Polycyclic Aromatic Hydrocarbon Analogues - American Chemical

Chapter 17

Polycyclic Aromatic Hydrocarbon Analogues Another Approach to Environmental Separation Problems 1

1

1,3

A. Cary McGinnis , Srinivasan Devanathan , Gabor Patonay , and Sidney A. Crow 2

Downloaded by NATL UNIV OF SINGAPORE on May 4, 2018 | https://pubs.acs.org Publication Date: October 27, 1992 | doi: 10.1021/bk-1992-0509.ch017

1Department of Chemistry, Georgia State University, Atlanta, GA 30303 2Department of Biology, Georgia State University, Atlanta, GA 30303

Aryl dyes containing different aromatic groups may be prepared using simple synthetic steps, e.g., by condensation of an arylcarboxaldehyde with a quaternary salt of a heterocycle having a methyl group in the 2or 4-position. The chromophore in these dyes, due to the more extensive conjugation, has much longer absorption and emission wavelengths than the aryl compound itself allowing the researcher to utilize the lower interference spectral region, e.g., the red or near-infrared region. The spectral properties of aryl dyes may be used to significantly reduce separation needs when the fate of aryl compounds is followed in the environment. This paper focuses on the spectral properties and environmental utility of aryl dyes prepared in our laboratory for decreasing separation requirements.

The worldwide exploration and shipment of crude oil and petroleum products have led to a number of catastrophic environmental disasters. A wealth of information exists on these events; however, long-term consequences are poorly understood. In addition, the processing and utilization of coal and petroleum fuels invariably lead to low levels of pollution in the terrestrial and marine environment by a variety of molecules including polycyelic aromatic hydrocarbons (PAHs). Pollution occurs at different levels which can either be acute or chronic. The effects of the latter are the most difficult to discern. The importance of studying the fate of PAHs in the environment can not be understated. PAHs are a ubiquitous, diverse group of organic compounds containing one or more fused aromatic rings. PAHs are found in air, water and soil samples due to contamination from combustion of hydrocarbons and from petroleum spills. But the fate of PAHs in the environment is hard to follow as a result of detection and 3

Corresponding author

0097-6156/92/0509-0229$06.00/0 © 1992 American Chemical Society

Vandegrift et al.; Environmental Remediation ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


230

ENVIRONMENTAL REMEDIATION

separation difficulties. Simple spectroscopic and chromatographic methods are severely limited because of interference in biological systems. Current mechanisms to evaluate the degradation and detoxification of anthropogenic PAHs in the environment frequently depend on complex analytical schemes which involve the extraction of large volumes of material. There are several possible chemical and biological routes through which PAHs are changing in the environment. For example, bacteria and fungi have been known to metabolize polycyelic aromatic hydrocarbons [1,2]. Owing to the extremely complicated chemical and biological processes, a large number of derivatives of PAHs may be found in the environment. This fact may explain the difficulties associated with environmental determination of PAHs. Advancements in separation science are major factors in the recent development of PAH research in the environmental sciences. In spite of the advances in separation sciences, however, there are still major difficulties due to significant interference from other environmentally important compounds [3-5]. The development of appropriate analogues is one possible means of decreasing interference and reducing the need for complicated separation procedures in studies of the environmental fate of PAHs. These analogues can be used for predicting the fate of PAHs in the environment using much simpler analytical procedures than otherwise required. We are in the process of developing an even simpler analytical approach to studying the fate of PAHs in the environment by using red and near infrared analogues of PAHs. Using these compounds, we can simplify the analytical procedure in environmental analyses by moving the absorption wavelengths away from the interference of the sample where the underivatized PAH molecules are normally seen. The direct use of absorption or fluorescence spectroscopy for the study of the biologically and environmentally important PAHs may be less valuable due to interference. PAHs are spectroscopically active in the region where interference is more likely. These difficulties warrant the investigation of molecules that may serve as model compounds or analogues for studying biological systems and the fate of PAHs in biological systems. These needs led us to the investigation of aryl dyes. Aryl dyes may be prepared by forming a methine chain between a quaternary salt of a heterocyclic compound having a methyl group in the 2- or 4-position and an aryl compound, e.g. PAHs [6]. The best known compounds in this group are the styryl dyes. Following a synthetic route similar to that used in the preparation of styryl dyes, other dyes containing aryl groups may be prepared. For example, styryl dyes are prepared by the condensation of benzaldehydes with the quaternary salt of heterocyclic compounds [6]. Pyrene is one of the best characterized PAH molecules. Its spectral and chemical properties as well as its role and fate in the environment have been thoroughly investigated [3]. For example, pyrene has been found to be a useful fluorescence probe for studying microenvironmental changes since it is very sensitive to different quenching processes and changes in microhydrophobicity [7-9]. In this manuscript, the spectroscopic characteristics and the interaction with the microbial population of a new aryl dye, will be discussed. The information gained by spectroscopic techniques provides an alternative approach to separation problems.


