Fluorescence quenching of protonated β-carbolines in water and microemulsions: evidence for...

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www.rsc.org/pps

1474-905X(2011)10:8;1-0

ISSN 1474-905X

An international journal

www.rsc.org/pps Volume 10 | Number 8 | August 2011 | Pages 1257–1366

Photochemical &Photobiological Sciences

View Article OnlineView Journal

http://dx.doi.org/10.1039/c3pp50044f

http://pubs.rsc.org/en/journals/journal/PP

1

Fluorescence Quenching of Protonated β-carbolines in Water and

Microemulsions: Evidence for Heavy-Atom and Electron-Transfer

Mechanisms

Souad A.Mousa*a,b

, Peter Douglasb,c

, Hugh D. Burrowsd, Sofia M. Fonseca

d

a) Chemistry Department, College of Science for Women, Baghdad University, Iraq

E-mail: [email protected]

b) Chemistry Group, College of Engineering, Swansea University, Singleton Park, Swansea, SA2 8PP, UK

c) School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban 4000, South Africa

E-mail: [email protected]

d) Departamento de Química da Universidade de Coimbra, Rua Larga, 3004-535 Coimbra, Portugal

E-mail: [email protected] ; [email protected]

Abstract

The fluorescence quenching of protonated β-carbolines has been investigated in acidic aqueous solutions and in w/o

microemulsions using I-, Br-, Cu2+, SCN-, and Pb2+ as quenchers. It was found that fluorescence quenching by these compounds

is much more efficient in water than in microemulsions since quenching in microemulsions depends on the simultaneous

occupancy of the water droplets by both fluorophore and quencher. Linear Stern-Volmer plots were obtained in all cases, leading

to quenching rate constants of ca. 108-1010 M-1 s-1 in water and ca. 107-108 M-1 s-1 in microemulsions. In the case of quenching by

SCN−, ns flash photolysis studies indicate formation of (SCN)2•- showing that at least part of the quenching process involves an

electron transfer mechanism. This indicates that the singlet excited states of the protonated β-carbolines can act as relatively

strong oxidants (Eº > 1.6 V), capable of oxidizing many species, including the biologically relevant DNA base guanine. The

observation of the (SCN)2•-

transient in microemulsions demonstrates that it is possible to have the protonated β-carboline and at

least two thiocyanate ions in the same water pool.

Keywords: fluorescence quenching, β-carbolines, protonated β-carboline excited state oxidation potential,

microemulsions.

1. Introduction

β-carbolines (Fig.1) are a group of naturally occurring alkaloids, whose pharmacological and biological properties have attracted

great interest.1-3 β-carbolines are found in a diverse range of plant families and genera,4,5 in food,6-8 and tobacco,9 and in

human10,11 and animal tissues12. The photophysical properties of β-carbolines have been quite widely studied, notably absorption

and emission spectra in solution13-20 and microemulsions,21 triplet state behaviour14,22 and singlet oxygen generation23. Because of

their attractive absorption and emission spectra, protonated β-carbolines have been suggested as fluorescence standards.24

Page 1 of 16 Photochemical & Photobiological Sciences

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Fig.1 Structures of protonated β-carbolines used in this work.

Water-in-oil (w/o) microemulsions can act as good models systems for the compartmentalization of biologically relevant

molecules, such as β-carbolines. We have been interested in the mechanisms of the fluorescence quenching in these molecules by

a range of species. In this work, results from fluorescence quenching of protonated β-carbolines (norharmane, harmane, and

harmine) by halides, the pseudo-halide thiocyanate ion, as well as heavy atom metal ions, both in aqueous solutions and

microemulsions are presented. The distinction between the kinetics of fluorescence quenching of β-carbolines in aqueous solution

and in AOT microemulsions seems to depend on the nature of these media. AOT microemulsions consist of three domains: water

droplets, bulk organic solvent (in this case cyclohexane), and an interfacial region of AOT. The existence of these phases gives

the systems the ability to modulate chemical reactivity because of the compartmentalization of the reactants in the different

domains.25 This makes them good models for certain biological systems.26

2. Material and methods

Norharmane, harmane, and harmine were purchased from Sigma-Aldrich, NaI, NaBr, CuCl2, Pb(CH3COO)2, KSCN, Ba(NO3)2,

