Energy trends in indole spectra
Substitution at any one of seven indole ring positions (Scheme 1, top) or variation in solvent from hydrophobic (cyclohexane) to hydrophilic (water), can induce energy shifts in the 1La and 1Lb absorption transitions, whose vectorial position relative to the indole ring plane is shown in Scheme 1, bottom. Measured spectral energy shifts for indole derivatives in response to both factors are given in the histogram in Fig. 1. The identification key for the indole species' acronyms used in Fig. 1 is given in Table 1. Absorption maxima for the 1La transition in cyclohexane (diamond) and water (filled triangle) and the 1Lb transition band origin in cyclohexane (filled square) are given, as well as the fluorescence emission maxima for cyclohexane (triangle) and water (filled circle) solutions. Results for 14 indole derivatives and indole vapor were acquired. Peaks for an additional eight indoles are also included . The peaks were ordered in an identity-blind fashion such that a rough linear trend in peak energy, measured in units of wavelength (nm), could be identified for almost all types of maxima. As a result, pyrrole ring derivatives are mostly found on the left side of Fig. 1, whereas benzyl ring derivatives are found on the right.
Scheme 1. Molecular details for the indole molecule. Top: atomic numbering for ring position. Bottom: orientation of the 1La and 1Lb electronic energy transitions adapted from .
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Table 1. Key for indoles in Fig. 1 histogram
|23M 2,3-dimethyl indole ||6M 6-methyl indole|
|235M 2,3,5-trimethyl indole ||5F 5-fluoro indole|
|12M 1,2-dimethy indole ||5M 5-methyl indole |
|3M 3-methyl indole||5MO2M 5-methoxy-2-methylindole |
|2M 2-methyl indole||5CN 5-cyano indole|
|I Indole||6MO 6-methoxy indole |
|3CN 3-cyano indole||6OH 6-hydroxyindole|
|I vap indole vapor||5Br 5-bromo indole|
|7M 7-methyl indole ||5Cl 5-chloro indole|
|4OH 4-hydroxyindole||5MO 5-methoxy indole |
|7OH 7-hydroxyindole||5OH 5-hydroxyindole|
Aggregation is always a consideration for molecules dissolved in an incompatible solvent. For such a scenario, red shifts in emission would be expected due to fluorescence reabsorption. Absorption band red shifts would also be expected for aggregated molecules. The spectral results for methyl indoles on the left side of Fig. 1, taken from , show large emission red shifts in aqueous solution. However, the 1La absorption maxima for the molecules in water are not greatly red-shifted relative to their maxima for molecules in cyclohexane. To first approximation, aggregation is not apparent.
Only the absorption spectra for indoles in cyclohexane showed sufficient vibronic structure to locate the 1Lb lowest energy or 0–0 band (Fig. 1 (filled square). The 1La 0–0 band is not identifiable in absorption spectra for either solvent due to spectral overlap between the 1La and 1Lb transitions. Instead, the short wavelength absorption peak maximum is chosen as the 1La transition marker. Indeed, for many indole derivatives in a variety of solvents, the 1La and 1Lb transitions are energetically degenerate. The following indoles did not fluoresce or fluoresced very weakly: 4- and 7-hydroxy and 5-bromoindoles in cyclohexane, and 3-cyano and 7-hydroxyindoles in water and so no point appears on the histogram for these species. Trend lines were fit to the spectral data where linear trends were apparent. This process yielded three near-parallel lines for pyrrole-substituted indole spectral data (Fig. 1, left side): through the 1La absorption maxima of aqueous indoles (filled triangle), the 1Lb absorption maxima of indoles in cyclohexane (filled square) and the emission maxima of indoles in cyclohexane (triangle). In general, the energy of these maxima decrease with increased methyl substitution to the pyrrole ring. A trend line is also applied to the emission maxima for pyrrole derivatives in aqueous solution (Fig. 1, left, filled circle). The slope of the trend line to these emission maxima is 1.8 times larger than those fit to the absorption maxima for pyrrole derivatives.
Trend lines were also fit to selected sets of spectral data for benzyl-substituted indoles, shown on the right side of Fig. 1. Fits were made to the 1La absorption maxima, cyclohexane solvent (diamond), the 1Lb absorption maxima, cyclohexane solvent (filled square) and to the aqueous emission maxima (filled circle). These trend lines reveal a different energy relationship between the absorption and emission transitions for benzyl-substituted indoles. The energy separation between the 1La and 1Lb absorption maxima is roughly two times greater for the benzyl-substituted indoles than the pyrrole-substituted ones, placing the 1La absorption maxima for benzyl-substituted indoles at higher energy. The trend line fit to the aqueous emission maxima for the benzyl-substituted indoles is not parallel to the corresponding absorption trend lines. For 5-methoxy and 5-hydroxyindole at the right edge of the plot, the energy separation between the aqueous and cyclohexane emission maxima is less than the Stokes shift between the 1Lb absorption maximum and the emission maximum in cyclohexane solvent. Another noteworthy feature of the plotted results is the lack of Stokes shift for 6-hydroxyindole.