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PAH Analogues and Separation Problems

231


Materials and Methods The methanol used in this study was obtained from the Aldrich Chemical Company (Milwaukee, WI) in spectrometrie grade. Other chemicals used in the synthesis of the aryl dye were also obtained from Aldrich. The chemical structure of the red absorbing pyrene analogue is given in Figure 1 along with some other aryl dyes that may be easily synthesized from commercially available precursors. Only preparation of the pyrenyl dye is given below because other aryl dyes may be prepared similarly using the appropriate aryl carboxaldehyde [6]. The starting material was 3-ethyl-2-methylbenzothiazolium iodide obtained by heating a solution of ethyl iodide and 2-methylbenzothiazole in dimethylformamide at 150 °C for 12 hours. Workup of this reaction mixture included cooling to 23 °C and slow addition of diethyl ether to crystallize the product. The crystals were filtered, washed with diethyl ether, recrystallized and dried under reduced pressure to give the analytically pure 3-ethyl-2-methylbenzothiazolium iodide, m.p. 241-243 °C. The subsequent transformation of this iodide was as follows: A mixture of 1 g (3.8 mmol) 3-ethyl-2-methylbenzothiazolium iodide, 0.898 g (3.9 mmol) of 1pyrenecarboxaldehyde and 0.726 g (7.2 mmol) of dry triethylamine was refluxed in 5 ml of ethanol for 30 minutes. The reaction mixture was cooled and the solvent was removed under reduced pressure. The crude solid thus obtained was dissolved in dichloromethane and precipitated with ether and further purified with recrystallization. Chromatographic purification may be necessary if crystals do not form readily. The m.p. of the analytically pure compound is 225-228 °C, decomposing. The structure of the dye, given in Figure la, was fully consistent with the measured NMR spectrum (CDC1 ). HPLC analysis has been performed for this series of dyes. A chromatogram of the phenanthrenyl dye on a C-8 column is shown in Figure 2. This chromatogram is representative of the aryl dyes and indicated retention times are similar to that of underivatized PAHs. 3

Instrumentation. Absorption spectra for the pyrenyl dye were obtained by using a Perkin-Elmer Lambda 2 UV/Vis/NIR spectrophotometer. The spectrophotometer is interfaced to a 286 computer to store spectra and control the instrument. Each spectrum was recorded using a PECS S program (Perkin-Elmer). Fluorescence spectra were recorded on an SLM 8000 spectrofluorometer interfaced to an IBM PS/2 computer. The 10 M stock solutions of the pyrenyl dye were prepared in spectrophotometrie grade methanol or ethanol. For the studies, solutions were prepared by pipetting the required amount of dye stock solution dissolved in alcohol into a volumetric flask which was filled with water or culture media. The preparation of dye solutions has been found to be much easier when this method is followed instead of completely removing the methanol from the aliquot of the stock solution by nitrogen gas. The final solutions contained a negligible amount of methanol which did not have any effect on the results. 3

In the process of developing this analytical approach to studying the environmental fate of PAHs using red and NIR absorbing aryl dyes as analogues, we




232

Figure lb. Chemical structure of different aryl dyes.



