J. Phys. Chem. 1992, 96, 10290-10296
10290
@-CarbolinePhotosensitizers. 1. Photophysics, Kinetics and Excited-State Equilibria in Organlc solvents, and Theoretical Calculations A. Dias,t A. P.Varela,: M. da G.Miguel,: A. L.Macanita,t and Ralph S.Becker*$g Departamento de Quimica de I.S.T, Centro de Tecnologia Quimica and Biologica, Oeiras, Portugal, Departamento de Quimica, Uniuersidade de Coimbra, Coimbra, Portugal, and Department of Chemistry, University of Houston, Houston, Texas 77204-5641 (Received: May 21, 1992; In Final Form: September 15, 1992)
Four &carbolines have been examined regarding absorption, emission, and excited-state equilibria in several classes of organic solvents. In hydrocarbon and nonprotic solvents, excitation results only in an excited-state neutral tautomer and fluorescence. In a neutral protic solvent (methanol), excitation of the neutral tautomer (except harmaline) results in multiple excited-state tautomers-neutral, cation, and zwitterion-and emission. The excited cation and zwitterion are formed from the neutral in and during the lifetime of the lowest ( r , r * )singlet state. A kinetic model has been developed for the complex decays, and an analysis is presented for the neutral protic solvent case. Absorption, emission, and kinetic decay data for the cations are also presented. Only a single excited-state tautomer exists and emits independent of the solvent. Theoretical calculations are carried out regarding (1) state orders and (2) ground- and excited-statecharge densities, the latter of which helps rationalize the existence of multiple excited-state tautomers. The impact of the results on photosensitizer aspects of the compounds is given.
Introduction Because of the presence of significantly different types of nitrogen atoms (pyridine and pyrrole) in 8-carbolines, excitation could result in signifcant and differentialchargedensity changes, resulting in new and unique species in neutral protic solvents. This represents a comprehensive study of the absorption, emission, theory, and kind of tautomers in equilibrium in the ground and fmt excited singlet states as a function of the nature of the solvent (nonpolar vs protic). In particular, norharmane (Norh), 1, harmane (Hara), 2, and harmine (Hari), 3, have been the most extensively examined. In addition, harmaline (hydrogenated Hari), 4, has been studied but to a somewhat less extent.
1, R1 = R2 = H 2, R1 = CH3; R2 = H 3, R1 = CH3; R2 = OCHj
4, R j = CHj; R2 = OCH3
8-Carbolines are important in several respects including their use as fluorescent standards as cations,' their use as photosensitizers of biological interest, for example, refs 2-4, and the fact that interesting tautomeric equilibria occur in water solutions as a function of pH (some pK, data exist in the ground and/or excited states, for example, refs 5-7). In some cases,fluorescmce quantum yields and/or lifetimes are known in water, at various pHs; for example, see refs 1 and 7-9. There is some quite limited data on absorption and emission of some compounds in benzene' and protic solvent^;^^^^^ however, no analysis of the possibility of excited-state tautomer equilibria and emission properties as a function of emitting wavelength exists. Furthennore, no extensive calculations of the transition energies, orbital origins of the transitions, oscillator strengths, and charge densities in both the ground and excited states and their sisnificance are available. The latter piece of data are important to establish the nature and order of the excited states as related to the emission properties, as well as aiding in the assignment of the identity of any tautomers existing in the excited state, their origin, and their associated fluorescence emission properties. We shall also consider the important impact of our findings on the biological photosensitization aspects. Centro de Tecnologia Quimica and Biologica.
* Universidadc de Coimbra. I University of Houston.
