Spectroscopic Evaluation of Novel Adenine/Thymine-Conjugated Naphthalenediimides: Preference of Adenine-Adenine over Thymine-Thymine Intermolecular Hydrogen Bonding in Adenine- and Thymine-Functionalized Naphthalenediimides

Article abstract of DOI:10.1007/s10895-018-02340-6

The synthesis and spectroscopic characterization of novel nucleobase (adenine/thymine)-conjugated naphthalenediimides (NDIs), namely, NDI-AA, NDI-TT, and NDI-AT have been successfully achieved. NDI-AA, NDI-TT and NDI-AT have similar absorption in the 300–400?nm region. The effect of solvent on the absorption spectrum indicates aggregation, either through intermolecular π-σ interaction among the main chromophore or through intermolecular hydrogen bonding between adenine and adenine group. Addition of water does not assist hydrogen bond formation between thymine-thymine, rather increasing the polarity of the solvent encourages π-σ interaction among NDI-TTs. No spectral change for NDI-TT with increasing temperature confirms hydrogen bonding is not playing a crucial role in NDI-TT. A fluorescence study on NDI-AA also establishes excimer formation along with ground state aggregation. As the water content in the solvent mixture increases, aggregation of NDI-AA is discouraged due to adenine-adenine hydrogen bonding in accordance with earlier results. At the same time, the NDI-TT emission spectrum does not shift to the blue region and the intensity of the peak around 535?nm increases at the expense of fluorescence in 411?nm. Thus, increasing water content in the solvent mixture facilitates aggregation through π-σ interaction in NDI-TT as thymine-thymine hydrogen bonding is less pronounced.

Full text of DOI:10.1007/s10895-018-02340-6

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-02340-6
ORIGINAL ARTICLE
Spectroscopic Evaluation of Novel Adenine/Thymine-Conjugated
Naphthalenediimides: Preference of Adenine-Adenine
over Thymine-Thymine Intermolecular Hydrogen Bonding in Adenine-
and Thymine-Functionalized Naphthalenediimides
Digambara Patra1 & Nadine Al Homsi1 & Sara Jaafar1 & Zeina Neouchy1 & Jomana Elaridi2 & Ali Koubeissi1
&
Kamal H. Bouhadir1
Received: 26 September 2018 /Accepted: 26 December 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The synthesis and spectroscopic characterization of novel nucleobase (adenine/thymine)-conjugated naphthalenediimides (NDIs),
namely, NDI-AA, NDI-TT, and NDI-AT have been successfully achieved. NDI-AA, NDI-TT and NDI-AT have similar absorption
in the 300–400 nm region. The effect of solvent on the absorption spectrum indicates aggregation, either through intermolecular π-σ
interaction among the main chromophore or through intermolecular hydrogen bonding between adenine and adenine group. Addition
of water does not assist hydrogen bond formation between thymine-thymine, rather increasing the polarity of the solvent encourages
π-σ interaction among NDI-TTs. No spectral change for NDI-TT with increasing temperature confirms hydrogen bonding is not
playing a crucial role in NDI-TT. A fluorescence study on NDI-AA also establishes excimer formation along with ground state
aggregation. As the water content in the solvent mixture increases, aggregation of NDI-AA is discouraged due to adenine-adenine
hydrogen bonding in accordance with earlier results. At the same time, the NDI-TTemission spectrum does not shift to the blue region
and the intensity of the peak around 535 nm increases at the expense of fluorescence in 411 nm. Thus, increasing water content in the
solvent mixture facilitates aggregation through π-σ interaction in NDI-TT as thymine-thymine hydrogen bonding is less pronounced.
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Keywords Naphthalenediimide Adenine Thymine Absorption Fluorescence Hydrogen bonding
Introduction
Application of naphthalenediimides (NDIs) in supramolecular
and materials chemistry has been extensively reported and
reviewed in the literature for synthesis of new materials
[1–5]. In many cases, NDI derivatives have demonstrated po-
tential use in the field of molecular sensors [6–8] and in
conducting films [9, 10]. These molecules can self-assemble
and intercalate in larger multicomponent complexes [1, 11].
NDI self-assembly is facilitated by hydrogen bonding and π-π
stacking [12]. NDI derivatives may have future applications in
the biomedical field due to their gelling properties and high
versatility [1, 5, 6, 8]. Attempts to expand the family of NDIs
through functionalization of the naphthalene core or the
diimide nitrogens have resulted in a variety of molecules with
diverse absorption and emission properties [1, 13].
The design of supramolecular structures often incorporates
aromatic molecules because spectroscopic and redox proper-
ties of aromatic molecules can be easily predicted [1].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-018-02340-6) contains supplementary
material, which is available to authorized users.
* Digambara Patra
dp03@aub.edu.lb
* Kamal H. Bouhadir
kb05@aub.edu.lb
Attempts to design self-organizing NDI derivatives have
included the synthesis of adenine and thymine functionalized
NDIs [14]. This idea was inspired by the compelling ability of
nucleobases, the building blocks of deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA), to form hydrogen-
1
Department of Chemistry, American University of Beirut,
P.O. Box 11-0236, Beirut, Lebanon
2
Department of Natural Sciences, Lebanese American University,
P.O. Box 13-5053, Beirut, Lebanon