Resolution of the 1La and 1Lb transitions
The absorption and fluorescence excitation spectra for 5- and 6-hydroxyindole in cyclohexane are given in Fig. 2a,b, respectively. These indoles are singled out because of the apparent resolution of their 1La and 1Lb transitions. The 5-hydroxyindole absorption spectrum (Fig. 2a, solid line) consists of a low energy band with three distinct peaks at 297, 302 and 308 nm and a shoulder at 291 nm. This is the 1Lb transition. A more intense, higher energy band is resolved into two peaks at 263 and 270 nm with a shoulder at 281 nm. This is the 1La transition. The lower energy band of the corresponding fluorescence excitation spectrum (Fig. 2a, bold line) duplicates the peak position and intensity of the lower energy absorption 1Lb band with little exception. The higher energy band, however, only has the peak at 267 nm of the 1La transition, and shoulder at 281 nm with a broad shoulder at 264 nm.
A similar pattern of bands and peaks is found for 6-hydroxyindole, shown in Fig. 2b. Herein, the low energy absorption band (solid line), the 1Lb transition, is the more intense. Peaks are blue-shifted 6–7 nm relative to those in the absorption spectrum for 5-hydroxyindole. The higher energy 1La absorption band for 6-hydroxyindole also has two peaks, centered at 260 and 267 nm. The low energy band of the excitation spectrum for 6-hydroxyindole (Fig. 2b, bold line) also recapitulates the 1Lb transition of the 1La absorption spectrum. A high energy peak at 267 nm corresponds to the 1La absorption peak at the same wavelength.
Figure 2c originates from reference , and is included herein for comparison with the results of Fig. 2a,b. It illustrates both experimental (dashed line) and calculated (solid line) 1La and 1Lb absorption transitions for indole. The experimental spectra were obtained from fluorescence anisotropy measurements for indole in propylene glycol at −58°C . The calculated spectra were derived from CIS/4-31G excited state and HF/4-31G ground state geometries . Comparison of the resolved transitions from Fig. 2c with the absorption and excitation spectra in Figs. 3a,b reveals a strong resemblance between the lower energy bands of the hydroxyindole spectra (Fig. 2a,b) and the calculated 1Lb transition (Fig. 2c, solid line) where more vibronic detail is evident. The higher energy absorption band for the hydroxyindoles (Fig. 2a,b) more strongly resembles the band shape of the experimental 1La transition (Fig. 2c, dashed line) where, again, there is resolution of distinct peaks. The high energy peak of the excitation spectra for the hydroxyindoles at 267–270 nm (Fig. 2a,b bold line) corresponds to the 1La transition peak at 280 nm (Fig. 2c, dashed line).
Figure 3. Absorption (room temperature, dashed line) and fluorescence excitation (77 K, solid line) spectra and fluorescence excitation anisotropy measurements (77 K, –■–) in poly(ethylene glycol), 400 MW avg./ethylene glycol (1:2 vol/vol) for (a) 5-hydroxyindole; 332 nm excitation for fluorescence measurements. (b) 6-hydroxyindole; 334 nm excitation for fluorescence measurements.
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Absorption and fluorescence excitation spectra for indole vapor at 75°C are given in Fig. 2d. These results are included because individual vibronic bands are particularly well-resolved in the vapor phase. The resolved 1La and 1Lb transitions for the 5- and 6-hydroxyindoles now make possible assignment of peaks where the transitions overlap. There is a one-to-one correspondence between the 1Lb peaks in the 6-hydroxyindole absorption spectrum (Fig. 2b) and the absorption and fluorescence excitation spectra peaks of indole vapor (269–284 nm). Likewise, the 253 and 259 nm peaks in the absorption spectrum of the indole vapor match the 1La transition peaks at 263 and 270 nm (Fig. 2a). The indole vapor excitation spectrum shows only the 259 nm peak of the 1La transition.
Comparison of absorption and excitation spectra for indoles can be extended to tryptophan containing proteins where vibronic bands are mostly obscured. The absorption and fluorescence excitation spectra for the single tryptophan in Pseudomonas aeruginosa azurin are shown in Fig. 2e. The broad, relatively featureless peaks are typical of spectral results for tryptophans in proteins, even for those buried in a hydrophobic environment . Although there is a weak vibronic peak at 291 nm that indicates the 1Lb origin, the remainder of each spectrum is without detail. The absorption and excitation peaks at 281 and 283 nm (Fig. 2e), respectively, are similar to the featureless calculated 1La band in Fig. 2c, but also suggest a fair degree of overlap for the 1La and 1Lb transitions for tryptophan in azurin.