Experimental Section Harmine, harmane, and norharmane were used as purchased (Aldrich). Harmaline (Aldrich) was purified by column chromatography (ethyl acetate-ethanol, silica gel Merck 7754). Benzene (Merck, P.A.), methanol (Merck, Uvasol), cyclohexane (Merck, Uvasol), and dioxane (Riedel, Chromasolv) were used without further purification. For some solutions, methanol was dried by distillation over magnesium and chromatographed on an AlzOl column. The solutions of 8-carbolines were prepared by setting the M and deoxygenated by bubbling with concentration at Ar or NZ. Absorption and fluorescence spectra were measured with a Beckman DU-70 spectrometer and a SPEX Fluorolog spectrofluorometer, respectively. All fluorescence spectra were corrected. The fluorescence quantum yields of the neutral and cationic forms of 8-carbolines were measured using several compounds as fluorescence standards: quinine bisulfate (4 in NH2S04 = 0.546),1°anthracene (4 in ethanol = 0.27)," methyl 1-pyrenoate (4 = 0.827 in methylcyclohexane),l* and methyl 1-naphthoate (4 = 0.4 in methylcyclohexane).12 Fluorescence decays were obtained using the time-correlated singlsphoton-counting technique as previously describtd.13J4The excitation wavelength was 337 nm or 356 nm. Alternate measurements ( lo3 counts at the maximum per cycle) of the pulse profile and the sample emission were done until 3 X lo4 counts at the maximum were reached. The photomultiplier wavelength shift was 1 ps/nm in the wavelength region of interest. The fluorescence decays were deconvoluted using a V A X 2000 computer using the modulation functions method with shift correction." Calculations of the intermediate neglect of differential overlap type (INDO) were performed with the lNDO/S-CI model.16 The configuration interaction (CI) consisted of 196 selected single excited canfigurations. The twcwxnter electron repulsion integrals for both INDO/S and PPP were estimated using the MatagaNishimoto approximation. Calculations in the r-electron-only approximation followed that of Pariser et al. (PPP),I7 which included a singly excited CI calculation. Parameters used in the PPP calculations of the various molecules are shown in Table I. The h and k are the usual Hiickel parameters, Z is the ionization potential, y is the one-center repulsion integral, and 8 s is ~the~ core integral. Molecular coordinates were commonly obtained from minimum energy geometries determined by a PC model molecular modeling software program (Serena Software, Bloomington, IN) employing an MMX force field including r system routines.
-
0022-365419212096-10290303.00/0 0 1992 American Chemical Society
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10291
&Carboline Photosensitizers TABLE I: Comparison of Experimental (Methanol) aod Theoretical Data theory CPd exp/nma INDO (0" PPP (fy Hari 331, 317b(1.0) 321 (0.06)c 324 (0.19)d
292 (0.22) 287 (0.01;n,r*) 269 (0.06) 227 (0.39) 342,329' (1 .O) 320 (0.08)' 287 (3.4) 308 (0.01;n,r*) 248, 238 287 (0.15) 233 (8.0) 265 (0.01) 244 (1.52) 234 (0.12) 225 (0.33) 214 (0.10) 343, 331* (1.0) 318 (0.06)' 309 (0.01;n,r*) 288 (3.5) 250, 238 (shs) 286 (0.18) 264 (0.01) 232 (7.6) -21 1 241 (1.51) 233 (0.19) 223 (0.35) 377 (1) 379 (0.16)' 334 (0.41) 303 (3.5) 250 (5.8) 266 (0.40) 200-225 260 (0.44) 240 (0.28) 226 (0.12)
279 (0.20) 259 (0.12) 217 (0.28) 202 (0.32) 326 (0.23)' 268 (0.03) 257 (0.07) 240 (1.47) 223 (0.17) 216 (0.38) 202 (0.23)
298 (3.2) 260 (sh) 240 (7.8)
Hara
Norh
Norh-Ht
326 (0.23)' 268 (0.02) 257 (0.06) 240 (1.47) 223 (0.17) 216 (0.38) 202 (0.23)
