photokinetic aspects of specific hydrogen bonding in all-trans retinal at room temperature

Photochem. Photohiol. Vol. 36. pp. 455 to 461, 1982 Printed in Great Britain. All rights reserved

003 1-8655/82/100455-07$03.00/0 Copyright 0 1982 Pergamon Press Ltd

PHOTOKINETIC ASPECTS OF SPECIFIC HYDROGEN

ROOM TEMPERATURE BONDING IN ALL-TRANS RETINAL AT

P. K. DAS* and G. L. HUG Radiation Laboratory, University of Notre Dame, Notre Dame, I N 46556, USA

(Receiced 24 February 1982; accepted 24 May 1982)

Abstract-Room-temperature hydrogen-bonding of all-trans retinal (ATR) with 1,1,1,3,3,3-hexaAuoro-2- propanol (HFIP) in cyclohexane results in fluorescence enhancement and triplet yield quenching; these effects, as well as the associated absorption spectral changes in both ground and triplet states and the kinetics of H-bonding in the triplet state, have been studied by steady-state absorption-emission and laser flash-photolytic transient measurements. The fluorescence enhancement is predominantly controlled by the H-bonding in the ground state (static interaction) and gives a value of 440 M - ' for the corresponding equilibrium constant which is very similar to the value (420 M - ' ) obtained from the analysis of absorption spectral data as a function of [HFIP]. The quantum yield of triplet occupation (q5T) of the H-bonded complex, ATR-HFIP, in cyclohexane is non-negligible and is about one-third of 4T of free ATR. The kinetic data of H-bonding equilibration in the triplet state, observable on a nanosecond time scale, indicate that the triplet ATR is a stronger base than the ground state as far as H-bonding with HFIP is concerned.

INTRODUCTION

Hydrogen bonding in molecules containing chromo- phores that are perturbed by interaction through a proton are often useful in obtaining insight into phenomena related with both ground and excited states (Mataga and Kubota, 1970; Joesten and Schaad, 1974). All-trans retinal (ATR, Scheme I), a long-chain polyenal of considerable biological interest, has been the subject of a number of studies where hydrogen bonding manifested itself in the form of fluorescence enhancement as well as triplet yield lowering; both these effects have been attributed to state order changes involving the low-lying 1,3(n, n*) and 's3(n, n*) states. The fluorescence enhancement has been observed by using specific hydrogen bonding agents such as water and phenol in a nonpolar glass at 7 7 K (Takemura et a/ . , 1976, 1978) as well as by comparison of emission behavior in nonpolar vs. polar-protic solvents at both room and low tempera- tures (Veyret et al., 1978; Becker et a/., 1971). The decrease in triplet yield has been studied by flash- photolytic methods at room temperature, both in terms of solvent effect, e.g. methylcyclohexane vs. methanol (Fisher and Weiss, 1974) and by using water and alcohols as quenchers (Dawson and Abrahamson,

Scheme 1. All-trans retinal (ATR).

I n this paper we present the results of a quantitative study concerning the effect of 1,1,1,3.3,3-hexa-

*To whom correspondence should be addressed.

fluoro-2-propanol (HFIP), a strong hydrogen-bonder, on the room-temperature photophysical behavior of all-trans retinal (Scheme 1). 1,1,1,3,3,3-Hexafluoro-2- propanol has been used both as a specific hydrogen- bonding agent in cyclohexane and as a neat solvent. In particular, we have attempted to sort out the static and dynamic aspects of the interaction, related with the ground and the singlet excited states, respectively. Furthermore, the kinetics of H-bond formation in the triplet state has been investigated on a ns time scale with results that show that the polyenal is a stronger base in the triplet state than in the ground state.

MATERIALS AND METHODS

All-trans retinal (Sigma) was chromatographed on a sil- ica gel column using petroleum ether + 10% diethylether as the eluent and then crystallized from n-hexane. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP, Aldrich) was used as received. Cyclohexane (MC/B, spectral grade) was stored over 3A molecular sieves before use.

