F4 derivative no emission was observed under excitation at 463 nm. But in buffered solutions of pH 8–10 we have found a very weak emission with a maximum at 670 nm, resulted from the excitation of 524 nm. 3.3.7. F4gl emission For F4gl under excitation at 463 nm we have observed an appreciable fluorescence with a maximum at 522 nm. Fig. 9 shows the effect of pH on the F4gl fluorescence intensity at the band maximum. In the region from pH 4 to pH 10 the emission intensity is pH independent. The fluorescence polarization degree, P, of F4gl is found to be 0.06. It is larger than those for F1 and F2 derivatives by factors of four and two, correspondingly. The decrease of pH from 4 to 0 is accompanied by complete quenching of fluorescence. The ground state protonation pKa value was estimated to be 1.7 from the dependency in Fig. 9. Under excitation of the ionized form at 520 nm no F4gl fluorescence was found.

Fig. 9. Effect of pH on the fluorescence intensity of F4gl (䊉) at maximum of band, λfl = 522 nm, in buffered aqueous solution at room temperature, λex = 463 nm. The data are normalized to the intensity at pH 7. The solid line represents the fitting according to the Eq. (3), where pKa = 1.7.

3.3.8. Phenazine For phenazine we have not detected any emission neither in acidic nor in neutral or base buffered solutions. 3.3.9. FES For FES under excitation at 463 nm no fluorescence was observed in the acidic buffered solution. In the neutral solution of pH 7 a weak emission appeared with a maximum at 524 nm (P = 0.06). The increase of pH from 7 to 12 was accompanied by a rise of the fluorescence intensity, the red shift of its maximum to 530 nm, and a change of the fluorescence polarization degree (P = 0.03). We believe that at high pH values the process of phenazinium oxidation takes place in the presence of oxygen from air. Therefore, this emission can be caused by either FES or its oxidation products. 3.4. Discussion The molecular structure of phenazine derivatives F1–F4 differs from the phenazine one (see Fig. 1) by the presence of additional heterocyclic planar pentagonal ring conjugated with the ␲-electronic system of the phenazine part. In case of F1–F3 this is the imidazole ring containing two heteroatoms, whereas in the F4 molecule this is the triazole one containing three nitrogen atoms. The p–␲ conjugation takes place for these dyes, and their electronic absorption spectra, located in UV and visible regions, are red shifted in comparison with that for the phenazine [26]. Our investigation show that F1–F4 are polar compounds. For F1, F3 and F4 the values of ground state dipole moments and charge distributions have been obtained earlier [18]. Quantum chemical calculations have shown that, unlike the phenazine with zero dipole moment [27], the molecules of F1, F3 and F4 are the polar ones in the ground state, S0 . Their ground state dipole moments, μg , appeared to be equal to 3.56 D, 3.18 D and 3.73 D, respectively [18], and directed almost across the long axis of a molecule. The optical excitation to the S1 state causes a large increase of the dipole moment (μ = 9 ± 1 D).

