Photochemical mechanisms responsible for the versatile application of naphthalimides and naphthaldiimides in biological systems

Article abstract of DOI:10.1021/ja971993c

Despite the number and variety of their biological applications, the mechanisms of action of the photoactive naphthalenic imides have not yet been fully elucidated. In order to provide mechanistic insight, the photochemistry of several N-substituted 1,8-naphthalimides (NT) and 1,4,5,8-naphthaldiimides (NDI) has been studied using absorption and fluorescence spectroscopy and by laser flash photolysis (λ(exc) = 355 nm). The lowest singlet state (S₁) is mainly ππ* in nature for NI whereas nπ* character predominates for the NDI. This difference exerts a profound effect on subsequent reaction mechanisms: upon irradiation, only the NDI molecules can undergo intramolecular γ hydrogen abstraction. In the case of NP-III, a bishydroperoxy NDI derivative, this photoprocess (Φ = 0.03) leads to concomitant formation of an oxygen-centered radical (ε = 21,600 M^-1 cm^-1 at 465 nm in acetonitrile) and release of the hydroxyl radical (^.OH). All the compounds studied produce the triplet state (in acetonitrile, ε(T) ~ 10,500-11,500 M^-1 cm^-1 at 470 nm for NI and 485 nm for NDI). The quantum yield of intersystem crossing was determined to be close to unity except where intramolecular γ hydrogen abstraction was possible (Φ(isc) < 0.5). The triplet states were found to efficiently sensitize the formation of singlet oxygen (with S(Δ) > 0.8 for NI and > 0.5 for NDI). In the absence of quenchers, the triplet states react with the ground-state of starting material via electron-transfer with a high rate constant [k = (4-6) x 10⁹ and 5 x 10⁸ M^-1 s^-1 for NDI and NI, respectively] to give the radical anion and radical cation of the corresponding naphthalenic derivative. The high reactivity of the triplet states toward electron donors such as DABCO and their low ability for hydrogen abstraction are typical of a ππ* configuration. These mechanistic photochemistry results are discussed with regard to the photobiological effects observed for these compounds and show that the actual reaction leading to biological damage will depend on the microenvironment of the naphthalenic molecule.

Full text of DOI:10.1021/ja971993c

J. Am. Chem. Soc. 1997, 119, 11785-11795
11785
Photochemical Mechanisms Responsible for the Versatile
Application of Naphthalimides and Naphthaldiimides in
Biological Systems
Be´atrice M. Aveline,† Seiichi Matsugo,‡ and Robert W. Redmond*,†
Contribution from the Wellman Laboratories of Photomedicine, HarVard Medical School,
Massachusetts General Hospital, Boston, Massachusetts 02114, and Department of Chemical and
Biochemical Engineering, Faculty of Engineering, Toyama UniVersity, Gofuku 3190,
Toyama 930, Japan
ReceiVed June 20, 1997X
Abstract: Despite the number and variety of their biological applications, the mechanisms of action of the photoactive
naphthalenic imides have not yet been fully elucidated. In order to provide mechanistic insight, the photochemistry
of several N-substituted 1,8-naphthalimides (NI) and 1,4,5,8-naphthaldiimides (NDI) has been studied using absorption
and fluorescence spectroscopy and by laser flash photolysis (λexc ) 355 nm). The lowest singlet state (S1) is mainly
ππ* in nature for NI whereas nπ* character predominates for the NDI. This difference exerts a profound effect on
subsequent reaction mechanisms: upon irradiation, only the NDI molecules can undergo intramolecular γ hydrogen
abstraction. In the case of NP-III, a bishydroperoxy NDI derivative, this photoprocess (Φ ) 0.03) leads to concomitant
formation of an oxygen-centered radical (ꢀ ) 21 600 M-1 cm-1 at 465 nm in acetonitrile) and release of the hydroxyl
radical (•OH). All the compounds studied produce the triplet state (in acetonitrile, ꢀT ≈ 10 500-11 500 M-1 cm-1
at 470 nm for NI and 485 nm for NDI). The quantum yield of intersystem crossing was determined to be close to
unity except where intramolecular γ hydrogen abstraction was possible (Φisc < 0.5). The triplet states were found
to efficiently sensitize the formation of singlet oxygen (with S∆ > 0.8 for NI and > 0.5 for NDI). In the absence
of quenchers, the triplet states react with the ground-state of starting material Via electron-transfer with a high rate
constant [k ) (4-6) × 109 and 5 × 108 M-1 s-1 for NDI and NI, respectively] to give the radical anion and radical
cation of the corresponding naphthalenic derivative. The high reactivity of the triplet states toward electron donors
such as DABCO and their low ability for hydrogen abstraction are typical of a ππ* configuration. These mechanistic
photochemistry results are discussed with regard to the photobiological effects observed for these compounds and
show that the actual reaction leading to biological damage will depend on the microenvironment of the naphthalenic
molecule.
Naphthalenic imides constitute a very versatile class of
compounds which have been used in a large variety of areas.
Their most recent applications have been oriented toward the
fields of biology and medicine. Substituted 1,8-naphthalimides
and bisnaphthalimides1 have been shown to demonstrate
dramatic anticancer activity,2,3 and members of both series have
already entered into clinical trials.4 Although the precise
mechanism of their tumoricidal efficacy has not yet been fully
understood, several lines of evidence suggest that it is linked
to their ability to intercalate into DNA with high affinity and
sequence specificity.5 Sulfonated naphthalimides and bisnaph-
thalimides have also been reported to be very promising antiviral
agents with selective in Vitro activity against the human
immunodeficiency virus, HIV-1.6
inhibition of enveloped viruses in blood and blood products.7-9
Photoexcitation of the bichromophoric derivatives has also been
found to efficiently induce cross-linking of proteins such as
collagen.10-12 This property has led to the investigation of their
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In addition to the above “dark” properties, photochemical
activation of these naphthalenic derivatives has been used to
further broaden the range of biological applications. In this
respect, brominated mono- and bisnaphthalimides have been
proposed as good candidates for the photochemotherapeutic
† Harvard Medical School.
‡ Toyama University.
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X Abstract published in AdVance ACS Abstracts, November 1, 1997.
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with a chemical bridge. (b) Brana, M. F.; Castellano, J. M.; Moran, M.;
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F.; Schlick, E. Anti-Cancer Drug Design 1993, 8, 257-268.
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S0002-7863(97)01993-8 CCC: $14.00 © 1997 American Chemical Society

11786 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
AVeline et al.
the apolipoproteins of human low density lipoprotein (LDL)26
and bovine serum albumin (BSA).27 This compound is impor-
tant considering the lack of convenient sources for the study of
•OH reactions in biologically relevant systems28,29 and the
general effort put into the development of new, selective
•
photochemical OH generators.30-41
We report here the results of a study of the photochemistry
of the bishydroperoxy naphthaldiimide, NP-III, and related
molecules. In order to elucidate the potential bioreactivity of
NP-III, it was necessary to investigate the structural effects
(nature of the substituent on the nitrogen atom, mono- or
bifunctionality of the molecule) on the primary and subsequent
photochemistry of these compounds. In this manner, we have
demonstrated a variety of possible mechanisms by which
photoactivation of NP-III could result in biological damage and
shown that the exact mechanism in operation will depend on
the microenvironment of the excited molecule.
Figure 1. Chemical structures of N-(2-hydroperoxy-2-methoxyethyl)-
1,8-naphthalimide (NP-II) and N,N′-bis(2-hydroperoxy-2-methoxy-
ethyl)-1,4,5,8-naphthaldiimide (NP-III).
potential application in light-induced tissue-repair and welding
of meniscal cartilage, articular cartilage and cornea has been
achieved in Vitro.13,14 The combination of high photoactivity
and DNA-intercalation has also prompted the use of some
naphthalenic derivatives as sequence-specific DNA-photo-
nucleases.15 Depending on the chemical structure of the
compound used, various mechanisms have been proposed,
involving free radical formation,16 photogeneration of carbo-
cation,17 electron-transfer from oxidizable guanine residues
(G),18 or hydrogen abstraction from thymine,19 but a lack of
experimental evidence precludes the confirmation of these
proposals.
