Water-soluble naphthalene diimides as singlet oxygen sensitizers

Article abstract of DOI:10.1021/jo401347z

Bromo- and/or alkylamino-substituted and hydrosoluble naphthalene diimides (NDIs) were synthesized to study their multimodal photophysical and photochemical properties. Bromine-containing NDIs (i.e., 11) behaved as both singlet oxygen (¹O₂) photosensitizers and fluorescent molecules upon irradiation at 532 nm. Among the NDIs not containing Br, only 12 exhibited photophysical properties similar to those of Br-NDIs, by irradiation above 610 nm, suggesting that for these NDIs both singlet and triplet excited-state properties are strongly affected by length, structure of the solubilizing moieties, and pH of the solution. Laser flash photolysis confirmed that the NDI lowest triplet excited state was efficiently populated, upon excitation at both 355 and 532 nm, and that free amine moieties quenched both the singlet and triplet excited states by intramolecular electron transfer, with generation of detectable radical anions. Time-resolved experiments, monitoring the 1270 nm ¹O₂ phosphorescence decay generated upon laser irradiation at 532 nm, allowed a ranking of the NDIs as sensitizers, based on their ¹O₂ quantum yields (Φ_Δ). The tetrafunctionalized 12, exhibiting a long-lived triplet state (τ ～ 32 μs) and the most promising absorptivity for photodynamic therapy application, was tested as efficient photosensitizers in the photo-oxidations of 1,5-dihydroxynaphthalene and 9,10-anthracenedipropionic acid in acetonitrile and water.

Full text of DOI:10.1021/jo401347z

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Article
Water Soluble Naphthalene Diimides as Singlet Oxygen Sensitizers
Filippo Doria, Ilse Manet, Vincenzo Grande, Sandra Monti, and Mauro Freccero
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Jul 2013
Downloaded from http://pubs.acs.org on July 19, 2013
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The Journal of Organic Chemistry
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Water Soluble Naphthalene Diimides as Singlet Oxygen
Sensitizers
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Filippo Doria,a Ilse Manet,b* Vincenzo Grande,a Sandra Monti,b and Mauro Frecceroa*
aDipartimento di Chimica, Università di Pavia. V.le Taramelli 10, 27100 Pavia, Italy.
bIstituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, via Gobetti 101,
40129 Bologna, Italy
mauro.freccero@unipv.it
ilse.manet@isof.cnr.it
Hydrosoluble naphthalene diimides exhibiting multimodal photophysical and photochemical properties.
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Abstract: Br and/or alkylammino substituted and hydrosoluble naphthalene diimides (NDIs) were
synthesized to study their multimodal photophysical and photochemical properties. Br containing NDIs
(i.e., 11) behaved as both singlet oxygen (1O2) photosensitizers, and fluorescent molecules, upon
irradiation at 532 nm. Among the NDIs not containing Br, only 12 exhibited similar photophysical
properties to BrꢀNDIs, by irradiation above 610 nm, suggesting that for these NDIs both singlet and
triplet excited state properties are strongly affected by length, structure of the solubilizing moieties and
pH of the solution. Laser flash photolysis confirmed that the NDI lowest triplet excited state was
efficiently populated, upon excitation at both 355 and 532 nm, and that free amine moieties quenched
both the singlet and triplet excited states by intramolecular electron transfer, with generation of
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detectable radical anions. Timeꢀresolved experiments, monitoring the 1270 nm O2 phosphorescence
decay generated upon laser irradiation at 532 nm, allowed a ranking of the NDIs as sensitizers, based on
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their O2 quantum yields (Φ ). The tetraꢀfunctionalized 12, exhibiting a longꢀlived triplet state (τ ~ 32
ꢁs) and the most promising absorptivity for photodynamic therapy application, was tested as efficient
photosensitizers in the photoꢀoxidations of 1,5ꢀdihydroxylnaphthalene and 9,10ꢀanthracenedipropionic
acid in acetonitrile and water.
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Introduction
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Photodynamic therapy (PDT) is based on the use of a lightꢀactivatable chemical, called
photosensitizer (PS), and irradiation with light of appropriate wavelength to impart cytotoxicity via the
generation of reactive molecular species.1ꢀ3 Typically, the useful range of wavelengths for therapeutic
activation of the PS is 600ꢀ800 nm in order to optimize tissue penetration. The main process occurring
upon excitation of the photosensitizer is the formation of the PS triplet excited state and energy transfer
to molecular oxygen yielding singlet oxygen (1O2),4 a very cytotoxic species. It is important to stress
that photosensitizers that preserve their fluorescence, due to incomplete singletꢀtriplet intersystem
crossing (ISC), can act also as fluorescent agents enabling contemporarily localization of the PS in the
biological tissue and occurrence of the therapeutic action.5 This is a very appealing objective in the
frame of multimodal theranostic applications.
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The triplet sensitizers actually used for PDT are limited to transitionꢀmetal complexes of
porphyrinoids, reduced porphyrins and phthalocyanines with high ISC efficiency due to the heavy atom
effect of the transition metal.2,3,6ꢀ8 Among their main drawbacks are low solubility and tendency to
aggregate in aqueous environment leading to non radiative decay pathways of their excited states and
consequently inefficient energy transfer to oxygen. More recently, porphyrinꢀlike photosensitizers have
been included by or bound covalently to nanoparticles with size dimension in the nanometer range to
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optimize delivery and O2 formation.9,10 Even though nanoꢀsized delivery systems for photosensitizers
offer several advantages, they suffer from drawbacks due to their complexity and often lower efficiency
in the 1O2 generation. Therefore the design and synthesis of water soluble organic triplet sensitizers with
intense absorption of red light, high photochemical stability and a longꢀlived triplet excited state is still
an interesting issue, particularly because very few have been reported yet. Recently, a bodipyꢀbased
triplet sensitizers soluble in water has been described as singlet oxygen sensitizer exhibiting anyway a
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rather low efficiency (yield in O2, 10%).11 During the last years, our attention has been focused on
naphthalene diimides (NDIs) and coreꢀsubstituted NDIs12 as selective nucleic acids (NAs) ligands and
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fluorescent probes. In more detail, Neidle’s group and our research unit have shown that triꢀ and tetraꢀ
substituted NDIs are potent and reversible ligands13ꢀ15 as well as alkylating agents targeting guanine rich
NAs folded into Gꢀquadruplex structures (G4s).16ꢀ19 Gꢀrich sequences able to fold into G4 are present in
biologically relevant protoꢀoncogenes and oncogenes promoters (like c-kit and c-myc)20ꢀ22 as well as
human telomeres and participate in biological processes crucial for cell replication and survival.21,23
Consequently they represent a very appealing target and propelled the research for the development of
new therapeutic approaches based on their selective targeting. Interestingly, the optoelectronic
properties of the NDI core can be effectively tuned by substitution,24ꢀ26 as electron rich substituents on
the aromatic core give origin to strong absorption and emission in the red spectroscopic window useful
for PDT applications. In addition, the NDI’s binding properties toward G4s14,17 may also be exploited
for potential selective photocleavage as suggested recently for cationic Znꢀphthalocyanines.27 This
prompted us to start from the NDI core carrying amine substituents on the naphthalene unit to engineer
efficient water soluble 1O2 photosensitizers, by structural modifications.
