Optical study of the formation of pyrrolo[2,3-d]pyrimidine-based fluorescent nanoaggregates

Article abstract of DOI:10.1016/j.tet.2013.09.044

Pyrrolo[2,3-d]pyrimidine derivatives possessing different sterically-hindered end-groups at position 7 of the heterocycle were studied and compared with respect to nanoaggregate formation ability by the reprecipitation method in aqueous solutions. The emergence of nanoaggregates with an increasing water fraction in THF/water mixture was traced by observing sudden changes in spectral and transient fluorescence dynamics accompanied by fluorescence efficiency turn-on. The aggregation induced emission with a maximal 20-fold emission efficiency enhancement was obtained. Tuning of the nanoaggregates sizes from about 50 nm to 600 nm by increasing the THF/water ratio was revealed by electron microscopy. Almost perfect spherical shapes of the nanoaggregates and their structureless fluorescence bands similar to those of their neat amorphous films suggested an amorphous-like nature of the pyrrolo[2,3-d]pyrimidine-based nanoparticles.

Full text of DOI:10.1016/j.tet.2013.09.044

Accepted Manuscript
Optical study of the formation of pyrrolo[2,3-d]pyrimidine-based fluorescent
nanoaggregates
Lina Skard˛iūtė, Karolis Kazlauskas, Jelena Dodonova, Jonas Bucevičius, Sigitas
Tumkevičius, Saulius Juršėnas
PII:
S0040-4020(13)01466-X
10.1016/j.tet.2013.09.044
TET 24819
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 20 March 2013
Revised Date: 4 September 2013
Accepted Date: 16 September 2013
Please cite this article as: Skard˛iūtė L, Kazlauskas K, Dodonova J, Bucevičius J, Tumkevičius
S, Juršėnas S, Optical study of the formation of pyrrolo[2,3-d]pyrimidine-based fluorescent
nanoaggregates, Tetrahedron (2013), doi: 10.1016/j.tet.2013.09.044.
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Graphical Abstract
Optical study of the formation of
Leave this area blank for abstract info.
pyrrolo[2,3-d]pyrimidine-based fluorescent
nanoaggregates
Lina Skardžiūt÷a*, Karolis Kazlauskasa, Jelena Dodonovab, Jonas Bucevičiusb, Sigitas Tumkevičiusb, and
Saulius Jurš÷nasa
a Institute of Applied Research, Vilnius University, Saul÷tekio 9-III, LT-10222 Vilnius, Lithuania
b Department of Organic Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania
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1
Tetrahedron
journal homepage: www.elsevier.com
Optical study of the formation of pyrrolo[2,3-d]pyrimidine-based fluorescent
nanoaggregates
Lina Skardžiūt÷a*, Karolis Kazlauskasa, Jelena Dodonovab, Jonas Bucevičiusb, Sigitas Tumkevičiusb, and
Saulius Jurš÷nasa
a Institute of Applied Research, Vilnius University, Saul÷tekio 9-III, LT-10222 Vilnius, Lithuania
b Department of Organic Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pyrrolo[2,3-d]pyrimidine derivatives possessing different sterically-hindered end-groups at
position 7 of the heterocycle were studied and compared with respect to nanoaggregate
formation ability by the reprecipitation method in aqueous solutions. The emergence of
nanoaggregates with an increasing water fraction in THF/water mixture was traced by observing
sudden changes in spectral and transient fluorescence dynamics accompanied by fluorescence
efficiency turn-on. The aggregation induced emission with a maximal 20-fold emission
efficiency enhancement was obtained. Tuning of the nanoaggregates sizes from about 50 nm to
600 nm by increasing the THF/water ratio was revealed by electron microscopy. Almost perfect
spherical shapes of the nanoaggregates and their structureless fluorescence bands similar to
those of their neat amorphous films suggested an amorphous-like nature of the pyrrolo[2,3-
d]pyrimidine-based nanoparticles.
Available online
Keywords:
Keyword_1 pyrrolo[2,3-d]pyrimidines
Keyword_2 fluorescent organic nanoparticles
Keyword_3 fluorescence turn-on
Keyword_4 oligoarylenes
leads to an effective fluorescence turn-on.13 The fluorescence
enhancement phenomenon in the aggregated form is termed
1. Introduction
aggregation induced emission (AIE).13-15 The AIE effect in some
cases can further be enhanced by the favorable molecule packing
(similar to that in J-type aggregates) facilitated by special
molecular design. However, generally, planar and rigid
molecules tend to form tightly packed H-type aggregates
(crystallites) with strong intermolecular interaction resulting in
emission quenching.16-18 In contrast, the non-planar molecules
bearing bulky peripheral groups can constitute amorphous-like
aggregates with significantly weakened intermolecular
interactions, and thus, can promote AIE property.19,20
Single molecules and bulk materials usually demonstrate
distinct electronic, optical and chemical features, and therefore,
special attention must be paid to the molecule arrangement and
intermolecular interactions, which essentially determine material
properties in the aggregated state. Recently, much attention has
been drawn to organic nanoparticles due to their unique optical
and electrochemical characteristics.1-3 As opposed to the
extensive studies of metal and traditional inorganic
semiconductor nanoparticles, the so called quantum dots, the
research of the organic nanoaggregates is still in the early stages
and is mainly focused on the structure – property relations of the
aggregated nanostructures.3 The possibility of tailoring electronic
and optical nanoparticle properties via chemical engineering of
the constituent molecules, as well as an abundance of organic
synthesis methods for altering molecule structure, imply a variety
of nanoparticle applications. Organic nanoparticles have already
been proven to perform well not only in the areas of
photovoltaics,4 organic light-emitting diodes5,6 and field-effect
transistors,7 but also in chemical or biological applications such
as fluorescent labels8,9 and sensors.10-12
In this work, we investigate the formation and photophysical
properties of the FONs based on pyrrolo[2,3-d]pyrimidine
derivatives. The pyrrolo[2,3-d]pyrimidines have been extensively
studied for medical applications due to their antiviral,
antibacterial, antifungal and antitumor properties.21-26 Moreover,
pyrrolo[2,3-d]pyrimidine, due to its structural resemblance to
biogenic purines, is a bio-compatible heterocycle.27 Some of the
pyrrolo[2,3-d]pyrimidine derivatives with fluorescent tags were
shown to be suitable substrates for DNA polymerases and also
useful for labeling of nucleic acids.28-32 It is thus expected that
pyrrolo[2,3-d]pyrimidine-based FONs could find application in
cellular or immunofluorescence labeling by benefiting from high
A special class of organic nanoparticles are the fluorescent
organic nanoparticles (FONs) distinguished by high fluorescence
efficiency. The molecules forming FONs are usually weakly or
non-fluorescent in the isolated state due to the efficient
nonradiative deactivation occurring via intramolecular torsions,
while the suppression of these motions in the aggregated state
emission
efficiency,
ready
chemical
modification,
biocompatibility and non-cytotoxicity.9 Previously, we have
reported the synthesis of a number of pyrrolo[2,3-d]pyrimidine

