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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Although tryptophan is a natural probe of protein structure, interpretation of its fluorescence emission spectrum is complicated by the presence of two electronic transitions, 1La and 1Lb. Theoretical calculations show that a point charge adjacent to either ring of the indole can shift the emission maximum. This study explores the effect of pyrrole and benzyl ring substitutions on the transitions' energy via absorption and fluorescence spectroscopy, and anisotropy and lifetime measurements. The survey of indole derivatives shows that methyl substitutions on the pyrrole ring effect 1La and 1Lb energies in tandem, whereas benzyl ring substitutions with electrophilic groups lift the 1La/1Lb degeneracy. For 5- and 6-hydroxyindole in cyclohexane, 1La and 1Lb transitions are resolved. This finding provides for 1La origin assignment in the absorption and excitation spectra for indole vapor. The 5- and 6-hydroxyindole excitation spectra show that despite a blue-shifted emission spectrum, both the 1La and 1Lb transitions contribute to emission. Fluorescence lifetimes of 10 ns for 5-hydroxyindole are consistent with a charge acceptor-induced increase in the nonradiative rate (1).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

As tryptophan is an intrinsic fluorescent probe of protein structure and environment, understanding its photophysics is of paramount importance. Complicating factors include two overlapping, electronic transitions, 1La and 1Lb, and the solvent sensitivity of the 1La transition, which allows its tuning to an energy lower than that of 1Lb. This stabilization makes 1La the emitting transition. The resulting emission band is energetically red-shifted, broad and featureless. In a hydrophobic solvent, the tryptophan emission spectrum is blue-shifted, and some vibronic structure may be present suggesting emission from the 1Lb transition. This has been difficult to prove [2]. Emission from 1La is difficult to rule out because the energetic position of the 1La origin remains unknown, and is hidden by overlap with the 1Lb transition. Various spectroscopic techniques [2-9], theoretical calculations [10-15] and tryptophan model compounds [3, 6, 16-20] have been applied to resolve this conundrum.

Callis and coworkers [12, 14] have shown through hybrid quantum/classical simulations of tryptophan fluorescence that the emission maximum can be tuned to higher energy (blue-shifted) by placing either a negative charge near the benzyl ring or a positive charge near the pyrrole ring of indole. Reversing the charge placement yields a red-shift in emission maximum. Together with the solvent, these point charges create an electric field, constituting an internal Stark effect.

An experimental approach to studying both charge placement and solvent effects on the relative energy of indole 1La and 1Lb transitions is given herein. Absorption, fluorescence emission and excitation spectra were recorded for 14 indole derivatives in solution with water and cyclohexane at room temperature and indole in the vapor phase. The focus is on electrophilic substitution on the benzyl ring and methylation at pyrrole sites. A histogram of 1La absorption and 1Lb origin maxima for derivatives in cyclohexane reveal that the 1Lb transition is especially sensitive to substitution on the benzyl ring. 1Lb energy decreases, whereas 1La energy increases for benzyl substitution, resulting in a large energy separation between transitions. In the case of 5- and 6-hydroxyindole, the 1La and 1Lb transitions are essentially resolved. For 6-hydroxyindole in cyclohexane, the Stokes shift is absent, and the emission band is characteristic of emission spectra recorded for tryptophan in a hydrophobic environment, i.e. a blue-shifted emission maximum with resolved vibronic peaks. The fluorescence excitation spectrum for 6-hydroxyindole in cyclohexane demonstrates that some emission emanates from the 1La transition, even in this case, where the 1La and 1Lb transitions are resolved. A general pattern of mixed 1La/1Lb fluorescence emission is demonstrated for indole vapor as well. Because of the resolution of the 1La and 1Lb transitions for 5- and 6-hydroxyindole in cyclohexane, additional spectroscopic data was collected to illuminate the nature of these transitions. Fluorescence excitation anisotropy measurements necessarily carried out at low temperature required use of a hydrophilic medium. For 6-hydroxyindole, excitation anisotropy showed that although the 1La transition did not completely overlap with the 1Lb, the 1La transition possessed sufficient intensity at the red edge of the excitation spectrum to become the emitting dipole. Fluorescence lifetime measurements yielded 10 ns lifetimes that are consistent with published results [1, 21-23]. An increase in charge transfer to the benzyl ring and an increase in the nonradiative rate are discussed as tandem effects of electrophilic substitution on the benzyl ring.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

