Spectroscopic studies of a near-infrared absorbing pH sensitive aminodienone—carbocyanine dye system

0039-9140/93 $6 00 + 0 00 Copyright 0 1993 Pergamon Press Ltd

Tahta, Vol 40, No 6, pp 935-942, 1993 m&d m GreatBntam All nghts resewed

SPECTROSCOPIC STUDIES OF A NEAR-INFRARED ABSORBING pH SENSITIVE AMINODIENONE-CARBOCYANINE DYE SYSTEM G PAMNAY, G. A. CASAY, M. LIPOWSKAand L. STREKOWSKI

Department of Chemistry, GeorBa State Umverslty, Atlanta, GA 30303, U S A (Receloed 1 September 1992 Rewed

13 November 1992 Accepted 15 November 1992)

Summary-Synthetrc red and near-infrared absorbing dyes may lx used as probe molecules m a large number of apphcations. Dyes etihtmg spectral changes wtth hydrogen ion concentration are useful as pH probes. Those dyes which have their absorption and fluorescence maxuna m the long wavelength regon of the vlslble spectral regon are especially valuable because of decreased interference and semiconductor laser applications In tis paper we have evaluated an ammodrenone dye 1 which demonstrates pH dependent absorption and fluorescence spectra as well as solvent polarity dependence. In organic solvents the long wavelength absorption band of the dye IS in the reduced interference repon The absorption maxlmum 1sat 535 nm m neutral or alkahne solutions m methanol The absorption spectra undergo a strong bathochronuc sluft m the presence of acids (.4, = 709 nm) with a conconutant change m the fluorescence spectra This pH senatlve dye was found to be especially useful for orgamc solvents The analflcal utdlty of tis and smular near-infrared absorbing dyes IS dIscussed

determination of hydrogen ion concentration (pH) is an important part of many analytical procedures. In previous years numerous dyes have been studied as to their potential for use as acid/base indicators. Sometimes only the color change is utilized, but more sophisticated methods use absorption or fluorescence spectroscopy. A large number of available weak organic acids and bases exhibit different absorption/fluorescence spectra or colors when m undissociated and ionic forms. The color changes or variations in absorption and fluorescence spectra are usually associated Hrlth the alteration in the degree of conjugation upon ionization of the functional groups present in the indicator molecule. The pH region of utility 1s usually determined by the pK, of the indicator molecule. Peterson was the first to report the use of an indicator that related the change in the absorption spectra to PH.~ Seitz used a pH indicator based on changes in the fluorescence spectra of fluoresceinaminem4 Fluorescence has become the preferred method because of its selectivity and sensitivity. Selectivity arises from the fact that not all chromophores that absorb will fluoresce, which is especially true in the NIR region where only a few classes of compound exhibit fluorescence. The use of fluorescent dyes as pH indicators, mostly azo or fluorescein derivatives, is well documented in the literature.5-‘0 AppliThe

cations include analysis of blood,’ spinal cord tissue,6 brain pH’ and more.8-10 The utility of the indicator is often greatly enhanced if the pH change has a large effect on the fluorescence quantum yield of the indicator. Fluorescent indicators can be especially valuable in determining the pH of samples which have significant absorbance. In particular, the near-infrared spectral region of the electromagnetic spectrum has proven to be useful in the characterization of biological samples. The long wavelength red and short-wave near-infrared spectral region of 650-1000 nm exhibits minimal interference from biological matter. This attractive feature of the spectral region has intensified interest m development of new probe molecules. The analytical applications related to NIR absorbing chromophores have increased dramatically durmg the last few years. The use of semiconductor lasers as light sources further enhances the advantages of this spectral region. The absorption and fluorescence properties of many pH sensitive indicators have been investigated in the past, including their use as fiber optic probes. Most of these dyes are weak acids or bases with ionizable functions such as carboxylic acid, phenol or amine. The appropriate pH indicator dye must have pH sensitive functional groups that reversibly change from the ionized to the non-Ionized form with a 935