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Our measurements show that F1–F4 are amphoteric compounds. On the one hand, their molecules can form anionic species as a result of deprotonation of NH-group in the pentagonal ring. This process is accompanied by appearance of the excess negative charge, as well as the free lone pair of electrons. It also results in the hyperchromism and the red shift of the absorption bands. The investigation of pH-induced changes in absorption spectra for F1–F3 imidazophenazine derivatives has shown that the proton releasing ability of the NH-group correlates with electron-accepting properties of the substituent group at the position 2 of imidazole ring in the ascending order: CH3 < H < CF3 . Based upon absorption changes data and the fluorescent spectra of F1–F4 in organic solvents of various polarities and hydrogen bonding abilities, as well as quantum chemical calculations [18,19], one can expect the considerable change of proton mobility in the NH-group for F3 and F4 derivatives, in comparison with F1 and F2 ones. On the other hand, because of the presence of lone pair electrons at the nitrogen atoms of phenazole, imidazole or triazole ring, it was expected that protonation could occur in the acidic medium. As it was mentioned earlier, the dyes F1–F3 have four, and F4 has five potential sites of protonation. And here we have shown that the protonation actually takes place, and the dye proton-accepting properties depend on a type of the substituent group. At first, let us consider the pH-induced spectral changes of F3, for which the least number of distinct ionic species were observed. The analysis of absorption spectra in the pH range between 0 and 12 shows the existence of three different molecular ionic forms (see Table 1): (i) neutral form in the pH range from 3 to 6; (ii) protonated (cationic) one in the pH range from 0 to 1; (iii) deprotonated (anionic) form in the pH range from 9 to 12. For intermediate pH values we have observed a mixture of the appropriate ionic forms. The absorption spectra of protonated and deprotonated forms appeared to be red shifted with increased molar absorptivity, in relation to the neutral form. For the F4 derivative we observed the existence of the same ionic forms as for F3. Also, the spectral changes induced by transitions between the corresponding forms of F3 and F4 appeared to be similar. The ranges of pH and positions of absorption maxima for each form are summarized in Table 1. A comparison with analogous pH-induced transformations of F absorption spectra, as well as the absence of any FES absorption spectra changes with decrease of solution pH value, allowed us to conclude, that acidification of F3 and F4 results in the protonation of one nitrogen atom in the phenazole ring. For F1 and, especially, for F2 derivatives, the ranges in which neutral and anionic species can be observed shift towards higher pH values (see Table 1). Thus, for F2 the mixture of neutral and anionic species is found at pH 12. Further, the changes of intensities and spectral shifts of absorption bands under transitions from neutral to anionic forms for F1 and F2 are similar to those of F3 and F4. As to appearance of phenazole ring protonated species (cationic form 2), similar to those for F3 and F4, for F1

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and F2 this cationic form has been observed at pH <0, whereas in the ranges of 2< pH <3 for F1, and near pH 3 for F2, the appearance of one more cationic species is revealed. This cationic form 1 is characterized by a small hypochromism and a blue shift of absorption bands in comparison with the neutral ones. Since in acidic/neutral range for both F1 and F2 there are no sharp isosbestic points in absorption spectra, we can suppose that overlapping of multiple protonation equilibria takes place, and the values of corresponding equilibrium constants are very close. The existence of the sharp isosbestic points in neutral/basic regions for F1–F4 derivatives is resulted from the availability of the single possible site of deprotonation. The analysis of absorption spectral transformations for these dyes unambiguously shows that values of deprotonation equilibrium constants pKa are shifted in ascending order of pH: F3 < F4 < F1 < F2. This is in accord with electron-accepting properties of the substituent group at position 2 of the imidazole ring. The measurements of F and FES absorption versus pH have revealed that absorption spectrum of the F protonated form is red shifted, and its intensity is increased relative to the neutral form. The positively charged FES, having one possible protonation site blocked by the ethyl group, exhibits the pH-independent spectra of absorption. From comparison of F1–F4 spectral changes in acidic/neutral region with those of F and FES it is follows, that appearance of cationic species at pH <1 is caused by a protonation of the nitrogen atom in the phenazole ring. The appearance of cationic species 1 for F1 and F2, characterized by absorption bands positioned in more shortwave region in comparison with the neutral ones, can be produced by a protonation of some nitrogen atom of the imidazole ring. This protonation results in exclusion of a lone pair of electrons from conjugation with other part of the molecule. Such cationic species has not been detected for F3 and F4. Possibly, this is due to the reduced protonation ability of N1 and N3 atoms, related to the electron-acceptor effect of the p-type trifluoridemethyl group for F3, as well as N2 for F4, strongly decreasing the electronic density at neighbor nitrogen atoms. Since similar F1–F4 cationic species manifest themselves in comparable pH regions, we can conclude that some details have negligible influence on the protonation of the phenazole ring. These details are found to be the types of substituents in the position 2 of imidazole ring, the substitution of the imidazole ring by the triazole one, and the protonation of pentagonal ring. However, the acceptor properties of abovementioned substituents affect substantially the value of the deprotonation equilibrium constant. Our investigation shows that in aqueous solution of investigated dyes the fluorescence is observed only for neutral species of F1, F1rib, F2, F2rib and F4gl. No emissions of cationic and anionic forms were found. Since the investigation was carried out in diluted dye solutions, the emission intensity is proportional to concentration of the neutral form. A substitution of a hydrogen atom near N1 in the pentagonal heteroring by sugar (ribose or glucose) induces a small blue shift of the fluorescence maximum and a significant rise of its intensity for F1rib and F2rib, in comparison with F1 and F2, as well as the appearance of the emission for F4gl. It can be noted that fluorescence polar-