All indole derivatives were purchased from Gold Biotechnology, (St. Louis, MO) with the exception of indole, 3-methyl, 5-chloro, 5-fluoro and 4-hydroxyindoles and l-tryptophan, which were obtained from Acros Organics (Fair Lawn, NJ). 7-Hydroxyindole was purchased from Chem-Impex International Inc. (Wood Dale, IL). Indoles were used without further purification. Deionized water was used in all aqueous solutions. Cyclohexane was “spectranalyzed” grade (for use in the UV range; Fisher Scientific, Fair Lawn, NJ). Pseudomonas aeruginosa azurin was purchased from Sigma–Aldrich (St. Louis, MO).

All indoles, where soluble, were made up at a concentration of 1 mg mL−1 and subsequently diluted to a concentration of 0.1 mm for absorbance measurements. For less soluble indoles, concentration was determined by absorbance using an extinction coefficient of 4900 M−1 cm−1 at the 1Lb 0–0 maximum, and then diluted to 0.1 mm for recording of the absorption spectrum if necessary. Indole solutions were diluted to 0.01 mm for fluorescence measurements. The path length for the indole vapor measurements was 1 cm. For the absorbance measurement, the indole vapor concentration was 4 mg cm−3; for fluorescence measurements, the indole vapor concentration was 0.2 mg cm−3.

Steady state fluorescence spectra and fluorescence anisotropy measurements were acquired on a PTI QM-4/206 SE Spectrofluorometer (PTI, Birmingham, NJ) with right angle detection of fluorescence. Thermostatting of the cuvette holder with a constant temperature bath set to 75°C allowed for fluorescence measurements on indole vapor. The excitation wavelength for indole emission spectra in Fig. 1 is 285 nm except for 5-fluoro and 5-chloroindole in water (λex = 290 nm). Solvent blank spectra were subtracted where necessary. For the fluorescence anisotropy measurements, 5- and 6-hydroxyinoles were dissolved in a solution of poly(ethylene glycol; 400 molecular weight average)/ethylene glycol (1:2 vol/vol). Solutions were then frozen to a glass at 77 K in a 3 mm diameter quartz tube. The sample compartment was purged with dry nitrogen to eliminate fogging. Anisotropy was collected every 3 nm with a dwell time of 30 s at each point. Anisotropy, r0, is given by [24]:

  • display math(1)
image

Figure 1. Absorption and fluorescence emission maxima (wavelength, nm) histogram for indole derivatives in cyclohexane and water (25°C), and for indole vapor (75°C). Results for indoles are ordered to show the effect of substituent ring position on band energy. The indole species acronym key is given in Table 1. Absorption band maxima for: 1La, cyclohexane (diamond); 1La, water (filled triangle); 1Lb band origin, cyclohexane (filled square); Emission: cyclohexane (triangle); water (filled circle). For pyrrole-substituted indoles (left side of chart), trend lines are fit to the 1La and 1Lb absorption maxima in cyclohexane and the emission maxima in both solvents. For benzyl-substituted indoles (right side of chart), trend lines are fit to the 1La and 1Lb absorption maxima in cyclohexane and the emission maxima in water. Data for eight indole species from Lami [16] are also included.

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where = the measured light intensity, the subscripts (h = horizontal, v= vertical) give the orientation of the polarizer in the excitation path and the emission path, respectively. The instrument bias correction factor, G, is given by: G = Ihv/Ihh.

Fluorescence lifetime measurements were acquired on a Horiba Fluorolog model FL-1000 fluorometer using 281 nm light-emitting diode excitation (Horiba Inc., Edison, NJ). The instrument response was 1.8 ns long, full-width half maximum. Measurements were corrected for instrument response using a casein scattering suspension. The fluorescent count maximum was set to 20 000 for all measurements.