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Results and Discussion
Design and synthesis. In this work we have designed and synthesised a class of water soluble NDIs
(Table 1) with unique optoelectronic properties that can be used as efficient waterꢀsoluble singlet
oxygen photosensitizers, and fluorescent probes.
Table 1. Structures of the water soluble NDIs (1ꢀ12) investigated as singlet oxygen sensitizers.
X
n
Y
X=NMe2
X=NMe3+Cl-
(CH2)n
O
O
N
O
2
H
1
2
3
7
8
9
X
NH
Br
(CH2)n
NH(CH2)2X
Y
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H
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N
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(CH2)n
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NH(CH2)3X
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To synthesize the above triꢀ and tetraꢀsubstituted NDIs sketched in Table 1, we followed two
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different threeꢀsteps synthetic protocols. The commercially available anhydride was brominated with
dibromocyanuric acid and the resulting product mixture containing the 2,6ꢀdibromoꢀ1,4,5,8ꢀ
naphthalenetetracarboxylic acid dianhydride (13) was used for a classical imidation procedure under
acidic condition yielding the 2,6ꢀdibromoꢀsubstituted NDIs 14–15 [step (a) in the Scheme 1].28,29
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SCHEME 1. Synthesis of water soluble triꢀ and tetraꢀsubstituted NDIs. a) N1,N1ꢀdimethylethaneꢀ1,2ꢀ
diamine (n=2) or N1,N1ꢀdimethylpropaneꢀ1,3ꢀdiamine (n=3) in acetic acid, 90°C, 30 min, under N2;
43% yield; b) N1,N1ꢀdimethylethaneꢀ1,2ꢀdiamine (n=2) or N1,N1ꢀdimethylpropaneꢀ1,3ꢀdiamine (n=3),
acetonitrile, 70 °C, 4 h; c) N1,N1ꢀdimethylethaneꢀ1,2ꢀdiamine (n=2) or N1,N1ꢀdimethylpropaneꢀ1,3ꢀ
diamine (n=3), dry DMF, 135°C, 3 min; MW assisted and closed vessel synthesis; d) MeI, acetonitrile,
r. t., 16 h.
The following nucleophilic aromatic substitution (SNAr) is the key step of the whole synthetic
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protocol. Recently, regioselectivity of the SNAr on polyꢀbromosubstituted NDIs with aniline has been
successfully achieved in the presence of fluoride.30 The choice between two different conditions [(b)
and (c), Scheme 1] allows to discriminate the reactivity of the aromatic core, yielding mainly monoꢀ (c)
or diꢀfunctionalization (b). To synthetize the tetraꢀsubstituted NDIs (3, 6) in good yields, we performed
a oneꢀpot microwaveꢀassisted (MW) functionalization in DMF at 135°C for 3 minutes, in closeꢀvessel.
Differently, the monoꢀsubstitutions were obtained under milder conditions in acetonitrile at 70°C for 4h
using N1,N1ꢀdimethylethaneꢀ1,2ꢀdiamine, or N1,N1ꢀdimethylpropaneꢀ1,3ꢀdiamine. Under these milder
conditions only regioselective monoꢀfunctionalization of the 2,6ꢀdibromoꢀNDIs was efficiently achieved
to give mainly the new Brꢀderivatives 2, and 5. These BrꢀNDIs were prepared in order to improve
intersystem crossing by the presence of the heavy atom and efficiently populate the triplet excited
state.31 In addition, in order to increase the lifetime of both the singlet and triplet excited states,
inhibiting their deactivation by intraꢀmolecular electron Transfer (eT) quenching, the corresponding
quaternary ammonium salts 7ꢀ12 have been synthesized.
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The quaternary ammonium salts 7ꢀ12 were synthesized by exhaustive methylation of the
corresponding amines 1ꢀ6, and subsequently purified by HPLC as trifluoroacetate salts. For the NDIs
embedding the ethylene spacer (1ꢀ3), the reaction carried out in acetonitrile with an excess of methyl
iodide gave the desired products 7-9 with quantitative yields after 24 h at r.t. Following the same
strategy for the NDIs with the propyl amine substituents (4ꢀ6), a mixture of quaternary ammonium salts
partially methylated, were obtained. In order to get the NDIs 10ꢀ12 a suspension of sodium carbonate
was required to achieve quantitative conversion. Such a difference in reactivity is likely the result of a
different basicity of the amines. All the NDIs 1ꢀ12 have been purified by HPLC using ACN:H2O +
0.1% CF3COOH, and characterized as chloride salts.
Photophysical characterization: Figures 1a and 1b show the absorption and corrected fluorescence
spectra of the NDI quaternary ammonium salts 7ꢀ12 (Scheme 1).
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Figure 1. UVꢀvis absorption (solid lines) and fluorescence spectra (dotted lines) of the NDIs (a) 7-9 and
(b) 10-12. Excitation at 485 nm and 600 nm for 7, 8, 10, 11 and 9, 12, respectively (1 cm path length).
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The absorption spectra of the amines (1ꢀ6) in solutions of different pH are very similar to those of the
quaternary ammonium salts, so the protonation state of the terminal amine groups (NMe2) does not
influence the absorption spectra (see Supp. Inf.). The absorption band with vibronic signature in the
300ꢀ400 nm range is typical of the NDI core, and is not substantially affected by substitution.32 It clearly
emerges how the introduction of just one amine substituent is able to generate a second absorption band
in the visible of comparable intensity to that with maximum at 370 nm.