17.

PAH Analogues and Separation Problems 233

McGINNIS ET M*

6β+086 +

4β+ββ6 +

2m+BBÛ 4-

\

1

,

1

1

1

1

5

10

15

20

25

30

35

Retention Time

Figure 2. HPLC chromatogram of phenanthrenyl dye on C-8 column (gradient: 15%-80% isopropanol in water, both being 5mM in pentane sulfonic acid).


234


expect to significantly reduce the separation requirements during environmental analysis. By using these aryl dyes, we hope simply to follow metabolitic changes of PAHs in the environment without complicated separation steps. This is achieved by moving the absorption away from the interference of environmental samples where the underivatized PAH molecule is normally seen. The effectiveness of this approach may be evaluated by studying the effect of dye on yeast cultures that are known to metabolize PAHs.


Studies of biological interaction of the PAH analogues Using the pyrene analogue, preliminary toxicity and binding studies were conducted. Fresh liquid biological cultures were prepared from stock cultures Mycological agar (Difco) for yeasts and Saboraud's Dextrose agar for filamentous fungi by transferring sufficient inoculum to an appropriate broth. Yeasts were routinely cultured on 67% yeast nitrogen base (Difco) supplemented with 0.5% glucose (GYNB). Filamentous fungi were cultured on Saboraud's Dextrose broth (Difco) because of the more complex requirements and slower growth rates. In-depth studies of biological activity were conducted with the yeasts. Toxicity. Initial assessment of the toxicity of these aryl dye molecules using a modification of the agar disk diffusion antibiotic sensitivity was inconclusive because of the limited diffusion of the compound and its intense binding to the cellulose disks. Liquid cultures supplemented with 1 mg of aryl dye dissolved in 1 ml of DMF were used to assay toxicity. Comparison of the growth of Candida lipolytica (GSU 37-1) and Candida maltosa (GSU R-42) was made by observing the optical density at 595 nm in a Turner spectrophotometer model 380. Determination of the absorption by compound was made for uninoculated GYNB. The increase in absorbance in cultures with and without analogue was compared. Uptake. Association of a molecule with a non-growing cell in a non-growth supporting environment is an important parameter for biologically active molecules. Because of differences in the absorption spectra of our analogues in solvents of different hydrophobicity, we were interested in seeing the effects in biological systems of effectively different physiological and morphological nature. Cultures were grown under similar basal conditions but supplemented with either 0.5% or 5.0% glucose, 0.5% ethanol, or 0.5% tetradecane. Cultures were inoculated as described above and incubated on an R. Braun gyrotary shaker (autoshake model U) at 120 cycles per minute. Cultures were incubated at room temperature (approximately 24 °C) for appropriate intervals sufficient to produce the necessary cell mass. Following incubation, an aliquot of cells was removed from the culture. The cells were sedimented by centrifugation (10 min at 3000 times G) and washed in 0.9% saline 3 times and resuspended to the original 10 ml. Different concentrations of aryl dye were added to the cell suspension in DMF (.5mg/.5ml). The mixture was allowed


17. McGINNIS ET AL.

PAH Analogues and Separation Problems 235

to incubate in the dark for 30 min. Following incubation the cells were again removed by centrifugation and the concentration of aryl dye was determined spectrophotometrically on a Perkin Elmer Lambda 2 UV/Vis/NIR spectrophotometer.