A Cary 100 Bio UV–Vis spectrophotometer (Varian Instruments Inc., Walnut Creek, CA) with a built-in Peltier/cuvette holder provided for absorbance measurement on indole vapor at 75°C. All spectra are referenced against solvent.

Baseline fits to the 1La/1Lb peak(s) of the absorption spectra were made using the freeware program, Fityk, v. 0.9.4 [25] to remove contribution from the more intense S0 [LEFTWARDS ARROW] S2 band. An example of such a fit is given in Figure S1 (see Supporting information). This program was also used to determine the absorption and emission spectra peaks given in Fig. 2. A Gaussian band shape was used. Curve fits to the absorption spectra of 5- and 6-hydroxyindole in cyclohexane (Figures S2 and S3, respectively) are likewise executed using the Fityk program; the band shapes are Gaussian. Trend line fits to the data in Fig. 1 are carried out using the Microsoft Excel program.

image

Figure 2. Absorption (solid line) and fluorescence excitation (bold line) spectra for indole species with computed and experimentally determined 1La and 1Lb transitions for indole reproduced from reference [26] with permission. Absorption spectra have been offset for ease of comparison. (a) 5-hydroxyindole in cyclohexane; 325 nm emission wavelength. (b) 6-hydroxyindole in cyclohexane, 320 nm emission wavelength. (c) 1La and 1Lb transitions resolved from fluorescence excitation anisotropy measurements on indole in propylene glycol at −58°C (dashed lines) from reference [27]. Calculated 1La and 1Lb transitions from [28]. Reproduced with permission from reference [26]. (d) indole vapor, 295 nm emission wavelength. (e) Pseudomonas aeruginosa azurin, 100 mm ammonium acetate, pH 5.3, 320 nm emission wavelength.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

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 [16]. 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.

image

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 [26].

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Table 1. Key for indoles in Fig. 1 histogram
23M 2,3-dimethyl indole [16]6M 6-methyl indole
235M 2,3,5-trimethyl indole [16]5F 5-fluoro indole
12M 1,2-dimethy indole [16]5M 5-methyl indole [16]
3M 3-methyl indole5MO2M 5-methoxy-2-methylindole [16]
2M 2-methyl indole5CN 5-cyano indole
I Indole6MO 6-methoxy indole [16]
3CN 3-cyano indole6OH 6-hydroxyindole
I vap indole vapor5Br 5-bromo indole
7M 7-methyl indole [16]5Cl 5-chloro indole
4OH 4-hydroxyindole5MO 5-methoxy indole [16]
7OH 7-hydroxyindole5OH 5-hydroxyindole
Trp l-tryptophan

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 [16], 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 [26], 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 [27]. The calculated spectra were derived from CIS/4-31G excited state and HF/4-31G ground state geometries [28]. 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).

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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 [2]. 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]:

  • display math(2)

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 [31], 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 [27]. 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].

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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 [22]. 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 [22], 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)
MoleculeLifetime (amplitude)/nsχ2
281 nm293 nm  281 nm293 nm
  1. * 285 nm excitation [1].

    † Excitation wavelength not available [23].

    ‡ 305 nm excitation [38].

    § 285 nm excitation [21]

    .

    ¶ 285 nm excitation [22].

5-OH indole
 Cyclohexane4.24.36.0*0.981.40
 Water3.03.03.21.41 (0.35)1.031.04
4.74 (0.65)
 He jet expansion11.1§
6-OH indole
 Cyclohexane0.98 (0.51)0.90 (0.32)0.851.27
1.8 (0.48)1.8 (0.48)
Water0.40 (1.0)1.29
 4.8 (0.15)
 He jet expansion2.8

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Substituent sensitivity of 1Lb transition

The solvent sensitivity of the 1La transition is well-documented [14, 26, 33-35], and is the source of the large Stokes shift observed for indoles in a polar environment, as illustrated by the emission maximum data points for pyrrole-substituted indoles in aqueous solution on the left side of Fig. 1. The effect of solvent relaxation is to lower the energy of 1La, allowing it to become the emitting dipole. The trend lines to the pyrrole-substituted indole spectral data show that methylation changes the energy of 1La and 1Lb absorption maxima in tandem with the emission maxima in both solvents.