Experimental Section
Chemicals. All reagents used were supplied by Sigma Chemical
Co. (St. Louis, MO), Fisher Chemical (Pittsburgh, PA) or Aldrich
Chemical Co. (Milwaukee, WI). Organic solvents were of spectro-
scopic grade from Fisher, deuterated acetonitrile (CD3CN: 99.6 atom
% D) was from Aldrich and Phosphate Buffered Saline Salt Solution
(PBS, pH ) 7.2) was purchased from Gibco BRL (Grand Island, NY).
Figures 1 and 2 show the chemical structures of the naphthalimide
and naphthaldiimide derivatives studied. 1,8-Naphthalimide (1) was
provided by Aldrich and used as supplied. NP-II, NP-III, and
compounds 2 and 3 were synthesized and purified as previously
described.20,25,42 All the naphthalenic derivatives were stored in solid
form at -20 °C. Solutions were prepared immediately before use and
were protected from light at all times. In case of low hydrophilicity,
a concentrated solution of naphthalenic derivative in acetonitrile was
diluted with PBS (the pH of the resulting solution was verified to be
7.2, and the percentage by volume of acetonitrile in the final sample
was <1%).
The most intriguing molecules of this family are the hydro-
peroxy derivatives NP-II and NP-III (presented in Figure 1),
which have been designed with the goal of generating the
hydroxyl radical (•OH) upon near-UV irradiation and named
“photo-Fenton reagents”.20,21 Photoexcitation of these com-
pounds has been shown to cause DNA-strand breaks20,21 and
in the case of the naphthaldiimide (NP-III), a sequence-specific
cleavage at the 5′G of 5′-GG-3′ sites has been observed.21,22
NP-III has also been used to investigate oxidative modifications
General Techniques. Ground-state absorption properties were
studied using a Cary 2300 UV-visible spectrophotometer or a Hewlett-
(24) Matsugo, S.; Kodaira, K. I.; Saito, I. Bioorg. Med. Chem. Lett. 1993,
3, 1571.
•
induced by OH in cells,23,24 in the eye lens proteins25 and in
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Biochemistry 1992, 31, 4296-4303.
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Michalek, G. R.; Utecht, R. E.; Skiles, H.; Chang, S. C.; Matthews, J. L.
AntiViral Res. 1994, 25, 133-146.
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Biophys. Res. Commun. 1995, 208, 161-167.
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D. E.; Utecht, R. E. Proc. Int. Conf. Lasers 1993, 15, 774-776.
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SPIEsInt. Soc. Opt. Eng. 1993, 1882, 305-308.
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Proc. SPIEsInt. Soc. Opt. Eng. 1994, 2128, 506-509.
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D. E.; Utecht, R. E. Proc. SPIEsInt. Soc. Opt. Eng. 1993, 1876, 175-
179.
(28) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press: Oxford, 1989.
(29) Bensasson, R. V.; Land, E. J.; Truscott, T. G. Excited States and
Free Radicals in Biology and Medicine; Oxford University Press: Oxford,
1993.
(30) Saito, I.; Takayama, M.; Matsuura, T.; Matsugo, S.; Kawanishi, S.
J. Am. Chem. Soc. 1990, 112, 883-884.
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6869-6872.
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E.; Utecht, R. E.; Yuan, D. Proc. SPIEsInt. Soc. Opt. Eng. 1996, 2671,
251-255.
(32) Sako, M.; Nagai, K.; Maki, Y. J. Chem Soc. Chem. Commun. 1993,
750-751.
(15) Bailly, C.; Brana, M.; Waring, M. J. Eur. J. Biochem. 1996, 240,
195-208.
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R. Angew. Chem., Int. Ed. Engl. 1995, 34, 2156-2158.
(34) Adam, W.; Cadet, J.; Dall’Acqua, F.; Epe, B.; Ramaiah, D.; Saha-
Mo¨ller, C. R. Angew. Chem., Int. Ed. Engl. 1995, 34, 107-110.
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Photochemistry of Naphthalimides and Naphthaldiimides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11787
ꢀT ) ꢀT(D)(∆AT/∆AT(D) (2)
)
where ꢀT(D) is the molar absorption coefficient of the triplet state of the
sensitizer,48 ∆AT(D) is the maximum absorbance of the triplet donor in
the absence of the acceptor and ∆AT is the sensitized triplet absorbance
of the naphthalimide or naphthaldiimide studied. This latter value
corresponds to the observed triplet absorbance (∆ATobs) corrected for
underestimation due to incomplete (< 100%) quenching of sensitizer
triplet states by the naphthalenic derivative. This correction was carried
out using
∆AT ) ∆AT kobs/(kobs - k1)
(3)
obs
where k1 and kobs are the rate constants for the decay of the triplet state
of the sensitizer in the absence and in the presence of the acceptor
molecule, respectively (kobs is given by eq 4, where kET is the
bimolecular rate constant for triplet energy transfer from the sensitizer
to the naphthalenic derivative, N). This correction was never greater
than 10%.
Figure 2. Chemical structures of 1,8-naphthalimide (1), N,N′-bis(2,2-
dimethoxyethyl)-1,4,5,8-naphthaldiimide (2), and N,N′-bis(2,2-dimeth-
ylpropyl)-1,4,5,8-naphthaldiimide (3).
Packard HP8451A UV-visible diode array spectrophotometer. Cor-
rected steady-state emission spectra were recorded with a Fluoromax
spectrofluorimeter (Spex Industry, Edison, NJ). Fluorescence quantum
yields (Φf) were determined at λexc ) 330 nm with quinine bisulfate
(Φf ) 0.55 in 0.5 M sulfuric acid)43 as reference. Standard and samples
in air-saturated solutions had absorbances below 0.05. Fluorescence
quantum yields were calculated using the following relationship:
kobs ) k1 + kET[N]
(4)
Quantum yields (Φ) of photoprocesses undergone by the naphtha-
lenic molecules were determined by comparative actinometry as
previously described,35,49 using benzophenone in deaerated benzene (for
which Φisc ) 1.050 and ꢀT ) 7220 M-1 cm-1 at 530 nm51) as standard.
Photoconductivity. Transient photoconductivity measurements were
performed with samples contained in a 10 mm × 10 mm path length
quartz cuvette equipped with a Teflon insert holding three horizontal
platinum foil electrodes (4 × 4 mm2) spaced exactly 4 mm apart. The
volume between the common center electrode and one of the outer
electrodes was excited by the laser beam whereas the unirradiated
volume between the other outer electrode and the common provided a
measure of the background signal. A DC voltage pulse, of variable
duration (1-4 ms) and amplitude (e400 V), was applied across the
electrodes, prior to the laser pulse. Signals were taken to a differential
amplifier to increase the signal to noise and observe only photoinduced
changes in conductivity of the sample. The differential signal was
passed to the digital oscilloscope and processed as in the laser flash
photolysis experiments. The electrode arrangement was such that
optical and conductivity detection could be carried out on the same
sample.
Φf(S) ) Φf(R)(FSn2S/FRn2R)
(1)
where the S subscript refers to the sample and R to the reference. F
represents the slope of the integrated area under the emission band
Versus the absorbance of the solution at 330 nm, and n is the index of
refraction at the sodium D line at 20 °C.
Laser Flash Photolysis. The nanosecond laser flash photolysis
apparatus has been described in detail elsewhere.44,45 For most of the
experiments, excitation was provided by the frequency-tripled output
of a Quantel YG660 Nd:YAG laser (355 nm, 8 ns duration pulse).
Laser intensities were attenuated to e18 mJ cm-2 pulse-1 through the
use of neutral density filters. Quenching rate constants were measured
using static samples. Transient absorption spectra and kinetic signals
under aerated conditions were recorded using a flow system ensuring
irradiation of a completely fresh volume of solution with each laser
pulse. When nitrogen- or oxygen-saturated conditions were needed,
experiments were carried out with a static sample which was shaken
between laser shots. The samples were contained in a 10 mm × 10
mm quartz cuvette. Photosensitization experiments were performed
using the frequency-quadrupled output of the Nd:YAG laser (266 nm,
8 ns duration pulse, up to 10 mJ cm-2 pulse-1).
Infrared Luminescence. Singlet oxygen (O2 (1∆g)) formation was
detected using the time-resolved infrared luminescence technique using
a (EOSS G-050) germanium diode-based system,52,53 equipped with a
Judson PA-100 pre-amplifier. Singlet oxygen quantum yields (Φ∆)
were calculated by comparison of the slopes of the linear energy
dependence plots of the maximum luminescence emission obtained for
the naphthalenic derivatives and phenazine used as reference (Φ∆
)
0.8454). Standard and samples in air-saturated deuterated acetonitrile
were optically matched at λexc ) 355 nm.
