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The conformation of some nitro-polycyclic aromatic hydrocarbons
Journal of Molecular Structure 550–551 (2000) 217–223 www.elsevier.nl/locate/molstruc
The conformation of some nitro-polycyclic aromatic hydrocarbons 夽 Y.S. Li a,*, P.P. Fu b, J.S. Church c a
Department of Chemistry, University of Memphis, Memphis, TN 38152, USA b National Center for Toxicological Research, Jefferson, AR 72079, USA c CSIRO Textile and Fibre Technology, Belmont, Victoria, 3216 Australia Received 6 July 1999; accepted 17 November 1999
Abstract The Raman spectra of some nitro-polycyclic aromatic hydrocarbons, including 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, 6-nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, have been recorded in the frequency region 1100–1700 cm ⫺1. The stretching vibrational modes for the nitro group in each of these compounds were assigned based on group frequencies, the measured infrared and Raman spectra, the relative spectral intensities, and the Raman depolarization data. From the measured Raman depolarization data, the orientation of the nitro-substituent with respect to polycyclic aromatic hydrocarbon was determined for each of these compounds except 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene. The results were correlated with the direct-acting mutagenicity of the nitro compounds according to the hypothesis suggested by P.P. Fu and his co-workers. 䉷 2000 Elsevier Science B.V. All rights reserved. Keywords: Nitro-polycyclic aromatic hydrocarbons; Raman spectra; Depolarization ratio; Conformation; Structure and mutagenicity relationship
1. Introduction Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are a class of widespread environmental contaminants that are present in different sources including diesel emissions, combustion emissions from kerosene heaters, gas fuel, liquefied petroleum, airborne particulates, coal fly ash, and food [1–3]. Many nitro-PAHs have been determined to possess a large spectrum of biological and genotoxic activities 夽
Dedicated to Professor James R. Durig on the occasion of his 65th birthday. * Corresponding author. Tel.: ⫹ 1-901-678-2621; fax: ⫹ 1-901678-3447. E-mail address: [email protected] (Y.S. Li).
[1]. Because of their widespread presence in the environment and genotoxic activities, including mutagenicity and carcinogenicity, many of these compounds may pose a health risk to humans. It has been reported that the structural and electronic features of nitro-PAHs may be useful in predicting the biological activities of nitro-PAHs [3–9]. The particular structural feature that has been well demonstrated to be useful in correlation with direct-acting mutagenicity and tumorigenicity is the orientation of the nitro group [3–9]. A general finding is that nitro-PAHs with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in Salmonella typhimurium strains TA98 and TA100 and possess a tumorigenicity weaker than that of the
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aromatic nitro compounds, including some nitroPAHs, but no structural or conformational information could be derived from the studies. Vo-Dinh and his coworkers [14] have reported the surfaceenhanced Raman (SER) spectra of 1-nitropyrene, 9nitroanthracene, 2-nitronapthalene, and 2-nitrofluorene; the major purpose of their studies was to develop SER scattering-active substrates for analysis. Vibrational spectroscopy is a useful and convenient method to work out the conformation of molecules. In the present study, we have measured Raman depolarization data for 2-nitroanthracene (A), 7-nitrobenz[a]anthracene (B), 6-nitrochrysene (C), 1-nitropyrene (D), 6-nitrobenzo[a]pyrene (E), and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene (F) (see Scheme 1 for their structural formulas) and tried to derive the conformation of the nitro-PAHs. The conformational results have been correlated with the direct-acting mutagenicity of the compounds.
2. Experimental
Scheme 1.
parent PAH [3,8,9]. Therefore, in order to predict and/ or interpret the biological activities of nitro-PAHs, it is important to determine the orientation of the nitro functional group. X-ray crystallographic and 1H NMR spectroscopic analyses have been employed for determining nitro group orientation [9–11]. X-ray crystallographic study of 9-nitroanthracene and 7nitrodibenz[a,h]anthracene indicated dihedral angles of 85 and 80.6⬚, respectively between the nitro oxygens and the aromatic ring [9,10]. When a nitroPAH adopts a perpendicular or a nearly perpendicular orientation, the anisotropy of the nitro group causes a slight upfield shift of the adjacent aromatic peri proton(s) in their 1H NMR spectra; whereas the coplanar or nearly coplanar nitro group will cause a marked downfield NMR shift of the adjacent peri protons [11]. Passingham et al. [12] have reported the Raman spectra of nitrobenzene and it derivatives; Juchnovski and Andreev [13] have measured IR spectra of 38
1-Nitropyrene was purchased from Aldrich Chemical Company. 2-Nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 6-nitrobenzo[a]pyrene, and 6nitro-7,8,9,10-tetrahydrobenzo[a]pyrene were synthesized as reported previously [7,15–17]. All the compounds were purified by column chromatography and/or recrystallization and determined to be ⬎99% pure based on HPLC analysis. The corresponding PAH samples of A, B, C, D, and E were obtained commercially. Carbon disulfide, chloroform, chloroform-d, carbon tetrachloride, dimethyl sulfoxide, and dimethyl sulfoxide-d6 were obtained from commercial sources and used as solvent for preparing sample solutions without further purification. All Raman analyses were carried out with the sample solutions sealed in glass capillaries. Conventional Raman spectra were recorded with a Spex Model 1403 double monochromator fitted with a Hamanatsu R928-07 photomultiplier tube (PMT) held at 243 K with a thermoelectric refrigerated chamber (Model TE177-RF). The Raman spectra were acquired at a resolution of 2 or 4 cm 1 plotted with a Spex DM 3000 R computer system loaded with spectroscopy software. A Lexell XL-3000 argon ion laser tuned at 488.0 or 514.5 nm was used
Y.S. Li et al. / Journal of Molecular Structure 550–551 (2000) 217–223
219
Fig. 1. Different orientations of the nitro group with respect to the PAH plane in 2-nitroanthracene. u represents the angle between the nitro group and the PAH plane. The point group symmetry is shown in the bottom of the figure.