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ization degree of F4gl is four times larger than that of F1 and F1rib. The ground state protonation pKa values for F1 and F4gl were also estimated from the fluorescence measurements. Obviously, they characterize the transition of dye from neutral to cationic form. For F1 derivative there is a transition to the cationic form 1, for which it is difficult to determine the protonation constant from absorption transformations due to the mixture of different cationic forms. The value of pKa for F4gl, revealed from fluorescent measurements, appeared to be somewhat higher than that from the absorption. Since the F1 and F1rib fluorescence intensities rises steeply up in the pH range of 1–7, these derivatives can be used as indicator dyes in optical pH sensors for investigation of processes, accompanying by acidification. The ratio of fluorescence intensities IpH 7 /IpH 1 amounts to 60 for F1rib, 30 for F2, and 10–20 for F4gl, correspondingly. Since in the wide range of pH (from 4 to 10) the emission intensity of F4gl is independent on pH, and the fluorescence polarization degree of this dye is four times higher than that of F1 and F1rib, this phenazine derivative is the most suitable for use as a fluorescent probe in studies of molecular hybridization of nucleic acids. 4. Conclusions The effect of pH changes on electronic absorption and fluorescence spectra of tetracyclic phenazine derivatives was studied. It is shown, that as the pH increases from 0 up to 12, the phenazine F1–F4 derivatives undergo reversible transitions from cationic to anionic species through the neutral one. It has been shown that chemical substitution of imidazophenazine results in a shift of absorption and fluorescence maxima, as well as in changes of the fluorescence ability and pKa values. The analysis of obtained results shows, that the change in donor–acceptor properties of substituent group in the second position of pentagonal ring substantially affects not only the values of protolitical dissociation constants for NH-group of the ring, but also the character of protonation for chromophore. The values of deprotonation equilibrium constants, pKa , were found to be shifted in ascending order of pH: F3 < F4 < F1 < F2. A substitution of a hydrogen atom in NH-group by the sugar residue blocks the formation of the anionic species. It is confirmed by invariability of dye absorption spectra under the increase of solution pH from neutral values to alkaline ones. Both in neutral and in acidic medium, the absorption spectra of F1rib and F4gl are identical with those of F1 and F4, correspondingly. Also, such substitution induces a significant rise of F1rib and F2rib fluorescence intensity in comparison with F1 and F2, as well as the appearance of emission for F4gl. The absence of F3 fluorescence in aqueous buffered solutions does not exclude a possibility to emit under substitution of hydrogen in the NH-group of this dye by some radical. This assumption is based upon the appearance of fluorescence for F4gl and existence of F3 fluorescence in organic solvents [18]. For F1, F1rib, F2 and F4gl dyes the fluorescence was found only for neutral species. Since the fluorescence for ionic forms