The absorption spectra were recorded on a Cary 219 spectrophotometer with 1 nm band pass. For fluorescence spectral measurements, a photon-counting spectrofluori- meter from SLM corporation was used. The description of this set-up is available in a separate publication (Chatto- padhyay et a/., 1982). The excitation and the observation of emission were carried out in 1 x 1 cm square cells (quartz) with a right-angle geometry between them.

The laser flash-photolytic observation of triplet-related phenomena was carried out using pulses (337.1 nm, 8 ns, 2-3 mJ) from a Molectron UV-400 nitrogen laser system. The essential features of the computer-controlled kinetic spectrophotometer with ns response are described else- where (Das et al., 1979; Das and Bhattacharyya, 1981). The excitation by laser pulses was carried out in 1 x 0.2cm quartz cells with optically flat surfaces; the monitoring light from a pulsed 450 W Xe lamp was directed along the 0.2 cm path length at - 15' to the direction of the exciting laser pulses.

455

456 P. K. DAS and G. L. Huci

I .o

0.8

0.6 0 0

0.4

0.2

O'O 350 400 450 500 550

WAVELENGTH, NM Figure 1. Absorption spectra of ATR (21.3pM) in cyclohexane in a 1 crn cell at [HFIP] = 0.0 (I) , 0.458 (2). 0.687 (3), 1.60 (4). 3.20 (S), 5.03 (6), 7.32 (7), 10.1 (8) and 27.5 (9) mM. The inset shows Benesi-Hildebrand plots based on absorption spectral data at 460 (A), 454 (B) and 450 (C) nm.

RESULTS

Absorption spectral changes

Figure-1 shows the absorption spectra of ATR (21.3 pM) in cyclohexane in the presence of varying concentrations of HFIP in the range 0--28mM. It is seen that the spectra intersect each other at an isosbestic point at 386nm for [HFIP] 5 IOmM. We attribute these spectral changes to the formation of a 1 : 1 hydrogen-bonded complex between ATR and HFIP. Since the absorption in the spectral region 450-490nm is totally due to the H-bonded complex, it is possible to obtain the equilibrium constant ( K J for H-bond formation in the ground state (see Eqs. 1 and 2, HFIP = H, ATR = A) through Benesi-Hilde- brand (1949) plots. The inset of Fig. 1 shows three of

A + H & A . . . H ( 1 )

K g ' = [A] [H]/[A.. . H]

these plots based on absorption spectral data at 450, 454 and 460nm, respectively. The linearity of these plots supports the 1 : 1 stoichiometry of the H-bonded complex. From the average of the intercept-to-slope ratios, a value of (4.2 & 0.4) x 10' M-' is obtained for K,.

It is noted that at [HFIP] > 10mM, the absorption spectra become more and more red-shifted with- out passing through the isosbestic point (Fig. 1). Also at these high H F I P concentrations, the Benesi-Hilde- brand plots deviate from linearity in a sublinear fash- ion (i.e. bend downwards). Clearly, a different species is formed at high [HFIP]; this could be a H-bonded complex of ATR where more than one molecule of HFIP participates, or it could be a protonated form of ATR having a structure close to

250 300 350 400 450 500 550

WAVELENGTH, NM Figure 2. Absorption spectra of ATR in cyclohexane (A) and HFIP (C) and of ATR-HFIP complex in cyclohexane

(B).

ATR-H' .0--CH(CF3),. Formation of multiple species between ATR and HFIP is also indicated by the emission behavior of this system in 3-methylpentane glass (77 K) where two different emission spectra at low ( t O . l mM) and relatively high (> 1 mM) [HFIP]. respectively, are observed with the maximum of thc latter emission being red-shifted by -2700cm-' relative to the former (P. K. Das and R. S. Becker. unpublished results).