for excitation; a laser power of 100 mW was employed. For some nitro-PAHs, fluorescence seriously affected the quality of Raman spectra preventing the observation, identification and depolarization measurement of Raman bands. Attempts to solve this problem were made by purifying the samples and by trying different organic solvents as well as different wavelengths of laser excitation. FT-Raman spectra were obtained at a resolution of 4 cm ⫺1 using a Bruker RFS-100 FT-Raman spectrometer equipped with an Adlas Nd:YAG laser operating at 1.064 mm and a Germanium diode detector. A laser power of 400 mW was found to be optimum. Depolarization measurements were made at the 90⬚ sampling geometry with the use of a 1/4-wave plate. Data acquisition was performed using Bruker OPUS software (version 2.1). Noise reduction was achieved by averaging 4 spectra of 2048 scans each. A Blackman-Harris 3-term apodization function was used. The infrared spectra of nitro-PAH solutions were recorded at a resolution of 4 cm ⫺1 and 64 scans using a Matteson Polaris FT-IR spectrophotometer equipped with a TGS detector. Data acquisition was achieved with Mattson FTIR FIRST software. A conventional liquid infrared cell with KBr windows was used.
3. Results and discussion For all the nitro-PAHs studied in the present work, one may assume that the structure of the PAH part is coplanar. Since the axis bisecting the ONO angle in the nitro group does not belong to the C2 symmetry element of the PAH in any of these nitro-PAHs, the molecules must have either Cs or C1 point group symmetry, depending on the angle between the nitro
group and the PAH plane (see Fig. 1). If the NO2 plane is parallel u 0⬚ or orthogonal u 90⬚ to the PAH plane, the molecule will have a conformation with Cs point group symmetry. In either conformation, the NO2 symmetric stretching mode will give rise to a polarized Raman band belonging to the A 0 irreducible representation. However, the NO2 antisymmetric mode will behavior differently depending on the conformation. If the NO2 plane is perpendicular to the PAH plane, the NO2 antisymmetric stretching mode will belong to A 00 irreducible representation with a depolarized Raman band. If the molecule has a planar conformation, the same vibrational mode will be an A 0 species with a polarized Raman band. In the third possible case, if the angle between the two planes is larger than zero but smaller than 90⬚, the molecule will have a C1 point group, and all the Raman bands will belong to the A species and be polarized. From the above group theoretical analysis, it is possible to determine the molecular conformation of the nitro-PAHs from the measured Raman depolarization data. Raman and infrared spectra of 2-nitroanthracene, 7nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, 6-nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10tetrahydrobenzo[a]pyrene were recorded in the wider. frequency region 1000–1700 cm ⫺1or Stretching vibrations of the nitro group in each of these nitro-PAHs were assigned based on the group frequencies [18,19], relative spectral intensities, and the IR and Raman spectra of the corresponding parent PAHs. From previous studies of nitro compounds [18–24], the symmetric NO2 stretching mode has relatively intense Raman bands while the antisymmetric NO2 stretching mode has strong IR bands. From the group frequencies, the symmetric NO2 stretching is expected to have frequency in the 1330–1370 cm ⫺1 region, and the antisymmetric NO2 stretching in the 1500–1550 cm ⫺1 region [19].
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Fig. 2. The conventional Raman spectra of: (A) 2-nitroanthracene; (B) 7-nitrobenz[a]anthracene; (C) 6-nitrochrysene; (D) 1-nitropyrene; and (E) 6-nitrobenzo[a]pyrene.