Molar absorption coefficients of the triplet states (ꢀT) of naphthalenic
derivatives were obtained by the sensitization method. Pulsed excitation
(λexc ) 266 nm) of 4-methoxyacetophenone in the presence of
naphthalimide or naphthaldiimide is followed by energy transfer to form
the triplet state of the acceptor molecule.46 Under the conditions
employed for these experiments, the light was absorbed predominantly
by the sensitizer (g90%).47 The ꢀT values were determined using the
following relationship:
Cyclic Voltammetry. The reduction potentials (Er1e/2d) of the naph-
thalenic derivatives were measured in anhydrous acetonitrile containing
e1 mM naphthalimide or naphthaldiimide and 0.1 M tetra-n-butyl-
ammonium fluoroboride (TBAF). Solutions were bubbled with nitrogen
(48) (a) ꢀT(D) for 4-methoxyacetophenone (4-MAP) in acetonitrile was
determined to be 16 100 M-1 cm-1 at 380 nm by comparative actinometry
using benzophenone in deaerated acetonitrile (for which Φisc ) 1 and ꢀT )
6250 M-1 cm-1 at 530 nm)48b as standard and assuming a triplet state
quantum yield of unity for 4-MAP. (b) Murov, S. L.; Carmichael, I.; Hug,
G. L. Handbook of Photochemistry, 2nd ed.; Marcel Dekker, Inc.: New
York, 1993.
(43) Melhuish, W. H. J. Phys. Chem. 1961, 65, 229.
(44) Krieg, M.; Srichai, M. B.; Redmond, R. W. Biochem. Biophys. Acta
1993, 1151, 168-174.
(45) Aveline, B.; Hasan, T.; Redmond, R. W. Photochem. Photobiol.
1994, 59, 328-335.
(49) Bensasson, R. V.; Land, E. J. Trans. Faraday Soc. 1971, 67, 1904-
1915.
(50) Inbar, S.; Linschitz, H.; Cohen, S. G. J. Am. Chem. Soc. 1981, 103,
1048-1054.
(46) This method is based on the assumption that the triplet state of
4-MAP is exclusively quenched by energy transfer. No electron transfer
(51) Hurley, J. K.; Sinai, N.; Linschitz, H. Photochem. Photobiol. 1983,
38, 9-14.
3
between (4-MAP)* and the naphthalenic derivatives was observed.
(47) To correct for the absorbance due to direct excitation of the
naphthalimide or naphthaldiimide, two sets of experiments were carried
out: one for the naphthalenic derivative alone in solution (where a small
amount of triplet state is generated by direct excitation) and the other one
in the presence of sensitizer (where further naphthalenic triplet state is
produced by sensitization). The ∆AT value used in eq 3 was calculated as
the differential between the ∆A valueosbsobtained from these two experiments.
(52) Rodgers, M. A. J.; Snowden, P. T. J. Am. Chem. Soc. 1982, 104,
5541-5543.
(53) Keene, J. P.; Kessel, D.; Land, E. P.; Redmond, R. W.; Truscott,
T. G. Photochem. Photobiol. 1986, 43, 117-120.
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Photosensitisation: Molecular, Cellular and Medical Aspects 1988, H15,
93-95.

11788 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
AVeline et al.
broad band in the case of the naphthaldiimides (∆λ ) 8 nm,
from benzene to methanol) and a red-shift in the case of the
naphthalimides (∆λ ) 6 nm). These results suggest that the
lowest singlet state (S1) is mainly ππ* in nature for naphthal-
imides whereas nπ* character predominates for the naphthal-
diimides.55 It is worthwhile noting that the ground-state
absorption properties of NP-III in aqueous buffer solution do
not follow the trend observed in organic solvents: in comparison
to that recorded in methanol, the spectrum of NP-III in PBS
was found to be red-shifted (∆λ ) 7 nm), indicating strong
hydrogen-bonding between the molecules of NP-III and water.
In the case of NP-II, as expected for a ππ* singlet state, the
broad absorption band undergoes a red-shift in aqueous media
(∆λ ) 14 nm, from methanol to PBS).
Figure 3 also shows the fluorescence emission spectra of NP-
II and NP-III recorded in acetonitrile. These spectra, which
are the mirror images of the corresponding long-wavelength
absorption band, were found to be identical irrespective of the
excitation wavelength used, which demonstrates the existence
of only one fluorescing species. Energies of the lowest singlet
state (ES) were estimated from the overlap of the emission and
absorption spectra. In the naphthaldiimide family, similar ES
values of ≈ 75 kcal mol-1 were obtained whereas ES of ≈ 80
kcal mol-1 were measured in the naphthalimide series. This
latter value is identical to that previously reported for N-methyl-
1,8-naphthalimide in acetonitrile.56 Determination of the fluo-
rescence quantum yields of the naphthalenic derivatives in
acetonitrile57 led to Φf values of 0.027 and 0.147 for compound
1 and NP-II, respectively and 0.005 and 0.010 for compound 3
and NP-III, respectively. These results show the naphthalimides
to be more fluorescent than the naphthaldiimides. The proticity
of the solvent was observed to strongly influence the fluores-
cence properties of all compounds (Φf increases from 0.027 in
acetonitrile to 0.18 in methanol to 0.44 in PBS for compound
1, from 0.145 to 0.27 to 0.63 for NP-II, and from 0.01 to 0.04
to 0.45 for NP-III). A similar effect of the solvent proticity
has already been reported in the case of several substituted and
unsubstituted 1,8-naphthalimides.56,58-60
Figure 3. Ground-state absorption (s) and fluorescence emission
(- - -) spectra of NP-III (A) and NP-II (B) in acetonitrile. The emission
spectra were recorded under air-saturated conditions using λexc ) 330
nm.
prior to the measurement. The cyclovoltammograms were obtained
using a potentiostat (model 273, EG&G Princeton Applied Research)
coupled to a recorder (model RE0091, EG&G Princeton Applied
Research) and platinum and silver (reference) electrodes. The reactions
at the electrodes were reversible in all cases and the redox potentials
were obtained as the arithmetic average of the cathodic and anodic
waves. The ferrocene/ferrocenium couple (0.43 V Vs SCE in aceto-
nitrile, 0.1 M TBAF) was used to calibrate the reference electrode.
Transient Absorption Spectroscopy. Figure 4A shows the
transient absorption spectrum recorded at times g40 µs after
laser flash photolysis (λexc ) 355 nm) of NP-III, in oxygen-
saturated acetonitrile. It displays negative bands with maxima
around 360 and 380 nm which present a mirror image symmetry
with the ground-state absorption spectrum. A large absorption
band at 465 nm with a shoulder around 530 nm and other weaker
bands at 600, 660, and 725 nm, which all decay following the
same kinetics, are also observed. The corresponding species
appears to be produced within the duration of the laser pulse
and to decay slowly with a lifetime of 250-300 µs at low laser
intensity. The decay of this primary long-lived species, denoted
X, was found to become rapidly more second-order in character
with increasing laser energies or ground-state concentration.
Results
Absorption and Fluorescence Properties. The ground-state
absorption spectra recorded for NP-II and NP-III in acetonitrile
are shown in Figure 3. All the naphthaldiimides studied were
found to display similar absorption spectra consisting of a
narrow band at 236 nm, a broad absorption band composed of
a shoulder around 340-345 nm and two distinct maxima
(situated at 355-365 nm and 375-385 nm, depending on the
solvent used). Other higher vibronic bands are also visible
between 250 and 325 nm. In addition to the 236 nm-band, all
the naphthalimides exhibit a broad absorption, which is less
structured than that of the naphthaldiimides and presents a
maximum which is blue-shifted (∆λ ≈ 15 nm) compared to
that of the bifunctional derivatives.
In all solvents studied (benzene, dichloromethane, ethyl ether,
acetone, acetonitrile, methanol, and PBS), the ground-state
absorption of the naphthalenic molecules followed linear Beer-
Lambert behavior in the measured range of concentrations (from
1 to 100 µM). In organic solvents, an increase in solvent
polarity had no effect on the wavelength maximum of the
stronger absorption at 236 nm but produced a blue-shift of the
(55) The solvent shifts observed are surprising, given the rather large
absorption coefficients which one would not expect from an nπ* state. One
might speculate the existence of a weaker nπ* absorption masked by a
dominant overlapping ππ* absorption.