TABLE 11: Fluorescence m h r m Yields ( 4 ~ and ) Lifetimes ( T F ) of Harmine,Harmane, Norhamaw, and Harmaline at 20 OCQ 4F r/ns ro/ns kF/1O8S-' &,,/Io8 s-I Benzeneb 0.30 3.2 1 1 0.93 2.2 Norh Hara 0.33 3.4 10 0.96 2.0 4.6 10 0.99 1.2 Hari 0.46 0.06 2.3 1.8 X lo2 harmaline 0.23 X lo-' 4.2 ded rlns Methanole 0.34 2.8 Norh Hara 0.51 4.6 Hari 0.53 3.5 "The radiative (kF)and the radiationless (knJ rate constants are also shown. Data are for monitoring wavelengths 350-370 nm. Data are for monitoring wavelengths 350-370 nm. Total fluorescence quantum yield-cationic and zwitterionic components are not subtracted. TABLE III Fluorescence Decay Time8 (7,) and Reexponential Factors (a,) rt Four Emission Wavelengths in Methanol at 20 O C &,,,/nm q/ns r2/ns r3/ns a, a2 a, Norharmane 365 2.8 absent absent 1.00 0.00 0.00 absent 0.95 0.05 0.00 410 2.8 23
460 500
"All transitions of INDO and PPP are r .-,r* u n l w otherwise noted. This is also true for experiment. Relative intensities in parentheses based on OD (or e) at maxima for experimental data. benzene, for the first transition; 337 nm in methanol and 333 nm in acetonitrile and dioxane. The band of 317 nm is nearly the same intensity as that at 331 nm, although subtraction of the adjacent strong band would favor 331 nm as the maximum. Relative intensities given in parenthem based only on OD or e at maxima for this and all other compounds. MMX used for coordinates. Also pseudo-harmine was calculated where OCH, is OH and CH, is H. In this case, the comparative data are quite similar. dParameters are hc = 0,ho = 0.5, h~ = 0.5 (pyridine), hNH = 1.2;IC = 11.20,y c 11.10;Io 27.20,yo 14.60,IN 17.10,YN * 12.20;IN, 23.10,YNH 14.00,@ 2.4 for all Coupled atoms; kCNH 0.9,kcc = 1.0,&C.N = 1.0,k a = 0.6. 'In benzene for the first transition; red shifted -3-6 nm in methanol. Bands very close to the same intensity but 342 nm appears to be maximum. Relative intensities given in parentheses based on OD at maxima (or e). fMMX coordinates. #Pseudo-harmine (CH, is H)with MMX coordinates using the same parameters as in d above. *In benzene for the first transition, red shifted -6-7 nm in methanol. Bands have nearly same intensity; 343 nm appears to be maximum. 'MMX coordinates. PPP calculations use the same parameters as in d above. JMMX coordinates; all transitions of the r r* type.
-
The absorption spectra of the compounds in a nonpolar (benzene) and neutral polar protic solvent (methanol) are shown in Figure 1. In methanol, note particularly for harmaline that a new weak absorption occurs in the long-wavelength region, -360 and 390 nm, respectively. This phenomenon does not occur in nonpolar solvents (cyclohexane, benzene, dioxane) or in polar aprotic solvents such as acetonitrile. However, the longer wavelength absorption is strongly enhanced in the presence of water (as in a mixtureof dioxane and water, 3:2) or in the presence of an acid, as in acidified methanol. In the latter protic solvents or mixtures, other &carbolines also show the shoulder band. Figure 2 shows the absorption spectra of all the compounds in methanol and HCL-acidifled methanol. It can be s e n that additional absorption a a m at considerably longer wavelengths for the cationic species (proton on pyridine nitrogen) compared to that found in aprotic solvents (except for harmaline). Fluorescence of the compounds in benzene and methanol is given in Figure 3 with excitation at 337 nm (neutral-form absorption). Nonetheless, note the significant differences in the fluorescence spectra betwan benzene and the protic solvent
2.8 2.6
23 24
&m/nm
r,/ns
350 400 450 500
4.6 4.7 4.9 4.2
X,,/nm
r,/ns
350 400 450 500
3.6 3.6 3.2 3.3
5.4 5.7
-0.38 -0.91
r2/ns Harmane absent
a1
r3/ns Harmine absent absent 6.7 absent
1.00 0.48 4.81 -0.91
28 28
0.44 0.84 a2
1.00 0.96 -0.02 -0.90 aI a2
23 22 23
r2/ns
6.5 7.5
0.56 0.16
0.00 0.04 1.00 1.00 a,
0.00
0.00
0.52 0.92 0.41
0.00 0.08 0.59