The conventional Raman spectra of 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1nitropyrene, and 6-nitrobenzo[a]pyrene are displayed in Fig. 2. Carbon disulfide was used as solvent for 2nitroanthracene and 7-nitrobenz[a]anthracene solutions; deuterated chloroform was used for the rest sample solutions. The IR spectra of 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, and 6-nitrobenzo[a]pyrene are shown in Fig. 3. The suggested vibrational assignments for the NO2 stretching modes of the five nitro-PAHs are indicated by arrows in Fig. 2 and dotts in Fig. 3, and the measured frequencies are summarized in Table 1. These assignments were made based on the features expected for the vibrations as described earlier in this section. Additionally, the assigned bands are clear from the recorded IR and Raman spectra of the corre-
Fig. 3. The IR spectra of: (A) 2-nitroanthracene; (B) 7-nitrobenz[a]anthracene; (C) 6-nitrochrysene; (D) 1-nitropyrene; and (E) 6nitrobenzo[a]pyrene.
sponding PAHs. For 7-nitrobenz[a]anthracene, 6nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, there are two IR bands and two or three Raman bands that may possibly be assigned to the symmetric stretching mode. There is no clear information that allows us to specifically assign any one of these to the mode. Three Raman bands of 6nitrobenzo[a]pyrene observed at 1495, 1509 and 1520 cm ⫺1 can possibly be assigned to the antisymmetric stretching mode. As all these three Raman bands are depolarized, assignment of any of these bands would result in the same molecular conformation. It should be noted that all the nitro-PAHs have
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Table 1 Stretching vibrational modes (cm ⫺1) of nitro groups in nitro-PAHs (abbreviations used: vs, very strong; s, strong; m, moderate; w, weak; p, polarized; dp, depolarized) Nitro-PAH
Symmetric stretching
Antisymmetric stretching
IR
Raman
IR
Raman
1342 (vs) 1342 (m) 1370 (m) 1342 (vs)
1342 (s, p) 1346 (m, p) 1370 (m, p) 1344 (m, p)
1518 (s) 1524 (vs)
1516 (w, p) 1506 (w, dp)
Parallel Perpendicular
1529 (w, p)
Parallel
1-Nitropyrene 6-Nitrobenzo[a] pyrene
1334 (vs) 1324 (m) 1388 (m)
1335 (s, p) 1342 (w, p) 1383 (s, p)
1509 (vs) 1527 (vs) 1511 (s) 1524 (vs)
Parallel Perpendicular
6-Nitro-7,8,9,10-tetrahydrobenzo-[a]pyrene
1374 (m) 1394 (m)
1373 (w, p) 1402 (s, p)
1504 (w, p) 1495 (s, dp) 1509 (s, dp) 1520 (n, dp) 1520 (w, dp) 1526 (w, p) 1534 (vw, p)
2-Nitroanthracene 7-Nitrobenz[a]-anthracene 6-Nitrochrysene
intense IR bands arising from the antisymmetric NO2 stretching (see Fig. 3). With an exception of 6-nitrochrycene, the vibrational mode is well isolated from other moderate IR bands giving a confidence of the assignments. The IR spectrum of 6-nitrochrysene (Fig. 3C) shows two intense bands at 1509 and 1527 cm ⫺1. The corresponding Raman spectrum has only a weak singlet at 1529 cm ⫺1. In the present study, no attempt was made to explain the splitting although it might arise from Fermi resonance or other couplings. In the IR spectrum of 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene (not shown in Fig. 3), an intense band was observed at 1523 cm ⫺1, which could be assigned to the NO2 antisymmetric stretching mode. However, in the corresponding Raman frequency shift region, three weak bands were observable at 1520, 1526, and 1534 cm ⫺1. The 1520-cm ⫺1 band was depolarized while the other two were polarized. Without a confirmed assignment of the Raman band to the vibration, we would be unable to determine the conformation of 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene from the current vibrational data. From the experimental depolarization data of the nitro-PAHs, it is concluded that 7-nitrobenz[a]anthracene and 6-nitrobenzo[a]pyrene have the NO2 plane perpendicular or nearly perpendicular to the PAH; 2nitroanthracene, 6-nitrochrysene, and 1-nitropyrene have Cs point group symmetry with coplanar geometry because at least one of the Raman bands
1523 (s)
Conformation
Perpendicular (?)