Results and Discussion Representative absorption and fluorescence spectra for the phenanthrenyl dye in methanol and water are shown in Figure 3. Detailed spectroscopic studies of the pyrenyl dye compound in the presence of organized media has been described earlier [10,11]. As can be seen from the data, these dyes can change their absorption spectra depending upon the hydrophobicity of the environment similar to the pyrenyl dye [10,11]. These changes, which have been associated with dimerization [10,11], may result in color changes of the dye solution when the hydrophobicity of the solvent is changing. Since the absorption spectra of the dyes indicate whether the dye molecule is in monomeric or dimeric form, useful information may be gained concerning its interaction with cell membrane. It is interesting to compare the absorption spectra of these dyes to that of the PAH molecules [10,11]. If we compare the region of the spectra where PAH molecules absorb (250-400 nm), we can observe interesting similarities [10,11]. Figure 4 illustrates these similarities. These indicate that the spectral properties of the aryl group are retained to a great extent. The spectroscopic similarities between these dyes and their underivatized PAH counterparts actually go beyond the ones mentioned above and have been discussed earlier [10,11]. These similarities, however, do not indicate whether we can utilize these compounds as PAH analogues. To evaluate this aspect, we studied the interaction of these dyes with different yeast strains. Table 1 reflects the growth of cultures with and without the aryl dye following adjustment for the PAH analogue absorption. There is no evidence of growth inhibition in either of these cultures. Furthermore, subsequent studies with the filamentous fungi; Aspergillus niger, Cunninghamella elegans, and Svncephalastrum racemosa have demonstrated no growth inhibition by the analogue. Uptake is distinctly influenced by the growth substrate (Table 2). The greatest specific binding (micrograms/10 cells) was with Candida lipolytica grown on ethanol, followed by tetradecane grown cells. Candida maltosa demonstrated a slightly different pattern (greatest binding in low glucose and very similar activity for tetradecane, and ethanol substrates). Least efficient binding for either culture was from high glucose conditions. These preliminary studies have demonstrated the utility of the study of analogues in discerning biologically significant processes such as toxicity and uptake. The differences noted suggest major differences in the uptake of our analogues as a function of growth substrate and a significant species-dependent component of the initial encounter with these analogues, similar to underivatized PAHs [1,2]. A wider study of these analogues and their interaction with microbial cultures and communities under a variety of conditions will enhance our ability to extrapolate from analogue to PAH effects. Detailed studies comparing the analogue behavior to PAH and studies to determine other facets of the biological effects of PAH are in progress. 6


236



0,10

0.00 400

460

520

580

640

700

Wavelength [nm]

Figure 3a. Representative absorption spectrum of the phenanthrenyl dye (10~ M) in methanol. 6

0.03

0.00 400

460

520

580

640

700

Wavelength [nm]

Figure 3b. Representative absorption spectrum of the phenanthrenyl dye (10~ M) in water. 6


McGINNIS ET AL.



17.


237



238


17.

McGINNIS ET AL.


239

0 15 r

I


a

0.00 250

290

330

370

410

450

W a v e l e n g t h [nm]

0.20 ι

1

^

0.16 h

b

250

290

330

370

Wavelength

[nm]

410

450

Figure 4. Representative absorption spectra in the UV region of the pyrenyl dye (a) and pyrene (b) in methanol.

Very little or no separation is required during determination of these aryl dyes in the media. Although the media exhibit a broad absorption peak severely interfering with the underivatized aryl compounds, very little interference is observed in the region where the chromophore absorbs, indicating the utility of using these aryl dyes as analogues to study the environmental fate of PAHs. Syntheses of additional analogues with a different number of fused rings are in progress. A systematic evaluation of the activities of these molecules in biological systems will provide a sound basis for the application of red and near-infrared laser Vandegrift et al.; Environmental Remediation ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

240


Table 1 Effect of NIR pyrene-analogue on growth of yeast in 0.5% GYNB Candida maltosa (R-42) 0.5%GYNB + analogue

Candida lipolvtica (37-1 ) OJ^oGYNB + analogue

1


Time [hours]

1

Absorption

Absorption

0

.01*

.01"