MnSO4, and tetra-butyl ammonium iodide, were from BDH chemicals. The photochemical study of β-carbolines can be

complicated by their acid-base equilibra since pKa values for protonation of the pyridinic nitrogen are around 7-8 in the ground

state and around 11-14 in the singlet excited state S1.16 As a consequence, acidified solutions were used through this work to

ensure that only the protonated species are present to any significant extent. Also an excitation wavelength of 370 nm was used

for fluorescence quenching studies, and 373 nm for fluorescence lifetime studies, which effectively limits excitation of β-

carbolines to their protonated form and avoids interferences arising from the complex excited state acid-base behavior, thus

simplifying data treatment.27

Aqueous solutions: β-carbolines, 1 × 10-5 M, were prepared in 0.01 M hydrochloric acid. Quencher concentrations were 0.002-

0.01 M for NaI and KSCN, and 0.02- 0.1 M for NaBr, Pb(CH3COO)2, CuCl2, MnSO4, and Ba(NO3)2.

Page 2 of 16Photochemical & Photobiological Sciences

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Microemulsions: w/o microemulsions were prepared using the surfactant AOT [28] with cyclohexane as the dispersion medium.

Stock solutions of 0.001M β-carbolines were prepared in 0.01 M hydrochloric acid. At this concentration of acid, the β-

carbolines will be almost exclusively present as the highly water-soluble protonated form.21 The following procedure was used in

the preparation of microemulsions. 2.22 g of AOT was dissolved in 100 ml of cyclohexane, to give a 0.05 M solution. To obtain

a w/o microemulsion with a value of water to surfactant molar ratio, ωo, approximately 10 (i.e. ωo= [H2O]/ [AOT] =10), 50 µl of

β-carboline at 0.001 M in 0.01 M hydrochloric acid was added to 5 ml of the solution of AOT in cyclohexane, and the system

kept in a ultrasonic bath until the final solution had a clear aspect. As a check on ωo, the amount of water was measured using

Karl Fischer titration, (Metrohm), which shows the presence of a small amount of additional water from surfactant and solvent29

giving a final value of ωo = 12.5. The concentration of β-carbolines in the added water phase was 0.001M, giving a concentration

in bulk microemulsions of 1 × 10-5 M. The quenchers were added to the acidic β-carbolines solutions before these were added as

aqueous phase, with concentrations of 0.02-0.1 M in the added aqueous phase, i.e. 2-10 × 10-4 M in bulk. Note that, because of

the small amount of water from the surfactant and solvent, final microemulsion aqueous phase concentrations were 0.8 those of

added aqueous phase. Concentrations given in the text are added aqueous phase values. The presence of the quenching salts at the

concentrations studied can lead to changes in shape of the microemulsion pools and, eventually, to phase separation.30 This is

likely to be particularly important with the divalent ions copper(II) and lead(II). However, there was no obvious visible evidence

for phase separation and the Stern-Volmer curves and data are consistent with the persistence of the microemulsion structures.

Apparatus and Methods: For the steady state measurements, fluorescence spectra were recorded with a Horiba-Jobin-Ivon SPEX

Fluorog 3-22 spectrometer and were corrected for instrumental response. As indicated above, all of the samples were excited at

370 nm. Fluorescence decays were measured using a home-built Time-Correlated Single Photon Counting TCSPC apparatus with

an N2 filled IBH 5000 coaxial flash lamp as excitation source (λexc=373 nm), Jobin-Ivon monochromator, Philips XP2020Q

photomultiplier, and Canberra instruments Time-to-amplitude converter TAC and Multichannel Analyzer MCA. Alternate

measurements (1000 c.p.c.), controlled by Decay® software (Biodinamica-Portugal), of the pulse profile at 373 nm and the

sample emission were performed until 1- 2 × 104 counts at the maximum were reached. The fluorescence decays were analyzed

using the modulating functions method of Striker with automatic correction for the photomultiplier “wavelength shift”.