methanol. In methanol, new band shoulders or a whole new band (harmaline) appears compared to benzene. Also in benzene, the entire nature of the fluorescence spectra is independent of excitation wavelength. In methanol, for harmaline only, excitation beyond the normal absorption in the 365-375-nm region results in emission at -490 nm, which is highly dominant. The emission shoulder at the longer wavelength of -415 nm (for harmaline at -490 nm) is similar in all respectf to that of the cationic species; see the following discussion. Table I1 presents data on quantum yields (+F), lifetimes (7F)r and rate constants in benzene and in methanol. The fluorescence spectra of the cationic forms, compared to the neutral forms, are shown in Figure 4. It can be seen that only one type of emission occurs with a maximum at -415 nm for Norh, Hara, and Hari, which is significantly red shifted from that of the neutral form at -350 nm. Also, this maximum correspondsto that component around 4 15 nm of the composite emission spectrum of the neutral species of these three compounds in methanol. Note that in the case of the harmaline cation, only one emission is observed at 490 nm, which corresponds to the -490-nm emission seen in methanol. The fluorescence decay3 of all of the compounds in benzene are single exponentials irrespective of the emission wavelength monitored. The same is observed in other nonprotic solvents (dioxane, cyclohexane), which means that only the neutral form emits in these solvents independent of the exciting wavelength used. On the other hand, the fluorescence decays of the neutral compounds Norh, Hara, and Hari monitored in methanol, Figure 5, strongly depend on the emission wavelength monitored with excitation at 337 nm in the neutral absorption region (or elsewhere). At &, = 350-370 nm (neutral-form emission), the decays are single exponentialsfor all compounds, Tables I1 and 111, but they
-
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
Dias et al.
Figure 1. Absorption spectra of norharmane (I), harmane (II), harmine (111), and harmaline (IV) in benzene (-) and methanol (---), TABLE Iv: FI~OWSWM% Ufetba ( T ) , FIuorcsceac~QIuntum Yields (b,), and RuUative (kF)rad Radiatiodtas (k,) Rate Conrrtrnb of Humine, Humaoe, Norhunrme, and H.nualine Catioar in Acidic (HCI) Methanol at 20 O C Norh Hara Hari harmaline
A,, r/ns -430 24.4 21.8 -425 ~ 4 1 5 6.8 8.0 -470
bF 0.79 0.96 0.62 0.86
io
31 23 11 9
kf/108 s-l 0.32 0.44
0.91 1.08
k,,/108 s-I 0.08 0.02 0.56 0.18
are double or triple exponentials at longer wavelengths, Table 111 and Figure 5. For example, in the case of harmine, the decay at X, = 400 nm can only be fittcdwith a sum of twoexponcntials, Table 111. One decay time (3.5 ns) is associated with the neutral form 71, and the second one, 72 (6.7 ns), is identical with the lifetime determined for the cation in acidic methanol or water, Table IV. At longer emission wavelengths (A = 450-500 nm), three component lifetimes arc required to fit the decay, with the , 28 ns, Table 111. The remaining two lifetimes, longest, T ~being 71 and 72, arc identical in magnitude to those observed at shorter wavelength, Table 111,but now the 3.5-ns component appears as a rise time (Le., negative pretxponential). For more concerning the nature of the species associated with the 28-11s lifetime, the zwitterion, see the Discussion section. In the case of harmane in methanol, Tables I1 and 111,the decay of 350 nm is single exponential ( T ~= 4.6 ns). At the remaining emission wavelengths, a sum of two exponents (4.6 and -23 ns), is sufficient to fit the data. Note that the -23-ns lifetime (72) closely corresponds to the lifetime of the emission of the cation in acidic methanol, -22 ns (or water) Table IV. Again, at the longest emission wavelength (500 nm), the neutral-form c o m p nent (4.6 ns) appears as a rise time and the sum of the preexponential factors approaches zero. Note that one component present in harmine and norharmane (see below) appears to be absent in harmane (but see later discussion).