in the experimental frequency region is depolarized in the Raman spectrum for each of these three nitroPAHs. Summarized in Table 2 are the experimental conformation and direct-acting mutagenicity of twevlve nitro-PAHs. It is of interest to note that there is a correlation between the conformation and direct-acting mutagenicity of nitro-PAHs. Thus, a determination of the nitro-PAH conformation should be very useful in predicting the direct-acting of a nitro-PAH. The conformational results obtained for the nitro-PAH compounds from the Raman depolarization measurements are in accord with those derived from the NMR method. Molecules with the nitro groups perpendicular to the poly-aromatic moiety, including 7-nitrobenz[a]anthracene and 6-nitrobenzo[a]pyrene, are expected to have less or no conjugation between the p-electrons of the nitro group and the p -electrons of the poly-aromatic moiety. Chemically, they are not easily reduced to the corresponding amino-PAHs. The present study has demonstrated the usefulness of the vibrational spectroscopy in determining the orientation of the nitro group in nitro-PAHs. Besides the symmetry of the PAH constituent with respect to the NC bond in the nitro-PAH, one major limitation is fluorescence that may occur with some sample solutions. Fluorescence may bury underlying Raman signals. The use of different solvent such as chloroform-d, carbon tetrachloride, carbon disulfide,
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Table 2 Conformation and direct-acting mutagenicity tested in Salmonella typhimurium strain TA 98 and TA100 of nitro-PAHs (corresponding reference numbers are given in square brackets) Compound
2-Nitroanthracene 7-Nitrobenz[a]anthracene 6-Nitrochrysene 1-Nitropyrene 6-Nitrobenzo[a]pyrene 6-Nitro-7,8,9,10-tetrahydro-benzo[a]pyrene 7-Nitrodibenz[a,h]anthracene 9-Nitroanthracene 4-Nitropyrene 1-Nitrobenzo[a]pyrene 3-Nitrobenzo[a]pyrene 3-Nitro-7,8,9,10-tetrahydro-benzo[a]pyrene a
Conformation
Direct-acting mutagenicity (revertants/nmol)
Conformation
Method of determination
TA98
TA100
Parallel Perpendicular Parallel Parallel Perpendicular Perpendicular Perpendicular Perpendicular Parallel Parallel Parallel Parallel
NMR [6,25], Raman a NMR [6,11,25], Raman a NMR [6,25], Raman a NMR [6,25], Raman a NMR [6,25], Raman a NMR [6,25] X-ray crystallography (80.6⬚) [9] NMR [6,25] NMR [6,25] NMR [6,25] NMR [6,25] NMR [25]
1625 [15] ⬍ 1 [25] 129 [25] 543 [25] ⬍ 1 [25] ⬍ 1 [25] ⬍ 1 [9] ⬍ 1 [25] 2475 [25] 713 [25] 1931 [25] 34603 [25]
– ⬍ 1 [25] 174 [25] 296 [25] ⬍ 1 [25] ⬍ 1 [25] ⬍ 1 [9] ⬍ 1 [26] 58 [25] 2376 [25] 3119 [25] –
Present study.
dimethyl sulfoxide and aceton-d6 for preparing 1nitropyrene solutions has been tried. It was found that different solvents might give different spectrum background levels. Higuchi et al. [27] have studied the influence of solvents on the fluorescence background in the Raman spectra of PAH samples. Although the use of an appropriate solvent may help somewhat to correct the problem of the fluorescence interference for some samples, it is not a universal and effective solution in the present study. For example, in attempts to collect an acceptable and useful Raman spectrum of 6-nitrobenzo[a]pyrene, we have tried different solvents including chloroform-d, carbon tetrachloride, carbon disulfide and dimethyl sulfoxide, but the fluorescence interference insisted when a visible laser was applied for scattering excitation. Changing the visible laser wavelength (488.0 and 514.5 nm) for excitation showed no significant and major improvement in eliminating fluorescence. However, the use of near IR laser for Raman scattering excitation has promised to be an effective solution to the fluorescence problem of nitro-PAHs.
Acknowledgements YSL would like to thank the University of Memphis for the Faculty Research Grants and the
College of Arts and Sciences at the UM for the Professional Development Assignment.
References [1] International Agency for Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risks to Humans. Diesel and Gasoline Engine Exhausts and some Nitroarenes. IARC, Lyon, Publication no. 46, 1989, p. 359. [2] P.P. Fu, Drug Metab. Rev. 22 (1990) 209. [3] P.P. Fu, D. Herreno-Saenz, Environ. Carcinogen. Ecotoxicol. Rev. C17 (1) (1999) 1. [4] W.A. Vance, D.W. Levin, Environ. Mutag. 6 (1984) 797. [5] G. Klopman, H.S. Rosenkranz, Mutation Res. 126 (1984) 227. [6] P.P. Fu, M.W. Chou, F.A. Beland, in: S.K. Yang, B.D. Silverman (Eds.), Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relationships, CRC Press, Boca Raton, FL, 1988, p. 37. [7] H. Shaikh, A.U. Jung, R.H. Heflich, P.P. Fu, Environ. Mol. Mutagen. 17 (1991) 169. [8] D.-J. Zhan, L.-H. Chiu, L.S. Von Tungeln, E. Cheng, D. Herreno-Saenz, F.E. Evans, P.P. Fu, Mutation Res. 379 (1997) 43. [9] P.P. Fu, L.S. Von Tungeln, L.-H. Chiu, D.-J. Zhan, J. Deck, J.C. Wang, Chem. Res. Toxicol. 11 (1998) 937. [10] J. Trotter, Acta Crystallogr. 12 (1959) 237. [11] D.W. Miller, F.E. Evans, P.P. Fu, Spectrosc. Int. J. 4 (1985) 91. [12] C. Passingham, P.J. Hendra, C. Hodges, H.A. Willis, Spectrochim. Acta 47A (1991) 1235. [13] N. Juchnovski, G.N. Andreev, Bulg. Acad. Sci. 16 (1983) 389.