The transient absorption spectra and decay kinetics were obtained with an Applied Photophysics LKS.60 flash photolysis

spectrometer which uses a pulsed 150W Xe lamp with a Hamamatsu R928 photomultiplier, and a Tektronix TDS 3052B (500

MHz, 5GSa/s) oscilloscope, for detection and recording of transient signals. A Spectra Physics Nd:YAG laser was used for

excitation, with the frequency tripled, 355 nm, excitation beam at right angles to the detection system. An HP digital analyzer

was used to analyze the obtained signal which was transferred to a computer where optical densities were collected at different

wavelengths by using the software provided (Applied Photophysics). Solutions were deoxygenated by bubbling with nitrogen.

All the experiments were carried out at room temperature, which was 20-22 ºC.

Data analysis: straight line fits of Stern-Volmer plots were obtained using TableCurve 2D automated curve fitting software from

Jandel Scientific, and error estimates in Stern-Volmer constants and derived rate constants are reported as one standard deviation.


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3. Results and discussion

In both aqueous solution and microemulsions, for every quencher/β-carboline combination used, and irrespective of quencher

concentration, the shape of the emission spectra remained the same, corresponding to emission from protonated β-carbolines

exclusively, but the intensity of emission was reduced as quencher concentration was increased.

3.1 Fluorescence quenching in water

3.1.1 Halides as quenchers:

Fig. 2 shows steady state fluorescence spectra of the protonated β-carbolines and Stern-Volmer plots for I-, and Br- quenching in

aqueous solution. This quenching is described by the Stern-Volmer equation,31

F0/F = 1 + kqτ0[Q] (1)

where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, kq is the bimolecular

quenching constant, τ0 is the lifetime of the fluorophore in the absence of a quencher, and [Q] is the concentration of quencher. A

plot of F0/F versus [Q] yields an intercept of 1 on the y-axis and a slope equal to the Stern-Volmer constant, Ksv, i.e. kqτo (Fig.2).

The variation of F0/F with quencher concentrations (I-, Br-) was linear for all three of the ß-carbolines, with a decreasing slope in

the order norharmane > harmane > harmine which is expected from the lifetimes (τo) of 21.4, 17.9, and 6.21 ns (this work) for

protonated norharmane, harmane, and harmine, respectively (Table 1).

kq values, calculated from the Stern-Volmer constants and lifetimes, are in the range of ≈ 1010 M-1s-1 for I- and ≈ 108-109 M-1s-1 for

Br- in aqueous solution. The diffusion controlled rate constant for neutral species in water at 25 oC is 7.0 × 109 M-1 s-1. However

the diffusion controlled rate constant depends on the relative charges, ZAZB, of the reacting species, and at zero ionic strength

these charge factors are 7.08, 3.65, 1.00, 0.106, and 0.006 for ZAZB= -2, -1, 0, +1, +2, respectively.32 Generally, quenching of

fluorescence by the halides in solution is in the order of effectiveness I- >> Br- > Cl- > F-.33 Fluorescence quenching by the larger

halogen anions such as bromide and iodide is usually ascribed to the heavy-atom effect i.e., intersystem crossing promoted by

spin-orbit coupling of the excited (singlet) β-carboline and the halide,33-36 but electron transfer has also been proposed as a

quenching mechanism37. As we will show in the next two Sections, we have used quenching studies with heavy metals and flash

photolysis to distinguish between these two.


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Table 1 Lifetimes, steady-state Stern-Volmer constants, and calculated bimolecular rate constants, for the quenching of β-