Norharmane behaves like harmane with respect to the number of exponentials at -400 nm and beyond 450 nm, Table 111. The decay time, 72, of 23-24 ns corresponds to the cation decay time of -24 ns as found in acidic methanol, Tables IV. This lifetime, T ~is, essentially the same as that found for the same component of harmane, but not for harmine (72 7 ns). The lifetime of the long-wavelength component 73, 500 nm, is now -5.5 118. This is assigned to the zwitterion species, see Discussion section, in which the lifetime is much longer in harmine, -28 ns. Again, as in the other c a m , the decay at 365 nm is single exponential. The results of the calculations are given in Table I regarding electronic transitions and their intensities. In addition, we have determined that the lowest energy singlet transition of harmaline is of the n r* type, whereas for the other three molecules, it is of the r r* type. Another interesting and important feature concerns the charge densities in the ground and excited states. The case of norharmant is typical of all of the others. Consider the following diagram for reference.
-
--
:&& 4
y1:
10
H
We will be particularly concerned about changes in the N,,(py) and N13(NH)charge densities upon electronic excitation using the PPP approximation.” In the ground state, the total electron charge density on Nll(py) is 1.49, while for NI3(NH)it is 1.66. In the first excited singlet state, these become 1.59 and 1.51, respectively. Thus,there is a significant inmcrpe in electron charge density (0.10) on the pyridine Nll(py), whik there is a sisnificant decreuse in charge density (0.15) on the pyrrole N13(NH) upon excitation to S1. The carbon atoms also undergo changes in electron charge density, of course, with atoms C3 1 0.06, C4 I 0.10, CB20.10, and CI2< 0.08 being the largest. There is also
The Journal of Physical Chemistry, Vol. 96, No.25, 1992 10293
fl-carboline Photosensitizers
111
\
\ \ \
200
250
3 00
\
350
I 400
4
A (nm) Figwe 2. Absorption spectra of norharmane (I), harmane (11), harmine (111), and harmaline (IV) in methanol (-) and acidic (HCL)methanol (---).
a corresponding increase in the dipole moment from a calculated 3.5 D (ground state) to 5.7 D.
Discuseion Norh and Hara are very similar in their spectra. The calculated results are also similar for them, including the n a* transition, Table I. In the case of Hari, the first transition is blue shifted from the other two cases, but this is not predicted by theoryexcept possibly for the PPP calculation, Table I. The first two major transitions of Hari are clearly more jammed together than for the other two, Figure 1. The spectrum of harmaline is significantly different than all others, Figure 1, as might be expected since the pyridine ring has been partially hydrogenated. In the a* calculations, recall from Table I that for Hari, the n transition was the third transition and some 3600 cm-I higher in energy than the lowest transition, which is a a*. In the case of Hara and Norh, the dif€mnceis considerably leap at 1100 cm-', the n a * is the second transition, and a a * is lowest as for the other two cam. In the case of harmaline, the lowest transition is calculated to be n a * vs a a * for the others. Experimentally based on the absorption spbctra and 70 in benzene (from 4Fand sh in Table 11), it appears that indeed the lowest excited singlet state is r,a* for Hari, Hara, and Norh. However, note that the rofor harmaline is 10 times longer than for the other three molecules. Thus, although the n r* transition cannot be observed directly, the rogivca strong indication that an '(n,a*) state is indeed lowest. This statborder change for harmaline compared to the other three molecules must be because of the reduced conjugation, nsulting in increased energy of the lowest (a,**) state compared to the '(n,a*) state. In an acidified solvent, a uniquely absorbing ground-state cationic species is present in all caw, Figures 1 and 4. Again, theory suaxssfully predicts the substantial difference in the absorption of the cation compared to the free base, Table I. The
-
-
- -
-
-
-
SCHEME I N-C
N*
I
-
kZ
ki
C*
1/q*
Z*-Z-N
relative intensities are also well predicted (in some cases an observed transition is considered to correspond to two close lying predicted ones, as the 250-nm observed one corresponding to the 266- and 255-nm calculated ones in Norh-H+, Table I). Because of the significant complexity of the kinetic data as a function of monitoring wavelength in methanol, it is obvious that with the excitation of the neutral form, new tautomers are created in and during the lifetime of the lowest excited singlet state. Also, for harmaline, the cation tautomer can cocxist in the ground state in methanol. For acidic solutions, solely a cation emission exists for all so that no similar problems exist for the acidic solutions/cation. Here we shall consider harmine and norharmane decays in methanol. More detailed considerations of the kinetics of the excited state of harmine and norharmane in water as a function of pH will appear later elsewhere. Since the excited-state tautomerization complexity affects the nature and number of the emissions/tautomers, we need to first consider the deductions discernible from the kinetic data. The triple-exponential fluorescence decay at long wavelength (450-500 nm) for harmine and norharmine in methanol can be rationalized as a complex system containing three species which are kinetically coupled in the excited state, Scheme I, where N, C, and Z represent the neutral, cationic, and zwitterionic (proton
10294 The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
Dias et al.