Substitution on the benzyl ring with electron withdrawing groups; however, simultaneously raises the energy of the 1La absorption and lowers that of the 1Lb absorption maxima as indicated by the nonparallel trend lines fitted to the absorption maxima for 1La and the 1Lb origin. For 5-methoxy and 5-hydroxyindole at the right hand edge of Fig. 1, the energetic difference between the 1La and 1Lb absorption transitions is sufficient to obviate any large polar solvent-induced relaxation of the solvent-sensitive 1La transition. Thus, the aqueous fluorescent emission maxima for these two indoles are close to their emission maxima in cyclohexane. The insensitivity of 5-hydroxyindole to solvent polarity has also been documented by two other groups [23, 35].

The energy separation between the 1La and 1Lb transitions for both 5- and 6-hydroxyindole presents the unique opportunity to study the resolved 1La and 1Lb transition bands without resorting to any spectral manipulation. A comparison of the absorption spectra for 5- and 6-hydroxyindole reveals that the 1La transition is more intense for 5-hydroxyindole, and there are peak intensity differences between the two absorption spectra, e.g. the 270 nm peak of 5-hydroxyindole is of equal intensity to the 263 nm peak, whereas the corresponding peak intensities differ in the 6-hydroxyindole spectrum. Such differences are to be expected as the difference in hydroxyl substitution site effects the ring charge distribution. Remarkably, the 1La peak profiles for 5- and 6-hydroxyindole both recapitulate features of the anisotropy-resolved 1La band shape for indole in Fig. 2c. The 1Lb band for these hydroxyindoles exhibit vibronic detail that is not resolved in the anisotropy-derived 1Lb transition, but appears in the calculated 1Lb band. In fact, the calculated 1Lb transition and those of the hydroxyl indole absorption and excitation spectra differ only in the resolution between the two lowest energy peaks and the relative intensity of the higher energy peak for 6-hydroxyindole. As the 1La and 1Lb absorption bands for 5- and 6-hydroxyindole show a small overlap, a curve fit to each has been carried out, and these are shown in Figures S2 and S3, respectively (see supporting information). The small overlap of the red edge of 1La with the blue edge of 1Lb for both hydroxyindoles shows that excitation of 1Lb only is possible.

As vibronic peaks can now unambiguously be assigned, the extent of 1La and 1Lb overlap for other indole spectra can be determined. In the case of indole vapor (Fig. 2d), several vibronic peaks are apparent. Although there is still significant overlap of the 1La and 1Lb transitions, it is clear that peaks at 253 and 259 nm belong to the 1La transition, whereas those at 273, 278 and 284 nm belong to 1Lb. The peak at 268 nm may well be a superposition of the low energy shoulder from 1La and the high energy shoulder from 1Lb. The lack of vibronic detail in spectra for azurin (Fig. 2e)—only the 1Lb peak at 291 nm is resolved—and the compressed bandwidth in the absorption (Fig. 2e, solid line) and excitation (Fig. 2e, bold line) spectra suggest that the 1La and 1Lb transition energies are degenerate.

Excitation spectra clarify the 1La/1Lb contribution to blue-shifted emission spectra

The spectral overlap of the 1La and 1Lb transitions for pyrrole-substituted indoles, including aqueous tryptophan, and the solvent sensitivity of the 1La transition, which leads to a featureless emission envelope due to solvent relaxation, obfuscates determination of the emitting dipole.

The separation of the 1La and 1Lb transitions for 5- and 6-hydroxyindoles in cyclohexane clarifies the source of emission in a hydrophobic environment. The fluorescence excitation spectra for 5- and 6-hydroxyindole in cyclohexane (Fig. 2a,b) make plain the contribution of both the 1La and 1Lb transitions. While the primary source of fluorescence emission is the 1Lb transition, there is undeniably a contribution from 1La as the high energy peaks at 267 (Fig. 2b) and 270 nm (Fig. 2a) in the excitation spectra align directly with an 1La absorption band. The same reasoning can be applied to the fluorescence excitation spectrum of indole vapor (Fig. 2d).