Figure 2B shows the absorption spectrum of the ATR-HFIP complex in cyclohexane, calculated from the composite spectrum 5 (at [HFIP] = 3.2mM) of Fig. 1 on the basis of the assumption that the total absorption in the latter is the sum of the contri- butions from free ATR and ATR-HFIP complex, the equilibrium concentration of both being determined by K , (Eq. 2). Comparison of Fig. 2A with Fig. 2B shows that while the absorption spectra of ATR- HFIP complex and free ATR are very similar in shape and broadness, hydrogen bond formation results in a red-shift (ATa) of -910cm-' in the spectral maximum as well as a reduction in the absorption intensity (by -20%). Using the approximate relationship (Weller, 1957; Mataga and Kubota, 1970)

0.625 T

log K , = log K , + __ AV, (3)

we estimate that the hydrogen-bonding equilibrium constant ( K , ) in the Franck-Condon excited state ('B:) is higher than that ( K , ) in the ground state by about two orders of magnitude.

Figure 2C is the absorption spectrum of ATR in neat HFIP. Evidently, the spectrum is broader (ATl,2 = 6100cm-' in HFIP relative to 5400cm-' in cyclohexane) and very much red-shifted. Because of the large red-shift in the main absorption band. a shoulder becomes noticeable on its short-wavelength side in the spectral region 3 W 3 5 0 nm. This shoulder corresponds to the band-system I1 (Das and Becker, 1978) of retinal-related polyenes and appears as more intense band systems with well-defined maxima in the ci., isomers of retinal and in the lower homologues (Das and Becker, 1978: Honig er ul., 1980). As ob-

H-bonding in retinal 451

served in other polyene systems, the location of band- system I1 is affected very little by solvent or hydrogen bonding interactions.

Fluorescence spectral changes

At room temperature ATR fluoresces only very weakly. Previous studies have reported on the room- temperature emission of ATR, with excitation by laser pulses in various solvents (Veyret et nl., 1978), as well as under steady state irradiation in polar solvents using a lamp source (Becker et al., 1976). In our spectrofluorimetric set-up, we could observe ATR fluorescence in polar solvents such as methanol, 2,2,2-trifluoroethanol and HFIP, but not in cyclohexane. Figures 3B,B' show the fluorescence spectrum of ATR in neat HFIP. Unfortunately, the maximum (-650nm), as well as the major portion of the fluorescence spectrum, is at a region where the detector system has a relatively poor response.

Of a particular interest in the present study is the fact that in cyclohexane, fluorescence can be induced in ATR through H-bonding with HFIP. Figures 3A,A' show the emission spectrum (i,,, = 510 nm) of the ATR-HFIP complex in cyclohexane at room temperature. As expected, the intensity of this emission increases steadily with increasing [HFIP]. The monitoring of the enhanced emission of ATR-HFIP complex as a function of [HFIP] permits us to detect a dynamic contribution, if any, to the emission behavior from complex formation/dissociation in the lowest-energy singlet excited states. With excitation at the isosbestic point (386 nm) where the extinction CO-

efficients are the same for free and H-bonded ATR, the scheme is as follows (ATR = A , HFIP = H, [HFIP] = h)

r

WAVELENGTH, NM Figure 3. Room-temperature emission spectra of the ATR- HFIP complex in cyclohexane with total retinal concentration at 2 0 p M and [HFIP] at 15mM (A, uncorrected and A', corrected for detector response) with = 386 nm and of ATR, 2 0 p M in HFIP (B, uncorrected and B , corrected for detector response) with i,, = 430 nm. The peak at 435 nm for A and A is due to a Raman band of cyclohexane. The inset shows the plot of reciprocal of relative emission intensity (at 530nm) vs. [HFIPI-'. For all measurements, a bandpass of 16nm was used for both

emission and excitation.

bution from the singlet excited state phenomena to the fluorescence behavior.