.02*

.02**

12

.16

.15

.09

.08

24

.60

.59

.47

.49

48

1.22

1.27

1.08

1.06

96

1.59

1.59

1.74

1.73

1

0.5% Glucose in 0.67 Yeast Nitrogen Base (YNB) * Absorption at 595nm ** Absorption at 595nm corrected for compound absorbance Table 2 Binding of NIR pyrene-analogue as influenced by culture media Candida maltosa (R-42) Media

Time

Candida lipolvtica (37-1)

% cell specific bound #'s binding*

% cell bound #'s

specific binding*

l

98 1.2E7

84

94 2.3E6

408

0.5%GYNB 24h

2

96 8.9E6

107

88 2.8E7

31

5% GYNB 24h

14 2.5E7

6

43

4.7E7

10

92 1.4E7

83

97

1.8E6

532

0.5%TYNB 24h

3

4

0.5%EYNB 24h

* micrograms binding for 1E6 cells (approx 1 microgram cells) 1 2 3 4

0.5 % Tetradecane in 0.67% Yeast Nitrogen Base (YNB) 0.5 % Glucose in 0.67% YNB 5 % Glucose in 0.67 YNB 0.5 % Ethanol in 0.67 YNB Vandegrift et al.; Environmental Remediation ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


17. McGINNIS ET AL.


241

spectroscopy to the evaluation of analyses where otherwise extensive separation would be required. The major advantage of this method is that the absorption maxima of the analogue may be chosen using appropriate heterocyclic derivatization. Using aryl dyes as analogues, not only may the separation aspect of environmental analyses be simplified, but it may also improve our understanding of microbial detoxification processes in cases where the environmental damage has already occurred. We have evaluated Candida lipolvtica and Candida maltosa as to the utility of using red or near-infrared absorbing aryl dyes to decrease separation requirements in environmental analyses. These, as well as other bacteria and fungi, are well documented as degraders of hydrocarbons and PAHs in marine environments. First aryl dyes were introduced into cell cultures to study the recovery and mass balance of the systems. These measurements may be conducted easily since very little or no separation is required due to significantly decreased spectral interference. Before we can fully utilize aryl dyes as PAH analogues, however, several important points need to be addressed. Further studies are needed to determine the biotic and abiotic degradation routes of these aryl dyes. These studies are presently under way in our laboratory. Acknowledgement This work was supported in part by a grant from the National Science Foundation (CHE-890456) and in part under a cooperative agreement from EPA (R818696). References 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

D.T. Gibson, Crit. rev. Microbiol., 1, (1972) 199. D.T. Gibson, "Biodegradation of Aromatic Petroleum Hydrocarbons," In, D.A. Wolfe ed., "Fate and Effects of Petroleum Hydrocarbons, Pergamon Press, New York, N Y (1977). M.C. Bowman, "Handbook of Carcinogens and Hazardous Substances," Marcel Dekker, New York, N Y (1982). H.V. Gelboin, P.O.P. Ts'o, "Polycyclic Hydrocarbons and Cancer," Academic Press, New York, NY (1978). C.E. Cerniglia and S.A. Crow, Arch. Microbiol., 129, (1981) 9. Rodd's Chemistry of Carbon Compounds, Vol IV, Part B., Ed: S. Coffey, Elsevier Scientific Publishing Company, (1977), pp. 370-422. G . Patonay, K . Fowler, A . Saphira, G . Nelson and I.M. Warner, J. Incl. Phenom., 5, (1987) 717. K. Kalyanasundaram and J.K. Thomas, J. Am. Chem. Soc., 99, (1977) 2039. R.C. Benson, H.A. Kues, J. of Chem. Eng. Data, 22, (1977) 379. M . D . Green, G. Patonay, T. Ndou and I.M. Warner, J. Incl. Phenom, in press. A . E . Boyer, S. Devanathan, and G. Patonay, Anal. Lett. 24, (1991) 701.

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Polycyclic Aromatic Hydrocarbon Analogues - American Chemical

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