Overlap of excitation and emission spectra for 6-hydroxyindole reveals band symmetry

The fluorescence emission spectra in Fig. 4 suggest a contribution of both the 1La and 1Lb transitions to blue-shifted emission is possible. While the fluorescence emission spectral envelope can exactly mirror that of the excitation spectrum for some molecules, that is generally not the case for indole derivatives. As shown by the emission spectra for indoles in Fig. 4, even such blue-shifted emissions—whereas featuring vibronic details at the short wavelength edge and suggesting 1Lb as the source—are featureless at the long wavelength edge, obscuring the transition involved. The emission spectrum for 6-hydroxyindole in cyclohexane, however, does not exhibit a Stokes shift and hence presents a unique opportunity to compare the excitation and emission spectra. This phenomenon indicates that the molecule is not subject to vibrational relaxation induced by either solvent or collision [24]. Absence of Stokes shift has been observed in excitation and emission results for anthracene confined and isolated within cyclodextrin cavities [36].

The excitation and emission spectra for 6-hydroxyindole are shown superimposed in Fig. 5. The spectral overlap extends from 290 to 305 nm. Clearly, this emission originates from the 1Lb transition. The band profiles of the two spectra also possess an atypical mirror symmetry in the 1Lb vibronic details at 293 and 301 nm, and the peak maxima. The spectral overlap also exposes differences: the full-width at half-maximum for the excitation spectrum is 25 nm, whereas that for the emission spectrum is 29 nm. The inset in Fig. 5, where the excitation spectrum has been flipped vertically, shows the difference in peak width more clearly. The excitation spectrum also has a second peak at 267 nm, which lines up with the low energy peak of the 1La transition. This peak is not observed in the emission spectrum, but most likely contributes to the increased emission bandwidth. The evidence then points to 1Lb emission with an energetic contribution from 1La for 6-hydroxyindole in cyclohexane.

image

Figure 5. Fluorescence spectra for 6-hydroxyindole in cyclohexane, showing spectral overlap due to the absence of a Stokes shift. Excitation spectrum, 320 nm emission wavelength (dashed line) and emission spectrum, 284 nm excitation wavelength (solid line). These spectra have been superimposed in the inset for band comparison.

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Although the emission spectra for 5-hydroxyindole in cyclohexane and indole vapor (Fig. 4a,d) are Stokes-shifted with respect to their excitation spectra, their band shapes and vibronic details bear a similar relationship with their excitation spectra as discussed above. Their excitation spectra feature peaks that align with both 1La and 1Lb absorption peaks. These features imply emission from the 1Lb transition with energetic contribution from 1La for both 5-hydroxyindole and indole vapor.

Fluorescence excitation anisotropy for 5- and 6-hydroxyindoles under hydrophilic conditions

While a solution of poly(ethylene glycol) and ethylene glycol forms a low temperature glass, it does not create the desired hydrophobic environment necessary for optimal resolution of the 1La and 1Lb transitions. A more hydrophobic solution of diethyl ether/2-methyl butane/ethanol, 5/5/2 by volume, has also been reported as forming a transparent glass at low temperature [37]. Monitoring the 240–350 nm absorbance of 5-hydroxyindole in this hydrophobic solution, however, showed a shift of peaks to 297 and 310 nm for the 1La and 1Lb absorption bands, respectively, over the time span of a few minutes (data not shown). This shift to lower energy of the absorption bands suggests dimerization. The reaction, nevertheless, eliminates the possibility of anisotropy measurements in this hydrophobic solution.

The fluorescence excitation anisotropy for 5-hydroxyindole (Fig. 4a, –■–) is similar in curve shape to that reported by Wong and Eftink [38] for 5-hydroxyindole in a 50% glycerol–phosphate buffer solution at 77 K, although the extreme values are greater. Their reported 5-hydroxyindole anisotropy has a high value of 0.25 at 315 nm and diminishes linearly to a minimum of 0.5 at 270 nm. The hydroxyindole anisotropy spectra reported here and elsewhere [38] differ from those reported for indole and tryptophan. Large swings (increases and decreases) in anisotropy value over the 280–295 nm wavelength range are seen in the anisotropy spectra for indole [27] and tryptophan [2] where the 1La and 1Lb absorption bands overlap. The absence of this feature in the anisotropy spectra of 5- and 6-hydroxyindole may be taken as a signature of at least partial 1La and 1Lb absorption band resolution.