I 1
1
I
\
I
I
I I I
.. - -.
I I I
-- - ----
I I
I
- 5
I
A(nm)
1
I
,'
I/
'\.
IV t
c
v)
t z
c
..._ 4 00
300
500
700
600
AhnI Figure 3. Fluorescence emission spectra of norharmane (I), harmane (II), harmine (111). and harmaline (IV) in benzene (-) and methanol (---).
on pyridine nitrogen, proton of pyrrole nitrogen) forms, r e a p tively, and * indicates the lowest excited singlet state of *,** character. Furthermore, in Scheme I, a is the fraction of light T ~ * and , T ~ are * the absorbed by the neutral form, TP, fluorescence lifetimes of the neutral, cationic, and zwitterionic forms, respectively, and k, and k2 are the pseudo-first-order rate constants (k = klMeOH]). The approach requires the solution of a unique set of differential equations which ultimately give some preexponential terms whose magnitude and sign permit interpretation of complex d w y differential equations d[N*]/dt = -(1/7N* kl + k2)[N*]
+
d[C*]/dt
k,[N*]
d[Z*]/dt
k,[N*]
- l/Tc*[C*] - 1/7z*[z*]
and the solutions are,with a = 1 (selective excitation of the neutral form), ZN*(t)
uN(x)e-f/rl
(1)
Ic*(t) = -uC(X)e-'/'l
+ uc(A)e-f/rz
(2)
Iz*(t) = -uZ(X)e-'ITl
+ aZ(k)e-'/'3
(3)
with 1/71
= 1/7N*
-
+ kl + kz
1/72 = 1/7c* 1/ 7 3 1/7z*
(4) (5)
(6)
At 350 nm, only the neutral form emits, and therefore, the experimental decay is given by eq 1, monoexponential, as is ob served, Table 111.
At 400 nm, both the neutral N* and cationic C* (produced from N*) forms emit; Le., 14(Jr)(t)= [ZN*(t) + Ic*(t)] = aN(400)e-'lrl (-e+Z) = [aN(400) - uc(400)]e-'/rl
+ ac(400) x + ~ ~ ( 4 0 e0+Z)
(7)
giving biexponential behavior, as is observed. The neutral-form emission is dominant at 400 nm, meaning aN(400) 2 ac(400), and therefore, both precxponentials are positive, Table 111. At 450 nm, there is now a small contribution of Z* emission (produced from N*), and we have
14&
= [aN(450) - ~ ~ ( 4 5 -0 uz(450)]rf/'l )
+
aC~450)e-'/'2+ aZ(450)e-'/'3 (8)
giving triexponential behavior, as is observed in Table 111,but now ~ ~ ( 4 5I 0 4) 4 5 0 ) , leading to a negative pnexponential term for uN. Finally, at 500 nm, the neutral-form emission is very small, and there is a very significant contribution from Z* (and some from C*) and the decay approaches zso0(t) = - [ a c ( 5 ~ )
-
+ ~~(5OO)le-'/~l +
ac(500)e-f/rz + ~~(5OO)e-~/~3 (9)
In the case of harmane in methanol, only two exponentials arc observed,Table 111,because the assigned zwitterion lifetime (e.g., T ~SEE * T~ 19 ns in 3-H@-7dioxaae at pH 12.5) is nearly qual to the cation lifetime ( T ~ = * T~ = 21.8 ns in methanol + HCL, Table IV). Under these conditions, the dcconvolution program is not able to split apart these two componcnta and the total decay appears to have only two components vs thrce components as in Norh and Hari. Even if N is excited in methanol for any of the @-carbolines, emission from C*can be seen so that C* can obviously arise from N*; see Scheme I. Furthemore, it is important to note that there are no back reactions from C* to N*,since the decays at 350 nm
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992 10295
&Carboline Photosensitizers
*
c, m
t.
