Photochemistry and Photobiology, 1997, 65(3): 536-542

The Excited States of Melatonin

Mary King and J. C. Scaiano* Department of Chemistry, University of Ottawa, Ottawa, ON, Canada

Received 30 October 1996; accepted 11 December 1996

ABSTRACT

Melatonin, a hormone that controls the circadian rhythm, has a strongly fluorescent singlet state. While it can sensitize the formation of singlet oxygen, the yield is limited by inefficient intersystem crossing. The triplet en- ergy is ca 72 kcal/mol, sufficient to transfer efficiently to a variety of acceptors, including biphenyl, which has been used as a tool for triplet characterization.

INTRODUCTION

Melatonin has been receiving a great deal of media attention (1). The synthesis of melatonin is known to occur in the pineal gland and the retina of most vertebrates. It has been claimed that melatonin possesses antioxidant properties sim- ilar to vitamin E, ascorbic acid and glutathione in the body (24) . While melatonin's antioxidant role remains to be sub- stantiated (and has been severely criticized) (3, it has been reported that this indoleamine reacts rapidly with hydroxyl (6) and alkoxyl radicals (7). Melatonin functions as a hor- mone whose circulating levels follow a circadian rhythm (8). The role of retinal melatonin, however, seems to be confined locally to functions within the retina. It is believed that ret- inal melatonin is responsible for photoreceptor disc shedding via a light-mediated process in which pineal melatonin plays little or no role (9,lO). Retinal melatonin also functions as a neuromodulator effecting the release of the neurotransmitter dopamine at a specific concentration of about M (1 1). Pigment migration, the movement of the rods and cones, as well as microtubule separation within the eye appear to be other local functions of retinally synthesized melatonin ( 10,12,13). Menaker suggests that the role of melatonin may have evolved to incorporate these light-mediated retinomoter tasks within the eye in addition to its original role as neu- romodulator (1 1). It is possible that retinal melatonin, if sub- stantially concentrated in relation to blood levels (14), may also assume a custodial, protective role in the retina by ac- tively participating in specific photochemical processes such as scavenging photoinduced radicals or quenching singlet oxygen (15).

By virtue of its capacity as the light receptor, the eye is

*To whom correspondence should be addressed at: Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, ON KIN 6N5, Canada. Fax: 613-562-5170; e-mail: tito@photo.chem.uottawa.ca.

0 1997 American Society for Photobiology 0031-8655/97 $5.00+0.M)

predisposed to many photochemical effects including pho- tooxidative processes. The photochemical and photooxida- tive processes of light absorbers within the eye such as rho- dopsin, retinal, melanin, cytochrome-c and lipofuscin have been well documented, but little is known about the photo- chemistry of melatonin. In this paper we explore the behav- ior of the excited states of melatonin in solution. We have investigated fluorescence and phosphorescence properties, including quantum yields, lifetimes and kinetics.

H 'N-COCH~

H Scheme 1

MATERIALS AND METHODS

Melatonin

Melatonin, biphenyl and 2-(4-biphenylyl)-5-phenyl- 1,3,4-oxadiazole (PBD)? were used as received from Aldrich (Milwaukee, WI), ben- zophenone (Aldrich) was recrystallized from ethanol, and 1,3-cy- clohexadiene and acetophenone were cold-trap distilled. Cyclohex- ane, benzene, methanol, ethanol and acetonitrile were Omnisolv grade and were used as received.

Phosphorescence and fluorescence (emission and excitation) spec- tra were recorded on a Perkin Elmer LS-50 spectrofluorometer. Phosphorescence was measured in a 4: 1 ethanolhethanol glass at 77 K. Fluorescence spectra and quantum yield measurements were performed on samples degassed with N,. Singlet oxygen generation was measured using a 308 nm excimer laser (described below) as the excitation source and a germanium diode was used to detect the infrared emission of lo2. The experimental set-up is the same as that employed in earlier work (16).