The fluorescence excitation anisotropy results for 6-hydroxyindole (Fig. 3b, –■–), reach a high limiting anisotropy value of 0.37 at 321 nm, close to the theoretical limit of 0.4. The highest anisotropy value reached for the anisotropy results of 5-hydroxyindole is only 0.21 so the results for 6-hydroxyindole are used to calculate 1La and 1Lb bands according to the equation [24, 27]:

  • display math(3)

and

  • display math(4)

where r0x(λ), x = a,b is the anisotropy contribution to the total anisotropy, r0(λ), by each transition at a given wavelength, λ, and fx(λ) is the transition band shape at the wavelength, λ. The individual transition contributions to the excitation band shape are calculated from

  • display math(5)
  • display math(6)

Replacing the theoretical maximum anisotropy, 0.4, in Eq. (2) above, with the limiting anisotropy of 0.37, and assuming orthogonality of 1La and 1Lb (α = 90°), the limiting anisotropy of 1La, r0a(λ), is 0.185 [24, 27]. The resulting calculated 1La and 1Lb bandshapes, fa(λ) and fb(λ) (Data not shown), have equal amplitude from 250 to 310 nm, and diverge only at the long wavelength edge, where the 1Lb transition reaches a maximum while the 1La transition falls to zero. These results are not in agreement with band shapes observed experimentally (Fig. 2a,b) or calculated (Fig. 2c). Band shapes were recalculated using a limiting anisotropy of 0.034 for r0a(λ), obtained at 276 nm, for the measured 6-hydroxyindole anisotropy, and r0b(λ) = 0.37. The band shapes recalculated with these limiting anisotropies, given in Figure S4, show a 1La transition that dominates from 250 to 303 nm with a gradual drop in intensity from 312 to 320 nm. The 1Lb transition has little intensity from 250 to 309 nm, but peaks at 321 nm. These anisotropy-derived band shapes are in general agreement with the experimental band shapes given in Fig. 2a,b and the calculated band shapes in Fig. 2c. The 1La transition overlaps with 1Lb in the longer wavelength region, but is apparently the only contributing transition at shorter wavelengths. At 321 nm, the 1La transition still contributes. The 334 nm emission maximum for 6-hydroxyindole in the hydrophilic, glycol glass at 77 K (data not shown) is consistent with emission from the solvent-sensitive 1La transition.

Charge acceptors on benzyl ring enhance charge transfer and nonradiative decay

In this study, single fluorescence lifetimes were obtained for 5-hydroxyindole in water and cyclohexane, whereas two lifetimes were obtained for 6-hydroxyindole in cyclohexane, and essentially a single lifetime was obtained for this indole in water (Table 2). The lifetimes obtained for 5-hydroxyindole in water (3.0 ns at two different excitation wavelengths) are in close agreement with the single 3.2 ns lifetime obtained by Sengupta et al. [23]. Wong et al. [38], exciting at 305 nm, obtained two lifetimes for 5-hydroxyindole in water. They report an amplitude-weighted average lifetime of 3.55 ns. Using 285 nm excitation and cyclohexane as solvent, Arnold et al. [1] obtained a 50% longer lifetime (6.0 ns) for 5-hydroxyindole than that reported herein. In He jet-cooled gas expansion, a longer lifetime of 11.1 ns. is obtained for 5-hydroxylindole. In general, longer lifetimes are expected at lower temperatures.