The inset of Fig. 3 shows a plot of the reciprocal of relative fluorescence intensity due to the ATR-HFIP complex (monitored at 530nm) vs. the reciprocal of [HFIP]. Clearly, the plot is linear; this strongly sug- gests that the contribution of the excited state dynamic processes represented by Eqs. 4c and 4d is relatively small. More importantly, the intercept-to-slope ratio of the plot is found to be 4.4 x 1O2AK1; this value is well within the errors of the value obtained for K , from Benesi-Hildebrand plots of absorption spectral data (vide supra). This result establishes that

In the above scheme, the hydrogen-bonded complex, A ' . . H, is the only emitting species (because no fluorescence is detected under the conditions of our experiment in the absence of HFIP). Applying steady- state conditions for 'A* and 'A* ' . . H, we obtain for the observed quantum yield of fluorescence (dF,obs)

where q5F.H = k i / ( k i + k , , + kb) and & = kb/(k: + k i r + kb). The expression in the square brackets on the right-hand side of Eq. 5 is a measure of the contri-

the fluorescence enhancement is predominantly controlled by the hydrogen-bonding in the ground state (i.e. static interactions).

The conclusion arrived at in the previous para- graph is not surprising. The observed fluorescence lifetimes of H-bonded complexes of ATR with water or phenol (Takemura et al., 1978), as well as with HFIP (P. K. Das and R. S. Becker, unpublished results) in 3-methylpentane glass at 77 K, are found to be in the range 1-2x1s. At room temperature, the lifetimes are expected to be shorter. From the data available for various H-bonded complexes in the excited

458

-0.08-

P. K. DAS and G. L. H1.b

'

Q n 0 r]r] 445 NM 480NM

0 I00 200 300 400 0 100 200 300 400 TIME, NS TIME, NS 8-

WAVELENGTH, NM Figure 4. Triplet-triplet absorption spectra (difference) of 4TR 2 O p M . in cyclohexane (A. with direct excitation) and HFIP (C. anthracene-sensitized). and o f ATR-HFIP complex in cyclohexane [B, with direct excitation of 20 p M (total) solution of ATR in the presence of 1 2 m M HFIP]. The inset shows the plot based on Eq. 8b for determination

of 4T of the ATR- HFIP complex.

state, k , 5 lo 's- ' (Mataga and Kubota, 1970). This means that for ATR--HFlP complex qbb (Eq. 5 ) is small compared to unity. As far as the competition between k, , and k , (for free ATR) is concerned, a ps photophysical study (Hochstrasser et al., 1976) has shown that the excited state of ATR at room temperature can be extremely short-lived ( 1 3 4 ps). Thus. even with k , as high as 10" M - I s - (see Discussion). k,h is negligible in comparion to k , , (for h in the range @ 20 m M ) .

In neat. HFIP, direct laser excitation of ATR does not lead to triplet formation to any significant extent (& < However, the triplet can be generated in this medium by energy transfer. Figure 4C shows the triplet--triplet (T -T) absorption spectrum of ATR in HFIP obtained by sensitization with anthracene. I t is noted that the red-shift of the T-T absorption maxima (-4300cm-') of ATR on going from cyclohexane to HFIP is even larger than that observed for the ground state absorption maxima (-3400cm-I). An analogous observation is also made for the ATR. . .HFIP complex in cyclohexane, that is. relative to free ATR in cyclohexane, the T--T absorption maximum of the ATR. . ' H F I P complex is more red- shifted (by - 1400cm-') than the ground state absorption maximum (by -910 cm- I ) . Figures 4A and B show the T-T absorption spectra of free and H-bonded ATR in cyclohexane, the latter being obtained by direct excitation in the presence of 12mM HFIP. Based on the large value of the

later) in the triplet state, more than 907,) of ATR triplets are H-bonded at [HFIP] = 12mM.