Kinetic measurements for the excited states of melatonin were conducted on a laser flash photolysis system that utilizes the 308 nm pulses from a Lumonics EX-530 excimer (-50-60 mJ, 6 ns in duration). The signals from the monochromator/photomultiplier sys- tem were captured by a Tektronix 2440 digitizer and transferred to a PowerMacintosh computer equipped with software developed in the LabVIEW 3.1 . I environment from National Instruments. Other aspects of the system are similar to those described earlier (17.18).

Fluorescence lifetimes were measured using the fourth harmonic (266 nm) of a YAG-pulsed picosecond continuum laser system with a Hamamatsu Streakscope detector controlled by a Macintosh com- puter. Static and flow-through cells were used to obtain spectral information.

TAbbreviafions: IRF, instrument response function; PBD, 2(4-bi- phenylyl)-5-phenyl- 1,3,4-oxadiazole.

538

Photochemistry and Photobiology, 1997, 65(3) 539

800

600 0

2 400 .- u3 c E .-

200

0 300 350 400 450 500

wavelength, nm

Figure 1. Phosphorescence spectrum (A) of melatonin in a 4: 1 eth- anolhnethanol glass at 77 K (excitation at 280 nm) and fluorescence in solution at room temperature in water (0) and methanol (m) (excitation 290 nm).

RESULTS AND DISCUSSION Absorption spectrum

Melatonin absorbs light only in the UV region with a max- imum absorption at -278 nm. Little or no shift in the ab- sorption maxima was apparent with any of the solvents used.

Fluorescence spectroscopy

The fluorescence spectrum of melatonin is strong in the near UV with a maximum at -340 nm, showing minimal overlap between the excitation and fluorescence spectrum. Although the absorption characteristics of melatonin appear to be un- affected by solvent, the fluorescence spectrum undergoes a bathochromic shift in water (Fig. 1). This Stokes shift ap- pears to be characteristic of other indoles ( 1 9) and is most likely due to the stabilizing effect of hydrogen bonding be- tween the indolic N-H and water.

The PBD in cyclohexane was used as a reference to mea- sure the fluorescence quantum yields for melatonin in vari- ous solvents. The PBD was selected because its absorption and fluorescence spectral characteristics are very similar to those of melatonin, and PBD has a fairly high fluorescence quantum yield (0.69) in cyclohexane (20).

Quantum yields were calculated using Eq. 1:

where @ is quantum yield, n is the refractive index of sol- vent, OD is absorbance, I is the integrated area under the fluorescence spectrum, M is melatonin and A is the reference PBD. The quantum yields obtained are given in Table I . Neutral density filters were used as required to bring the strong fluorescence into the instrumental range. The rela- tively low quantum yields of fluorescence in water may be due to intramolecular quenching caused by a charge transfer mechanism between the excited electron-rich indole ring and the electrophilic carbonyl group of the amide side chain, in a conformation specifically favored in an aqueous environ- ment. Such intramolecular processes have been proposed by Robbins et al. to describe similar decreases in fluorescence quantum yields of tryptophan derivatives that possess side

Table 1. in both protic and aprotic polar solvents and a nonpolar solvent

Fluorescence lifetimes and quantum yields for melatonin

Solvent % T, ns

Acetonitrile 0.60 6.1 Methanol 0.52 5.6 Water (buffer at pH 7.0) 0.20 3.6 Benzene 0.54 -* 1 : 1 Acetonitrilelwater - 5.4

*The 266 nm laser employed in these studies cannot be used with benzene as solvent.

chains including an electrophilic carbonyl group but no NH3' (19).

Fluorescence lifetimes

The fluorescence lifetimes of melatonin in various solvents listed in Table 1 range from 4.7 ns in dioxane to 6.1 ns in acetonitrile. Fluorescence decay was approximately monoex- ponential regardless of solvent, except in unbuffered water where a biexponential fit provided for an improved fitting. A typical fluorescence decay trace is illustrated in Fig. 2. The similarities to the photophysics of tryptophan are once again evident with respect to fluorescence decay kinetics. Szabo and Raynor have reported biexponential decay of tryptophan in aqueous solutions of pH 7.0 with lifetimes of 3.14 ns and 5.1 ns (21). They attributed the two lifetimes to the presence of two stable conformations. It is possible that in the case of melatonin, more detailed studies will unveil additional components, a characteristic multiexponential be-

0.2 , , , I ' " " ' ' 1 ~ 1

1 o4

0 4 8 12 16 time, ns

Figure 2. Fluorescence decay for melatonin in buffer at pH 7 fol- lowing 266 nm ps excitation. Top: plot of residuals. Bottom: fluo- rescence decay with monoexponential fit and instrument response function (IRF).