Compared to the Hꢀderivative bromination of the NDI core shifts the absorption band in the visible
spectrum to the red by ca. 10 nm, due to the Br atom acting as weak electron donating substituent. The
most significant shift of ca. 90 nm is anyway obtained by introduction of the second amine substituent
on the NDI core. With the introduction of the second amine substituent the NDI derivative complies
with the PDT requirements of strong absorption in the 600ꢀ800 nm spectroscopic window. As
underlined in several studies these interesting electronic properties arise from a charge transfer (CT)
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transition involving the doublet of the aromatic amines.25,33
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Table 2 collects the photophysical data of all the quaternary ammonium salts (7ꢀ12), in water, and
their related amines (1ꢀ6), under acid buffered conditions. Measurements were carried out under acidic
conditions in an attempt to protonate all terminal amine groups. Protonation of the aromatic amines was
negligible at pH above 1, as the absorption spectra did not change (see Supp. Inf.).
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Table 2. Photophysical properties of the NDI compounds in water for the salt derivatives in phosphate
buffer of pH 2 for the neutral derivatives.
a
c
λ
(nm)
ε
(Mꢀ1cmꢀ1)
Φ
τ (ns)b
τ
(ꢁs)d
T
Φ
NDIs
max
max
F
f
ꢀ
4
522
522
509
511
616
613
598
598
531
530
518
517
11000
11000
7100
0.19
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0.29
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0.17
0.17
0.26
0.27
0.11
0.12
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0.11
5.60
5.50
7.36
7.35
4.40
4.00
7.00
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3.50
3.33
3.40
3.00
ꢀ
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ꢀ
ꢀ
ꢀ
10
1
0.34
ꢀ
7
7100
0.18
ꢀ
6
7400
12
3
10700
13000
13000
6400
0.30
0.04
0.07
0.39
0.46
0.48
0.63
32
ꢀ
9
7.4
26
22
30
23
5
11
2
7600
7200
8
7300
aUsing Ru(bpy)3+ as reference with value of 0.028 in aerated water for Br and H derivatives and compound 11 in water as
reference for 3, 6, 9 and 12. bExcitation at 373 nm. cDetermined from phosphorescence of 1O2 at 1270 nm in D2O, under airꢀ
equilibrated conditions. All solutions are isosbestic at the 532 nm excitation wavelength. dTriplet lifetime in Argonꢀsaturated
solution. Excitation at 532 nm.
As expected the lowest fluorescence quantum yields and the shortest lifetimes have been observed for
the Brꢀderivatives. In fact the presence of the bromide substituent directly on the NDI core lowers both
parameters due to the heavy atom effect. Concerning the triꢀ and tetraꢀderivatives we observe an
unexpected effect of the length of the alkyl chain. In fact the ethyl derivatives (1, 3, 7 and 9) have better
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fluorescence performance compared to the propyl derivatives (4, 6, 10, 12). Considering the quaternary
ammonium salts having all aliphatic amines positively charged and not available for eT processes one
could conclude that the nonꢀradiative deactivation processes are more efficient for compounds with the
longer and more flexible alkyl spacer (n=3), as the fluorescence lifetimes are significantly reduced
compared to the those of the compounds with shorter spacer (n=2, see Table 2). Indeed radiative rate
constants (kr = Φ/τ) are very similar for H and tetraꢀderivatives, while the rate constant for nonꢀradiative
processes of the compounds with long spacer is twice that of the compounds with shorter spacer. The
fluorescence spectra of 5-7 at three different pH values are reported in ESI (Figures 4Sꢀ6S). At pH 7 we
observe lower relative ΦF values, suggesting deprotonation of an ammonium moiety and subsequent
quenching of the emitting singlet excited state by intramolecular eT. The fluorescence decay is
monoexponential at all pH values and the lifetimes values do not vary significantly. For a quantitative
evaluation of the acidity of the ammonium moieties and the remarkable effect of the spacer length on it,
fluorescence and potentiometric titrations have been performed.
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Evaluation of the ground state NDIs’ acidity by fluorescence and potentiometric titrations: The
mode of protonation of the solubilizing amino moieties tethered to the NDI core was investigated by
potentiometric and fluorescence titrations, as it controls the quenching of the excited states by eT. The
NDIs 3 and 6 were chosen as model, as their fluorescence quantum yields dropped passing from pH 2 to
pH 7. Fluorescence intensity change as a function of pH was analyzed for both the amines (Figure 2).
The resulting sigmoids clearly suggests that the deprotonation occurs at a lower pH for 3. The
absorption spectra of the NDIs 3 and 6 in water solutions are very similar at different pH, so the
protonation state of the terminal amine groups does not influence the absorption spectra at pH ≤ 9.
Under more basic conditions (pH≥9), the amine 3 exhibited an additional absorption band at longer
wavelength (λmax 625 nm) than that observed under acid and neutral conditions (λmax 597 nm). These
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spectroscopic data suggest an aggregation of 3 in dimer and/or oligomer aggregates, rather than a
deprotonation of the aromatic amine, as the latter has never been reported on similar NDIs in water. As
at pH > 10 precipitation of 3 occurred, we were unable to measure its pKa4 . Therefore, the
potentiometric titration of 3 yielded only three pKa values: pKa1 = 5.6, pKa2 = 7.4, and pKa3 = 7.7,
suggesting that when the amine moiety is fairly close to the NDI core, due to a short alkyl spacer, the
ammonium salts are rather acidic. In fact, the NDI 6 was much more basic and did not show any
detectable aggregation into dimers or oligomers at pH≤12. Therefore, we were able to measure all the
pKa constants for 6 (pKa1 = 8.1, pKa2 = 8.6, pKa3 = 8.8 and pKa3 = 11.3).
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Figure 2. Fluorescence intensity (If) vs pH. The sigmoids defined by the (a) empty squares and (b)
black circles describe the fluorescent quenching of 3 (at 640 nm) and 6 (at 661 nm), respectively, both
5x10ꢀ6M in aqueous solution, 0.1 M in NaNO3.
The speciation analysis in Figure 3, resulting from the potentiometric titrations describes the distribution
of tetraꢀcationic, vs triꢀ, bisꢀ, monoꢀcationic and neutral NDIs (NDIꢀH4, NDIꢀH3, NDIꢀH2, NDIꢀH and
NDI, respectively) as function of the pH. These data suggest that the amine 6 is mainly fully protonated
at pH 7 (94% of 6ꢀH4), unlike the amine 3, which exists as a mixture of both triꢀ (68%, 3-H3) and diꢀ
cationic (26%, 3ꢀH2) species.