Fluorescence anisotropy reveals the relative orientation of the 1La and 1Lb transitions
Fluorescence excitation anisotropy is a spectroscopic technique that reveals the orientation of the excitation transition dipole relative to the emission dipole. In the case of indole derivatives, where there are two absorption transition dipoles, 1La and 1Lb, of variable energetic overlap, calculated orthogonality ([29, 30]; Scheme 1, bottom), and highly variability in the energy of the 1La transition. Anisotropy measurements have the potential to resolve the relative contribution of these transitions to fluorescence emission. The requirement of a static population of oriented dipoles for anisotropy measurements requires a medium that can be frozen to low temperature and yet maintain optical transparency. A glycol glass is one such medium. However, it is not as apolar as cyclohexane and so transition resolution matching that obtained in cyclohexane (Fig. 2a,b) is not expected due to solvent-induced inhomogeneous broadening.
Absorption and fluorescence excitation spectra for 5-hydroxyindole and 6-hydroxyindole in poly(ethylene glycol)/ethylene glycol solution are given in Fig. 3a,b (dashed and solid line, respectively); the absorption results were acquired at room temperature, whereas the excitation results were obtained at 77 K. These spectra reveal the position of each transition in the more hydrophilic glycol solvent environment, and therefore aid in the interpretation of the anisotropy measurements. As the glycol solvent is more hydrophilic than cyclohexane, vibronic detail is lacking in both hydroxyindole absorption and excitation spectra (Fig. 3a,b dashed and solid line, respectively). The solvent-sensitive 1La transition maximum for 5-hydroxyindole appears at 275 nm in the excitation spectrum (Fig. 3a, solid line) and at 273 nm in the absorption spectrum (Fig. 3a, dashed line). The 1Lb origin appears at 309 nm in the excitation spectrum (Fig. 3a, solid line) and at 310 nm in the absorption spectrum (Fig. 3a, dashed line). Relative 1La/1Lb absorption and excitation band intensity for 5-hydroxyindole in glycol solution (Fig. 3a, dashed and solid line, resp.) follows that for cyclohexane (Fig. 2a, solid line and bold line, resp.).
Vibronic detail is also absent in the absorption and excitation spectra of 6-hydroxyindole in glycol solution (Fig. 3b, dashed and solid lines, respectively). Peaks are located at 295 (1Lb) and 273 nm (1La) in both absorption and excitation spectra. The relative intensity of the 1La and 1Lb absorption and excitation bands for 6-hydroxyindole in glycol solution mirror those for cyclohexane solution (Fig. 2b, solid and bold lines, resp.).
Fluorescence anisotropy measurements for 5-hydroxyindole and 6-hydroxyindole in the glycol glass at 77 K are also given in Fig. 3a and b (–■–). Anisotropy values, r0, follow the relationship [24, 27]:
where α is the angle between the excitation and emission dipoles. The values of r0 range from 0.4, corresponding to collinear excitation and emission dipoles (α = 0°) to −0.2, where the excitation and emission dipoles are orthogonal. At wavelengths shorter than 250 nm, overlap with the higher energy 1Bb transition is possible , and hence, the anisotropy above 250 nm will not be considered.
The anisotropy measurements for both hydroxyindoles follow a general pattern of decreasing value with decreasing wavelength. For 5-hydroxyindole (Fig. 3a, –■–), the anisotropy at 321 nm is at a maximum value of 0.21, corresponding to a 34° angle between the emission and excitation dipole. At 309 nm, the location of the 1Lb origin, the anisotropy drops to 0.15. The anisotropy continues to decrease with wavelength, “plateauing” twice more at 305 and 291 nm. The anisotropy for 5-hydroxyindole finally reaches a minimum of −0.03 at 275 nm near the 1La transition absorption maximum.
The anisotropy for 6-hydroxyindole (3b, –■–) at the short wavelength edge of the excitation band is 0.37, a limiting anisotropy that is close to the theoretical limit of 0.4 . This anisotropy value corresponds to a 13° angle between the excitation and emission dipoles; that is, they are nearly collinear. At 300 nm, the apparent 1Lb origin, r0 = 0.13. Near the 1La maximum at 272 nm, a minimum value in anisotropy is found: r0 = 0.034, yielding α = 51°. An angle of 54.7°, the so-called “magic angle,” indicates a random orientation of dipoles.