J Fluoresc
bonded molecular assemblies. Adenine-NDI-Adenine (NDI-
AA) and Thymine-NDI-Thymine (NDI-TT) have been suc-
cessfully synthesized and shown to self-assemble on tem-
plates of peptide nucleic acid (PNA) dimers whose role is to
mimic the nucleobases [14]. Complexes with a greater affinity
have been observed compared to the self-assembly of non-
conjugated nucleobases on a PNA template [3].
Furthermore, the NDI-nucleobases self-assemble in a unique
and well-defined manner on the PNA templates [3]. NDI-AA
conjugates assemble into nanoribbons while with complemen-
tary PNA-TT templating, 2D-microstructures have resulted.
For the NDI-TT conjugated, it has self-organized into porous
spheres whereas complementary PNA-AA templating have
resulted in fibers and petal-like 2D sheets [3]. Hence, the
difference in the strength of self-assembly has reflected by a
change in the resulted nanostructures [12, 14, 15].
5 nm using Jobin-Yvon-Horiba Fluorolog III fluorometer
and the FluorEssence program. The excitation source was a
100 W Xenon lamp, and the detector used was R-928 op-
erating at a voltage of 950 V. The concentration used for
fluorescence measurement was 10 μM.
Preparative column chromatography employed ACROS
silica gel (60 Å, 200–400 mesh). Preparative thin layer chro-
matography employed ALLTECH silica gel 60 F254 plates.
Reagents used in the syntheses were purchased from the
Aldrich Chemical Company (Milwaukee, WI, USA),
ACROS Chemicals (Belgium), Fisher Scientific Company
(Fair Lawn, NJ, USA), and were used as received.
Synthesis 1-(2-(Carboxyethyl)ethyl)thymine (2): Thymine 1
(2.00 g, 16 mmol) was dissolved in an ethanol:benzene mix-
ture (8:1, 60 mL) in the presence of a catalytic amount of
sodium metal (33 mg) and ethyl acrylate (2.2 mL, 20 mmol)
was added dropwise after hydrogen evolution ceased. The
mixture was heated at reflux overnight and subsequently con-
centrated under reduced pressure. The flask was immersed in
an ice-water bath and the resultant precipitate was filtered,
washed with cold ethanol and dried under reduced pressure
to afford 2 as a colorless solid (3.10 g, 86%). Mp: 167–168 °C;
1H NMR (300 MHz, CDCl3): δ 1.25 (t, J = 6.9 Hz, 3H), 1.91
(s, 3H), 2.77 (t, J = 6.0 Hz, 2H), 3.95 (t, J = 6.0 Hz, 2H), 4.15
(q, J = 7.1 Hz, 2H), 7.21 (s, 1H), 9.41 (bs, 1H); 13C NMR
(75 MHz, CDCl3): δ 12.2, 14.1, 33.1, 45.0, 61.1, 110.2,
141.6, 150.6, 164.1, 171.4. The spectroscopic data are in
agreement with those reported in the literature [16].
In this paper, we report the synthesis and photophysical
properties of novel nucleobase-functionalized NDIs, specifi-
cally NDI-AA and NDI-TT. The purity of the final product
1
was assessed, and the structure was elucidated using H-
NMR, 13C-NMR, DEPT, COSY, NOESY, FT-IR, and MS.
The collected data confirmed the chemical structures of all
the products in this study. In addition, extensive photophysical
properties for NDI-AA and NDI-TT were realized in different
solvents and at various proportions.
Experimental
Instrumentation Melting points were determined on a
Fisher-Johns apparatus and were uncorrected. NMR
spectra were determined in deuterated solvents with
TMS as the internal standard on a Bruker AM 300
NMR spectrometer. Chemical shifts are reported in ppm
(δ) downfield relative to TMS. Infrared spectra were re-
corded as KBr pellets using a Nicolet AVATAR 360
FTIR ESP spectrometer with a Hewlett Packard Desk
jet 840C plotter.
Attenuated total reflectance Fourier transform infrared
(ATR-FTIR) spectrometry was performed on dry samples.
ATR-FTIR spectra (4 scans, 4 cm−1 resolution, wave num-
ber range 4000–650 cm−1) were obtained using a Perkin
Elmer FTIR Spectrum 2000 Spectrophotometer with a
diamond/ZnSe crystal window. All spectra were recorded
under ambient conditions. SPECTRUM software (Perkin
Elmer) was used for data collection. The IR bands are
reported in wavenumbers (cm−1). Thin layer chromatogra-
phy (TLC) was performed on polygram Sil G/UV254 silica
gel sheets. The absorption spectra were recorded at room
temperature using a JASCO V-570 UV–VIS–NIR spectro-
photometer. The concentration used for absorption mea-
surement was 100 μM. The fluorescence measurements
were documented with resolution increment 1 nm and slit
1-(2-Hydrazidoethyl)thymine (3): Thymine-ester 2 (3.50 g,
15.4 mmol) was dissolved in ethanol (77 mL) followed by
dropwise addition of hydrazine monohydrate (N2H4.H2O,
2.3 mL, 46.2 mmol) and the resulting mixture was heated at
reflux overnight. The solvent was evaporated to dryness under
reduced pressure and the solid was triturated in cold ethanol
and filtered to afford 3 as a colorless solid (2.84 g, 86%). Mp:
188–190 °C; FT-IR (ATR, cm−1): 1224,1636, 1662, 3347; 1H
NMR (300 MHz, D2O): δ 1.86 (s, 3H), 2.58 (t, J = 6.4 Hz,
2H), 4.00 (t, J = 6.3 Hz, 2H), 7.42 (s, 1H); 13C NMR
(75 MHz, D2O): δ 14.1, 35.6, 48.2, 113.6, 145.9, 154.9,
169.9, 174.6. The spectroscopic data are in agreement with
those reported in the literature [16].
NDI-TT (4): 1,4,5,8-Naphthalenetetracarboxylic
dianhydride (0.5, 1.86 mmol) was added to a solution of
thymine-hydrazide 3 (0.794 g, 3.72 mmol) in DMF (20 mL).
The resulting mixture was heated at 110 °C for 6 days. The
reaction mixture was cooled to room temperature and the pre-
cipitate was filtered and washed with ethanol. The solid was
triturated with hot ethanol (20 mL), filtered and washed with
ethanol. The product was dried under reduced pressure at
80 °C for 2 days to furnish 4 as a pale yellow solid (0.89 g,
79%). Mp: 333–336 °C; FT-IR (KBr, cm−1): 1250, 1668,
1
1692; H NMR (300 MHz, DMSO): 1.76 (s, 6H), 2.84 (t,