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Figure 3. Speciation analysis resulting from the potentiometric titrations for the NDIs (a) 3 (tetrꢀ
cationic 3ꢀH4, triꢀcationic 3-H3, and diꢀcationic 3ꢀH2 distribution is reported) and (b) 6 (the distribution
of 6ꢀH4, 6ꢀH3, 6ꢀH2, 6-H and 6 are reported). The diamonds describe the fluorescent quenching.
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These data clearly indicate the NDI 6 is a better fluorescent probe than 3 under physiological
conditions; being fully protonated, it is not quenched by intraꢀmolecular eT. Surprisingly, the
fluorescence quenching of 3 occurs during the deprotonation of the second ammonium moiety in the
specie 3ꢀH3, probably because the first free amino group is not enough electron donor due to the closer
NDI core. On the contrary, the fluorescence quenching of 6 follows the deprotonation of the
tetracationic specie 6ꢀH4.
Spectroscopic and Kinetic Characterization of the NDI Triplet State: To provide a full
characterization of the synthesized NDI as singlet oxygen sensitizers, it is mandatory to examine the key
features of their triplet states. In fact, both the population of the triplet excited states and their lifetimes
have a direct effect on the efficiency for singlet oxygen production. Therefore, we set out to examine
these issues in detail for several of our NDIs, in acetonitrile and in water solution under neutral and
slightly acidic conditions (pH 5 and 7), by nanosecond laser flash photolysis using a NdꢀYAG laser,
operating at both 532 and 355 nm. Upon laser excitation at both 355 and 532 nm, transient absorption
spectra in the range of 380ꢀ750 nm, with λmax at 430 nm, were observed for all the substituted NDIs
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investigated. A second broad band exhibiting λmax centered at 530, 630 and 670ꢀ690 nm was recorded
for the monoꢀsubstituted (1 and 4), Brꢀsubstituted (2 and 5), and NHR diꢀsubstituted NDIs (9 and 12)
(Figures 4 and 5).
1
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Figure 4. Timeꢀresolved difference absorption spectra flashing a 8x10ꢀ6M solution of 1, 2 (at 355 nm),
3 (at pH 5) and 9 (at pH 7), both at 532 nm, in argon purged 10ꢀ2 M phosphate buffer.
Figure 5. Timeꢀresolved difference absorption spectra flashing a 8x10ꢀ6M solution of 4, 5 (at
355 nm), 6 (at pH 5) and 12 (at pH 7), both at 532 nm, in argon purged 10ꢀ2 M phosphate buffer.
Likely the electron donor character of the second substituent on the NDI core, passing from H, Br to
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NHR, resulted in a progressive red shift of the second band, which also became more intense. Bleaching
at 510ꢀ550 nm and 550ꢀ600 nm for the NDIs 1, 2, 4, 5 and 3, 6, 9, 12, respectively was due to the
depletion of the ground state of the NDIs upon excitation to the triplet excited state. All transient
absorption profiles monitored at both 430 nm and 680 nm decay monoexponentially in airꢀequilibrated
solutions with a lifetime of ~ 2 ꢁs (Figure 6).
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In argonꢀsaturated solutions lifetimes increase to ca. 20ꢀ30 ꢁs, indicating that at pulse end we are
very likely observing the triplet state absorption. The transient absorption spectra in Arꢀsaturated
solution observed from the pulse end to 80 ꢁs after (Figures 3 and 4) have been assigned to the triplet
state of each NDI, on the basis of its sensitivity to O2 and similarity to the T1ꢀTn absorption of nonꢀwater
soluble NDIs.34 The amines 3 and 6 exhibit a fairly similar spectra at pulse end to their quaternary
ammonium analogues 9 and 12. Nevertheless, at pH 5 both the amines 3, and 6 reveal also the
generation of a more stable species on a longer time scale (>200 ꢁs). This second species, which was
completely bleached by oxygen also exhibits an absorption spectra (λmax 480ꢀ490 nm and 660 nm) fairly
similar to the stable species generated by NDI monoꢀelectronic reduction. Therefore it can be assigned
to the NDI radical anions 3.- and 6.- generated by eT from free amine to the excited NDI core.35ꢀ37 In the
hypothesis that the NDIs 3 and 9 and the homologues 6 and 12 have similar molar absorption
coefficients for TꢀT absorption at 430 nm, we see that initial absorbance is much higher for 12
compared to the other tetraꢀsubstituted compounds 3, 6 and 9 (Figure 6). This allows us to conclude that
the triplet is likely formed with much higher efficiency in the case of 12. It is somewhat surprising that
its analogue 6 at pH 2 did not efficiently produce the triplet excited state. In spite of very similar
fluorescence properties of 6 and 12 the triplet formation yield seems much lower. A possible
explanation for this behavior may be the presence of the carbonyl group enabling excited state
intramolecular proton transfer (ESIPT) from the protonated aliphatic amine to the carbonyl.38
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Figure 6. Differential absorption decay monitored at a) 430 nm and b) 660 nm for 3, 6, 9, and 12 (8x10ꢀ
5M) and c) 430 nm for 2, 5, 8, and 11 (4x10ꢀ5M), in air equilibrated neutral D2O solution (9, 12, 8 and
11) or acidic D2O of pH 2 (3, 6, 2, and 5). All the solution were excited at 532 nm where they exhibited
identical absorbance of 0.3.
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Singlet Oxygen Generation Efficiency: The fluorescence data are coherent with the ability of
ꢀ
singlet oxygen production of these compounds. The singlet oxygen quantum yields (Φ ) reported in the
present study, and listed in Table 2, were obtained in timeꢀresolved experiments by comparing the initial
intensity of the 1270 nm singlet oxygen phosphorescence decay (Figure 7) produced upon irradiation at
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532 nm, (by means of a NdꢀYAG laser) of the given NDIs, to that of 5,10,15,20ꢀtetrakis(4ꢀ
1
2
3
4
39,40
ꢀ
sulfonatophenyl) porphyrin (TPPS) as standard sensitizer (Φ =0.76).
As expected the Brꢀderivatives (2, 5, 8 and 11) produce 1O2 in water. Our approach to introduce a Br
5
6
7
8
9
ꢀ
substituent favoring intersystem crossing gave thus positive results. The Φ values are good ranging
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from 0.39 to 0.63, even though they are lower compared to the TPPS value. From the point of view of
application in PDT their absorption extending up to 550 nm represents, however, a drawback. Under the
ꢀ
very same experimental conditions the Brꢀderivatives exhibit higher Φ than that of the another
conventional photosensitizer Rose Bengal (Figure 7). Such a difference may be due to aggregation and
TꢀT annihilation known to occur for Rose Bengal.