Y.S. Li et al. / Journal of Molecular Structure 550–551 (2000) 217–223 [14] P.D. Enlow, M. Buncick, R.J. Warmack, T. Vo-Dinh, Anal. Chem. 58 (1986) 1119. [15] P.P. Fu, R.H. Heflich, L.S. Von Tungeln, D.T.C. Yang, E.K. Fifer, F.A. Beland, Carcinogenesis 7 (1986) 1819. [16] P.P. Fu, L.S. Von Tungeln, M.W. Chou, Mol. Pharmacol. 28 (1985) 62. [17] K.B. Delclos, R.P. Walker, K.L. Dooley, P.P. Fu, F.F. Kadlubar, Cancer Res. 47 (1987) 6272. [18] D. Lin-Vien, N.B. Colthup, W.G. Fateley, J.G. Grasselli, The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules, Academic Press, Boston, MA, 1991, p. 179. [19] N.B. Colthup, L.H. Daly, S.E. Wiberley, Introduction to Infrared and Raman Spectroscopy, 3rd ed. 1990 (346p). [20] J.H.S. Green, W. Kynaston, A.S. Lindsey, Spectrochim. Acta 17 (1961) 486.
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[21] C.V. Stephenson, W.C. Coburn Jr., W.S. Wilcox, Spectrochim. Acta 17 (1961) 933. [22] I.N. Juchnovski, G.N. Andreev, C. R. L’Acad. Bulg. Sci. 29 (1976) 1637. [23] G.N. Andreev, I.N. Juchnovaki, Bulg. Acad. Sci. 13 (1980) 166. [24] Z. Zheng, S. Higuchi, S. Tanaka, Spectrosc. Lett. 15 (1982) 773. [25] H. Jung, A.U. Shaikh, R.H. Heflich, P.P. Fu, Environ. Mol. Mutag. 17 (1991) 169. [26] P.P. Fu, L.S. Von Tungeln, M.W. Chou, Carcinogenesis 6 (1985) 753. [27] S.E. Higuchi, E.J. Yu, S. Tanaka, Appl. Spectrosc. 41 (1987) 413.
The conformation of some nitro-polycyclic aromatic hydrocarbons 夽 Y.S. Li a,*, P.P. Fu b, J.S. Church c a
Department of Chemistry, University of Memphis, Memphis, TN 38152, USA b National Center for Toxicological Research, Jefferson, AR 72079, USA c CSIRO Textile and Fibre Technology, Belmont, Victoria, 3216 Australia Received 6 July 1999; accepted 17 November 1999
Abstract The Raman spectra of some nitro-polycyclic aromatic hydrocarbons, including 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, 6-nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, have been recorded in the frequency region 1100–1700 cm ⫺1. The stretching vibrational modes for the nitro group in each of these compounds were assigned based on group frequencies, the measured infrared and Raman spectra, the relative spectral intensities, and the Raman depolarization data. From the measured Raman depolarization data, the orientation of the nitro-substituent with respect to polycyclic aromatic hydrocarbon was determined for each of these compounds except 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene. The results were correlated with the direct-acting mutagenicity of the nitro compounds according to the hypothesis suggested by P.P. Fu and his co-workers. 䉷 2000 Elsevier Science B.V. All rights reserved. Keywords: Nitro-polycyclic aromatic hydrocarbons; Raman spectra; Depolarization ratio; Conformation; Structure and mutagenicity relationship
1. Introduction Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are a class of widespread environmental contaminants that are present in different sources including diesel emissions, combustion emissions from kerosene heaters, gas fuel, liquefied petroleum, airborne particulates, coal fly ash, and food [1–3]. Many nitro-PAHs have been determined to possess a large spectrum of biological and genotoxic activities 夽
Dedicated to Professor James R. Durig on the occasion of his 65th birthday. * Corresponding author. Tel.: ⫹ 1-901-678-2621; fax: ⫹ 1-901678-3447. E-mail address: [email protected] (Y.S. Li).
[1]. Because of their widespread presence in the environment and genotoxic activities, including mutagenicity and carcinogenicity, many of these compounds may pose a health risk to humans. It has been reported that the structural and electronic features of nitro-PAHs may be useful in predicting the biological activities of nitro-PAHs [3–9]. The particular structural feature that has been well demonstrated to be useful in correlation with direct-acting mutagenicity and tumorigenicity is the orientation of the nitro group [3–9]. A general finding is that nitro-PAHs with their nitro substituent oriented perpendicular to the aromatic system exhibit either very weak or no direct-acting mutagenicity in Salmonella typhimurium strains TA98 and TA100 and possess a tumorigenicity weaker than that of the
0022-2860/00/$ - see front matter 䉷 2000 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(00)00519-6
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Y.S. Li et al. / Journal of Molecular Structure 550–551 (2000) 217–223
aromatic nitro compounds, including some nitroPAHs, but no structural or conformational information could be derived from the studies. Vo-Dinh and his coworkers [14] have reported the surfaceenhanced Raman (SER) spectra of 1-nitropyrene, 9nitroanthracene, 2-nitronapthalene, and 2-nitrofluorene; the major purpose of their studies was to develop SER scattering-active substrates for analysis. Vibrational spectroscopy is a useful and convenient method to work out the conformation of molecules. In the present study, we have measured Raman depolarization data for 2-nitroanthracene (A), 7-nitrobenz[a]anthracene (B), 6-nitrochrysene (C), 1-nitropyrene (D), 6-nitrobenzo[a]pyrene (E), and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene (F) (see Scheme 1 for their structural formulas) and tried to derive the conformation of the nitro-PAHs. The conformational results have been correlated with the direct-acting mutagenicity of the compounds.