G PATONAY et al

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concomitant change in the degree of conjugation of the double bonds in the molecule as the hydrogen ion concentration changes. The use of pH indicators that are suitable for tissue and blood pH measurements in the physiological range has become of interest since their first application in the late 1970s.‘A The chemistry of pH sensitive molecules has been of immense interest. One group of fluorescent dyes, howeven, that has not been fully investigated as pH indicators is the aminoenones.“*” Junek reported in 1973 the synthesis and properties of dialkyl aminomethylene substituted tetralones, indanediones and indanones.” In the following year he reported these dyes as a new group of pH indicators.‘* Nevertheless, few reports have addressed the use of aminoenone indicators that can be useful in the physiological range.“*i4 We report here the spectral characteristics and the pH dependence of a new symmetrical aminodienone dye system 1 in methanol and in a series of organic solvents. The data presented reveal dramatic effects of protonation of the dye on its spectral characteristics. The dye is stable in alcohols under acidic and basic conditions and the dye spectra are dependent on the solution pH. Also, the dye exhibits high spectral sensitivity to the presence of protons in aprotic organic solvents. EXPERIMENTAL

Reagent and chemrcals

The chemical structure of the pH sensitive dye, 2,6-Bis[2’-( 1”-ethyl-3”,3”-dimethylindolin-2”-ylidene)ethylidene]cyclohexanone (1, CMH400N2, MW = 492.68) used in this study, is

shown in Fig. 1. We recently presented an efficient synthesis of 1 and its purification.” A sample of 1 used in this work was homogenous by TLC on silica gel with several mobile phases, gave good microanalytical results (C, H, N), and its 400 MHz proton NMR spectrum was fully consistent with the high purity of the dye. For the experiments, spectrophotometric grade methanol was obtained from Baker Chemical Co. Isopropyl alcohol, dimethyl sulfoxide, dichloromethane, tetrahydrofuran, acetonitrile, hydrochloric acid, sodium hydroxide and buffer components were obtained from Fisher Scientific Co. The absorbance data were determined by using a disposable PS cuvette (Fisherbrand). The fluorescence intensity was determined in a quartz cuvette (Fisherbrand). Method

Stock solutions of the dye (1O-3 M) were prepared in spectrophotometric grade methanol, isopropanol and DMSO (Baker analyzed). For the pH studies, small amounts of stock solution were diluted to arrive at the desired concentrations for absorbance and fluorescence measurements. Since the dye is less soluble in water, the aqueous solutions were prepared by dissolving the dye in methanol and then diluting with water to obtain dye solutions in a methanol/water (1: 9) mixture. No spectral differences were observed in the presence of up to 10% methanol. Dye 1 is stable in methanol/water at room temperature for 24 hr. If kept in the dark at -20°C the dye solutions are stable for up to 20 days; however, they would turn orange if exposed to excessive sunlight. Dye 1 is stable in acetonitrile for 10 days, but undergoes slow

2-D Fig. 1 Structures of a neutral form of the pH sensitive dye 1 and its protonated form 2. A suggested mechanism for the selective hydrogen-deutenum exchange m poslhons 1’ and 7’ of the dye under acldlc con&trons ISalso shown. Note different numbering scheme-sfor the dlsubstituted cyclohexanone denvauve 1 and the cyanine dye 2, as reqmred by chenucal names of these systems

Near-infrared absorbing pH senatwe ammodlenone-carbocyanme dye system

decomposition in dimethyl sulfoxlde, even in the dark. Accordingly, fresh working solutions were prepared for each experiment. The solution pH was varied by slowly adding small amounts of hydrochloric acid or sodium hydroxide in a methanol-water mixture. The absorption spectra of the dye were taken before and after the pH measurements to examine the stability of the dye. The visual transition of color from basic (pink) to acid conditions (green) occurred at pH 3.0. Buffer solutions were used to calibrate the pH glass electrode (Fisher Scientific). Instrumentation Solution pH was determined by using an Orion Research Model 701A digital tonalyzer. A Perk&Elmer Lambda 2 UV/Vis/near-IR spectrophotometer interfaced to a Zenith 286 PC was used to obtain absorption measurements. The PECSS program supplied with the instrument was used to analyze, store and retrieve data. An SLM 8000 spectrafluorometer interfaced to a PS/2 IBM PC was used for fluorescence measurements, and the fluorescence spectra were stored on floppy disks for later analysis. The p& values were determined using a Schott Geraete Model 250 autotitrator and model T90/10 autoburet interfaced to a PC. RESULTS AND DISCUSSION