Figure 7. Timeꢀresolved phosphorescence signal of singlet oxygen in D2O (monitored at 1270 nm),
using the standards TPPS and Rose Bengal and the NDIs 2, 3, 5, 8, 9, 11, 12 as photosensitizers. For the
amines 2, 3 and 5 the pH was set to 2, by DCl addition. Excitation with NdꢀYAG laser at 532nm; energy
of 3.6 mJ/pulse; all solutions absorb equally at 532 nm.
0,020
TPPS
Rose bengal
8
2
5
0,015
11
12
3
0,010
9
0,005
0,000
0
1x10-4
2x10-4
3x10-4
4x10-4
time / s
More surprisingly, the quaternary ammonium salt 12 was the only NDI among those without Br, able
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to produce singlet oxygen in water with a very satisfactory yield (0.30). Such an efficiency was even
1
2
3
4
ꢀ
more striking in comparison to the very low Φ (0.07) for its analogue 9, exhibiting a shorter alkyl
5
6
7
8
spacer (n=2). The related ethylꢀ (n=2) and propylꢀ (n=3) amino analogues 3 and 6, produce singlet
oxygen with negligible yields (≤0.04), even at pH 2, which ensuring complete protonation of all the
amino moieties, erased the excited state quenching by intramolecular eT. As suggested above this
behavior may be due to the presence of the carbonyl group enabling excited state intramolecular proton
transfer from the protonated aliphatic amine to the carbonyl.38 This however does not explain the poor
behavior of compound 9 with respect to that of 12.
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Photooxidation of 1,5-dihydroxynaphthalene and 9,10-anthracenedipropionic acid in
acetonitrile and water by the tetra-cationic NDI 12. Sensitizers such as 12, which are characterized
by (i) intense absorption of red visible light, (ii) long living triplet and (iii) high singlet oxygen yields
are ideal for performing efficient photoꢀoxidations. In addition, the solubility of 12 in both acetonitrile,
and water allows to exploit its properties as singlet oxygen sensitizer in both solvents, also targeting
water soluble biomolecules. Thus, as a proof of concept, we decided to investigate 12 as singlet oxygen
sensitizer in the photoꢀoxidation of 1,5ꢀdihydroxynaphthalene (DHN) and 9,10ꢀanthracenedipropionic
acid (ADPA) in acetonitrile and water, respectively. Visible light at longer wavelength than 500 nm was
used. DHN was efficiently oxidized to its naphthoquinone derivative (5ꢀhydroxyꢀ1,4ꢀnaphthalenedione)
1
through the endoꢀperoxide, by O2.41 The resulting naphthoquinone absorbing at 360ꢀ440 nm, has been
efficiently monitored by the increase of its absorption, which appeared to be in the spectroscopic
window where the NDI shows negligible absorption (Figure 8a).
Unfortunately, the solubility in water of DHN is very poor, and we were able to perform a photoꢀ
oxidation of DHN only in aqueous acetonitrile (Supp. Inf.). Therefore, DHN has been replaced by
ADPA as water soluble 1O2 trap.42 Contrary to DHN, in this case, the photoꢀoxidation has been followed
by the decrease absorption at 400 nm due to the consumption of ADPA anthracene chromophore,
forming a colorless endoꢀperoxide (Figure 8b). Continuous photobleaching of the anthracene absorption
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was observed for 10 h by using excitation wavelength longer than 450 nm. Byproduct formation was not
detected by UVꢀabsorption during the irradiation. No decrease in the anthracene absorbance was
observed in the solutions without the sensitizer. To our knowledge this is the first fairly efficient photoꢀ
bleaching experiments of ADPA, performed in H2O, rather than in D2O, where the lifetime of singlet
oxygen is much longer. This evidence further suggests that 12 is a promising singlet oxygen sensitizer
for biological applications. A small blue shift (6 nm) of the absorption at 640 nm was detected during
the photooxidation, suggesting a weak interaction between ADPA and the NDI core, in water solution.
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Figure 8. Change in the absorption spectra by irradiation (at λ > 450nm) of (a) an acetonitrile solution
of DHN (1.2x10ꢀ4 M) and (b) an aqueous solution of ADPA (1.2x10ꢀ4 M), using 12 (2x10ꢀ5M and 6x10ꢀ5
M, respectively) as singlet oxygen photosensitizer.
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Conclusions
1
2
3
4
5
6
7
8
9
Naphthalene diimide derivatives with bromo and amino or quaternary ammonium substituents on
both the aromatic core and the imide moieties were prepared as water soluble singlet oxygen sensitizers,
in which water solubility derived from ionic substituents. Their photophysical properties were
thoroughly investigated by steadyꢀstate and timeꢀresolved spectroscopy. The red colored Brꢀcontaining
NDIs (2, 5, 8 and 11) were very efficient singlet oxygen sensitizers in water solution. Unfortunately
their absorbing wavelength was centered at 537 nm, slightly below the PDT spectroscopic window. On
the contrary, the blue colored tetraꢀcationic NDI 12 retains a comparable efficiency in the generation of
1O2, coupled to an absorption in the red. Moreover, all the NDI investigated, including the best singlet
oxygen sensitizers, also exhibited a remarkable red (for the BrꢀNDIs 2, 5, 8 and 11) and NIR (tetraꢀ
substituted NDIs 3, 6, 9 and 12) fluorescence emission with lifetimes that are longer than those of
intrinsic biological fluorophores. These evidences make them very appealing candidates for multimodal
applications as fluorescent reporters and efficient photosensitizers for PDT. Moreover, taking into
consideration that cationic NDIs have already been proved to be excellent and selective Gꢀquadruplex
ligands, NDIs such as 12 may be exploited as selective G4 ligand as well as agent to achieve
photocleavage footprinting of Gꢀquadruplex folding nucleic acids. These aspects are currently under
investigation.