Before we present our results concerning triplet yield decrease associated with specific hydrogen bonding, it is important to describe the kinetics of H-bond formation that is observable in the triplet state on a short time scale (ns). The insets of Fig. 5 show two typical kinetic traces obtained by monitoring transient absorbance changes at 445 and 480nm. respectively. in the presence of 0.025 M HFIP. At 445 nm (inset A, Fig. 5), a decay process, essentially complete within - 80 ns following the laser pulse. is observed. On the other hand at 480 nm (inset B. Fig. 5) , a growth process is seen to occur in the same time scale. Noting the facts that 445 nm is the wavelength where T-T extinction due to free ATR is higher than H-bonded ATR and that the situation is opposite at 480 nm, we attribute the transient processes to equilibration between the free ATR triplet and its H-bonded form following their formation in a non- equilibrium mixture through intersystem crossing from the singlet manifold. The non-equilibrium composition concerning free and H-bonded ATR triplet arises from a combination of three factors, namely, (i) K , is smaller than the hydrogen bonding equilibrium constant (Kr,T) in the triplet state (see later), (ii) the extinction coeficient of ground state absorption of frec ATR is higher than that of the H-bonded complex at the wavelength of excitation, 337.1 nm (see Figs. 2A.B). and (iii) the triplet yield (4,) of free ATR is about three times higher than that of the H-bonded complex (see below).

The H-bonding equilibration process, Eq. 6. in the triplet state following flash photolytic generation of a nonequilibriuni mixture of 'ATR* and 'ATR*. . 'H- FTP can be shown to follow a first-order kinetics with observed rate constant (k,,,,J given by Eq. 7a.b

(HFIP] , mM

Figure 5. Plot of rate constant (!iob,) of H-bonding equih- bration in the triplet state vs. rHFIP1, based on Eq. 7b. The insets show the decay ( A ) aid the growth (B) profile at

H-bonding equilibrium constant (> 800 M - see 445 and 480 nm. respectively. at 25 mM HFIP.

H-bonding in retinal 459

(ATR = A, HFIP = H. [HFIP] s h)

(6) 3A* + H &3A* . . . H i b T

AODZT - AODTT = kt~bbA~Tr/(kf,Th C3A*]0

- kb,T[3A*. ' . H],)exp( - k o b c t ) (7a)

(7b)

In the above relationships, 1 is the path length along the monitoring light, the subscript 0 designates triplet concentrations immediately after the laser pulse, AOD;' is the observed absorbance change due to combined T-T absorption of both free and H-bonded ATR at the end of the equilibration process, AODTT is the same at time t during the equilibration process, and AcTT is the difference in T-T extinction coefficients of free and H-bonded ATR at the monitoring wavelength. Given the conditions that the end-of-pulse composition of the two species is in favor of free ATR triplet (i.e. C3A*Io > t3A*. . .HI,) and that k,Th > kb.T, the sign of AcTT determines whether a growth or a decay kinetics would be observed.

Based on Eq. 7a, and using the decay profiles at 445nm, we have determined kabr at several concentrations of HFIP. It should be noted that at relatively low [HFIPJ (i.e. <3mM), the initial decay (or growth) process due to H-bonding nearly merges with the decay of the triplets themselves and hence reliable measurement of kob\ is not possible at low [HFIPJ. Figure 5 shows the plot of kobr against HFIP concentrations. From the slope and the intercept of the plot (Eq. 7b), we obtain a value of 2.5 x lo9 M - ' s - ' for kf,T and an estimate of 5 3 x 106s-' for kb,T. This means that the equilibrium constant for H-bond formation in the triplet state of ATR is greater than 8 0 0 M - ' . In spite of the approximate nature of this result, ATR appears to be a stronger base in the triplet excited state than in the ground state.

We have made a quantitative analysis of the depen- dence of triplet yield on the concentration of HFIP in cyclohexane. With [HFIP] in the range &-15mM, a systematic decrease in AODrT at 4 4 M 7 0 n m is observed with increasing [HFIP], but the decrease is not of the magnitude expected if the quantum yield of triplet occupation ($T) were negligible for the ATR- HFIP complex. At higher values of [HFIP], namely. >15mM, the observed triplet yield in terms of AODT' drops quite sharply, suggesting that at least one more species with very small 4;s is formed at high HFIP concentrations (one may recall that the absorption spectral data corroborate this). To analyse the data concerning the initially formed ATR-HFIP complex in the [HFIPJ range %15mM, we have made use of the AODT' data at 465nm, the latter being the isosbestic point for free and H-bonded ATR triplet. That 465nm is the wavelength where AcTT is practically zero is established by the criterion that neither growth nor decay kinetics due to H-bonding equilibration is observed at this wavelength on a