Published fluorescence lifetime studies of weakly fluorescent 6-hydroxyindole are scarce, hampering discussion. One study, by Sulkes et al. [22], reports a short lifetime of 2.8 ns for 6-hydroxyindole in a He jet expansion. Lifetimes measured herein for 6-hydroxyindole are 1.8 ns and 0.90–0.98 ns in cyclohexane and 0.4 ns (ca 100%) in water (Table 2). In their jet-cooled fluorescence lifetime studies of substituted indoles, Sulkes et al. [1, 21, 22] found that 4- and 6-hydroxyindoles display anomalously short lifetimes due to excited state formation of a keto tautomer. The effect is greatest for 4-hydroxyindole, which displays a subnanosecond lifetime in expanded He jet medium (0.23 ns), and somewhat less for 6-hydroxyindole (Table 2). Excited state tautomerism for 5-hydroxyindole is nil in an He-cooled expanded jet, as the 11.1 ns lifetime included in Table 2 suggests [21]. Our very short lifetime of 0.4 ns for 6-hydroxyindole in water and 1.8 ns lifetime for this species in cyclohexane suggest that the nonradiative process of excited state tautomerism is operative at room temperature as well. For this and other indoles substituted with charge acceptors on the pyrrole or benzyl ring, charge transfer to the substituent increases the nonradiative decay rate, k(nrad), [1]. Charge donating groups, such as CH3, placed on the pyrrole ring, enhance charge transfer to the benzyl ring, increasing k(nrad), whereas donating groups on the benzyl ring diminish charge transfer and decrease k(nrad). Regarding solvent effects, cyclohexane was found to stabilize charge through polarizability [1].

It is instructive to compare this relationship of charge acceptors and donors to nonradiative decay (Sulkes effect) with the Stark effect, discussed above. Recall that the Stark effect predicts that a negative charge near the benzyl ring or a positive charge near the pyrrole ring results in a blue shift (higher energy) of the fluorescence emission maximum. Converse charge placement yields a red shift in emission maximum. From the perspective of Sulkes, hydroxyl, carboxylates and halogens are charge accepting groups. From the perspective of the Stark effect, these are electron rich groups. Their summary effect is to promote charge transfer, increase the nonradiative rate (Sulkes effect; [1]) and increase the fluorescence emission energy (emission blue shift) when placed on the benzyl ring, as shown in Fig. 1.

The Sulkes effect also predicts that charge acceptors on the pyrrole will increase nonradiative decay. Emission data for indoles containing pyrroles substituted with charge acceptors are missing from Fig. 1, but the aqueous dipeptide zwitterion, tryptophanyl glycine (TrpGly), exhibits a fluorescence emission that is blue shifted relative to the dipeptide anion [39]. Molecular dynamics simulation shows that the terminal amine cation is positioned over the pyrrole ring for one dominant conformer [39]. From the perspective of the Sulkes effect, the terminal amine cation is a charge acceptor. The average fluorescence lifetime for the TrpGly zwitterion, 1.97 ns, is significantly shorter than that for the TrpGly anion, 6.95 ns [40]. This is further evidence of confluence between the Stark and Sulkes effect: the general trend seems to be that electron accepting groups on either indole ring decrease fluorescence lifetime or blue-shift the emission maximum. The results of Fig. 1 suggest that lowering of the 1Lb energy is concomitant with electron accepting group substitution on the benzyl ring.

The Sulkes/Stark effect analogy may be extended to electron donating groups, such as methyl groups, if the dielectric of the medium is considered. Sulkes found that methyl substitution on the pyrrole ring enhanced the nonradiative rate for measurements made in cyclohexane [1]. Emission maxima for indoles where pyrrole rings are methyl-substituted and the solvent is cyclohexane are significantly blue-shifted (Fig. 1, left side). As noted by Sulkes et al. [1], the combined effect of ring substitution with electron donor/acceptor groups and solvent polarizability can work to stabilize intramolecular charge transfer [1]. This combined effect is apparently also operable for fluorescence emission maxima shifts.