J Fluoresc
J = 6 Hz, 4H), 3.95 (t, J = 6 Hz, 4H), 7.49 (s, 2H), 8.75 (s, 4H),
11.06 (d, J = 8 Hz, 2H), 11.31 (bs, 2H);); 13C NMR (75 MHz,
D2O): δ 12.1, 32.2, 44.2, 108.2, 126.3, 126.5, 131.4, 131.4,
142.1, 150.9, 160.7, 164.5, 169.1; Mass spectrum (ESI−): m/z
[M-H+] calcd for C30H23N8O10: 655.1, found 655.0.
(41 mL) and KOH (1.5 M, 46 mL). The pH of this solution
was adjusted to 5.6 with acetic acid (2 M, ~30 mL). Adenine
hydrazide 7 (0.60 g, 2.90 mmol) was added to this solution and
the pH was adjusted again to 5.6. The cloudy light brown mix-
ture was heated at reflux overnight and was subsequently cooled
to room temperature and filtered. The filtrate was acidified to
pH 1.8 and the mixture was heated at ~80 °C for 30 min. The
product was dried at 80 °C under reduced pressure to yield the
coupled product 9 as a dark beige powder (0.37 g, 32%). Mp:
315–318 °C; FT-IR (ATR, cm−1): 1166, 1248, 1687; 1H NMR
(500 MHz, DMSO): 3.04 (t, J = 6.8 Hz, 2H), 4.46 (J = 6.8 Hz,
2H), 7.26 (s, 2H), 8.10 (s, 1H), 8.19 (s, 1H), 8.23 (d, J = 7.5 Hz,
2H), 8.61 (d, J = 7.5 Hz, 2H), 11.01 (s, 1H); 13C NMR
(125 MHz, DMSO): 32.9, 39.1, 124.4, 126.1, 128.8, 129.9,
131.5, 137.8, 141.0, 143.9, 147.1, 149.2152.0, 159.3, 161.3,
168.8. Mass spectrum (ESI−): m/z [M-H+] calcd for
C22H14N7O7: 488.1, found.
NMI-T (11): A solution of 1,4,5,8-naphthalenetetracarboxylic
dianhydride (3.75 g, 14 mmol) was prepared with water
(245 mL) and KOH (1.5 M, 276 mL). The pH of the solution
was adjusted to 5.6 with acetic acid 2 M (~170 mL). Thymine
hydrazide 3 (3.00 g, 14 mmol) was added to this solution and the
pH was adjusted again to 5.6. The brown mixture was heated at
reflux overnight and was subsequently cooled to room tempera-
ture and filtered. The filtrate was acidified to pH 1.8 and the light
brown mixture was heated at ~80 °C for 30 min. The product
was dried at 80 °C under reduced pressure to yield the coupled
product 11 as a light brown powder (6.05 g, 90%). Mp: 305–
307 °C; FT-IR (ATR, cm−1): 1186, 1237, 1285, 1374, 1670,
1684; 1H NMR (300 MHz, DMSO + TFA): 1.77 (s, 3H), 2.81
(t, J = 6.5 Hz, 2H), 3.96 (t, J = 6.3 Hz, 2H), 7.50 (s, 1H), 8.26 (d,
J = 7.5 Hz, 2H), 8.62 (d, J = 7.5 Hz, 2H), 11.04 (s, 1H), 11.32 (s,
1H); 13C NMR (125 MHz, DMSO + TFA): 12.4, 32.7, 44.6,
124.6, 126.1, 128.9, 129.9, 131.4, 137.8, 142.6, 151.3, 161.3,
165.0, 168.8, 169.5. Mass spectrum (ESI−): m/z [M-H+] calcd for
C22H15N4O9: 479.1, found.
NDI-AT (10): In a sealed tube, a mixture of naphthalene
mono-thymine-imide 11 (0.35 g, 0.7 mmol, 1 eq), adenine
hydrazide (0.15 g, 0.7 mmol) and three drops of TFA in
DMF (11 mL) was heated and for 6 days at 130 °C. The
mixture was cooled to room temperature and filtered under
reduced pressure. The precipitate was washed with ethanol
and triturated with hot ethanol. The product was dried at
80 °C under reduced pressure to yield NDI-AT 10 as a dark
grey powder (0.23 g, 53%). Mp: >355 °C. FT-IR (ATR,
cm−1): 1200, 1243, 1355, 1449, 1476, 1657, 1690; 1H NMR
(300 MHz, DMSO + TFA): 1.76 (s, 3H), 2.79 (t, J = 6.6 Hz,
2H), 2.91 (t, J = 6.6 Hz, 2H), 3.94 (t, J = 6.5 Hz, 2H), 4.48–
4.53 (m, 2H), 7.48 (s, 1H), 8.23 (d, J = 7.5 Hz, 2H), 8.42 (s,
1H), 8.50 (s, 1H), 8.61 (d, J = 7.8 Hz, 2H), 11.01 (s, 1H),
11.04 (s, 1H), 11.29 (s, 1H); 13C NMR (125 MHz, DMSO +
TFA): 12.4, 32.7, 33.1, 33.3, 44.6, 118.6, 124.6, 126.1, 126.8,
127.0, 128.8, 130.0, 131.5, 137.8, 142.6, 143.7, 147.6, 149.2,
1-(2-(Carboxyethyl)ethyl)adenine (6): Adenine 5 (16.7 g,
0.123 mol) was dissolved in an ethanol:benzene mixture (8:1,
562 mL) and the flask was immersed in an ice-water bath.
Sodium metal (175 mg) was carefully added followed by the
dropwise addition of ethyl acrylate (5 mL) and the mixture
was refluxed overnight. The solvent was evaporated under
reduced pressure and the residue was triturated with cold eth-
anol, filtered and dried under reduced pressure to afford the
ester 6 as a colorless solid (26.7 g, 92%). Mp: 167–168 °C;
FT-IR (KBr, cm−1): 1204, 1481, 1609, 1733, 3315; 1H NMR
(300 MHz, DMSO): 1.12 (t, J = 7.1 Hz, 3H), 2.94 (t, J =
6.8 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 4.38 (t, J = 6.8 Hz,
2H), 7.24 (s, 2H), 8.11 (s, 1H), 8.14 (s, 1H); 13C NMR
(75 MHz, DMSO): 13.8, 33.6, 38.6, 60.2, 118.6, 140.9,
149.4, 152.3, 155.9, 170.5. The spectroscopic data are in
agreement with those reported in the literature [16].
1-(2-Hydrazidoethyl)adenine (7): Hydrazine-hydrate
(9.58 g, 191 mmol) was added dropwise to a stirred solution
of 6 (15.0 g, 63.7 mmol) in ethanol (300 mL) at room temper-
ature. The mixture was then heated at reflux for 24 h. The
solution was cooled down to room temperature and the sol-
vent was removed under reduced pressure. The residue was
triturated with cold ethanol, filtered and dried under reduced
pressure to afford the hydrazide 7 as a colorless solid (13.9 g,
99%). Mp: 268–271 °C; FT-IR (ATR, cm−1): 1298, 1647,
1
3160, 3326; H NMR (300 MHz, D2O + D2SO4): 3.05 (t,
J = 6.5 Hz, 2H), 4.68 (t, J = 6.4 Hz, 2H), 8.41 (s, 1H), 8.47
(s, 1H); 13C NMR (75 MHz, D2O): δ 35.3, 42.5, 120.5, 147.0,
147.5, 151.1, 152.3, 172.9. The spectroscopic data are in
agreement with those reported in the literature [16].
NDI-AA (8): 1,4,5,8-Naphthalenetetracarboxylic
dianhydride (0.50 g, 1.86 mmol) was added to a solution of
1-(2-hydrazidoethyl)adenine 7 (0.822 g, 3.72 mmol in DMF
(20 mL). and the resulting mixture was heated at 130 °C for
6 days. The reaction mixture was cooled to room temperature
and the precipitate was filtered and washed with ethanol. The
solid was triturated with 20 mL of hot ethanol, filtered and
washed with cold ethanol. The product was dried under reduced
pressure at 80 °C for two days to afford a brown solid (0.962 g,
77%). Mp: 305–310 °C; FT-IR (KBr, cm−1): 1695, 1669, 1250;
1H NMR (300 MHz, DMSO): 3.06 (t, J = 6 Jz, 4H), 4.46 (t, J =
6 Hz, 4H), 7.25 (s, 2H), 8.09 (s, 2H), 8.18 (s, 2H), 8.75 (s, 4H),
11.05 (s, 1H), 11.10 (s, 1 H); 13C NMR (75 MHz, CDCl3):
32.8, 35.7, 113.5, 117.9, 126.2, 131.3, 144.3, 148.4, 149.8,
158.4, 159.0, 160.4, 168.6. Mass spectrum (ESI−): m/z [M-
H+] calcd for C30H21N14O6: 673.2, found.
NMI-A (9): A solution of 1,4,5,8-naphthalenetetracarboxylic
dianhydride (0.64 g, 2.40 mmol) was prepared with water