'oh = kb.T + k f , T h .

short time scale following the laser pulse. Thus monitoring AODTT at 465 nm obviates the difficulty arising from compositional change (free vs. H-bonded ATR triplet) following intersystem crossing. The following relationship can be easily derived for AODT' at 465 nm as a function of [HFIP]

AODZL = A r ( l - 10-Ar)-lAODTT (8a)

AOD:: = Const. ( 1 + K,h)-'

(8b) A, is the total absorbance in the photolysis cell due to the ground state absorption at 337.1 nm, eF and eH are the ground state extinction coefficients of free and H-bonded ATR. respectively. at 337.1 nm and, #T,F and $T,H are the corresponding triplet yields. The correction factor A T ( l - l O - " T ) ~ ' arises from the inner filter effect (Takemura et a/., 1978) due to the two absorbing species at the exciting wavelength (337.1 nm) and changes by less than 5% on going from [HFIP] = 0 to [HFIP] = 13 mhl, for a 3.3 x t 0 - 5 M solution of ATR in a photolysis cell with 2 m m pathlength. Equation 8b is derived with the assumption that dynamic hydrogen bonding phenomena in the singlet excited states are not important, as established by the fluorescence spectral behavior (Dine supra).

Equation 8b enables us to measure $T,H. The inset of Fig. 4 shows a plot of (1 + Kgh)AODzT, against h. Using the slope-to-intercept ratio (85 M - ') from this plot and the values of 4.2 x 10' M--' for K , and 0.75 for E H / E F at 337.1 nm, we obtain: +T,H/$T,F z 0.3. The literature value of 0.4-0.7 for $T,F of ATR in n-hexane or cyclohexane (Bensasson et al., 1973; Rosenfeld et a/., 1974; Fisher and Weiss, 1974; Azerad et a/., 1976; Bensasson et a/., 1975; Veyret et al., 1978) puts $T,H in the range 0.14.2.

Table t summarizes the spectral and photophysical data concerning free and H-bonded ATR.

DISCUSSION

In this study, we have examined a well-defined H-bonded complex of ATR with HFIP at relatively low concentrations of the alcohol in a nonpolar medium and have characterized its emission-absorption spectra and intersystem crossing behavior. The comparison of the spectral and kinetic properties of such a complex with those of free ATR is more mean- ingful than using data in hydrogen-bonding vs. non- hydrogen-bonding solvents. This is because in a hydrogen-bonding solvent, not only the environmental effect is superimposed on the specific hydrogen-bonding interaction but also there are complications from formation of multiple species. Specific hydrogen bonding with ATR in cyclohexane results in fluorescence enhancement, triplet yield lowering. and red- shifting of both ground state and triplet-triplet

460 P. K . D ~ s and G . L. Hcc;

Table I . Spectral and photopyhysical data of all-trrrn.s retinal and its H-bonded complex with 1.1.1.3.3,3-hexafluoro-2-propanol at room temperature

Max. T -T Absorption extinction Emisston absorption

max., coefficient max. tnax. T I

System Solvent (nm) ( 1 0 4 ~ b f - ' c t n - 1 ) (nm) (nm) ( i t s ) 4% ~- ~

ATR Cyclohexdne 372 4.8 530* 445 9.3 0.62t

ATR H F I P 425 4.3 650 5 50 74.2 < 10 .I

4.0 510 475 1 1 . 1 0.2 ATR-HFIP Cyclohexane 385 complex

~

*347 nm Laser stimulated (Veyret et ul.. 1978). tThe most recently reported value in n-hexane with 347 nm laser excitation (Veyret et ( I / . . 1978).