carbolines by various quenchers in aqueous solutions and microemulsions.a

β-c

τ/ns

Quencher KSV/M-1

Microemulsion

Apparent kq /M-1 s-1

Microemulsion

KSV/ M-1

Water

kq /M-1s-1

Water

Calculated

diffusion

controlled

rate constant /

M-1s-1

Nh

21.4

NaI 5.7± 0.6 2.7 ± 0.3×108 235± 3.0 1.1 ± 0.02×1010 2.6 ×1010

TBAIb 5.9± 0.2 2.8 ± 0.1×108 2.6 ×1010

NaBr 0.9 ± 0.3 2.2 ± 0.6×107 5.3 ± 0.3 2.5 ± 0.2×108 2.6 ×1010

CuCl2 5.0± 0.7 1.8 ± 0.2×108 15.5 ± 0.9 6.4 ± 0.4×108 4.7 ×107

(CH3COO)2Pb 1.7 ± 0.2 7.9 ± 0.9×107 7.3 ± 0.2 3.2 ± 0.1×108 4.7 ×107

KSCN 5.7 ± 0.6 2.7 ± 0.3×108 187 ± 8.0 8.8 ± 0.4×109 2.6 ×1010

Ba(NO3)2 ≤ 0.2

MnSO4 ≤ 0.2

Ha

17.9

NaI 3.2 ± 0.5 1.8 ± 0.3×108 207 ±8.0 1.2 ± 0.04×1010 2.6 ×1010

TBAIb 4.5 ± 0.9 2.5 ± 0.5×108 2.6 ×1010

NaBr 0.3 ± 0.04 1.9 ± 0.2×107 6.7 ± 0.2 3.8 ± 0.1×108 2.6 ×1010

CuCl2 2.6 ± 0.3 1.4 ± 0.2×108 17.6 ± 1.2 1.0 ± 0.6×109 4.7 ×107

(CH3COO)2Pb 0.7 ± 0.1 3.8 ± 0.2×107 3.7 ± 0.1 2.1 ± 0.1×108 4.7 ×107

KSCN 4.3 ± 0.9 2.4 ± 0.5×108 149 ± 8.0 8.3 ± 0.4×109 2.6 ×1010

Hi

6.21

NaI 2.0 ± 0.1 3.2 ± 0.2×108 145 ± 20 2.3 ± 0.1×1010 2.6 ×1010

TBAIb 3.17 ± 0.88 5.1 ± 1.4×108 2.6×1010

NaBr 0.86 ± 0.18 3.8 ± 0.8×108 24 ± 4 4.2 ± 0.2×109 2.6×1010

CuCl2 1.11 ± 0.18 1.8 ± 0.3×108 9.0 ± 0.7 1.5±0.1×109 4.7×107

(CH3COO)2Pb 0.43 ± 0.37 6.9 ± 6.0×107 7.4 ± 0.3 1.2 ± 0.1×109 4.7×107

KSCN - - 71 ± 6 1.1 ± 0.1×1010 2.6×1010

a kq values were calculated using KSV= kqτo using τo values of 21.4, 17.9, and 6.21 ns determined in this work for protonated

norharmane, harmane, and harmine; error estimates are one standard deviation. b tetra-butyl ammonium iodide.


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Fig. 2 Quenching of fluorescence spectra of cationic β-carbolines in acidic aqueous solutions. a1, a2 – norharmane; b1, b2 –

harmane; c1, c 2 harmine; using: 1- NaI; 2- NaBr, respectively. Quencher concentrations: [NaI]: 0.002, 0.004, 0.006, 0.008, 0.01

M. [NaBr]: 0.02, 0.04, 0.06, 0.08, and 0.1 M. Direction of arrows indicates increasing quencher concentration

3.1.2 Quenching of fluorescence by Cu2+ and Pb2+

Stern-Volmer data for fluorescence quenching by Cu2+ and Pb2+ are shown in Table 1. The order of KSV for β-carbolines is

harmane > norharmane > harmine for Cu2+, but it is harmine > norharmane > harmane for Pb2+. This reversal of order suggests

Cu2+ and Pb2+ may quench by different mechanisms. When β-carbolines are quenched by Cu2+ this may possibly involve

electron-transfer, i.e. reversible donation of an electron from the β-carboline to Cu2+.38 However, since Pb2+ does not have any

readily accessible oxidation or electronic states, quenching in this case can only be a result of the heavy atom effect via

promotion of intersystem crossing by spin-orbit coupling of the excited (singlet) fluorophore and the heavy atom Pb2+.33 With

both metal ions complexation with the β-carbolines may also be important although we have not examined this. Ba2+ and Mn2+

are inactive as quenchers of fluorescence of β-carbolines. Heavy atom quenching depends on atomic number, which may explain

the stronger quenching by Pb2+ (Z = 82) than by Ba2+ (Z = 56). The lack of quenching by Mn2+ probably stems from the fact that

it does not have any low-lying oxidation state. The lack of quenching by Ba2+ also suggests that with I-, which has a similar


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atomic number (Z = 53), the heavy atom effect can only make a minor contribution to quenching, and that electron transfer is the

dominant mechanism.