(56) Wintgens, V.; Valat, P.; Kossanyi, J.; Biczok, L.; Demeter, A.;
Berces, T. J. Chem. Soc., Faraday Trans 1994, 90, 411-421.
(57) As oxygen was found to have no significant effect on the
fluorescence of the naphthalenic derivatives, the Φf values were measured
under air-saturated conditions.
(58) Barros, T. C.; Molinari, G. R.; Berci Filho, P.; Toscano, V. G.;
Politi, M. J. J. Photochem. Photobiol. A: Chem. 1993, 76, 55-60.
(59) Samanta, A.; Saroja, G. J. Photochem. Photobiol. A: Chem 1994,
84, 19-26.
(60) The proticity of the solvent was explained to lead to a larger
separation between the lowest singlet state and the lowest triplet state making
the intersystem crossing less favorable while enhancing the fluorescence.

Photochemistry of Naphthalimides and Naphthaldiimides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11789
Figure 4. (A) Time-dependent transient absorption spectra recorded
for NP-III in oxygen-saturated acetonitrile 40 (b), 100 (O), and 400
µs (2) after pulsed excitation (λexc ) 355 nm). (B) Transient absorption
spectra recorded for NP-III in air-saturated acetonitrile 600 ns (b), 1.5
µs (O), and 4 µs (2) after the laser pulse. In both cases, the absorbance
of the solution at λexc was ≈ 0.3 (corresponding to [NP-III] ≈ 15 µM)
and the laser intensity was 8 mJ cm-2 pulse-1. The inset in B shows
Figure 5. (A) Concentration dependence of the decay of the triplet
state of NP-III monitored at 325 nm (b) and of the growth at 610 nm
(O). The observed rate constants (k) were plotted against the concentra-
tion of precursor ([NP-III]) and the data were fitted by a linear equation.
The inset shows the kinetic signal recorded at 480 nm after excitation
of a solution of NP-III in deaerated acetonitrile. (B) Transient absorption
spectra of the secondary long-lived species, Y, recorded 40 (b), 100
(O), 400 (2), and 700 µs (4) after the laser pulse (λexc ) 355 nm).
The absorbance of the solution at λexc was ≈ 0.3 ([NP-III] ≈ 15 µM),
3
the absorption spectrum of (NP-III)* in acetonitrile.
The absorption spectra recorded at shorter times (t g 600
ns) after 355 nm excitation of NP-III in air-saturated acetonitrile
are shown in Figure 4B. This figure reveals the presence of a
short-lived species in addition to the longer-lived intermediate,
X. This short-lived species decays by first-order kinetics with
a time constant of 450 ns. Its absorption spectrum (inset of
Figure 4B) displays a band at 485 nm with a shoulder around
455 nm. The presence of a positive absorption around 325 nm
in the transient difference spectrum indicates that this short-
lived species has another band with a maximum at lower
wavelength (λ < 400 nm).
and the laser intensity was 8 mJ cm-2 pulse-1
.
transient absorption spectrum was identical to that of the even
longer-lived X, observed under oxygen- and air-saturated
conditions.
Triplet State. The short-lived species detected in air and
3
absorbing at 485 nm was identified as (NP-III)*, the triplet
state of NP-III, through its efficient quenching61 by oxygen (k
) 2.5 × 109 M-1 s-1) and 2,5-dimethyl-2,4-hexadiene (k )
2.1 × 1010 M-1 s-1) and the observation of sensitization of the
triplet state absorption of perylene (k ) 1.8 × 1010 M-1 s-1).
3(NP-III)* was unequivocally generated by energy transfer from
the triplet state of 4-methoxyacetophenone or xanthone using
266 nm as excitation wavelength (Vide infra) and the transient
absorption spectrum recorded under these conditions was found
to be identical to that of the short-lived species produced by
direct excitation of NP-III in aerated acetonitrile.
Different behavior was observed in deaerated acetonitrile.
The transient absorption spectrum recorded at times < 3 µs after
the laser pulse was identical to that of the short-lived intermedi-
ate detected in aerated solution. However, the lifetime of the
corresponding species (which can be measured at 325 nm
without interference from other transients) was considerably
longer (7 µs at 15 µM of NP-III) and the decay of this species
was accompanied by the concomitant growth of an absorption
which maximizes at 480 nm (inset of Figure 5A). In addition
to the large absorption band at 480 nm, the absorption spectrum
of this secondary species displays a shoulder around 530 nm
and several weaker bands with maxima at 610, 680, and 760
nm (Figure 5B). This secondary species, Y, was found to decay
by second-order kinetics. Following its decay, the residual
3
The lifetime of (NP-III)* under deaerated conditions was
observed to depend on the concentration of NP-III present in
(61) In these experiments, the concentration of 2,5-dimethyl-2,4-hexa-
diene was varied from 10 µM to 1 mM, that of perylene from 12.5 to 150
µM. The reaction rate constant with oxygen was determined using the
approximations of complete removal of oxygen under N2-saturated condi-
tions and a concentration of 1.9 mM in O2 in air-saturated acetonitrile.

11790 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
AVeline et al.
solution. The decay kinetics of the triplet state absorption
(monitored at 325 nm) was identical to that of the growth of
the secondary long-lived species at 610 nm at all concentrations
of NP-III studied (between 3 and 45 µM). A plot of the
observed rate constant for either of these processes against [NP-
III] gave a straight line, the slope of which yields a bimolecular
rate constant of 5.5 × 109 M-1 s-1 (Figure 5A).
Triplet state formation was found to take place upon pulsed-
excitation of all the compounds studied, i.e. (1) mono- and
bifunctional molecules and, (2) all the N-substituted derivatives,
irrespective of the nature of the substituent. The triplet states
of all the naphthaldiimides have spectral features identical to
3
those of (NP-III)*. In the case of the naphthalimides, NP-II
and compound 1, the absorption spectrum of the triplet state
displays a band at 470 nm with a shoulder around 440-445 nm
and another band with a maximum at lower wavelength (λ <
370 nm).62 These spectral characteristics are similar to those
reported for the triplet states of N-methyl56 and N-phenyl63 1,8-
naphthalimides. In a given solvent, the red-shift between the
absorption of the triplet states of naphthaldiimides and that of
the triplet states of naphthalimides parallels the red-shift of the
corresponding ground-state absorptions. Triplet state formation
was also observed to take place in benzene, methanol and PBS.
In all cases, a decrease in solvent polarity results in a weak
blue-shift of the maximum of the absorption band (∆λ ) 10
nm, from PBS to benzene for both the naphthalimides and
naphthaldiimides).
The molar absorption coefficients (ꢀT) of the triplet states of
the naphthalenic derivatives were determined in acetonitrile by
the sensitization method using 266 nm as excitation wavelength
and 4-methoxyacetophenone as sensitizer.47 Similar ꢀT values
were obtained for the naphthalimides (ꢀT ≈ 10 500 M-1 cm-1
at 470 nm) and naphthaldiimides (ꢀT ≈ 11 250 M-1 cm-1 at
485 nm). These values are close to that reported for N-methyl-
1,8-naphthalimide in the same solvent (ꢀT ≈ 10 000 M-1 cm-1
at 470 nm).56
The quantum yields of intersystem crossing (Φisc) were
determined by comparative actinometry using 355 nm excitation
of optically-matched samples of naphthalenic derivative and
benzophenone in benzene. In acetonitrile, compounds 1 and 3
were found to have quantum yields of intersystem crossing close
to unity (0.95 and 1, respectively) whereas NP-II, NP-III64 and
compound 2 display lower Φisc values (0.42, 0.39, and 0.23,
respectively). In PBS, where intermolecular hydrogen bonding
with molecules of water is possible, Φisc was found to be
reduced to 0.43 for compound 1, 0.31 for NP-II and 0.24 for
NP-III.65
All the naphthalenic derivatives were observed to sensitize
the formation of singlet oxygen, O2 (1∆g). Comparison of the
quantum yields of singlet oxygen production (Φ∆) and of
intersystem crossing (Φisc) shows that in all cases, the efficiency
of O2 (1∆g) formation (S∆ ) Φ∆/Φisc) is high (S∆ > 0.8 for the
naphthalimides and >0.5 for the naphthaldiimides). Singlet
oxygen was also observed to react with the ground-state of the
naphthalenic molecules. In acetonitrile, reaction rate constants
Figure 6. Transient conductivity (A and C) and transient absorption
at 610 nm (B and D), recorded following pulsed excitation (λexc ) 355
nm) of NP-III in deaerated acetonitrile. The absorbance of the solution
at λexc was 0.6 ([NP-III] ≈ 30 µM), and the laser intensity was 10 mJ
cm-2 pulse-1
.
of (2-3) × 108 M-1 s-1 were determined for NP-II and
compound 1 and 1 × 109 M-1 s-1 for NP-III.66
Having characterized the triplet state behavior of NP-III, we
now turn our attention to the identification of the primary and
secondary long-lived species, (X and Y, respectively), which
are also formed on 355 nm excitation of the bishydroperoxy
naphthaldiimide, NP-III (Vide supra).