2. Experimental
Scheme 1.
parent PAH [3,8,9]. Therefore, in order to predict and/ or interpret the biological activities of nitro-PAHs, it is important to determine the orientation of the nitro functional group. X-ray crystallographic and 1H NMR spectroscopic analyses have been employed for determining nitro group orientation [9–11]. X-ray crystallographic study of 9-nitroanthracene and 7nitrodibenz[a,h]anthracene indicated dihedral angles of 85 and 80.6⬚, respectively between the nitro oxygens and the aromatic ring [9,10]. When a nitroPAH adopts a perpendicular or a nearly perpendicular orientation, the anisotropy of the nitro group causes a slight upfield shift of the adjacent aromatic peri proton(s) in their 1H NMR spectra; whereas the coplanar or nearly coplanar nitro group will cause a marked downfield NMR shift of the adjacent peri protons [11]. Passingham et al. [12] have reported the Raman spectra of nitrobenzene and it derivatives; Juchnovski and Andreev [13] have measured IR spectra of 38
1-Nitropyrene was purchased from Aldrich Chemical Company. 2-Nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 6-nitrobenzo[a]pyrene, and 6nitro-7,8,9,10-tetrahydrobenzo[a]pyrene were synthesized as reported previously [7,15–17]. All the compounds were purified by column chromatography and/or recrystallization and determined to be ⬎99% pure based on HPLC analysis. The corresponding PAH samples of A, B, C, D, and E were obtained commercially. Carbon disulfide, chloroform, chloroform-d, carbon tetrachloride, dimethyl sulfoxide, and dimethyl sulfoxide-d6 were obtained from commercial sources and used as solvent for preparing sample solutions without further purification. All Raman analyses were carried out with the sample solutions sealed in glass capillaries. Conventional Raman spectra were recorded with a Spex Model 1403 double monochromator fitted with a Hamanatsu R928-07 photomultiplier tube (PMT) held at 243 K with a thermoelectric refrigerated chamber (Model TE177-RF). The Raman spectra were acquired at a resolution of 2 or 4 cm 1 plotted with a Spex DM 3000 R computer system loaded with spectroscopy software. A Lexell XL-3000 argon ion laser tuned at 488.0 or 514.5 nm was used
Y.S. Li et al. / Journal of Molecular Structure 550–551 (2000) 217–223
219
Fig. 1. Different orientations of the nitro group with respect to the PAH plane in 2-nitroanthracene. u represents the angle between the nitro group and the PAH plane. The point group symmetry is shown in the bottom of the figure.
for excitation; a laser power of 100 mW was employed. For some nitro-PAHs, fluorescence seriously affected the quality of Raman spectra preventing the observation, identification and depolarization measurement of Raman bands. Attempts to solve this problem were made by purifying the samples and by trying different organic solvents as well as different wavelengths of laser excitation. FT-Raman spectra were obtained at a resolution of 4 cm ⫺1 using a Bruker RFS-100 FT-Raman spectrometer equipped with an Adlas Nd:YAG laser operating at 1.064 mm and a Germanium diode detector. A laser power of 400 mW was found to be optimum. Depolarization measurements were made at the 90⬚ sampling geometry with the use of a 1/4-wave plate. Data acquisition was performed using Bruker OPUS software (version 2.1). Noise reduction was achieved by averaging 4 spectra of 2048 scans each. A Blackman-Harris 3-term apodization function was used. The infrared spectra of nitro-PAH solutions were recorded at a resolution of 4 cm ⫺1 and 64 scans using a Matteson Polaris FT-IR spectrophotometer equipped with a TGS detector. Data acquisition was achieved with Mattson FTIR FIRST software. A conventional liquid infrared cell with KBr windows was used.