Other evidence for 1La and 1Lb contribution to blue-shifted indole emission

Both theoretical studies and measurements on indoles in van der Waals clusters [8, 18, 19, 41, 42] have found evidence of 1La/1Lb mixing. Gas phase geometrical parameters for the ground and 1La/1Lb excited states of 5-hydroxyindole have been calculated at the CASSCF and TDDFT levels of theory, respectively [43]. The 1Lb transition is centered on the benzyl ring in indole, 2,3-dimethyl indole and tryptophan [28, 44], with changes in bond length at the C4–C5 (+0.056 Å) and C8–C9 (+0.055 Å) bonds [43]. The 1La transition, however, is delocalized over both indole rings in indole and tryptophan [12, 13], with bond changes at the C2–C3 (+0.085 Å), C4–C5 (+0.045 Å) and C6–C7 (+0.063 Å) bonds [43]. As both transitions involve a bond length change at the C4–C5 bond and therefore an electronic redistribution along this bond, excitation of the 1Lb transition invariably leads to a change in 1La. A change in C4–C5 bond length for both the 1La and 1Lb transition has also been found by Eisenberg in ab initio quantum mechanical calculations for 5-OH indole (A. Eisenberg, pers. comm.). The excitation spectra for 5- and 6-hydroxyindole in cyclohexane and that of indole vapor (Fig. 2a,b,d) show that 1La contributes to emission even when energetically well-separated from 1Lb, which is consistent with mixed 1La/1Lb emission.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

This spectroscopic study of pyrrole and benzyl-substituted indoles under hydrophobic and hydrophilic conditions reveals specific substituent effects on the 1La and 1Lb transition energy. For each additional methyl substitution on the pyrrole ring, the energy of both transitions decreases in tandem, with emission maxima following the same trend of energy decrease. Addition of electrophilic or aliphatic groups to the benzyl ring raises the energy of the 1La transition, while decreasing the energy of the 1Lb transition. The effect on fluorescence emission is to increase the energy of the emission maxima even in aqueous solution. For indoles, such as 5-hydroxyindole, the emission maximum in a hydrophobic solvent, such as cyclohexane, is nearly coincident with the maximum in water. Hydroxyl substitution at either the 5- and 6-ring position of indole reveals experimentally resolved 1La and 1Lb transitions, providing for the first time, the energetic location of the 1La origin. Fluorescence excitation spectra for 5- and 6- indole in cyclohexane are also transitionally resolved, and reveal that the 1La transition makes an energetic contribution to fluorescence emission even when emission is from 1Lb. Fluorescence excitation anisotropy measurements have the potential to resolve the relative contribution of overlapping 1La and 1Lb transitions to fluorescence emission under favorable solution conditions. For 6-hydroxyindole in a hydrophilic glass, vibrational detail is missing and transition overlap is greater than in cyclohexane solution. Deconvolution of the anisotropy results reveal that the 1La is the dominant band under hydrophilic conditions, and whereas at somewhat higher energy than the 1Lb transition, contributes sufficiently at the excitation spectrum red edge. Lifetimes for 5- and 6-hydroxyindole in aqueous or cyclohexane solution are on the order of 100 ns. Combining Stark effects for fluorescence emission maximum shifts and Sulkes effects [1] for charge transfer and the resulting increase in nonradiative decay (lifetime shortening), it is observed that charge acceptor groups on either ring of indole blue shift the emission maximum while increasing charge transfer and the nonradiative decay rate [1]. In a hydrophobic environment, charge donor groups on the pyrrole ring blue shift fluorescence emission while promoting charge transfer to the benzyl ring [1].

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank Dr. Azaria Eisenberg for helpful comments. This research was supported by NIH Grant 5 SCZGM092291-02.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
php1219-sup-0001-FigureS1.docWord document39KFigure S1. Absorption spectrum for 6-hydroxyindole in cyclohexane showing baseline curve fit used to approximate contribution to absorbance above 240 nm from intense, S0 [LEFTWARDS ARROW] S1 peak.
php1219-sup-0002-FigureS2.docWord document42KFigure S2. Curve fit to the S0 [LEFTWARDS ARROW] S1 absorption spectrum for 5-hydroxyindole in cyclohexane.
php1219-sup-0003-FigureS3.docWord document40KFigure S3. Curve fit to the S0 [LEFTWARDS ARROW] S1 absorption spectrum for 6-hydroxyindole in cyclohexane.
php1219-sup-0004-FigureS4.docWord document40KFigure S4. 6-hydroxyindole transition bandshapes, 1La and 1Lb, calculated from eqns. (6) and (7), using limiting anisotropy values derived from the fluorescence excitation anisotropy measurement at 77 K, given in Fig. 4b: fa(λ), dashed line and fb(λ), solid line.

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