540 Mary King and J. C. Scaiano

0.003

0.002

0'00320 360 400 440 480 520 560 600 640 Wavelength, nm

Figure 3. Transient spectrum obtained by laser (308 nm) excitation of melatonin in acetonitrile.

havior long established in the case of tryptophan, that con- tinues to be a subject of attention (22). The values in Table 1 provide our initial determinations of these lifetimes.

Phosphorescence

The triplet state emission (phosphorescence) spectrum of melatonin (Fig. 1) was measured to determine its triplet en- ergy; Fig. 1 shows the (0,O) band to be at 409 nm, repre- senting a triplet energy of -72 kcdmol. The phosphores- cence spectrum of melatonin is similar to that of tryptophan in the same wavelength range; the (0.0) band of tryptophan emission is around 410 nm (23).

Singlet oxygen emission

From the point of view of its role in biology, it is also in- teresting to explore melatonin's sensitization efficiency. Generation of singlet oxygen is commonly used to confirm the involvement of a triplet state. Benzophenone was used as a reference standard because it is known to generate sin- glet oxygen with a quantum yield of 0.36 and absorbs light at the selected excitation wavelength (i.e. 308 nm) (24). We found that melatonin produced singlet oxygen with an effi- ciency of about 3%, suggesting that intersystem crossing from the singlet manifold into the triplet (vide infru), albeit inefficient, does occur.

Laser flash photolysis studies

In order to determine the triplet absorption characteristics of melatonin, the technique of laser flash photolysis was em- ployed. However, the resulting transient spectra of melatonin in various solvents were complex and difficult to interpret (see Fig. 3). Transient spectra of several substituted indolyl radical species are available in the literature (25) and char- acteristic absorptions at 320 and 520 nm for the neutral rad- icals and 330 and 580 nm for radical cations, with hypso- chromic shifts of about 30 nm for electron-attracting substi- tuted groups, appear to be typical (25,26). Examination of the spectra of Fig. 3 clearly shows absorptions in the 400- 500 nm region that cannot be due to radical species.

2.5

. . o ~ ' " ' ' " " ' ' ' ~ ' " ' ' " ' ' ~ " ' ~ 0 0.0005 0.001 0.0015 0.002 0.0025

[Biphenyl], M

Figure 4. Dependence of the rate constant for the growth of bi- phenyl triplet as a function of quencher concentration. The slope (7.7 X lo8 M-I s-I) corresponds to the rate constant for energy transfer. Inset: growth of the biphenyl triplet for 1.2 mM biphenyl.

The use of common triplet quenchers such as oxygen and 1,3-~yclohexadiene provided further insight into the identi- fication of the triplet state in the transient spectrum. While several of the transient peaks diminished upon the addition of quencher, it is clear that this (particularly in the case of oxygen) is not unequivocal proof for the triplet state. Thus, we employed a better diagnostic method to establish the trip- let state characteristics.

In order to exploit the advantages of triplet quenching, it is desirable to employ a quencher that, through energy trans- fer, leads to a readily detectable triplet state for the acceptor. The quencher should be unreactive toward melatonin, be transparent at the excitation wavelength, its triplet energy must be lower than that of melatonin and the triplet-triplet absorption of the quencher must be strong and easily re- solved to allow for the time-resolved monitoring of its growth. Biphenyl, with a triplet energy of 65.8 kcal/mol (27,28), met these criteria, and was the triplet quencher of choice. The growth of the biphenyl triplet was monitored at 360 nm and was easily measured (see inset in Fig. 4). In order to suppress interferences and unnecessary complica- tions arising from possible multiphoton absorption process- es, a light diffuser was used to attenuate the laser beam, thus providing a lower intensity, homogeneous excitation source. Multiphoton processes could lead to electron ejection and partial recapture by biphenyl.