Absorbance Representative absorption spectra of the pH sensitive dye 1 in methanol under acidic and basic conditions are shown in Fig. 2. Under basic conditions (dark pink solution), the dye is in a ketone form and shows a broad absorbance band with a maximum at 531 nm (Fig. 2B). The intensity of the 531 nm peak decreases with decreasing pH. At around pH 3 the dye solution turns a pale pink color. As the pH of the dye solution is decreased further, the color becomes pale green with the concomitant appearance of a narrow peak wrth a maximum absorbance at 709 mn (Fig. 2A). The intensity of the peak at 709 nm increases as the solution pH is lowered further while the color of the dye solution turns dark green. The change in the absorption spectra is attributed to the protonation of the oxygen atom to yield a cationic enol from 2 (Fig. 1). The formation of the enol function at the polymethine chain results in the development of a longer conjugated system. More specifically, the protonation results in the formation of a cat-

937

ionic cyanme dye systems 2. This structural change is responsible for the observed, large bathochromic shift. It is known from the chemistry of the polymethine cyanine dyes that the two major determining factors in the absorption maximum wavelength are the extent of conjugation and presence of a quaternary nitrogen in the heterocyclic moiety. Those dyes that have no quaternary nitrogen, commonly referred to as dye bases, have much lower absorption wavelength maxima than a similar molecule with quaternary nitrogen in the heterocyclic terminus. The absorption of the cationic form 2 is typical for a heptamethine cyanine dye system.16 Two forms of the dye, thus, are an aminodienone 1 and a cationic heptamethme cyanine system 2. The symmetrical cationic species 2 is the predominating form under acidic conditions. Dye 1 is more stable in acid conditions than m basic conditions, including light sensitivity. Under basic conditions the molecule undergoes deprotonation to form the neutral aminodienone 1. The dye can be converted back and forth between the two forms simply by adjusting the solution pH, and these changes are fully reversible. The dye is sparingly soluble in pure aqueous solutions; however, m the presence of a small amount of alcohol, the dye becomes soluble and it is stable in such a solvent system. The spectral behavior of the dye is markedly different in aqueous media. Under acidic conditions a broad absorbance peak with a maximum at 423 mu can be observed, while under basic conditions a very broad peak with a maximum absorbance of 484 nm is present. The molar absorptivity measured at pH 6 and pH 2, respectively, and other spectral properties of the dye are tabulated m Table 1. The different

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G PATONAYet al Table 1 Absorbance for 1 Absorbance Water*