3.1.3 Fluorescence quenching by thiocyanate ion

To test the possibility of electron-transfer quenching more fully, we have looked at the behavior with the pseudo-halide ion

thiocyanate. Table 1 shows Stern-Volmer data for quenching by SCN-. If the mechanism of fluorescence quenching by SCN-

involves electron-transfer, this may be expected to lead to formation of (SCN)2•- ,,

39-43 The relevant equations using protonated β-

carbolines (β-cH+) are as follows:

β-cH+* → β-cH+ + hν (2)

β-cH+* + SCN- → β-cH• +SCN• (3)

SCN• + SCN-→ (SCN)2•- (4)

β-cH• + (SCN)2•- → β-cH+ + 2SCN- (5)

Flash photolysis has been used to look for (SCN)2

•- formation. Absorption spectra of transients formed after laser excitation for

norharmane with and without thiocyanate are given in Fig. 3. The transient without quencher present shows a broad band with

absorption maximum around 440 nm which is assigned to the triplet state of protonated norharmane, in agreement with literature

data.22 In the presence of thiocyanate, there is a marked increase in absorption and a shift in the maximum to around 480 nm, in

addition to further absorption at longer wavelengths, suggesting quenching goes by some chemical reaction which generates new

transient species. To characterise these, we have taken the flash photolysis spectrum after adding the thiocyanate ion (spectrum 2

in Fig. 3), and corrected this by subtracting the absorbance due to triplet form of unquenched β-carbolines from the total spectrum

to leave just the absorption for the products of quenching, as shown as spectrum 3 in Fig. 3.The fraction of β-carbolines that is

quenched by thiocyanate ion in aqueous solution was calculated from the steady-state Stern-Volmer plot to be ca. 67 %. The

absorption maximum around 460 nm of the corrected transient absorption strongly suggests that the part of the absorption in

spectrum 3 in the region 390-590 nm is due to (SCN)2•-, (spectrum 4 in Fig. 3)44, in agreement with the above scheme. If this is

the case, the rest of spectrum is presumably due to the reduced β-carboline radical co-product. In contrast, if fluorescence

quenching were by a heavy-atom enhanced intersystem crossing then the spectrum in the presence of thiocyanate would be

identical to that in spectrum 1 of Figure 3, but with an increased yield. This is not seen.

This indicates that the singlet excited state of protonated norharmane (and probably the other β-carbolines) must be capable of

oxidizing SCN- and that its standard oxidation potential must be greater than the Eº for SCN•/SCN- (1.63 V)45. This is a lower

limit, and the fact that bromide ion also quenches the fluorescence of protonated β-carbolines suggests that it could be higher (Eº

for Br•/Br- 1.92 V). Electrochemical studies on β-carbolines have tended to concentrate on their oxidation processes,46,47 and we

have been unable to find any experimental data on their reduction. However, the results given here indicate that protonated β-

carbolines in their singlet excited states are strong oxidants, and are capable of oxidizing a variety of biologically relevant

compounds, including the DNA base guanine (Eox 1.41 V)48. This has clear implications for the role of β-carbolines in

photoinduced reactions in biological systems, and indicates that they may be involved in both direct and sensitized

photooxidation processes. This is supported by the observation that under acidic conditions, DNA damage by these compounds

involves a direct type-I photoreaction of the protonated β-carboline.49 It is worth noting that in aerated solutions, formation of

dimers and oligomers is observed upon photolysis of norharmane solutions.50 However, this involves oxidation of the β-


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carboline, and under our anaerobic conditions photolysis in the presence of SCN- would appear to give the reduced β-carboline

radical. There were no indications of formation of stable photoproducts under these conditions.