J Fluoresc
151.3, 159.2, 161.3, 165.0, 169.1, 169.9. Mass spectrum
nucleobase esters 2 and 6 and acylhydrazides 3 and 7 are
(ESI−): m/z [M-H+] calcd for C30H22N11O8: 664.2, found.
consistent with those previously reported in the literature
1
[16, 17]. Briefly, the H NMR spectra of 2 and 6 displayed
signals corresponding to the methylene (δ 4.15) and methyl (δ
1.25) hydrogens of the ethyl ester group, in addition to two
new triplets attributed to the newly incorporated ethylene
spacer. Similarly, conversion of the esters to the hydrazides
was supported by the disappearance of the ethyl hydrogens;
the NHNH2 protons did not appear due to exchange with the
D2O solvent. However, the IR showed C=O peaks at 1663 and
1637 cm−1 and an NH2 stretch at 3347 cm−1. Formation of the
NDI-TT derivative 4 was supported with the appearance of a
[M-H+] peak at 655.0 in the mass spectrum. Furthermore, the
1H NMR spectrum displayed a new aromatic signal at δ 8.75
attributed to the newly installed naphthalene core in addition
to chemical shift changes of the thymine protons and the eth-
ylene spacer. Similar observations were made in the spectro-
scopic analysis of NDI-AA 8: C=O stretch at 1695 cm−1, a
new aromatic proton signal at δ 8.75 and two carbonyl carbon
peaks at δ 160.4 and 168.6 corresponding to the hydrazide and
imide carbons respectively.
Results and Discussion
The preparation of nucleobase-naphthalenediimide conjugates 4
(NDI-TT) and 8 (NDI-AA) are presented in Scheme 1. The
novel NDI-derivatives were synthesized via a straightforward,
facile and high-yielding strategy from cheap, readily available
starting materials. Initially, the nucleobases thymine 1 and ade-
nine 5 underwent Michael addition upon reaction with ethyl
acrylate in ethanol in the presence of a catalytic amount of so-
dium metal. This led to the incorporation of a two-carbon eth-
ylene spacer that links the nucleobase to the new ester function-
ality. The thymine- and adenine-derived ethyl esters 2 and 6
were isolated as solids in 86% and 92% yields respectively.
Hydrazinolysis of the ethyl esters 2 and 6 in refluxing ethanol
overnight furnished the nucleobase hydrazides 3 and 7 in excel-
lent yields of 86% and 99% yields respectively. The nucleobase-
hydrazides 3 and 7 were condensed with 1,4,5,8-
naphthalenetetracarboxylic dianhydride (NDA) in a 2:1 mol ra-
tio in dimethylformamide (DMF) to give the nucleobase-
appended NDI-derivatives 4 and 8 in 79% and 77% yields.
The synthesized compounds were characterized by IR, 1H
and 13C NMR spectroscopy. Spectroscopic data of the
Synthesis of the adenine-thymine appended
naphthalenediimide conjugate 10 (NDI-AT) is outlined in
Scheme 2. NDI-AT 10 can be synthesized from either the
thymine 3 or adenine 7 derivative via two successive reactions
between the nucleobase hydrazides and 1,4,5,8-
O
Scheme 1 Reagents and
conditions: (a) Ethyl acrylate, Na,
EtOH, reflux, 24 h. (b)
N2H4.H2O, EtOH, reflux, 24 h.
(c) NDA, DMF, 110 °C, in sealed
tube, 10–12 h
HN
O
N
O
O
NH
N
O
O
N
O
N
O
HN
HN
HN
a
b
c
O
N
H
O
O
86%
86%
79%
NH2
4
N
(NDI-TT)
N
O
N
O
O
1
CO2Et
O
NH
N
HN
N
NH2
2
3
N
O
O
NH
N
O
O
NH2
N
NH2
N
O
NH
NH2
N
N
N
N
N
N
N
c
b
a
N
92%
99%
77%
CO2Et
N
H
N
NH
O
N
O
O
O
NH2
HN
7
6
5
8
(NDI-AA)
N
N
N
N
H2N