3. Results and discussion For all the nitro-PAHs studied in the present work, one may assume that the structure of the PAH part is coplanar. Since the axis bisecting the ONO angle in the nitro group does not belong to the C2 symmetry element of the PAH in any of these nitro-PAHs, the molecules must have either Cs or C1 point group symmetry, depending on the angle between the nitro
group and the PAH plane (see Fig. 1). If the NO2 plane is parallel u 0⬚ or orthogonal u 90⬚ to the PAH plane, the molecule will have a conformation with Cs point group symmetry. In either conformation, the NO2 symmetric stretching mode will give rise to a polarized Raman band belonging to the A 0 irreducible representation. However, the NO2 antisymmetric mode will behavior differently depending on the conformation. If the NO2 plane is perpendicular to the PAH plane, the NO2 antisymmetric stretching mode will belong to A 00 irreducible representation with a depolarized Raman band. If the molecule has a planar conformation, the same vibrational mode will be an A 0 species with a polarized Raman band. In the third possible case, if the angle between the two planes is larger than zero but smaller than 90⬚, the molecule will have a C1 point group, and all the Raman bands will belong to the A species and be polarized. From the above group theoretical analysis, it is possible to determine the molecular conformation of the nitro-PAHs from the measured Raman depolarization data. Raman and infrared spectra of 2-nitroanthracene, 7nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, 6-nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10tetrahydrobenzo[a]pyrene were recorded in the wider. frequency region 1000–1700 cm ⫺1or Stretching vibrations of the nitro group in each of these nitro-PAHs were assigned based on the group frequencies [18,19], relative spectral intensities, and the IR and Raman spectra of the corresponding parent PAHs. From previous studies of nitro compounds [18–24], the symmetric NO2 stretching mode has relatively intense Raman bands while the antisymmetric NO2 stretching mode has strong IR bands. From the group frequencies, the symmetric NO2 stretching is expected to have frequency in the 1330–1370 cm ⫺1 region, and the antisymmetric NO2 stretching in the 1500–1550 cm ⫺1 region [19].
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Fig. 2. The conventional Raman spectra of: (A) 2-nitroanthracene; (B) 7-nitrobenz[a]anthracene; (C) 6-nitrochrysene; (D) 1-nitropyrene; and (E) 6-nitrobenzo[a]pyrene.
The conventional Raman spectra of 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1nitropyrene, and 6-nitrobenzo[a]pyrene are displayed in Fig. 2. Carbon disulfide was used as solvent for 2nitroanthracene and 7-nitrobenz[a]anthracene solutions; deuterated chloroform was used for the rest sample solutions. The IR spectra of 2-nitroanthracene, 7-nitrobenz[a]anthracene, 6-nitrochrysene, 1-nitropyrene, and 6-nitrobenzo[a]pyrene are shown in Fig. 3. The suggested vibrational assignments for the NO2 stretching modes of the five nitro-PAHs are indicated by arrows in Fig. 2 and dotts in Fig. 3, and the measured frequencies are summarized in Table 1. These assignments were made based on the features expected for the vibrations as described earlier in this section. Additionally, the assigned bands are clear from the recorded IR and Raman spectra of the corre-
Fig. 3. The IR spectra of: (A) 2-nitroanthracene; (B) 7-nitrobenz[a]anthracene; (C) 6-nitrochrysene; (D) 1-nitropyrene; and (E) 6nitrobenzo[a]pyrene.
sponding PAHs. For 7-nitrobenz[a]anthracene, 6nitrobenzo[a]pyrene, and 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene, there are two IR bands and two or three Raman bands that may possibly be assigned to the symmetric stretching mode. There is no clear information that allows us to specifically assign any one of these to the mode. Three Raman bands of 6nitrobenzo[a]pyrene observed at 1495, 1509 and 1520 cm ⫺1 can possibly be assigned to the antisymmetric stretching mode. As all these three Raman bands are depolarized, assignment of any of these bands would result in the same molecular conformation. It should be noted that all the nitro-PAHs have
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Table 1 Stretching vibrational modes (cm ⫺1) of nitro groups in nitro-PAHs (abbreviations used: vs, very strong; s, strong; m, moderate; w, weak; p, polarized; dp, depolarized) Nitro-PAH
Symmetric stretching
Antisymmetric stretching
IR
Raman
IR
Raman
1342 (vs) 1342 (m) 1370 (m) 1342 (vs)
1342 (s, p) 1346 (m, p) 1370 (m, p) 1344 (m, p)
1518 (s) 1524 (vs)
1516 (w, p) 1506 (w, dp)
Parallel Perpendicular
1529 (w, p)
Parallel
1-Nitropyrene 6-Nitrobenzo[a] pyrene
1334 (vs) 1324 (m) 1388 (m)
1335 (s, p) 1342 (w, p) 1383 (s, p)
1509 (vs) 1527 (vs) 1511 (s) 1524 (vs)
Parallel Perpendicular
6-Nitro-7,8,9,10-tetrahydrobenzo-[a]pyrene
1374 (m) 1394 (m)
1373 (w, p) 1402 (s, p)
1504 (w, p) 1495 (s, dp) 1509 (s, dp) 1520 (n, dp) 1520 (w, dp) 1526 (w, p) 1534 (vw, p)
2-Nitroanthracene 7-Nitrobenz[a]-anthracene 6-Nitrochrysene
intense IR bands arising from the antisymmetric NO2 stretching (see Fig. 3). With an exception of 6-nitrochrycene, the vibrational mode is well isolated from other moderate IR bands giving a confidence of the assignments. The IR spectrum of 6-nitrochrysene (Fig. 3C) shows two intense bands at 1509 and 1527 cm ⫺1. The corresponding Raman spectrum has only a weak singlet at 1529 cm ⫺1. In the present study, no attempt was made to explain the splitting although it might arise from Fermi resonance or other couplings. In the IR spectrum of 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene (not shown in Fig. 3), an intense band was observed at 1523 cm ⫺1, which could be assigned to the NO2 antisymmetric stretching mode. However, in the corresponding Raman frequency shift region, three weak bands were observable at 1520, 1526, and 1534 cm ⫺1. The 1520-cm ⫺1 band was depolarized while the other two were polarized. Without a confirmed assignment of the Raman band to the vibration, we would be unable to determine the conformation of 6-nitro-7,8,9,10-tetrahydrobenzo[a]pyrene from the current vibrational data. From the experimental depolarization data of the nitro-PAHs, it is concluded that 7-nitrobenz[a]anthracene and 6-nitrobenzo[a]pyrene have the NO2 plane perpendicular or nearly perpendicular to the PAH; 2nitroanthracene, 6-nitrochrysene, and 1-nitropyrene have Cs point group symmetry with coplanar geometry because at least one of the Raman bands
1523 (s)
Conformation
Perpendicular (?)