Fig. 3 Transient absorption spectrum after 5 µs following laser flash photolysis of an acidic aqueous solution of norharmane

(absorbance ca. 0.2 at 355 nm) and KSCN (0.01 M). Spectrum 4 from reference 44.

3.2 Fluorescence quenching of β-carbolines in microemulsions

Time-resolved fluorescence decays of the protonated form of the three β-carbolines in w/o microemulsions have been recorded

using 373 nm excitation (at this excitation wavelength the cationic form absorbs while the neutral form does not), with emission

collected at 400 nm. Lifetimes were 21.4 ns for norharmane and 17.9 and 6.21 ns for harmane and harmine, figures which are

close to lifetimes in the literature: 23, 22, 6.7 for norharmane, harmane and harmine, respectively13.

In micellar systems, the β-carboline concentration and the excitation intensity must be kept as low as possible to facilitate

application of Poisson statistics to the observed behaviour since this is made simpler if there is not more than one excited β-

carboline present in any one individual microemulsion droplet.51

The probability distribution of solute molecules in microemulsion droplets is given by a Poisson distribution52:


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P(r) = [Mr]/[M] = e-µµr/r! (r = 0, 1, 2 … µ > 0) (6)

where [Mr] is the concentration of droplets containing r molecules of β-carboline, [M] is the total concentration of droplets (given

by [AOT]/N, where N is the aggregation number), µ is the average number of β-carbolines per droplet, and r is the number of β-

carboline molecules. It is expected that, since they are protonated, most of the β-carboline molecules will be in the aqueous phase

(water droplet).21 Taking an aggregation number from the literature of 116,53, (but noting this is for AOT = 0.1 M, whereas for

our experiments it is 0.05 M), values of P(r) have been calculated for occupation of β-carbolines which shows that, assuming all

β-carbolines molecules will be the aqueous phase (water droplet), ca. 98% of microemulsions droplets have no β-carboline

molecules and 2% of microemulsions have one molecule. Taking a quencher concentration of 0.0167 M in water droplets (1.67 ×

10-4 M in the bulk) as an example, at this concentration ca. 60% of microemulsion droplets have no quencher molecules, ca. 30%

of microemulsion droplets have one and ca. 10% more than one. As the concentration of quencher increases, the distribution of

quencher molecules within the microemulsion droplets changes significantly (Fig. 4). The amphiphile molecule (AOT) which

forms the w/o microemulsion system has a negatively charged head therefore, the positively charged protonated β-carboline and

also positively charged quencher molecules may associate electrostatically with the interfacial region.54. From the distribution of

β-carbolines and quenchers, it can be seen that some of the microemulsion droplets contain quencher molecules but no β-

carboline molecules, and vice versa, and it is for this reason that the efficiency of quenching of fluorescence in microemulsions is

less effective than that in water. There is a great similarity among the apparent Stern-Volmer quenching rate constants in

microemulsions in Table 1, because these are dependent on occupancy of the droplets by β-carbolines or quencher molecules as

well as the bimolecular quenching rate constants.

For microemulsion droplets that contain one molecule of quencher with nominal ωo = 12.5, if the aggregation number is 116, then

the concentration of quencher in that droplet aqueous phase is 0.038 M. If the rate constant of quenching is assumed to be the

same as that in bulk water, the fraction of β-carbolines quenched when in a droplet containing one quencher molecule, Q, can be

calculated from the following equation:

Fraction of β-carbolines quenched by quencher in droplet = kq[Q]/(kq[Q] + 1/τβ-c) (7)

where kq is the rate constant of quenching and τβ-c is the fluorescence lifetime of the protonated β-carbolines. Taking kq for

quenching by I-, then Eq. 8 gives the fraction of β-carboline quenched by the presence of one molecule of I- in a microemulsion

droplet to be ca. 0.9 for norharmane and harmane, and ca. 0.8 for harmine. When the number of quencher molecules increases in

the droplet, the fraction of β-carboline quenched is expected to be higher.


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Fig. 4 Poisson distribution of r quencher molecules per microemulsion droplet for the system AOT (0.05 M), nominal ωo = 12.5,

N = 116 with different concentrations of quencher.