absorption spectra; these effects, however, are not as large as those observed on going from cylohexane to HFIP (as a solvent). In terms of the usual model of competing first order processes from the lowest singlet excited state. the three-fold decrease in intrinsic & on H-bond formation with ATR could be the result of a decrease in the intersystem crossing rate constant. k ; , , , and/or an increase in the rate of internal conversion, ki, (any change in the radiative rate constant. k,. is immaterial because fluorescence quantum yield is very small in both free and H-bonded ATR). While the decrease in kist is quite plausible in view of de- creased contribution of favorable processes such as ' ( n , n*) -9 3(x . n*) or '(x, x*) + % I , x*) (El-Sayed. 1962, 1963. 1964) to the intersystem crossing in the H-bonded complex, an increase in ki, cannot be ruled out because of the additional vibrational degrees of freedom that may contribute to the Franck-Condon factors involved in the internal conversion process in the H-bonded complex.

Our analysis of the fluorescence data in the presence of varying concentrations of HFIP leads to the conclusion that the forming or the breaking of the H-bond in the lowest excited singlet states is not fast enough to affect the fluorescence enhancement behavior. In other words, the magnitude of the fluorescence enhancement can be explained primarily as a conse- quence of the ground state complexation. A parallel study concerning the effect of hydrogen bonding on the photophysics of the immediate higher homologue of ATR (all-trans C,, aldehyde) has shown that k , is of the order of 10'' M - ' s - ' for C,, aldehyde with HFIP as the H-bonder (P. K. Das and G. L. Hug. to be publsihed). Providing that k , is of similar magnitude in ATR, our results suggest that the singlet life- time of free ATR has to be less than ca. 0.2 ns in order that the dynamic interaction can be negligible. It should. however, be noted that the lowest excited state of free ATR has been shown to be primarily of (n, n*) character (Takemura et a/., 1976, 1978) in con- trast to the (n, .*) character of the CZz aldehyde (Das and Becker. 1978) and hence the excited state affinity for protons may be significantly smaller for ATR than for Czz aldehyde. Results of semiempirical calcu- lations (all-valence-electron CNDO/S SCF-MO-CI) show that dipole moments of retinals are small in

' ( n , n*) states and large in (x, n*) states ('B,*, *A; or *A:), both being relative to ground states (Weimann er a/., 1975).

The data from the ns kinetics of H-bonding equilibration in the triplet excited state demonstrate that with respect to HFIP triplet ATR is a stronger base than its ground state. Increase in pK, values by 0.1-2.9 units has been observed for such heterocyclic bases as quinoline. its bromoderivatives, and acridine (Jackson and Porter, 1961; Aaron et al., 1979) on going from the ground to the triplet state. For these systems. the change in acid-base behavior is even more dramatic on going from the ground to the singlet state, e.g. the pK, value of singlet (S,) acridine is higher than that of the ground state by 5.1 units (Weller, 1957; Jackson and Porter, 1961). Analogous behavior has also been observed for n-butylamine Schiff base of all-trans retinal; this retinal-related polyene system has been shown to be a very strong base in its fluorescing state (pK, - 16.95) relative to the ground state (pK, = 6.95) (Schaffer et a/.. 1975).

Our conclusion regarding K , < Kr,T is at slight variance with the finding of Dawson and Abraham- son (1962) who obtained nearly identical values for both triplet and ground state equilibrium constants for H-bonding of ATR with methanol. Another point of note is that in the work of Dawson and Abraham- son (1962). & of the H-bonded complex of ATR with methanol in methylcyclohexane has been assumed to be negligible. That this assumption may not be sound is shown by our result that & of the ATR . . 'HFIP complex is quite high (-0.2) in spite of the fact that the H-bonding interaction is stronger in this complex than in the complex with methanol.

Acknowledyement -The research described herein was sup- ported by the Office of Basic Energy Sciences of the Department of Energy. This is Document No. NDRL-2319 from the Notre Dame Radiation Laboratory.

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photokinetic aspects of specific hydrogen bonding in all-trans retinal at room temperature

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