c
z
300
II
400
500
600
700
I",
I I I I
I
\ I
I
' '
\
111
Figure 4. Fluorescence emission spectra of norharmane (I), harmane (11), harmine (III), and harmaline (IV)in methanol (-) and acidic (HCL) methanol (- -).
-
(N*) are single exponential. Finally, given the fact that the C* is produced by proton extraction from methanol, the pK,* of the neutral must be greater than 16 in the excited state. This value is consistent with the fact that the pyridine N undergoes a large increase in electron charge density in S1compared to the ground state. Recall that for harmine in methanol, for example, particularly at monitoring wavelengths 1 450 nm, three component lifetimes exist, Table 111. Two of these correspond to the neutral species 7 , (-3.5 ns) and the cationic species 72 (-6.8 ns) observed at shorter wavelengths, Table 111. However, at the wavelengths 2 450 nm (450-500 nm), the 3.5-ns component, N*, appears as a rise rime; that is, there is a negative preexponential, Table 111. This strongly suggests that the 73(-28-1s) component is associated with a s p i s formed from and during the lifetime of P. Moreover, the observation that the sum of the prcexponential factors approaches zero at -500 nm strongly indicates that the species associated with the -28-ns component is formed exclusiuely in the excited state (N*). Furthermore, recall that the the N by) increaxs in charge density calculations show that in SI, (stronger base) while the pyrrole N (NH) decreases in charge density (stronger acid). Therefore, formation of a zwitterion in S1 is probable and totally consistent with the kinetic results. Finally, 0thers~9~ have assigned emissions in the -490-nm region of harmane and harmine in water at high pH as due to the zwitterion basad on model studies of N-methyltetrafluoroboratcs. In our case, there in no back reactionfrom Z* to P, as there was none from C to P. The emission spectra in methanol are unique as one might expect basad on the above considerations. For the neutral compounds in a neutral protic solvent, as methanol, even with excitation in the neutral species absorption band region, there are multiple emissions/forms (except apparently for harmaline, where
only two emissions occur but one strongly dominates). This can be seen from the nature of the fluorescence spectra, Figure 3, which can be rationalized utilizing eqs 1-9. However, it is apparent that in a nonpolar solvent, only one form, the neutral one (N*), emits, including harmaline. The assignment of the band shoulders in the 415-nm region for Hari, Hara, and Norh in methanol is fortunately relatively straightforward based on parallelism to the emission from the cation, Figures 3 and 4. In the case of harmaline, a similar consideration is valid, but in addition here, the cationielike emission strongly dominates the neutral one, Figure 3. The emission in methanol in the -480-nm region of Hari, Hara, and Norh could be assigned as that from the zwitterion (via N*). The zwitterionic species Z* apparently is not present for harmaline and the -490-nm emission is from C (via N*). Others5+*believed that the emitting tautomer from methanol was only the neutral form and thought that some of the shoulders were only vibrational components of the spectrum of this species. Nonetheless, these same investigators did observe several emissions and tautomers as a function of pH (in water), which included neutral, cation, zwitterion, and anion. As noted earlier, the calculations regarding charge densities were critical in aiding in the prediction that a zwitterion and cation could exist, in the excited state upon excitation of the neutral. Moreover, the careful analysis of the fluorescence regarding the magnitude of preexponential factors and their sign was powerful regarding the determination of the existence of multiple species. Furthermore, the latter of these two considerationspermitted the determination that C* and Z* do originate from N*. Again, the fact that the emission lifetime of N* is solely monoexponential indicates that there is no back reaction from C* or Z* to N*. Note that the cation lifetime of Hari is much shorter than for Hara and Norh, Table IV, and that the zwitterion 73lifetime of
102%
The Journal of Physical Chemistry, Vol. 96, No. 25, 1992
channel
channel
chonnel
chenntll
Figure 5. Fluorescence decays of harmine in methanol, after excitation of the neutral form (hex= 337 nm), collected at 350,400,450, and 500 nm.