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Experimental Section
Synthesis and Purification. The NDIs 1, 3, 4 and 6 have been already synthesized and
characterized.14 In this study they have been synthesized following a more efficient protocol, starting
from the precursors 14 and 15. The anhydride 13 and the NDIs 14, 15 have been synthesized according
to published procedures.17,19 HPLC analysis and purifications were performed using both preparative
and analytical HPLCs. The analytical column was XSelect CSH PhenylꢀHexyl (150 x 4.6 mm). The
preparative column was XSelect CSH Prep PhenylꢀHexyl 5ꢁm (150 x 30 mm). Flows were 1 ml/min for
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analytical and 27 ml/min for preparative. An analytical method was used: method A (Aqueous solvent:
0.1% trifluoroacetic acid in water; organic solvent: Acetonitrile; Gradient: 95% aqueous, gradually to
0% aqueous over 14 minutes and at the end an isocratic flow over 2 minutes). Preparative HPLC were
performed using method B (Aqueous solvent: 0.1% trifluoroacetic acid in water; organic solvent:
Acetonitrile; Gradient: 95% aqueous, gradually to 20% aqueous over 18 minutes and at the end an
1
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1
13
isocratic flow over 4 minutes). Hꢀ, CꢀNMR spectra were recorded on a 300 MHz spectrometer and
the chemical shifts are reported relative to TMS. The structures of new compounds were deduced from
the results of 1Hꢀ, and 13CꢀNMR.
Nucleophilic aromatic substitution reaction at r.t., optimized for Br-NDI synthesis. The NDI 14-
15 (0.5 mmol) was dissolved into 40 ml of acetonitrile in a round bottom flask together with the
corresponding amine (N,Nꢀdimethylꢀethylamine for 1-2 and N,Nꢀdimethylꢀpropylamine for 4-5; 1.5
mmol). The mixture was stirred at 70°C for 4 h under argon. The resulting red solution was
concentrated under vacuum and a red solid was obtained. The crude product was purified by preparative
HPLC chromatography, using a Cꢀ18 reverse phase column, (CH3CN:H2O 0.1%TFA) according to
analytical method B. HCl 1 M solution was added to each chromatographic portion. Solvent evaporation
under vacuum at r.t. afforded the adducts 1 (99.4 mg, 33% yield), 2 (142.8 mg, 42%), 4 (93.7 mg, 29%),
and 5 (163.1 mg, 45%) as hydrochlorides.
N,N’-Bis-((dimethylamino)ethylamino)-2-bromo-6-((dimethylamino)ethylamino)-1,4-5,8-
naphthalenetetracarboxylic bisimide trihydrochloride (2ꢂ3HCl): The collected solid was purified by
1
preparative HPLC chromatography (CH3CN:H2O 0.1%TFA). Red solid; m.p. dec.>350°C. HꢀNMR
(300 MHz, D2O): 8.68 (s, 1H), 8.3 (s, 1H), 4.74 (m, 4H), 4.38 (m, 2H), 3.96 (m, 2H), 3.75 (m, 4H), 3.41
(bs, 18H). 13CꢀNMR (75 MHz, D2O): 165.1; 162.3; 161.6; 151.1; 137.6; 127.9; 126.7; 122.7; 121.1;
120.8, 120.4; 100.7; 64.1; 62.0; 61.7; 53.6; 53.3; 36.9; 34.7; 34.0. Anal. Calcd. for C26H36BrCl3N6O4: C,
45.73; H, 5.31; N, 12.31. Found: C, 45.82; H, 5.29; N, 12.24.
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N,N’-Bis-((dimethylamino)propylamino)-2-bromo-6-((dimethylamino) propylamino)-1,4-5,8-
naphthalenetetracarboxylic bisimide trihydrochloride (5ꢂ3HCl): The collected solid was purified by
1
2
3
4
5
6
1
preparative HPLC chromatography (CH3CN:H2O 0.1%TFA). Red solid; m.p. dec.>350°C. HꢀNMR
7
8
3
(300 MHz, CD3OD): 8.21 (s, 1H); 8.00 (s, 1H); 4.15 (m, 4H); 3.76 (t, J=7.6 Hz, 2H); 3.43 (m, 2H);
9
13
3.31 (m. 4H); 3.27 (s, 6H); 2.94 (s, 12H); 2.34 (m, 2H); 2.17 (m, 4H). CꢀNMR (75 MHz, CD3OD):
10
11
12
13
14
15
16
17
18
19
20
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22
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25
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166.8; 163.1; 162.4; 152.8; 138.4; 129.3; 128.3; 124.1; 123.9; 122.3; 121.5; 121.2, 101.0; 57.0; 56.9;
56.7; 43.8; 41.4; 39.5; 38.8; 26.1; 24.8; 24.7. Anal. Calcd. for C29H42BrCl3N6O4: C, 48.05; H, 5.84; N,
11.59. Found: C, 47.95; H, 5.82; N, 11.66.
General procedure for the Microwave assisted (MW) nucleophilic aromatic substitution,
optimized for NDIs containing no Br. The NDI 14-15 (0.5 mmol) was dissolved into 6 ml of dry DMF
in MWꢀvial together with the corresponding amine (N,Nꢀdimethylꢀethylamine for 3 and N,Nꢀdimethylꢀ
propylamine for 6; 1.5 mmol). The mixture was stirred and heated in a microwave reactor, according a
closed vessel protocol, at 135 °C, 200 psi, 200 W, for 3 min. The resulting darkꢀviolet solution was
cooled at r.t. and water (50 ml) was added to induce precipitation. The filtered crude product was
purified by preparative HPLC chromatography, using a Cꢀ18 reverse phase column (CH3CN:H2O
0.1%TFA, method B). HCl 1 M solution was added to each chromatographic portion. Solvent
evaporation under vacuum afforded the adducts 1 1 (152.5 mg, 51%, yield), 3 (97.8, 27%) and 4 (148.6
mg, 46%), 6 (125.2 mg, 32%) as hydrochlorides.
Exhaustive methylation of the NDI 1-5. The NDIs purified as hydrochlorides were dissolved in a
NaHCO3 solution and extracted 3 times with CH2Cl2. The recovered organic layers have been dried on
Na2SO4 and the solvent evaporated under reduced pressure. The collected amine (0.5 mmol) was
suspended in 50 ml of CH3CN and 0.24 g (1.7 mmol) of CH3I were added. This suspension was stirred
under nitrogen atmosphere for 12 h. After this period the solvent was removed under vacuum, and the
crude was purified by preparative HPLC, using a Cꢀ18 reverse phase column (CH3CN:H2O 0.1%TFA,
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method B). HCl 1 M solution was added to each chromatographic portion. Solvent evaporation under
1
2
3
vacuum at r.t. afforded the adducts to give the quaternary ammonium salts as chlorides 7-11.