Secondary Long-Lived Species. In aerated acetonitrile,
quenching of the triplet state of NP-III by oxygen (k ) 2.5 ×
109 M-1 s-1, [O2] ) 1.9 mM) dominates over ground-state
quenching (k ) 5.5 × 109 M-1 s-1, [NP-III] ) 15 µM) and the
secondary species Y is not observed. In the absence of oxygen,
however, 3(NP-III)* reacts efficiently with the ground-state with
a rate constant approaching diffusion control (Vide supra) to
give Y, the absorption spectrum of which is shown in Figure
5B.
To aid in the identification of Y, experiments were carried
out using 355 nm excitation with simultaneous optical and
conductivity detection. Figure 6 shows the time-resolved
absorption (at 610 nm) and conductivity signals recorded after
pulsed excitation of NP-III in deaerated acetonitrile. Good
agreement was observed between the kinetics of growth of the
secondary species absorbing at 610 nm (Figure 6B) and the
buildup of the charged intermediate in the photoconductivity
measurement (Figure 6A). The decays of the conductivity signal
(Figure 6C) and of the shorter-lived species absorbing at 610
nm (Figure 6D) recorded on longer time scales also show good
correspondence.67 The obvious conclusion is that Y is a charged
intermediate resulting from reaction between the triplet and
ground-state of the bishydroperoxy naphthaldiimide. In aerated
solution, where 3(NP-III)* is exclusively quenched by oxygen,
no conductivity signal could be detected. It should be noted
that this experiment also leads to the conclusion that, X, the
longer-lived species absorbing at 610 nm, is not charged.
(62) The absorption of the triplet state of compound 1 in acetonitrile is
presented in Figure 8.
(63) Demeter, A.; Biczok, L.; Berces, T.; Wintgens, V.; Valat, P.;
Kossanyi, J. J. Phys. Chem. 1993, 97, 3217-3224.
(64) In order to correct for the absorbance due to the primary long-lived
species X at the λmax of the triplet state, two sets of experiments were carried
out: one under N2-saturated conditions [where both 3(NP-III)* and X
absorb], the other one under O2-saturated conditions (where only the primary
long-lived species is produced).
(66) These reaction rate constants were determined by monitoring the
decay of singlet oxygen phosphorescence produced by excitation (λexc
)
355 nm) of phenazine in air-saturated acetonitrile in the presence of several
concentrations of naphthalenic derivatives (from 1 to 10 µM). The
luminescence signals were corrected for the initial fast spike due, at least
in part to fluorescence of phenazine, by subtracting the signal recorded for
the same solution under deaerated conditions.
(65) The Φisc determinations in aqueous media were carried out using
the assumption that the triplet absorption coefficients of the naphthalenic
derivatives were identical in acetonitrile and PBS.
(67) In addition to Y, the transient absorption signal recorded at 610 nm
using a longer time scale (Figure 6D) presents a contribution from the
primary long-lived species, X.

Photochemistry of Naphthalimides and Naphthaldiimides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11791
3(p-C)* + NP-III f (p-C)•- + (NP-III)•+
(7)
The spectrum resulting from this reaction appears to be almost
exclusively that of (p-C)•-, which confirms that the absorption
of (NP-III)•+ is very weak.70 It is, thus, not surprising that the
transient absorption spectrum observed following ground-state
quenching of the triplet state is dominated by that of the radical
anion, (NP-III)•-. From a plot of the observed growth at 680
nm as a function of the concentration of NP-III, the rate constant
for reaction 7 was determined to be 5 × 109 M-1 s-1 71
.
In deaerated solution, the triplet states of all naphthaldiimides
studied underwent electron-transfer with the ground-state, giving
rise to an observable radical anion. The rate constant for this
reaction (which also takes place in methanol and in water) is
high (in acetonitrile, k was determined to be 4.9 × 109 M-1 s-1
for compound 2, 5.5 × 109 M-1 s-1 for NP-III, and 6 × 109
M-1 s-1 for compound 3 and in PBS, a k value of 3.3 × 109
M-1 s-1 was obtained for NP-III).72 The transient absorption
spectra recorded for the radical anions of these compounds were
all very similar with a large absorption band at 480 nm, a
shoulder at 530 nm, and several weaker bands around 610, 690,
and 770 nm. The same reaction between the triplet state and
the ground-state was observed to occur in the naphthalimide
series. However, the rate constant of electron-transfer was found
to be an order of magnitude lower (k ) 4.8 × 108 M-1 s-1 for
compound 1 in acetonitrile)72 than in the case of the naphthal-
diimide derivatives. In addition, under identical conditions,
formation of the radical anion of NP-II or compound 1 was
less obvious in the transient absorption spectra, suggesting also
a lower efficiency of the electron-transfer reaction. Similarly,
the rate constant for electron-transfer from DABCO to the triplet
Figure 7. Transient absorption spectrum of (NP-III)•-, the radical anion
of NP-III resulting from reduction of the triplet state of NP-III by
DABCO (100 µM). The spectra were recorded 2 (b), 15 (O), 30 (2),
and 70 µs (4) after the laser pulse (λexc ) 355 nm). The absorbance of
the solution was ≈ 0.4 at λexc ([NP-III] ≈ 20 µM), and the laser intensity
was 8 mJ cm-2 pulse-1
.
In order to identify Y, the radical anion and radical cation of
the bis-hydroperoxy naphthaldiimide, were generated by inde-
pendent pathways. Excitation (λexc ) 355 nm) of a deaerated
solution of NP-III in acetonitrile containing the electron donor,
1,4-diazabicyclo[2,2,2]octane (DABCO, 100 µM) results in the
production of a species with the absorption spectrum shown in
Figure 7. This spectrum displays a large absorption band with
a maximum at 480 nm, a shoulder at 530 nm and other weaker
bands at 610, 680, and 760 nm.
state of compound 1 was determined to be 5 × 109 M-1 s-1 73
,
It can be attributed to (NP-III)•-, the radical anion of NP-III,
which is significantly lower than that obtained for the naph-
thaldiimides. The differences in the quenching of the triplet
states of naphthalimides and naphthaldiimides by electron-
transfer from DABCO or the corresponding ground-states, can
be explained by the much more negative reduction potentials
of the naphthalimides. In acetonitrile, Er1e/2d values of -1.195 V
Vs SCE and -1.23 V Vs SCE were measured for NP-II and
compound 1, respectively, whereas two reductions were ob-
3
formed by electron-transfer from DABCO to (NP-III)*, as
described by reaction 5. In acetonitrile, this reaction was found
to be diffusion controlled, by monitoring the growth of (NP-
III)•- at 610 nm as a function of DABCO concentration (from
1 to 100 µM).
3(NP-III)* + DABCO f (NP-III)•- + (DABCO)•+ (5)
served in the case of the naphthaldiimides with, for example,
red
1/2
E
values of -0.385 and -0.695 V Vs SCE for NP-III.74
The absorption spectrum of (NP-III)•- shows the same profile
as that of Y (Figure 5B). The absorption due to the radical
cation of DABCO is very weak in this region68 and does not
contribute significantly to the observed spectrum at λ > 400
nm. Thus, it can be concluded that the secondary long-lived
species is (NP-III)•-, resulting from the one-electron transfer
between the ground-state and the excited triplet state of the
hydroperoxy derivative, as shown in eq 6.