in the experimental frequency region is depolarized in the Raman spectrum for each of these three nitroPAHs. Summarized in Table 2 are the experimental conformation and direct-acting mutagenicity of twevlve nitro-PAHs. It is of interest to note that there is a correlation between the conformation and direct-acting mutagenicity of nitro-PAHs. Thus, a determination of the nitro-PAH conformation should be very useful in predicting the direct-acting of a nitro-PAH. The conformational results obtained for the nitro-PAH compounds from the Raman depolarization measurements are in accord with those derived from the NMR method. Molecules with the nitro groups perpendicular to the poly-aromatic moiety, including 7-nitrobenz[a]anthracene and 6-nitrobenzo[a]pyrene, are expected to have less or no conjugation between the p-electrons of the nitro group and the p -electrons of the poly-aromatic moiety. Chemically, they are not easily reduced to the corresponding amino-PAHs. The present study has demonstrated the usefulness of the vibrational spectroscopy in determining the orientation of the nitro group in nitro-PAHs. Besides the symmetry of the PAH constituent with respect to the NC bond in the nitro-PAH, one major limitation is fluorescence that may occur with some sample solutions. Fluorescence may bury underlying Raman signals. The use of different solvent such as chloroform-d, carbon tetrachloride, carbon disulfide,
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Table 2 Conformation and direct-acting mutagenicity tested in Salmonella typhimurium strain TA 98 and TA100 of nitro-PAHs (corresponding reference numbers are given in square brackets) Compound
2-Nitroanthracene 7-Nitrobenz[a]anthracene 6-Nitrochrysene 1-Nitropyrene 6-Nitrobenzo[a]pyrene 6-Nitro-7,8,9,10-tetrahydro-benzo[a]pyrene 7-Nitrodibenz[a,h]anthracene 9-Nitroanthracene 4-Nitropyrene 1-Nitrobenzo[a]pyrene 3-Nitrobenzo[a]pyrene 3-Nitro-7,8,9,10-tetrahydro-benzo[a]pyrene a
Conformation
Direct-acting mutagenicity (revertants/nmol)
Conformation
Method of determination
TA98
TA100
Parallel Perpendicular Parallel Parallel Perpendicular Perpendicular Perpendicular Perpendicular Parallel Parallel Parallel Parallel
NMR [6,25], Raman a NMR [6,11,25], Raman a NMR [6,25], Raman a NMR [6,25], Raman a NMR [6,25], Raman a NMR [6,25] X-ray crystallography (80.6⬚) [9] NMR [6,25] NMR [6,25] NMR [6,25] NMR [6,25] NMR [25]
1625 [15] ⬍ 1 [25] 129 [25] 543 [25] ⬍ 1 [25] ⬍ 1 [25] ⬍ 1 [9] ⬍ 1 [25] 2475 [25] 713 [25] 1931 [25] 34603 [25]
– ⬍ 1 [25] 174 [25] 296 [25] ⬍ 1 [25] ⬍ 1 [25] ⬍ 1 [9] ⬍ 1 [26] 58 [25] 2376 [25] 3119 [25] –
Present study.
dimethyl sulfoxide and aceton-d6 for preparing 1nitropyrene solutions has been tried. It was found that different solvents might give different spectrum background levels. Higuchi et al. [27] have studied the influence of solvents on the fluorescence background in the Raman spectra of PAH samples. Although the use of an appropriate solvent may help somewhat to correct the problem of the fluorescence interference for some samples, it is not a universal and effective solution in the present study. For example, in attempts to collect an acceptable and useful Raman spectrum of 6-nitrobenzo[a]pyrene, we have tried different solvents including chloroform-d, carbon tetrachloride, carbon disulfide and dimethyl sulfoxide, but the fluorescence interference insisted when a visible laser was applied for scattering excitation. Changing the visible laser wavelength (488.0 and 514.5 nm) for excitation showed no significant and major improvement in eliminating fluorescence. However, the use of near IR laser for Raman scattering excitation has promised to be an effective solution to the fluorescence problem of nitro-PAHs.
Acknowledgements YSL would like to thank the University of Memphis for the Faculty Research Grants and the
College of Arts and Sciences at the UM for the Professional Development Assignment.
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