4
5
N,N’-Bis-((trimethylamino)ethylamino)-2-((trimethylamino)ethylamino) -1,4-5,8-naphthalene
6
7
8
9
1
tetracarboxylic bisimide trichloride (7): Red solid, 309 mg, yield 96%; m.p.>350°C. HꢀNMR (300
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MHz, D2O): 8.59 (d, 3J=7.9 Hz, 1H); 8.33 (d, 3J=7.9 Hz, 1H); 8.24 (s, 1H); 4.63 (m, 4H); 4.27 (t, 3J=6.5
13
Hz, 2H); 3.84 (t, 3J=6.5 Hz, 2H); 3.71 (m, 4H); 3.31 (s, 27H). CꢀNMR (75 MHz, D2O): 165.7; 164.3;
164.1; 163.7; 151.8; 131.5; 129.2; 127.9; 125.9; 125.2; 123.2; 119.8; 118.7, 101.0; 64.2; 62.3; 62.0;
53.7; 53.3; 36.8; 34.3; 33.9. Anal. Calcd. for C29H43Cl3N6O4: C, 53.91; H, 6.71; N, 13.01. Found: C,
54.03; H, 6.70; N, 13.11.
N,N’-Bis-((trimethylamino)ethylamino)-2-bromo-6-((trimethylamino)ethylamino)
-1,4-5,8-
naphthalenetetracarboxylic bisimide trichloride (8): Red solid, 325 mg, yield 90%; m.p.>350°C. 1Hꢀ
NMR (300 MHz, D2O): 8.53 (s, 1H); 8.19 (s, 1H); 4.59 (m, 4H); 4.25 (t, 3J=6.3 Hz, 2H); 3.84 (t, 3J=6.3
Hz, 2H); 3.62 (m, 4H); 3.29 (s, 27H). 13CꢀNMR (75 MHz, D2O): 165.0; 162.2; 161.6; 151.0; 137.5;
127.9; 126.7; 122.6; 121.1; 120.8; 120.3; 100.6; 63.9; 61.8; 61.6; 53.5; 53.2; 36.8; 34.6; 33.9. Anal.
Calcd. for C29H42BrCl3N6O4: C, 48.05; H, 5.84; N, 11.59. Found: C, 48.22; H, 5.78; N, 11.35.
N,N’-Bis-((trimethylamino)ethylamino)-2,6-((trimethylamino)ethylamino)
-1,4-5,8-
naphthalenetetracarboxylic bisimide tetrachloride (9): Violet solid, 386 mg, yield 99%; m.p.>350°C.
1HꢀNMR (300 MHz, D2O): 8.10 (s, 2H); 4.59 (m, 4H); 4.11 (m, 4H); 3.71 (m, 4H); 3.59 (m, 4H); 3.21
(s, 36H). 13CꢀNMR (75 MHz, D2O): 167.4; 165.4; 164.6; 164.1; 150.0; 127.2; 123.1; 119.7; 115.9;
104.6; 65.7; 63.7; 55.0; 54.8; 38.0; 35.4. Anal. Calcd. for C34H56Cl4N8O4: C, 52.18; H, 7.21; N, 14.32.
Found: 52.31; H, 7.18; N, 14.27.
N,N’-Bis-((trimethylamino)propylamino)-2-((trimethylamino)propylamino)-1,4-5,8-
naphthalenetetracarboxylic bisimide trichloride (10): Red solid, 337 mg, yield 98%; m.p.>350°C.
1HꢀNMR (300 MHz, D2O): 8.2 (d, 3J=7.9 Hz, 1H); 7.97 (d, 3J=7.9 Hz, 1H); 7.79 (s, 1H); 4.11 (m, 4H);
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3.68 (t, 2H); 3.46 (m, 6H), 3.10 (s, 9H); 3.06 (s, 18H); 2.16 (m, 6H). 13CꢀNMR (75 MHz, D2O):
δ=165.3; 163.8; 163.6; 163.3; 151.7; 130.8; 128.6; 127.0; 125.1; 124.4; 122.3; 119.6; 118.6, 99.5; 63.9;
52.9; 39.5; 37.7; 37.1; 22.9; 21.4. Anal. Calcd. for C32H49Cl3N6O4: C, 55.85; H, 7.18; N, 12.21. Found:
C, 55.71; H, 7.27; N, 12.30.
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N,N’-Bis-((trimethylamino)propylamino)-2-bromo-6-((trimethylamino) propylamino)-1,4-5,8-
naphthalenetetracarboxylic bisimide trichloride (11): Red solid, 372 mg, yield 97%, m.p.>350°C.
1HꢀNMR (300 MHz, D2O): 8.18 (s, 1H); 7.92 (s, 1H); 4.05 (m, 4H); 3.68 (m, 2H); 3.49 (m, 2H); 3.38
(m, 4H), 3.10 (s, 9H); 3.03 (s, 18H); 2.24 (m, 2H); 2.10 (m, 4H). 13CꢀNMR (75 MHz, D2O): 165.0;
162.2; 162.0; 161.3; 151.2; 137.0; 127.5; 126.1; 121.9; 120.4; 119.9; 118.0; 114.1, 99.2; 63.8; 52.8;
39.6; 38.1; 37.2; 22.7; 21.2; 21.1. Anal. Calcd. for C32H48BrCl3N6O4: C, 50.11; H, 6.31; N, 10.96.
Found: C, 49.89; H, 6.35; N, 11.03.
General protocol for exhaustive methylation of tetra-substituted-NDI 6: The NDI 6 purified as
hydrochloride was dissolved in a Na2CO3 solution and extracted 3 times with CH2Cl2. The recovered
organic layers have been dried on Na2SO4 and the solvent evaporated under reduced pressure. The
collected amine (0.5 mmol) was added to a suspension of Na2CO3 (2 mmol) in 50 ml of CH3CN and
0.24 g (1.7 mmol) of CH3I were added. This suspension was stirred under nitrogen atmosphere for 12 h.
After this period the solvent was removed under vacuum to give the and the crude was purified by
preparative HPLC, using a Cꢀ18 reverse phase column (CH3CN:H2O 0.1%TFA, method B). HCl 1 M
solution was added to each chromatographic portion. Solvent evaporation under vacuum at r.t. afforded
the adducts to give the quaternary ammonium salt as chloride 12 (415 mg, 99%, yield).
N,N’-Bis-((trimethylamino)propylamino)-2,6-
((trimethylamino)propylamino)
-1,4-5,8-
naphthalenetetracarboxylic bisimide tetrachloride (12): Violetꢀdark solid, yield 99%; m.p.>350°C.