J Fluoresc
NH2
Scheme 2 Reagents and
N
conditions: (a) NDA, KOH, H2O,
reflux, overnight. (b) 3, TFA,
130 °C, sealed tube, 6 d. (c) 7,
TFA, 130 °C, sealed tube, 6 d
N
N
N
HO2C
CO2H
NH2
NH
N
O
O
N
O
N
O
N
O
O
N
O
O
NH2
N
N
HN
N
N
N
HN
N
a
b
c
a
O
74%
NH
N
O
O
O
N
O
O
N
O
NH
O
NH
NH2
O
HN
NH
NH2
O
7
3
N
O
11
(NMI-T)
HO2C
CO2H
NH
9
O
(NMI-A)
10 (NDI-AT)
naphthalenetetracarboxylic dianhydride (NDA). For example,
thymine hydrazide 7 was heated with NDA in aqueous potas-
sium hydroxide solution overnight to afford the thymine-
linked naphthalene mono-imide 11 (NMI-T) in 90% yield.
The appearance of two new aromatic doublets at δ 8.23 and
8.61 in the 1H NMR spectrum, characteristic of the two sets of
aromatic protons in the naphthalene core, in addition to signals
typical of the thymine-linker, provides evidence for formation
of the coupled product. Subsequent reaction of 11 with ade-
nine hydrazide 7 in a sealed tube at 130 °C furnished the
coupled product 10 (NDI-AT) in 53% yield.
The UV-visible spectra of NDI-AA 8, NDI-TT 4 and a 1:1
NDI-AA 8: NDI-TT 4 mixture in 60:40% DMSO/water are
presented in Fig. 1. Interestingly, NDI-AA 8 and NDI-TT 4,
and their mixture showed similar absorption in the 300–
400 nm region. The vibrational peaks at around 340 nm,
358 nm and 378 nm were well resolved in all cases. Since
there is no difference in positions of absorption spectra of 8
and 4 (and their mixture), the influence of adenine/thymine on
the absorption of the NDI chromophore is negligible and the
absorption is characteristic of π-π* transitions of NDI chro-
mophores polarized along the z-axis [14].
As seen in Fig. 2a, upon the addition of water in DMSO,
the absorption spectrum of NDI-AA 8 showed an increase
in absorbance at the maximum (at 358 nm and 378 nm) but
around 400 nm and 310 nm regions, the absorbance de-
creased. It was also observed that as the water concentra-
tion increased, the absorption spectrum of NDI-AA 8 was
found to be narrower. This observation is interesting. There
are two possibilities for two NDI-AA 8 molecules to ag-
gregate, either through intermolecular π-σ interaction be-
tween the main chromophore or through intermolecular
hydrogen bonding between adenine-adenine groups.
Aggregation generally decreases the absorbance of chro-
mophores, however, depending on the aggregation type, a
blue or red shift can be observed in the absorption spec-
trum. In this case, no additional peak was observed.
However, the increase in absorbance could be due to weak-
ening intermolecular π-σ interaction that is a result of
strong hydrogen bonding between adenine-adenine groups
facilitated by solvent molecules like water.
This was further examined by replacing water (protic sol-
vent) with acetonitrile (aprotic solvent) as depicted in Fig. 2b.
When acetonitrile was added in DMSO, the absorption spec-
trum of NDI-AA 8 showed a slight blue shift with an increase
in absorption intensity. However, the absorption spectra of 8
were also found to be narrower with increasing acetonitrile
concentration. The blue shift is due to low polarity of aceto-
nitrile and increasing absorbance is due to a weakening of π-σ
interaction in the relatively less polar solvent. Interestingly,
1.0
NDA
NDT
Mixture
0.5
0.0
300
350
400
450
500
Wavelength (nm)
Fig. 1 UV-visible absorption spectra of NDI-AA 8, NDI-TT 4 and their
mixture in 60:40% DMSO/water