Methanol VH A&K Baac

1 (nm) d (I mol-’ cm-‘) 709 535

2082E+5 776OE+4

1 (nm) 6 (1 mol-’ cm-‘) 425 484

2.651E + 4 3438E+4

Isopropanol L (nm) 6 (1 mol-’ cm-‘) 709 520

1 537E + 5 6218E+4

* 10% Methanol/!%% water

spectral behavior m aqueous systems might be attributed to two factors, namely, dimerization or higher order aggregation of the molecules m a less hydrophobic environment and covalent hydration of the dye system or dye aggregate under acidic conditions (Fig. 1). However, the absorbance spectra of dye 1 at concentrations Q lo-*M showed no dimerization effect. The hydration would result m shortening of the dye chromophore with a concomitant blue shift m the absorption spectra, as observed. This blue shift would be especially large for hydration of the cationic form 2, due to a facile nucleophilic addition reaction with the polyunsaturated cationic chromophore.‘5*16 The changes between the aqueous and organic solutions are fully reversible, however. After the aqueous solution of 1 was extracted with dichloromethane, the spectra of the organic extract taken under different pH conditions exhibited the same behavior as the spectra of a freshly prepared solution of 1 in dichloromethane. The same results were obtained with the extracts from alkaline and acidic solutions. The addition reaction of water to position 2(2”) of the indoline of dye 2 to give an adduct 3 (R = H) can be suggested (Fig. 1). Absorption maximum around 400 nm was estimated’7~‘8 for 3, which is in reasonable agreement with the experimental value of 423 nm. With methanol the equilibrium 2 + CH,OH # 3 must favor the substrate 2 because the increased stenc hindrance destabilizes the adduct 3 (R = Me) in comparison to its hydroxy analog (R = H). As a result, the absorption maximum of 2 in methanol is shifted to longer wavelengths relative to the absorption in aqueous media. support for the experimental Strong suggested addition reactions was obtained from proton NMR studies. The spectrum of 1 taken in deuterochloroform exhibited a characteristic AB pattern for Hl’ and H2’ at 6 5.46 and 8.17, respectively, with a coupling constant of 13.2 Hz.‘~ Upon acidification of the 1 ml sample solution in the NMR tube with 2 equivalents of

HCl in 0.05 ml of methanol, a similar absorption pattern was observed at 6 8.58 and 5.79, provided the spectrum was obtained immediately following acidification of the sample. The deshielding effect is attributed to the formation of cation 2. With deuterium chloride instead of HCI a highly selective deuteration at positions 1’ and 7’ was observed to give 2-D (note a different numbering scheme for this compound), as evidenced by the appearance of a singlet for H2 (6’) at S 8.58 and lack of absorption at S 5.79. This fast deuteration was complete within 5 min, a minimum time period necessary for obtaining the NMR spectrum. No additional changes m integration of the spectral signals were observed after the sample had been allowed to stand at 23°C for 24 hr. The facility of this acid-catalyzed hydrogen+leuterium exchange was further stressed by the isolation of non-deuterated 1 after the sample of 2-D had been treated with silica gel, a weak acid. These results are fully consistent with the proposed mechanism (Fig. 1) in which the adduct 3 is an intermediate product for 2-D. The NMR signals of the samples discussed above were gradually broadened with time, and no further broadening was observed after 1 hr after the addition of acid m methanol-d4 to the solutions in deuteriochloroform. The period of time required to reach the final line shapes at 23°C decreased with increased amount of methanol-d, m the solution and was 15 min for pure methanol-d, m the presence of 2 equivalents of acid. Unfortunately the NMR spectra in aqueous acidic methanol-d., could not be obtained due to low solubihty of the dye under these conditions. Interestingly, the neutral dye 1 gave sharp NMR signals regardless of the concentration, time, and solvent composition. The NMR signal broadening is indicative of a relatively slow aggregation of the hydrophobic dye molecules in a hydrophilic environment, a typical property of most carbocyanine dyes.” It should be noted that in all cases dye 1 was isolated quantitatively from solutions used for NMR studies.ig

Near-mfrared absorbing pH senattve ammodtenone-carbocyamne

In aqueous or methanol environment, under the influence of atmospheric oxygen, especially in the presence of intense sunlight, the dye decomposes and a color change can be observed with the absorption maximum changes from 535 nm (pink) in methanol (or 484 nm in aqueous solutions) to 445 nm (orange) in 25 min. The absorbance spectra of the dye m a 50% (v/v) methanol-water solution 1s shown in Fig. 3. This type of light sensitivity is typical for several members of the carbocyanine dye family and can be attributed to the reaction of the molecular oxygen with the conjugated system in the presence of short wavelength radiation. Accordingly, special care was taken not to expose our solutions to excessive sunlight during experiments. In isopropanol the dye showed properties similar those in methanol. Under basic conditions the dye gave an absorbance peak at 520 nm which is only slightly lower than the absorbance shown in methanol. In acid conditions the dye showed an absorbance band at 709 nm. These results are m good agreement with the general observation about carbocyanine dyes that alcohols are usually good solvent systems for studying electronic spectroscopic properties of cyanine dyes and there is very little variation from alcohol to alcohol The absorption properties of the dye were also examined in dichloromethane and tetrahydrofuran. When the solution was made acidic with the addition of acetic acid, a narrow peak with an absorption maximum at 709 nm was observed. This result is consistent with the formation of a cationic dye system 2. The intensity of this absorption increased with increased acetic acid concentration in both solvents.