Figure 8 shows the spectra recorded after laser flash pho-
tolysis (λexc ) 355 nm) of a deaerated solution of compound 1
in acetonitrile in the absence and presence of DABCO (1 mM).
The spectrum of the radical anion of 1 is very different from
that of (NP-III)•-, it displays a large absorption band at 415
nm and a small shoulder around 490 nm. It is similar to that
recorded for NP-II under the same conditions and to that
reported for the radical anion of N-phenyl-1,8-naphthalimide
formed by reaction with triethylamine in acetonitrile.63
3(NP-III)* + NP-III f (NP-III)•- + (NP-III)•+ (6)
(70) (a) The molar absorption coefficient of the radical cation of the
closely related naphthalene was reported70b to be 2970 M-1cm-1 at 685
nm. (b) Gschwind, R.; Haselbach, E. HelV. Chim. Acta 1979, 62, 941-
965.
This reaction also leads to simultaneous production of the
radical cation, (NP-III)•+. However, the similarity of the
absorption spectra obtained on reaction of 3(NP-III)* with either
DABCO or ground-state NP-III suggests that the contribution
of the radical cation to the spectrum formed by the latter reaction
is negligible. An attempt was made to generate (NP-III)•+
through 355 nm excitation of p-chloranil (p-C) to its strongly
oxidizing triplet state, which can then act as an electron acceptor
in the presence of NP-III.69
(71) At 680 nm, the absorption is mainly due to (p-C)•-. However, by
comparison to known radical cations of closely related compounds such as
naphthalene,70b a small part of the absorption at 680 nm can be due to
(NP-III)•+
.
(72) In these experiments, the concentrations of naphthaldiimides 2 and
3 were varied from 5 to 50 µM, that of compound 1 was varied from 20 to
700 µM. In PBS, the concentration of NP-III was varied from 5 to 25 µM.
(73) This reaction rate constant was determined by monitoring the growth
at 415 nm for DABCO concentrations between 5 and 500 µM.
(74) (a) The limits for the cyclic voltammetry experiments were -2 and
+2 V Vs SCE. This explains that the oxidation of NP-III, which is expected
to have a potential >2 V Vs SCE as estimated by the equation proposed by
Loufty and Sharp,74b was not observed. (b) Loufty, R. O.; Sharp, J. H.
Photogr. Sci. Eng. 1976, 20, 165-174.
(68) Shida, T.; Nosaka, Y.; Kato, T. J. Phys. Chem. 1978, 82, 695-
698.
(69) p-Chloranil has an ET value48b of 49.3 kcal mol-1 and therefore
cannot sensitize the formation of the triplet state of NP-III.

11792 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
AVeline et al.
material. The negative part in this spectrum (Figure 4A) shows
a mirror image symmetry with the ground-state absorption
spectrum. Under such conditions, the assumption is made that
X does not possess significant absorption in this region and the
absorbance change is a simple consequence of loss of the
ground-state, which has a known absorption coefficient at 380
nm, ꢀ(380 nm). The formation of X at the expense of the
ground-state allows estimation of the molar absorption coef-
ficient of X by direct comparison of the ∆A values of the signals
at the depletion region and the visible absorption of the transient.
Using such an approach and eq 8, an absorption coefficient of
21 600 M-1 cm-1 was obtained at 465 nm.77
ꢀ(X) ) ꢀ(380nm)∆A(465nm)/∆A(380nm)
(8)
The quantum yield of formation of the primary long-lived
species X was then determined by comparative actinometry
using benzophenone in benzene as standard. This experiment
yielded a Φ(X) value of 0.03.
Figure 8. Transient absorption spectra of the triplet state (b) and of
the radical anion (O) of compound 1 produced by electron-transfer from
DABCO (1 mM) to the triplet state of the naphthalenic molecule. Each
spectrum was recorded 8 µs after laser flash photolysis (λexc ) 355
nm) of a solution of 1 in deaerated acetonitrile. The laser intensity
was 8 mJ cm-2 pulse-1, and the absorbance of the sample was 0.4 at
355 nm.
Clues to the identity of X were obtained by comparison of
transient absorption spectra recorded in oxygen-saturated aceto-
nitrile for all the derivatives studied. Formation of a primary
long-lived species was also observed to take place in the case
of compound 2 but was not detected for any of the other
naphthaldiimides or naphthalimides investigated. From a
consideration of these findings and the chemical structures of
these molecules, it can be concluded that the generation of a
primary long-lived species requires the starting material (1) to
be a naphthaldiimide, and, (2) to possess a hydrogen atom at
the γ position.78
Further information on the nature of this species was obtained
from the photoconductivity experiments outlined above, which
led to the conclusion that X is an uncharged intermediate. The
decay of X was also found to be oxygen-independent. In the
presence of the hydrogen donor, 1,4-cyclohexadiene (1,4-CHD,
500 mM), the transient absorption spectrum recorded on 355
nm excitation of NP-III in oxygen-saturated acetonitrile, showed
spectral characteristics identical to those seen in the absence of
1,4-CHD, but a large increase in the signal intensity was
observed. This suggests that the species produced by hydrogen
abstraction from 1,4-CHD is structurally close to X. Under
these experimental conditions, the triplet state is quenched by
oxygen (k ) 2.5 × 109 M-1 s-1, [O2] ) 9.1 mM) rather than
by the hydrogen donor (k ) 2.9 × 105 M-1 s-1, [1,4-CHD] )
500 mM), therefore the singlet state of NP-III is the excited
intermediate involved in the hydrogen abstraction. This conclu-
sion is confirmed by the efficient fluorescence quenching
observed for NP-III in acetonitrile in the presence of 1,4-CHD
(50 mM to 1 M).
The radical anions of the naphthalimides and naphthaldiimides
were also found to display different reactivities toward oxygen.
The decay of (NP-III)•- was identical under air- and N2-saturated
conditions whereas a large quenching by oxygen was observed
in the case of (NP-II)•-, which suggests different electron
distributions in these intermediates.
By analogy with other carbonyl compounds, the high rate
constant observed for electron-transfer from DABCO to the
3
triplet state of NP-III suggests that (NP-III)* (as well as the
triplet states of the other naphthalenic derivatives studied)
behaves with typical ππ* characteristics. In order to verify this
statement, the reactivity of 3(NP-III)* toward hydrogen abstrac-
tion, a reaction characteristic of nπ* ketone triplets, was
examined. Relatively low rate constants for triplet quenching
by known hydrogen donors such as isopropyl alcohol (k < 1.6
× 104 M-1 s-1) and 1,4-cyclohexadiene (k ) 2.9 × 105 M-1
3
s-1) confirm that (NP-III)* is predominately ππ* in nature.75
Previous work has shown that this is also the case for a variety
of naphthalimides.56,76
Primary Long-Lived Species. As previously mentioned,
laser flash photolysis of NP-III in acetonitrile produces a primary
long-lived species, X, absorbing at 465 nm (see Figure 4) which
appears to be formed within the laser pulse. The amplitude of
the absorption, measured following decay of other transient
species, is identical whether irradiation is carried out in air- or
oxygen-saturated solution. It is also unaffected under conditions
where the addition of quenchers such as perylene induces
complete scavenging of the triplet state. These results indicate
that the production of X is not mediated by the triplet state but
more probably by the singlet state (S1) of NP-III.
Estimations of the molar absorption coefficient, ꢀ(X), and
quantum yield of formation, Φ(X), of the primary long-lived
species were determined in the following manner. Under
oxygen-saturated conditions, X is the only intermediate detected
at times > 100 ns (the triplet state, which is efficiently quenched
by O2, is not observed). Therefore, the spectrum recorded is
the difference between the spectra of X and the ground-state
Formation of the primary long-lived species was also found
to take place in other solvents such as ethyl ether, acetone and
methanol. The spectrum of X in ethyl ether was identical to
that recorded in acetonitrile, whereas the spectra obtained in
acetone and methanol were red-shifted and presented a large
absorption band at 480 nm instead of 460 nm. Different
behavior was observed in PBS, where formation of a primary
long-lived species was not detected for both NP-III and
(77) This value must be viewed as a lower limit due to the assumptions
made.