J Fluoresc
Fig. 2 UV-visible absorption
spectra of NDI-AA 8 in (a)
DMSO/water and (b)
2.5
2.0
1.5
1.0
0.5
0.0
a
NDA
DMSO/acetonitrile
DMSO/H2O 100:0
DMSO/H2O 80:20
DMSO/H2O 60:40
DMSO/H2O 40:60
DMSO/H2O 20:80
300
350
400
450
500
Wavelength (nm)
2.5
2.0
1.5
1.0
0.5
0.0
b
NDA
DMSO/CH3CN 100:0
DMSO/CH3CN 80:20
DMSO/CH3CN 60:40
DMSO/CH3CN 40:60
DMSO/CH3CN 20:80
300
350
400
450
500
Wavelength (nm)
(Fig. 3a), the absorbance of NDI-TT 4 decreased remarkably
with the addition of water in DMSO. This decrease was un-
expected as the interaction of 8 and 4 was predicted to be
similar. This will be discussed later on. The addition of aceto-
nitrile to DMSO (Fig. 3b), resulted in similar observations for
NDI-TT 4 and NDI-AA 8, which was expected because hy-
drogen bonding does not play any role in DMSO/acetonitrile
mixture. However, the absorption spectra of NDI-AT 10, as
depicted in Fig. 4a, behaved similar to NDI-AA 8 suggesting
conjugated aromatic rings of adenine has a dominant effect on
π-π* transitions of NDI chromophores.
To understand different species in the ground and excit-
ed state, we recorded the more sensitive fluorescence exci-
tation and emission spectra of NDI-AA 8 and NDI-TT 4 at
different possible excitation and emission wavelengths. At
excitation wavelengths 360 nm, 368 nm, 378 nm and