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Frg 4 Fluorescence for the pH senmhve dye 1 m methanol under actdtc (A), pH 2 (-) and basic (B), pH 6 5 ( ) condrttons

Fluorescence

Changes m the fluorescence spectra of dye 1 m methanol were studied under acidic and basic conditions. The representative spectra under basic conditions, shown in Fig. 4(B), exhibited a broad peak with a maximum emission at 606 nm (&x = 534 nm). The spectra under acidic conditions with an emission maxima at 743 nm, (Lax = 709 nm) is shown in Fig. 4(A). As can be seen, the emission spectra are determined by the solutton pH. A plot of fluorescence mtensity as a function of solution pH is shown m Fig. 5. The fluorescence emission was monitored at a constant wavelength of I,, = 620 nm (1,x = 530 nm) for the basic peak (Fig. 5B) and at AEM= 740 nm (&x = 709 nm) for the acidic peak (Fig. 5A). The fluorescence emission intensity for dye 1 in the cationic enol form is not as intense as in the ketone form. The fluorescence properties of the dye were found to be different in aqueous solutions compared to alcohols. Under basic conditions, the

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Rg. 3 Absorbance spectra for the pH sensrtrve dye 1 m a 50% methanol-water solutron

Rg 5. Fluorescence intensity m methanol momtored at a constant wavelength of A,, = 620 nm (A, = 530 nm) for the basic peak (B) and at I,, = 740 run (&,t = 709 nm) for the actdrc peak (A)

G PATUNAYet al.

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Fig. 6 Fluorescence for the pH sensitive dye 1 m LYO- Fig 7 The pH dependence of the wslble and NIR absorption peaks for dye 1 m methanol propanol under acldlc (A), pH 2 (-) and basuz (B), pH 6 ( ) conditions

dye showed a weak emission at lzrx = 495 nm (L = 425 nm) while in the presence of acid a weak emission at lZEM = 660 nm (&, = 545 nm) could be observed. The solvent hydrophobic&y dependence of fluorescence spectra was also studied using different methanol-water mixtures. The fluorescence intensity was the highest at high methanol concentrations indicating higher fluorescence quantum yield in alcohols (up to lOO-fold). No significant change in the fluorescence intensity was observed up to 10% water concentration. The fluorescence intensity decreased at higher water concentrations; the fluorescence quantum yield was very low above water concentrations higher than 70% in methanol (v/v). The fluorescence emission maximum of dye 1 in isopropanol was at 741 nm (Aax = 709 nm) under acidic conditions (Fig. 6A), while under basic conditions fluorescence maximum was at 606 nm (Fig. 6B). The dye fluorescence was also studied in dimethyl sulfoxide, acetonitrile and dichloromethane, and the results are shown in Table 2. These data indicate that the fluorescence spectra of this NIR dye are strongly dependent on the hydrophobicity of the solvent.