of these derivatives is quenched, and also the dependence of F1 and F1rib emission versus pH rises steeply up in the range of 0–7 (the ratio IpH 7 /IpH 1 = 60), these dyes are expected to be candidates for indicator dyes in luminescence based pH sensors for investigation of processes accompanied by acidification. In particular, they can be used in gastric pH-sensor [28]. The independence of F4gl fluorescence intensity in the wide range of pH from 4 to 10, as well as a relatively high fluorescence polarization degree for this dye (P = 0.06), in comparison with other dyes under investigation, makes this derivative the most suitable for use as a fluorescent probe under studies of molecular hybridization of nucleic acids. Acknowledgement This work is partially supported by Science and Technology Center in Ukraine (Project #3172). References [1] A.G. Ryder, S. Power, T.J. Glynn, Appl. Spectrosc. 57 (2003) 73. [2] Q. Chang, Z. Murtaza, J.R. Lakowicz, G. Rao, Anal. Chim. Acta 350 (1997) 97. [3] H.R. Kermis, Y. Kostov, P. Harms, G. Rao, Biotechnol. Prog. 18 (2002) 1047. [4] J.F. Callan, A.P. de Silva, J. Ferguson, A.J.M. Huxley, A.M. O’Brien, Tetrahedron 60 (2004) 11125. [5] B.H. Weigl, A. Holobar, W. Trettnak, I. Klimant, H. Kraus, P. O’Leary, O.S. Wolfbeis, J. Biotechnol. 32 (1994) 127. [6] R.D. Lillie, H.M. Fulmer, Histopathologic Technique and Practical Histochemistry, McGraw-Hill, New York, 1976. [7] C.E.J. van Rensburga, G.K. Joon´ea, F.A. Sirgelb, N.M. Matlolaa, J.F. O’Sullivan, Chemotherapy 46 (2000) 43. [8] M.E. Hernandez, A. Kappler, D.K. Newman, Appl. Environ. Microbiol. 70 (2004) 921. [9] A.K. Jana, J. Photochem. Photobiol. A: Chem. 135 (2000) 1. [10] X. Xu, J. Zhao, D. Jiang, J. Kong, B. Liu, J. Deng, Anal. Bioanal. Chem. 374 (2002) 1261. [11] J.-F. Gillion, G.-F. Begin, C. Marecos, G. Fourtanier, L. Haiying, Y. Tailin, S. Kang, L. Haihong, Q. Deyao, Anal. Chim. Acta 344 (1997) 187. [12] M.V. Kosevich, O.A. Boryak, V.V. Orlov, V.S. Shelkovsky, V.V. Chagovets, S.G. Stepanian, V.A. Karachevtsev, L. Adamowicz, J. Mass Spectrom. 40 (2005) 113. [13] W. Muller, D.M. Crothers, Eur. J. Biochem. 54 (1975) 267. [14] V. Zozulya, Yu. Blagoi, G. Lober, I. Voloshin, S. Winter, V. Makitruk, A. Shalamay, Biophys. Chem. 65 (1997) 55. [15] V. Zozulya, Yu. Blagoi, I. Dubey, D. Fedoryak, V. Makitruk, O. Ryazanova, A. Shcherbakova, Biopolymers (Biospectroscopy) 72 (2003) 264. [16] V.L. Makitruk, S.N. Yarmoluk, A.S. Shalamay, I.V. Alexeeva, Nucl. Acids Res. Symp. Ser. 24 (1991) 244. [17] V.L. Makitruk, et al., Biopolym. Kletka (Ukraine) 13 (6) (1997) 453. [18] O.A. Ryazanova, V.N. Zozulya, I.M. Voloshin, V.A. Karachevtsev, V.L. Makitruk, S.G. Stepanian, Spectrochim. Acta Part A 60 (2004) 2005. [19] O.A. Ryazanova, V.N. Zozulya, I.M. Voloshin, V.A. Karachevtsev, V.L. Makitruk, Biophys. Bull. (Ukraine) 1 (2003) 49. [20] C. Reichardt, Solvents and Solvent Effects in Organic Chemistry, third ed., Wiley-VCH, Weinheim/Germany, 2003. [21] C. Reichardt, Pure Appl. Chem. 76 (2004) 1903. [22] N.A. Vodolazkaya, N.O. Mchedlov-Petrossyan, G. Heckenkemper, C. Reichardt, J. Mol. Liq. 107 (2003) 221.

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