Dias et al. Finally, it is worthwhile to comment on the aspects dealing with the use of these compounds as photosensitizers. It is clear for the neutral base form that only a truly aprotic environment will provide a single excited-statetautomer (called N* in text) with irradiation (independent of the wavelength used). In such a case, any proposition regarding singlet oxygen and/or superoxide production, or electron or H atom transfer, can be truly related to the neutral/base tautomer and its excited state. However, in a protic environment, especially water, even though the principal absorbing tautomer may be the neutral/base form, multiple excited-state tautomers exist and the principal emitting tautomer(s) can be the cation or, the zwitterion at sufficiently high pH, >12 (also independent of the exciting wavelength used). Thus, the dominant tautomer existing in the excited state could be the cation (or zwitterion), even though the neutral/base species was the absorbing and first produced tautomer. At pHs of signifkantx.in biological systems, led us say pH 6-8, both the neutral and cation tautomers will be present in the ground state. Nonetheless, the principal existing and emitting excited-state tautomer will be the cation tautomer (also independent of the excitation wavelength). The facts here and immediately above engender severe complications in interpreting the nature and mechanism of photosensitization in a biosystems involving these or similar molecules. Acknowledgment. A.L.M. thanks the Alexander von Humboldt Foundation, Drs. K. Zachariasse and R. Busse for their support for the construction of the single-photon-counting equipment, and G.Striker for making his deconvolution programs available. The Instituto Nacional de Investigacao Cientifica is acknowledged for financial support. Registry NO. 1, 244-63-3; 2, 486-84-0; 3, 442-51-3; 4, 304-21-2; benzene, 71-43-2; methanol, 67-56-1.
Norh is much shorter than for Hari and Hara, Table 111. Also, the lifetime of the cation of harmaline is similar to that of Hari. Others9 have measured QF and T~ data of the cations of Hari, Hara, and Norh and harmalines in ethanol, and all are clearly of smaller magnitude than ours (for example, for harmane T F = 8.5 ns and QF = 0.67 vs our values of 21.8 ns and 0.96, respectively). Lifetime data also exist in acidic water solutions for Norh, T F = 22.0 ns7 and 22.03 I M , ~which are very close to our 24.4 ns. Moreover, for harmane and harmine cations, QFof 0.89 and 0.51, respectively, have been determineds#*in H 2 0 solution, which, although somewhat less than ours in methanol, are similar. In the case of the zwitterion, other data7 give a lifetime of 1.6 ns for Norh in basic water solution (vs our value of 5.4-5.7 ns in methanol). There are very little data on the lifetimes of the zwitterions in general and none in methanol. In the case of the fluorescence from the neutral species, note that our QF and lifetimes are quite similar in benzene and methanol, Table I1 (the QF seem a little greater in benzene). Others have QF values in the 0.3 area or in the 0.16 area1*in ethanol. particularly those in the 0.16 area1*are sign5cantly lower than ours, Table 11. Lifetimes previously determined range from 3.5 for norharmane?J8 3.1-3.8 for harmane,9J8 and 3.0 for harmine.g Our values are comparable for norharmane (3.2 and 2.8), comparable for harmane in methanol (longer in benzene), and longer for harmine, Table 11.
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