(78) Further evidence for the intramolecular hydrogen abstraction was
obtained using N,N′-bis-(2-methylpropyl)-1,4,5,8-naphthaldiimide. Pulsed
excitation of this molecule leads to the formation of a primary long-lived
species Via the singlet state, S1. The absorption spectrum of the oxygen-
independent species produced was observed to be similar to that generated
from compound 2. However, in the case of N,N′-bis(2-methylpropyl)-1,4,5,8-
naphthaldiimide, the quantum yield of this photoprocess was found to be
much lower than for NP-III and compound 2.
(75) In these experiments, the concentration of 1,4-cyclohexadiene was
varied from 0.1 to 1 M and that of isopropyl alcohol was increased up to
1 M.
(76) Demeter, A.; Berces, T.; Biczok, L.; Wintgens, V.; Valat, P.;
Kossanyi, J. J. Chem. Soc. Faraday Trans 1994, 90, 2635-2641.

Photochemistry of Naphthalimides and Naphthaldiimides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11793
Discussion
compound 2, suggesting that hydrogen-bonding between the
naphthalenic derivatives and water inhibits the production of
the primary long-lived species.
Generation of the Hydroxyl Radical (•OH). NP-III was
The results obtained show that the photophysical and
photochemical properties of the naphthalimides (NI) and naph-
thaldiimides (NDI) display a large, structurally-dependent
variation.83 A distinct difference in the absorption and fluo-
rescence behavior is observed between the NI and NDI families.
The long-wavelength absorption bands of these two series
undergo opposite spectral shifts with change in solvent polarity,
suggesting that the S1 states are of different configuration. For
NI, the red-shift caused by increasing solvent polarity is in
accordance with a ππ* configuration whereas the blue-shift in
the case of NDI more resembles nπ* behavior. This difference
in electronic configuration between NI and NDI can be expected
to exert a profound effect on subsequent reaction mechanisms.
Confirmation is provided by the fact that among the molecules
bearing a hydrogen atom at the γ position relative to the
carbonyl group, intramolecular hydrogen atom abstraction was
only seen to occur in the NDI compounds (NP-III and compound
2) but not in NP-II.
developed with the aim of efficient generation of the hydroxyl
•
radical (•OH) on near-UV irradiation. Since OH exhibits
negligible absorption in the visible range, its production cannot
be detected by direct spectroscopic means.28,29 Thus, indirect
trapping experiments were required to determine whether •OH
was produced by photolysis of NP-III. N,N-Dimethyl-p-
nitrosoaniline (RNO) has been used in this capacity as its
reaction with •OH leads to the photobleaching of its character-
istic 420 nm absorption band.79,80 Photolysis (λexc ) 355 nm)
of NP-III (15 µM) in oxygen-saturated acetonitrile in the
presence of RNO (50 µM) resulted in a loss of RNO absorption
at 420 nm which followed first-order kinetics (the rate constant
of RNO bleaching was determined to be k ) 9 × 109 M-1 s-1).
•
In order to verify that the bleaching of RNO was due to OH
generation (RNO also reacts with singlet oxygen) control
On excitation of NP-III and compound 2, a primary long-
lived species was observed to be formed and was demonstrated
to arise from the S1 state of the NDI. The photochemistry of
NP-III showed that the hydroxyl radical was produced from
the S1 state with a quantum yield identical to that of X,
suggesting a common pathway for the formation of these two
intermediates. A plausible mechanism for the generation of •OH
involves the intramolecular γ hydrogen abstraction to generate
the 1,4-biradical 4 followed by cleavage of the hydroperoxy
experiments were carried out as follows. Addition of tert-butyl
alcohol or DMSO, efficient OH quenchers, resulted in the
•
inhibition of the RNO photobleaching. Under oxygen-saturated
3
conditions, direct reaction between (NP-III)* and RNO was
ruled out since under the same conditions, the triplet state of
the structurally close compounds 2 and 3 did not show any
•
interaction with RNO. All the above demonstrate that OH is
indeed responsible for the photobleaching of RNO on excitation
of NP-III.81
•
bond to give OH and the ketyl radical 5, as shown by eq 9.
By monitoring the magnitude of the bleached RNO ab-
sorbance at 420 nm as a function of laser intensity and carrying
out comparative actinometry, the quantum yield of RNO
•
photobleaching, and therefore that of OH production, was
•
determined to be 0.03. That OH and X are both generated
under oxygen-saturated conditions and that their quantum yields
of formation are identical [Φ(•OH) ) Φ(X)] suggest that these
species are produced through the same process and have the
same precursor, i.e. the lowest singlet state S1 of NP-III. This
conclusion is supported by the fact that in the case of the
hydroperoxy derivatives, •OH is only detected when formation
of the primary long-lived species is also observed (no RNO
bleaching was seen for NP-II in acetonitrile, and pulsed
excitation of NP-III in PBS, in the presence of potassium
thiocyanate (KSCN) did not give rise to a detectable amount
of (SCN)2•- formed by reaction of •OH with SCN-, although a
lower value than 0.03 is close to our estimated detection limit
for the KSCN method).82
(79) Vidoczy, T.; Blinov, N. N.; Irinyi, G.; Gal, D. J. Chem. Soc. Faraday
Trans 1, 1988, 84, 1075-1081.
(80) Buxton, G. V.; Greenstock, W. P.; Helman, A. B. Critical Review
of Rate Constants for Reactions of Hydrated Electrons, Hydrogen Atoms
and Hydroxyl Radicals (•OH/O•-) in Aqueous Solution. J. Phys. Chem.
Ref. Data 1988, 17, 523.
(81) From a study of the literature80 it would appear that the rate constant
•
for reaction between OH and acetonitrile (CH3CN) is much higher than
estimated by Vidoczy et al. in ref 79. Under the conditions used here and
•
in their experiments, the reaction between OH and CH3CN should occur
•
much faster than that of OH with RNO. The solvent radical species thus
These two processes (intramolecular hydrogen abstraction and
O-OH bond cleavage) are both very rapid and the 1,4-biradical
4 is not observed with the detection system used in this study.
This is not surprising since singlet biradicals are expected to
have very short lifetimes.84 Ketyl radicals such as 5 are
formed must then react with RNO resulting in its bleaching. The rate
•
constants for reaction of OH with tert-butyl alcohol or DMSO are higher
than that for CH3CN and at the concentrations used here, they quench •OH
before its reaction with solvent and inhibit RNO bleaching. The reaction
rate constant measured here is therefore that of the solvent radical with
RNO but the quantum yield value should be correct providing all solvent
radicals react with RNO. This also explains why some of the rate constants
•
attributed to OH with a variety of compounds by this method were much
(83) A discussion of the differences between the photoproperties of NI
and NDI molecules will be published elsewhere. Here we wish to discuss
these differences in view of their consequences in the biological applications
of these compounds.
lower than expected.79
(82) The lowest quantum yield of OH production by direct excitation
•
of the hydroperoxy naphthalenic derivatives was estimated to be 0.005 for
the RNO method and 0.025 for the KSCN method.
(84) Johnston, L. J.; Scaiano, J. C. Chem. ReV. 1989, 89, 521-547.

11794 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
AVeline et al.
normally very reactive toward oxygen. One explanation for
the lack of oxygen reactivity observed for X is the rearrangement
shown in eq 10 to give the oxygen-centered radical 6.85
unaffected by O2 whereas the radical anion of NP-II is very
oxygen sensitive. The radical anions of related N-substituted
1,8-naphthalimides studied by Berces and co-workers76 exhibit
spectra similar to that of (NP-II)•- and are proposed to have
the structure shown in 8, based on theoretical calculations. The
presence of a carbon-centered radical in this intermediate
explains the reactivity toward oxygen.
In the case of (NP-III)•-, rearrangement may again occur along
the extended molecule to give the oxygen-centered radical anion
9. Supporting evidence for this assignment comes from the
absence of reactivity toward oxygen and the fact that addition
of a few drops of HCl to a deaerated solution of NP-III in
acetonitrile results in a change in the spectrum of (NP-III)•- to
a spectrum more in agreement with X produced from excitation
of NP-III in aerated acetonitrile. Protonation of (NP-III)•-
would give 10, the chromophore of which is essentially the same
as that of 6.
A similar mechanism involving hydrogen abstraction and O-OH
•
bond cleavage as a source of OH was previously suggested
3
for NP-III but proposed (NP-III)* as the precursor.22 The
experiments reported here provide a refinement of the mecha-
nism to involve the singlet state rather than triplet state as the
•
excited state intermediate responsible for OH generation.