Time-resolved studies of norharmane in microemulsions with 0.1 M NaI as quencher give the decay shown in Fig. 5 which is

well fitted using two lifetimes of 21.4 and 9.6 ns, with initial intensity ratios of 0.149 and 0.283 respectively. The longer lifetime

is essentially the same as free protonated norharmane, and the second we take as due to norharmane in droplets present with

quencher. The ratios of the lifetimes indicate that 54% of norharmane with an I- in the droplet is quenched. The ratio of intensities

indicates that around a third of the droplets appeared to have no quencher. Re-analysis using the Poisson distribution indicates an

aggregation number of ca. 60 is required for a third of droplets to contain no quencher at this quencher concentration. The

Poisson distribution for N = 60 can be seen in Fig. 6. It is interesting to note that the shorter lifetime, 9.6 ns, which we take to be

for norharmane with quencher present, is about 8× longer than we would expect if the kinetics in the droplet were exactly the

same as bulk aqueous solution. So that, even when there is a quencher present in the droplet, the quenching process itself in the

microemulsion droplets is less efficient than in water. We do not know the reason for this but it may be related to microviscosity,

or selective localisation or movement, of the β-carboline and/or quencher within the microemulsion structure.


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Fig. 5 Fluorescence decay of protonated norharmane in w/o microemulsions, ωo = 12.5, [AOT] = 0.05 M, using NaI 0.1 M as

quencher, after excitation of the cationic form (λex = 373 nm) with emission collected at 400 nm. Weighted residuals for fitting to

a double exponential function with the fitting parameters provided in the text are also shown.

Fig. 6 Poisson distribution of quencher molecules per microemulsion droplet for the system AOT (0.05M), ωo = 12.5, N = 60 at

various concentrations of quencher: 0.02, 0.04, 0.06, 0.08, and 0.1 M.

A calculation of the occupancy of droplets by β-carbolines and quencher molecules has also been carried out from steady-state

fluorescence data as follows.


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From the Poisson equation, the probability of a β-carboline in the droplet with no quencher in that microemulsion droplet is:

e-x (8)

where

x = [Q]/([AOT]/N) (9)

and when x is small

e-x = 1-x (10)

Taking the fluorescence intensity (F0) in the case where no quencher is present in a microemulsion droplet as 1 then:

F/F0 = (1-x) + fx (11)

where f is the probability of quenching with a droplet containing one quencher molecule. 1-x is the emission intensity from those

β-carbolines with no quencher present in the droplet and fx is the emission intensity from those β-carbolines with a quencher

present.

So,

F/F0 = 1 - (1-f)x (12)

By substitution of eq. 10 in eq. 12:

F/F0 = 1 – ((1 - f)N/AOT)[Q] (13)

A plot of F/Fo versus [Q] should yield an intercept of 1 on the y-axis and a slope equal to –((1-f)N/[AOT]). Using a value of 0.54

for f for norharmane, as obtained from time-resolved studies, gives an aggregation number of 37, in reasonable agreement with

that from direct analysis of time resolved data. Taking both values of N, the best estimate obtained is N= 48±12, although there

are considerable uncertainties in these calculations, and this is less than half the aggregation number value from the literature.

Three factors may be involved in this. Firstly, the surfactant concentrations are different in the two cases; the concentration of

AOT in this study is 0.05 M, while it was 0.1 M in the calculation of the aggregation number in the literature53. There are

indications in the literature that AOT microemulsion droplet aggregation number is dependent upon surfactant concentration.54

Secondly, the present studies were carried out in acidic solution, which may decrease droplet size. However, an important effect

is expected to stem from the polydispersity of droplet size.55 Droplet size in ref. 53 was determined by dynamic light scattering.

This will give a weight average, which will tend to favor observation of larger particles,56 while the fluorescence quenching

results will provide a number average droplet size, which will be considerably smaller.