Energy transfer from the triplet of melatonin to biphenyl was monitored by measuring the growth of the quencher triplet as the concentration of biphenyl was increased while that of melatonin remained constant. If interaction involves energy transfer, then the rate of decay of the triplet sensitizer (melatonin) should match the rate of growth of the quencher excited state. The rate constants for energy transfer to the triplet of the quencher are obtained by fitting the growth kinetic traces for each concentration of quencher (kobr) with first order kinetics, thus

( 3 )

where ko is the rate constant of melatonin triplet decay in

kobs = ko + k3 [biphenyl]

Photochemistry and Photobiology, 1997, 65(3) 541

the absence of quencher, and k3. the slope of the line, is the bimolecular rate constant for energy transfer to biphenyl. A plot of the triplet growth rate constant as a function of bi- phenyl concentration (Fig. 4) revealed that in the absence of quencher, the melatonin triplet decays with a rate constant of about 4.8 X los s-l, corresponding to a lifetime of 2.1 j .~s, and the rate constant of energy transfer from the mela- tonin triplet to form triplet biphenyl is 7.7 X lo8 M-' s-I. The inset in the same figure shows a representative growth trace for triplet biphenyl.

With this information at hand, we can now reexamine the transient spectrum recorded for melatonin (Fig. 3). The peak at 440 nm, with a decay rate constant of 4.2 X los s-l, matched well the triplet rate constant derived from the bi- phenyl studies. Thus we have assigned the absorption at 440 nm to the triplet state. This assignment is in line with other published indolyl triplet absorptions; for example, the indole and tryptophan triplets absorb at 440 nm and 450 nm, re- spectively (29).

The intersystem crossing yield for melatonin was deter- mined using acetophenone as the reference and biphenyl as quencher; the triplet of acetophenone absorbs at 320 nm, its triplet energy lies at 74.1 kcavrnol, and the quantum yield for intersystem crossing is - 1 .O (27,28). Based on the rate constants derived from quenching studies, the concentration of biphenyl required to quench approximately 99% of the melatonin triplets was calculated. The concentration of bi- phenyl used was 0.049 M with melatonin in acetonitrile and 0.050 M with acetophenone in acetonitrile. The magnitude of the biphenyl triplet signals were compared for these two solutions, which were optically matched at the laser wave- length (308 nm). The optical density of the biphenyl triplet in the melatonin sample compared to that in the acetophen- one sample was weak, and the intersystem crossing yield was found to be 0.22. This is partially responsible for the low efficiency with which melatonin generates singlet oxy- gen and is in line with intersystem crossing efficiencies for other indoles (30).

Because the human lens filters light below 400 nm in adults, and children have the added capability of transmitting a narrow band of light at 320 nm with about a 20% effi- ciency, it is unlikely that retinal melatonin participates as an ocular photosensitizer (3 1,32). We (7) have found that me- latonin quenches benzophenone triplets in acetonitrile with a rate constant of 7.6 X lo9 M-' s - I and traps photoinduced alkoxy radicals a t rates greater than 1 O7 M-I S K I . Reszka and coworkers have found that melatonin efficiently scavenges photosensitized singlet oxygen with a rate constant of about 6 X lo7 M-I s - I and does not undergo chemical alteration in the process (33). Protoporphyrin, which is present in the blood that directly feeds the retina, absorbs light at wave- lengths greater than 400 nm and can sensitize singlet oxygen efficiently, which in turn can cause ocular tissue damage. This indicates that the role of retinal melatonin may include, to some degree, that of protective radical trap and quencher for both singlet oxygen generation and any harmful photo- chemistry arising from the triplet excited state of other oc- ular chromophores.

Acknow[edgenrents-Thanks are due to the Natural Sciences and Engineering Research Council of Canada for generous support in

the form of research and strategic grants to J.C.S. Thanks are also due to Ms. Nadereh Mohtat for her help with the measurement of fluorescence lifetimes. This work was performed while J.C.S. was the recipient of a Killam Fellowship awarded by the Canada Coun- cil.

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