The pK, value for the dye was measured in 50%/50% water-methanol rmxture with an automated titrator using NaOH dissolved in 50%/50% water-methanol mixture. The titration curves were analyzed with first and second order derivatives and only one pK, was observed for the solution at 6.91. This value indicates that protonation is not the only factor that brings about changes in the absorption and fluorescence spectra of the dye with changing pH. When Figs 5 and 7 are compared to the pK, value of the dye we can see that there is very little effect due to excited state deprotonation. Solvent effect may be the dominating factor as evidenced by spectral dependence on hydrophobicity. One possible explanation is the effect of pH on the dye aggregation. Dye 1 as pH probe in organic solvents The data presented here indicate that the dye is suitable as a pH probe in organic solvent medium. The advantages of this dye over other pH probes are a significant change in color and a relatively long absorption wavelength of the principal absorption band. Both the visible or the NIR bands can be used for determining the

Table 2. Fluorescence for 1 Solution pH condlhons Pure solventt

Acldlc+

Methanol Water Isopropanol A&on&de Dlchloromethane Dlmethyl sulfoxlde

Lax (nm) 709 545 709

1EM (nm) 744 660 742

Int (E+O4) 128 0 0353 17

kx (nm) 520 425 520 505 469 515

1, (nm)

Int (E+O4)

606 495 606 571 544 574

4 54 0.0250 45 11 0 0578 26

*HCI

t&x correspond to the absorbance maximum measured m the pure solvent

Near-Infrared absorbmg pH sensttive a~n~~on~~~ne

pH of the environment around the dye molecule. Figure 7 can be used as a calibration curve for determining pH using the NIR absorption peak. Figure 7 (circles) indicates that this peak is not present in the dye spectra in basic solutions. In basic solutions, however, the visible absorption appears and the intensity of the peak increases with increasing basicity of the solution and levels off at pH 6-7. The appearance of this shorter wavelength absorption peak corresponds to a decrease in the NIR absorption peak (Fig. 2A), clearly Indicating the removal of the positive charge from the dye molecule. The dye base ketone which forms durmg this process has significantly lower absorption wavelength and blue shifted absorption maxims. The hyps~hro~c shift in absorption maximum is a result of two effects, less extensive conjugation and lack of positive charge in the chromophore. The combination of these two effects results in a significant spectral shift, which may be very advantageous when observing pH change. This large spectral shift and the pH range of spectral change indicate that additional processes have an influence on the dye spectral behavior. Further studies are under way to fully characterize this process. Dye 1 ashydrophobicityprobe The NIR dye 1 also exhibits spectral changes as the hydrophoblcity of the solvent is changed. The utility of this application was demonstrated for the dete~ination of water content in alcohols. Most likely the observed change in the absorption spectra with changing water concentration is a result of dimer formation. The dimenzation property of carbocyanine dyes has been documented in the llterature.‘9 As the water ~n~ntration increases, the carbocyanines tend to form dimers or higher aggregates because of the strong dispersion forces associated with the high polarizability of the polymethine chain. However, in most cases, the dlmer or higher aggregate band ISnot too well resolved from the monomer band, hindering the application. The utility of our NIR dye as a hydrophobicity probe is illustrated in Fig. 8. It shows a typical calibration curve obtained using the NIR pH sensitive probe as a hydrophobicity probe using a methanol-water mixture as the model solvent. It is important to point out, however, that the researcher must make sure that the two effects, pH and hydrophobicity, do not interfere.

dye system

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Water % (v/v) Fig 8 Typical cahbratxon curve for determmmg water concentrattlon in methanol by usmg dye 1 as a probe. CONCLUSIONS

In s~rna~, the NIR absorbing carbocyanine dye discussed in this paper can be valuable in determining analytical properties. The data presented illustrate the utility of this dye for determining pH and solvent hydrophobicity. The well separated absorption peaks of the protonated and non-protonat~ forms of the dye further enhance the analytical utility of this probe molecule. The dye is an especially useful probe organic solvents, e.g alcohols for the determination of PH. We are currently synthesizing and investigating similar NIR dyes for other probe and labeling applx~ations. As a result of these studies the design of new, tmproved NIR absorbing probes may be available. Acknowledgements-Tlus work was supported m part by grants from the NatIonal Science Foundation (CHE-890456 and CHE-~5~) and the National Instrtutes of Health (AI-28903) Acknowledgement 8salso made to the donors of the Petroleum Research Fund, admmlstered by the Amencan Chemical Society, for partial support of thu research

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