J Fluoresc
Fig. 3 UV-visible absorption
spectra of NDI-TT 4 in (a)
DMSO/water and (b)
DMSO/acetonitrile solvent
mixture
2.5
2.0
1.5
1.0
0.5
0.0
NDT
a
DMSO/H2O 100:0
DMSO/H2O 80:20
DMSO/H2O 60:40
DMSO/H2O 40:60
DMSO/H2O 20:80
300
350
400
450
500
Wavelength (nm)
2.5
b
NDT
2.0
1.5
1.0
0.5
0.0
DMSO/CH3CN 100:0
DMSO/CH3CN 80:20
DMSO/CH3CN 60:40
DMSO/CH3CN 40:60
DMSO/CH3CN 20:80
300
350
400
450
500
550
Wavelength (nm)
409 nm, both 8 (see Fig. 5), 4 (see Fig. 6) and 10 (see Fig.
4b) showed similar emission spectra with vibronic struc-
tures. However, the fluorescence intensity of the emission
band at around 541 nm remarkably increased at excitation
wavelength 409 nm. Similarly, at emission wavelengths
411 nm, 434 nm and 541 nm both NDI-AA 8 and NDI-
TT 4 gave similar excitation spectra but the fluorescence
intensity was remarkably high at around 405–410 nm. It
should be noted that the excitation spectra and absorption
spectra were different, which suggest the absorbing and
emitting species are different indicating aggregation of
chromophores in the excited state. The band at around

J Fluoresc
Fig. 4 a UV-visible absorption
spectra of NDI-AT 10 in
a
DMSO/acetonitrile mixture; b
Fluorescence excitation and
emission spectra of NDI-AT 10 in
different excitation and emission
wavelengths in 60:40
DMSO/water mixture
b
405–410 nm was not clearly observed in absorption spectra
but it is well resolved in the excitation spectra of 4 and 8.
This indicates excimer formation along with ground state
aggregation. The photo-stability of the NDI-AA 8: NDI-
TT 4 mixture was also investigated and there was no
change in spectral shape and intensity even after 70 min
(see Fig. 7).
Hydrogen bonding is sensitive to temperature. We ana-
lyzed absorption spectra of NDI-AA 8, NDI-TT 4 and the
NDI-AA 8: NDI-TT 4 mixture at temperatures between
20 °C and 80 °C in 60:40% DMSO:water to gain greater
insight into the interaction between 8 and 4. As seen in
Fig. 8, an increase in temperature decreased the absorbance
of NDI-AA 8. The decrease in absorbance of 8 with in-
creasing temperature could be explained based on the fact
that at elevated temperature, hydrogen bonding between
adenine and adenine is disrupted, thus, encouraging π-σ
interaction among NDIAAs. On the other hand, no such
change for NDI-TT 4 with increasing temperature indicates
hydrogen bonding is not playing a crucial role in this

J Fluoresc
Fig. 5 Fluorescence excitation
and emission spectra of NDI-AA
8 in different excitation and
emission wavelengths in 60:40
DMSO/water mixture
derivative. This is further supported by Fig. 3a, which
showed a decrease in absorbance with a greater water per-
centage in DMSO. The addition of water does not appear
to help hydrogen bond formation between thymine-thy-
mine, rather increasing polarity of the solvent encourages
π-σ interaction among NDI-TTs, thus, decreasing the ab-
sorbance. A mixture of 4 and 8 led to a decrease in absor-
bance with elevating temperature but it was less than NDI-
AA 8 alone, which is mainly due to the contribution from
NDI-AA 8 in the mixture.
Fluorescence spectral change with solvent environment of
NDI-AA 8 (Fig. 9) vs. NDI-TT 4 (Fig. 10) was investigated.
In NDI-AA 8, an increase in water content shifted the fluo-
rescence emission spectral position to the blue region and
the fluorescence emission peak at around 535 nm disap-
peared in above 70% water in the solvent mixture. In this
case a new peak around 390 nm and a maximum at 405 nm
appeared at the expense of the peak at around 475 nm and
550 nm. This 405 nm peak is similar to the spectral position
found in the excitation spectrum, which could be 0–0
Fig. 6 Fluorescence excitation
and emission spectra of NDI-TT 4
in different excitation and
emission wavelengths in 60:40
DMSO:water