In order to understand the behavior of β-carbolines inside the microemulsion droplet during their excited state lifetimes we have

made some rough estimates of their homogeneous diffusion distances. Since diffusion coefficients of β-carbolines could not be

found in the literature we have used the value for anthracene in a moderately viscous dioxane:water mix (η ~ 2 cp) of 0.4 × 10-5

cm2 s-1,57 as a rough lower estimate since anthracene is of a similar molecular size to β-carbolines, and we do not know either the

micro-viscosity in the microemulsion droplet or the effect of the charge on the protonated β-carbolines. The diffusion process is

described by the Gaussian distribution function;58


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C/M = [8(πDt)3/2]-1exp(-x2/4Dt) (14)

where M is the quantity of diffusing substance (taken here to be 1) at t = 0, C is the concentration at time t at a distance x from

the point source. Plotting C/M against x at any time t gives a distribution of the diffusing substance at different x values, and

multiplying by 4πx2 gives the radial distribution function (RDF). Fig. 7 shows RDF values for diffusion of β-carbolines after one

excited state lifetime, assuming homogeneous diffusion with a diffusion coefficient of 0.4 × 10-5 cm2 s-1. The calculations indicate

that norharmane and harmane at least can probably explore a large fraction of the droplet volume within the lifetime of their

excited states.

Fig. 7 Radial distribution functions for excited-state β-carbolines from point of origin after one singlet-state lifetime assuming

homogeneous diffusion with a diffusion coefficient of 0.4 × 10-5 cm2 s-1. Droplet diameter = 5 nm for N = 116.

Quenching of photoexcited protonated norharmane by thiocyanate has also been studied in microemulsions by flash photolysis.

As with aqueous solutions, formation of (SCN)2•- is indicated by the transient absorption around 480 nm (Fig. 8), suggesting

electron transfer from SCN- to excited norharmane cation, followed by reaction of the SCN• radical with a second thiocyanate

anion. Higher concentrations of SCN- (≈ 0.1 M) are needed to observe this species in microemulsions, which is consistent with

the Poisson distribution of quencher shown in Fig. 6, since formation of (SCN)2•- requires the presence of two thiocyanate anions,

as well as the β-carboline, in the microemulsion droplet.


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Fig. 8 Transient absorption spectrum observed 5 µs after laser flash photolysis of norharmane 1×10-5 M in the acidic aqueous

phase with 0.1 M KSCN as quencher in w/o microemulsions, ωo = 12.5, [AOT] = 0.05 M.

4. Conclusion

Bimolecular quenching constants have been obtained for fluorescence quenching of protonated β-carbolines (norharmane,

harmane, harmine) in water and microemulsions by various quenchers: halide and pseudo halide ions, Cu2+ and Pb2+, and SCN-.

Quenching rates in water are generally 0.1-1 of diffusion controlled values. Quenching of β-carbolines by halides and Cu2+

probably occur by electron transfer, with a minor contribution from enhanced intersystem crossing, while with Pb2+ heavy atom

increased intersystem crossing is probably the route. Evidence for an electron transfer mechanism comes with flash photolysis

studies in the presence of SCN-, where the (SCN)2•-

,radical anion is observed as an intermediate. This indicates that protonated β-

carbolines are relatively strong oxidizing agents. Quenching in water is faster than in w/o microemulsions. Quenching in

microemulsions depends on the occupancy of microemulsion droplets by β-carbolines and quencher molecules. Calculations of

occupancy have been carried out from time-resolved and steady state fluorescence studies giving aggregation numbers of 60 and

37, respectively. These are less than half that previously reported in the literature for this system. Various factors may contribute

to this difference, including changes in AOT concentration, pH effects and droplet polydispersity.

5. Acknowledgments

Grateful acknowledgment is made to the Iraqi government for financial support of this research. We are grateful to Professor J. S.

Seixas de Melo, Drs J. Pina and C. Serpa for assistance with the Time-Correlated Single Photon Counting and Flash Photolysis

studies. We also thank the Fundação para a Ciência e a Tecnologia (FCT) for the award of a postdoctoral fellowship (SMF,

grant FCT/SFRH/BPD/34703/2007).


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Fluorescence quenching of protonated β-carbolines in water and microemulsions: evidence for...

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Transcript of Fluorescence quenching of protonated β-carbolines in water and microemulsions: evidence for...