1HꢀNMR (300 MHz, D2O): 7.62 (s, 2H); 4.14 (m, 4H); 3.62 (m, 4H); 3.49 (m, 8H); 3.08 (s, 18H); 3.06
(s, 18H); 2.17 (m, 8H). 13CꢀNMR (75 MHz, D2O): 165.3; 163.3; 148.2; 124.6; 120.5; 117.3, 101.6;
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64.1; 53.1; 39.2; 37.3; 22.7; 21.6. Anal. Calcd. for C38H64Cl4N8O4: C, 54.41; H, 7.69; N, 13.36. Found:
C, 54.22; H, 7.79; N, 13.26.
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Sample Preparation for Solution Studies. For the spectroscopic measurements we used pure water
as well as 10 mM K+ phosphate buffer of pH 2 adjusted with aliquots of HCl 3N.
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Absorption and fluorescence spectra. UVꢀvisible absorption spectra were recorded on a standard
commercial spectrophotometer. Fluorescence spectra were measured using 1 nm steps and 0.5 s dwell
time. Slits were kept narrow to 1 nm in excitation and 1 or 2 nm in emission. Where necessary a cutꢀoff
filter was used. Right angle detection was used. All the measurements were carried out at 295 K in
quartz cuvettes with path length of 1 cm. All fluorescence spectra have been obtained for airꢀ
equilibrated solutions absorbing less than 0.1 at all wavelengths to avoid inner filter effects and reꢀ
absorption of emission. Furthermore, they have been corrected for wavelength dependent response of
the monochromator/PMT couple. The compound Ru(bpy)3Cl3.xH2O dissolved in airꢀequilibrated water
with known fluorescence quantum yield (ΦF) of 0.028 was used as standard for the determination of the
fluorescence quantum yield of the NDI samples. The ΦF value obtained for the compound 11 in water
was used as reference to determine the fluorescence quantum yield of the tetraꢀNDI compounds (3, 6, 9
and 12), excited at 546 nm. Using the same solvents for all compounds and isoꢀabsorbing solutions at
the excitation wavelength no corrections had to be made for absorbance neither solvent refraction index
and we calculated the fluorescence quantum yields, ΦF, using the formula below, with A being the
integrated area of the corrected fluorescence spectra:
ΦF= ΦFref × A/Aref
(1)
Fluorescence lifetimes. They were measured in airꢀequilibrated solutions with a time correlated
single photon counting system. A nanosecond LED source at 373 nm was used for excitation and the
emission was collected at right angle at 580 nm or 635 nm using a long pass cutꢀoff filter at 495 nm.
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Decay profiles were fitted using a monoꢀ or multiexponential function and deconvolution of the
instrumental response.
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I(t) = ∑i ai × exp(ꢀt/τi)
fi = (ai × ti)/ ∑j(aj × tj)
(2)
(3)
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Nanosecond laser flash photolysis. The pulse of a NdꢀYAG laser, operating at 355 and 532 nm (20
ns FWHM, 2 Hz), was suitably shaped passing through a 3 mm high and 10 mm wide rectangular
window, and providing a fairly uniform energy density of 2.7 mJ/pulse corresponding to 9 mJ/cm2
incident on the sample cell. A front portion of 2 mm of the excited solution was probed at right angle,
the useful optical path for analyzing light being 10 mm. A532 was ~ 0.3 over 1 cm. Arꢀsaturated or airꢀ
equilibrated solutions were used. The sample was renewed after few laser shots. Temperature was 295
K.
Singlet oxygen time resolved emission measurements. The pulse of a NdꢀYAG laser, operating at
532 nm (20 ns FWHM, 2 Hz), was used for excitation of the reference TPPS and the NDI samples
dissolved in airꢀequilibrated deuterated water. We used DCl to adjust pH values to 2 when necessary.
All solutions had identical absorbance of ca. 0.3 at the 532 nm excitation wavelength. A preamplified
(low impedance) Geꢀphotodiode cooled at 77 K ( Applied Detector Corporation, Model 403HS, time
resolution 300 ns) and equipped with a 5 mm thick AR coated silicon metal filter with wavelength pass
>1.1 ꢁm and an interference filter at 1.27 ꢁm was used to measure emission of singlet oxygen at 1270
nm in right angle geometry. The photodiode output current was fed into a digital oscilloscope. The
intensities of singlet oxygen emission of reference and sample can be compared directly with each other
and the calculation of the singlet oxygen emission quantum yield, Φꢀ, is based on the following
equation:
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ref
Φꢀ= Φꢀref × I0/I0
(4)
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where I0 is the emission intensity extrapolated at time zero upon single exponential fitting of the
emission decay with the exclusion of the initial part of the signal affected by scattered light, sensitizer
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fluorescence and formation kinetics. The standard value of 0.76 was used for Φꢀref of TPPS.40
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Fluorescence Quenching and Potentiometric titrations. Spectrofluorimetric were performed with
on a commercial spectrophotometer. The pHꢀmetric titrations were carried out with a commercial
titration system. All titrations were performed at 25.0 ± 0.1 °C. Protonation constants of ligand L were
determined in a water mixture, made 0.1 M in NaNO3. In a typical experiment, 15 mL of a 5×10−4
M
ligand solution were treated with an excess of a 1.0 M HNO3 standard solution. Titrations were run by
addition of 10 ꢁL aliquots of carbonateꢀfree standard 0.1 M NaOH, recording 80ꢀ100 points for each
titration. Prior to each potentiometric titration, the standard electrochemical potential (E°) of the glass
electrode was determined in the water, by a titration experiment according to the Gran method.43
Protonation titration data (emf vs. mL of NaOH) were processed with the Hyperquad package,44 to
determine the equilibrium constants.
Associated Content
Supporting Information. Additional Absorption and Fluorescence spectra (Figure 1ꢀ6S), Timeꢀ
correlated single photon counting experiments (TCSPC, Figure 7S), HPLC purity data and NMR spectra
of the new NDIs. This material is available free of charge via the Internet at http://pubs.acs.org
Author information
*Corresponding Author. Fax: +39 0382 987323. Tel: +39 0382 987668. Eꢀmail:
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mauro.freccero@unipv.it
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The authors declare no competing financial interest.
Acknowledgements
Financial support from MIUR, Rome (FIRBꢀIdeasRBID082ATK, and PRIN 2009MFRKZ8), and
University of Pavia, is gratefully acknowledged. I.M. likes to thank Dr. F. Manoli for assistance in
practical laboratory issues.
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