J Fluoresc
Fig. 7 Fluorescence emission
spectra of NDI-TT 4 in 60:40
DMSO:water at different time
intervals
NDT
0 Mins.
5 Mins.
10 Mins.
15 Mins.
20 Mins.
25 Mins.
30 Mins.
35 Mins.
40 Mins.
45 Mins.
50 Mins.
55 Mins.
60 Mins.
65 Mins.
70 Mins.
3x105
2x105
1x105
0
400
450
500
550
Wavelength (nm)
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 8 Fluorescence excitation
and emission spectra of NDI-AA
8 in different combination of
excitation and emission
wavelengths in 60:40
NDT in DMSO:H O 60:40
2
0.15
0.10
0.05
0.00
NDT in DMSO:H O 60:40
2
20oC
30oC
40oC
50oC
60oC
70oC
80oC
20oC
30oC
40oC
50oC
60oC
70oC
80oC
DMSO:water
300
350
400
450
500
300
350
400
450
500
Wavelength (nm)
Wavelength (nm)
1.0
0.9
0.8
0.7
0.6
NDA
NDT
NDA + NDT
Linear Fit to NDA
Linear Fit to NDT
Linear Fit to NDA + NDT
20
30
40
50
60
70
80
Temperature (oC)

J Fluoresc
Conclusion
A straightforward, facile and high-yielding strategy, synthesis of
nucleobase (adenine/thymine) conjugated naphthalenediimide
(NDI), namely, NDI-AA8 and NDI-TT 4, was presented. The
prepared compounds were characterized by various spectro-
scopic and analytical techniques. Photophysical study
established that NDI-AA 8, NDI-TT 4 and the NDI-AA 8:
NDI-TT 4 mixture have similar UV-visible absorption in the
300–400 nm region. It was observed that effect of adenine/
thymine group on the absorption of the NDI chromophore is
negligible and the absorption is due to π-π* transitions of NDI
chromophores polarized along the z-axis. The influence of sol-
vent polarity and temperature on absorption spectra suggested
aggregation formation, which can be either through intermolec-
ular π-σ interaction between the main chromophore or through
intermolecular hydrogen bonding between adenine-adenine
groups in NDI-AA 8. However, addition of water does not help
for hydrogen bond formation between thymine-thymine in NDI-
TT 4; rather an increase in solvent polarity encourages π-σ
interaction among NDI-TTs. No spectral change for NDI-TT 4
with increasing temperature confirmed hydrogen bonding is not
playing a crucial role in NDI-TT 4. A fluorescence study on
NDI-AA 8 indicated excimer formation along with ground state
aggregation. It was shown that the emission band around
475 nm and above is due to aggregation resulting from π-σ
interaction of NDI-AA 8. As the water content in the solvent
mixture increased, aggregation of 8 was discouraged due to
adenine-adenine hydrogen bonding. Nevertheless, the emission
spectrum of NDI-TT 4 did not shift to the short wavelength
region and the fluorescence intensity of the peak around
Fig. 9 Fluorescence emission spectra of NDI-AA 8 with increase
concentration of water in DMSO/water solvent mixture
transition of NDI-AA 8. Thus, the emission band around
475 nm and above is due to aggregation resulting from
π-σ interaction of NDI-AA 8. As the water content in the
solvent mixture increases, aggregation of NDI-AA 8 is dis-
couraged due to adenine-adenine hydrogen bonding similar
to results described earlier. At the same time, the emission
spectrum of NDI-TT 4 did not shift to the blue region and
the intensity of the peak around 535 nm increased with the
expense of fluorescence in 411 nm. In the case of NDI-TT 4,
increasing water content in the solvent mixture facilitates
aggregation through π-σ interaction as thymine-thymine hy-
drogen bonding is less pronounced.
Fig. 10 Fluorescence emission
spectra of NDI-TT 4 with increase
concentration of water in
DMSO/water solvent mixture

J Fluoresc
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535 nm increased at the expense of fluorescence in 411 nm.
Thus, an increase in water content in the solvent mixture facil-
itates aggregation through π-σ interaction in NDI-TT 4, as
thymine-thymine hydrogen bonding is less pronounced. The
photo-stability of the NDI-AA 8: NDI-TT 4 mixture was further
established as there was no change in spectral shape and inten-
sity even after 70 min.
Acknowledgements The authors are grateful to the Lebanese National
Council for Scientific Research (LNCSR), the University Research Board
(URB) and the Kamal Shair CRSL research fund at the American
University of Beirut for financial support.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
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