Fluorescence emission spectra for some indoles
The fluorescence emission spectra for 5- and 6-hydroxyindole in cyclohexane, indole vapor and P. aeruginosa azurin are shown in Fig. 4. The hydroxyindole spectra (Figs. 4a,b) are included to illustrate the very small Stokes shift for the emission of 6-hydroxyindole in cyclohexane versus the near identical Stokes shift for the emission of 5-hydroxyindole in cyclohexane and water (Fig. 1). The emission spectrum of indole vapor (Fig. 4c) is included because of the sharpness of the vibronic bands. The P. aeruginosa azurin emission spectrum (Fig. 4d) represents the case where tryptophan is buried in a hydrophobic protein environment and emission shows vibronic structure. The source of emission for such a buried tryptophan—1La or 1Lb transition—has been discussed at length in the literature [2, 26, 32].
Figure 4. Fluorescence emission spectra for indole species. (a) 5-hydroxyindole in cyclohexane, 300 nm excitation wavelength (b) 6-hydroxyindole in cyclohexane, 285 nm excitation wavelength. (c) Indole vapor at 75°C, 285 nm excitation wavelength. (d) Pseudomonas aeruginosa azurin, 100 mm ammonium acetate, pH 5.3, 275 nm excitation wavelength.
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The fluorescence emission spectrum for 5-hydroxyindole shows a maximum at 325 nm. The blue-edge 1Lb origin emission at 313 nm is Stokes-shifted 5 nm from the corresponding excitation band (Fig. 2a). 1Lb origin absorption wavelengths and emission maxima for other indoles, given in Fig. 1, show that this is not an atypical Stokes shift for indoles in cyclohexane. The fluorescence emission spectrum for 6-hydroxyindole in cyclohexane (Fig. 4b) exhibits the vibronic detail and blue-shifted emission maximum (304 nm) characteristic of indole in a hydrophobic environment. However, the 293 nm emission peak corresponds to the 301 nm excitation peak (Fig. 2b), which means that there is no Stokes shift for 6-hydroxyindole in cyclohexane. The excitation wavelength for the 6-hydroxyindole emission shown in Fig. 4b was 284 nm to collect the full emission band. Excitation at 299 nm shifted the emission maximum to 319 nm with a secondary vibronic band at 326 nm, but identical bandwidth (data not shown). The lack of Stokes shift for the emission of 6-hydroxyindole in cyclohexane will be discussed below.
The fluorescence emission spectrum of indole vapor (Fig. 4c) is narrow, extremely blue-shifted with an emission maximum at 295 nm and shows vibronic structure. The high energy vibronic detail at 290 nm is Stokes-shifted 14 nm from the corresponding excitation peak (Fig. 2d). The band shape, although very narrow, is similar in profile to that of 6-hydroxyindole.
The emission spectrum for the P. aeruginosa protein, azurin (Fig. 4d), shares the features of the other blue-shifted emission spectra just described. The emission peak is at 305 nm—close to that of 6-hydroxyindole—and the short wavelength vibronic peak appears at 295 nm. At the long wavelength edge, a shoulder is apparent, which is also defined in the indole vapor emission spectrum (Fig. 4c).
Fluorescence lifetime measurements for 5- and 6-hydroxyindoles
Fluorescence lifetime measurements were made for 5- and 6-hydroxylindoles in water and cyclohexane. All measurements were carried out at two excitation wavelengths, 281 and 293 nm. The results are given in Table 2 along with lifetime measurements from the literature for 5-hydroxyindole in water, cyclohexane and in a helium jet expansion [1, 21, 23] and for 6-hydroxyindole in helium jet expansion . Fluorescence lifetime measurements on 6-hydroxyindole in solution have not been found in the literature. The low fluorescence intensity of 6-hydroxyindole, resulting from excited state formation of a keto tautomer , a nonradiative process, discourages lifetime measurement. Single lifetimes were obtained for all but 6-hydroxyindole in cyclohexane, where two lifetimes were obtained. A ca. 10 ps lifetime attributed to scattering was necessary for acceptable fits (χ2 ~ 1) to the 6-hydroxyindole in cyclohexane data. A lifetime for 6-hydroxyindole in water with 293 nm excitation could not be obtained because the fluorescence decay was of the same timescale as the instrument response function. The lifetimes for each hydroxyindole—where measurable—did not vary significantly with the excitation wavelengths of 281 and 293 nm.
Table 2. Fluorescence lifetimes for 5- and 6-hydroxyindoles with goodness-of-fit (χ2)
|281 nm||293 nm|| || ||281 nm||293 nm|
| Water||3.0||3.0||3.2†||1.41 (0.35)‡||1.03||1.04|
| He jet expansion||–||–||11.1§||–||–||–|
| Cyclohexane||0.98 (0.51)||0.90 (0.32)||–||–||0.85||1.27|
|1.8 (0.48)||1.8 (0.48)||–||–||–||–|
| ||4.8 (0.15)||–||–||–||–||–|
| He jet expansion||–||–||2.8¶||–||–||–|