The naphthalimide analog NP-II does not exhibit formation
of such a primary long-lived species in aerated acetonitrile, even
though a γ hydrogen is available. There are three possible
explanations for this behavior: (1) the ππ* nature of the S1
state of NP-II reduces the efficiency of this reaction compared
to the nπ* S1 state of NP-III, (2) the hydrogen abstraction occurs
and the 1,4-biradical is formed but it then collapses back to the
starting material or gives cyclic products, (3) hydrogen abstrac-
tion takes place but the resulting 1,4-biradical and subsequent
products are transparent at λ > 300 nm. The absence of
hydroxyl radical generation from NP-II suggests that (1) and/
or (2) applies. The product of an intramolecular γ hydrogen
abstraction reaction and subsequent •OH liberation would be 7,
which is a ketyl radical and should be detectable, as would the
•
liberated OH.
The triplet states of both NI and NDI compounds behave in
a similar manner, typical of a ππ* configuration. Electron-
transfer reactions were observed to be very efficient between
all triplet states and electron donors such as DABCO. Ad-
ditionally, these triplet states are relatively unreactive toward
hydrogen abstraction, even with excellent hydrogen donor
molecule like 1,4-CHD (k ) 2.9 × 105 M-1 s-1 for NP-III in
acetonitrile). This rate constant can be equated to that exhibited
by a true nπ* triplet state such as that of benzophenone for
example (for which, in acetonitrile, a k value of 2.9 × 108 M-1
s-1 was reported for hydrogen abstraction from 1,4-CHD).86
The radical anions of NI and NDI display very different
absorption spectra (see Figures 5B and 8). They also display
different reactivity toward oxygen: (NP-III)•- was found to be
Given the versatility of NI and NDI molecules in biological
applications, it is of interest to compare the mechanistic
photochemistry presented here with the observed photobiological
effects of these compounds in order to arrive at informed
speculations concerning the mechanistic basis of the biological
phenomena. Triplet state formation is an efficient process in
NP-III (Φisc ) 0.39 in acetonitrile and 0.24 in PBS) and various
reactions can take place from this state. In the presence of
oxygen, energy transfer to O2 leads to the formation of singlet
oxygen (O2, 1∆g) with high efficiency for such ππ* triplet states
and as this species is highly oxidative, Type II biological damage
may ensue in the presence of suitable substrates. Alternatively,
(85) It is worthwhile noting that two structures can be drawn, one where
the radical is localized at the oxygen atom in the 4 position and the other
where the radical is borne by the oxygen atom in the 5 position.
(86) Encinas, M. V.; Scaiano, J. C. J. Am. Chem. Soc. 1981, 103, 6393-
6397.
3
it has been demonstrated that (NP-III)* is both a strong pro-
oxidant and reductant in one-electron transfer reactions and Type

Photochemistry of Naphthalimides and Naphthaldiimides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11795
Scheme 1. Possible Excited State Decay Channels for NP-III in the Presence and the Absence of an Electron-Donor Molecule,
D
I biological damage87 may be induced in this fashion. Singlet
state reactions also lead to the formation of reactive intermedi-
ates which must be considered in context of reactions in
biological media. Intramolecular hydrogen abstraction and
subsequent rearrangement give rise to hydroxyl radicals (one
of the most oxidative species known) and other free radical
species. Clearly then, NP-III is a highly photoreactive molecule,
capable of functioning by various means Via oxygen-dependent
and oxygen-independent mechanisms.
Bromo-substituted naphthalimides have been used in the
neutralization of viruses such as herpes simplex and HIV-1 to
sterilize blood and blood products.7-9 The same types of
compounds have also been shown to induce tissue repair on
irradiation with visible light (420 nm).13,14 In blood purification,
the photodynamic reaction can be mediated by either singlet
oxygen or by electron-transfer with viral protein leading to
denaturation and loss of protein function. Preliminary indica-
tions of oxygen independence suggest the latter effect to be
effective although the contribution of the singlet oxygen pathway
leading to protein damage and inactivation cannot be discarded
under the conditions normally needed for such a procedure. In
the tissue welding application, the formation of cross-linked
collagen appears to be necessary to impart strength to the weld.
Although singlet oxygen has been proposed to initiate crosslink-
ing in proteins, it is likely that the bis-naphthalimides used are
reacting directly with collagen at both reactive “ends” to form
a bridged crosslink. This reaction is likely of an electron-
transfer nature with available electron rich amino acids in the
collagen since the bisnaphthalimides were shown to be reactive
toward tryptophan, tyrosine, methionine, and cysteine in an
oxygen-independent manner.
Both NP-II and NP-III have been shown to induce sequence-
specific photonuclease activity by initiating piperidine-assisted
cleavage of DNA at the 5′G of 5′-GG-3′ sequences.20,21 Both
hydroxyl radical formation and electron-transfer reactions from
guanine residues have been proposed to be responsible for this
effect. Our results clarify this situation as NP-II does not
•
produce detectable levels (Φ < 0.005) of OH, in contrast to
NP-III. Guanine is the most easily (one-electron) oxidized base
and is a good electron donor which reacts readily with the triplet
state of NP-II (k ) 3.6 × 108 M-1 s-1) and that of NP-III (k )
2.5 × 109 M-1 s-1) in PBS, in the form of guanosine. The
electron-transfer reaction can be considered the most likely
pathway for this reaction given the 5′-GG-3′ sequence selectivity
which has been shown to be typical of electron acceptor
photonucleases such as methylene blue88 and anthraquinone
derivatives,89,90 in contrast to the random cleavage patterns
Conclusion
•
produced by other OH generating systems.91 This selectivity
This work has shown the mechanistic basis for the varied
uses of naphthalenic imides in photobiological applications.
These compounds can interact Via oxygen-dependent and/or
oxygen-independent mechanisms, thus, overcoming a potential
limitation to their use in photodynamic applications. Subtle
changes in the electronic character of the lowest excited states
have large effects on the photochemistry of these compounds.
NP-III, was confirmed to be a hydroxyl radical photogenerator,
although not in a specific fashion. However, alternative reaction
mechanisms such as singlet oxygen sensitization and electron-
transfer ability, provide this compound with a strong arsenal of
reactivity with which to affect and alter biological systems. The
reaction mechanisms operating in such milieu will likely be
determined by the environment of the compound and the
accessibility of reactive substrates for each pathway (Scheme
1).
has been explained recently to be due to the 5′G of 5′-GG-3′ to
be the most electron donating site in DNA and subsequent acts
as a “hole” sink.92 In model systems both NP-II and NP-III
were shown to generate 8-hydroxyguanosine (8-OHdG) from
irradiation in the presence of guanosine, the efficiency of this
reaction being much higher for NP-III than NP-II.21 8-OHdG
is a fairly nonspecific mechanistic indicator as it has been
•
demonstrated to arise from reaction of guanine with OH,
O2(1∆g) and by one-electron oxidative reactions.93 Saito and
co-workers have demonstrated the efficacy of a 1,8-naphthal-
imide derivative containing no hydroperoxy group in the
N-substituent in generating sequence-specific cleavage at 5′G
•
of 5′-GG-3′ sites. Obviously, OH cannot be involved in this
reaction and electron-transfer from guanine is probable.
(87) Foote, C. S. Photochem. Photobiol. 1991, 54, 659.
(88) Schneider, J. E.; Price, S.; Maidt, L.; Gutteridge, J. M. C.; Floyd,
R. A. Nucleic Acids Res. 1990, 18, 631-635.
Acknowledgment. We gratefully acknowledge Susan Blood
from the Worcester Polytechnic Institute (Worcester, MA) for
assistance in the cyclic voltammetry measurements. This work
was supported by the MFEL program of the ONR under
Contract N00014-94-1-0927.
(89) Armitage, B.; Yu, C.; Devadoss, C.; Schuster, G. B. J. Am. Chem.
Soc. 1994, 116, 9841-9889.
(90) Breslin, D. T.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 2311-
2319.
(91) Portugal, J.; Waring, M. J. FEBS Lett. 1987, 225, 195-200.
(92) Sugiyama, H.; Saito, I. J. Am. Chem. Soc. 1996, 118, 7063-7068.
(93) Kasai, H.; Yamaizumi, Z.; Berger, M.; Cadet, J. J. Am. Chem. Soc.
1992, 114, 9692-9694.
JA971993C