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Tetrahedron
derivatives possessing different polar substituents and featuring
intense blue emission.33,34
Figure 1 Chemical structures of the pyrrolo[2,3-d]pyrimidine
derivatives
Here we demonstrate the formation of the pyrrolo[2,3-
d]pyrimidine-based FONs by the reprecipitation method in
aqueous solution. The derivatives in this study were designed to
have identical pyrrolo[2,3-d]pyrimidine cores, yet different 4-
(diphenylamino)-, 4-(dimethylamino)- or 4-(9-carbazolyl)phenyl
end-groups. The photophysical and morphological properties of
the fluorescent nanoparticles were examined and compared
utilizing various techniques including absorption and
fluorescence spectroscopy, excited stated decay time and
quantum yield evaluation methods as well as scanning electron
and fluorescence microscopy.
Our previous studies have shown that the electronic ground
state of the carbazole-free pyrrolo[2,3-d]pyrimidine derivatives
(similar to compound 2) is determined by pyrrolo[2,3-
d]pyrimidine core, whereas upon excitation intramolecular
charge transfer between the core and the polar substituent at the
position 7 of the heterocycle (triphenylamine in the case of
compound 2) occurs.33 On the other hand, the ground state of the
derivatives containing carbazole groups (similar to compounds 3,
4 and 5) is mainly governed by the carbazole moiety, whereas in
the excited state molecular charge is localized predominantly on
the pyrrolo[2,3-d]pyrimidine core, where it is also strongly
affected by the polar character of the substituent at the 7th
position of the heterocycle (4-(diphenylamino)phenyl, 4-
(dimethylamino)phenyl or 4-(9-carbazolyl)phenyl in the case of
compounds 3, 4 or 5, respectively).
2. Results and discussion
Figure
1 shows the chemical structures of the four
pyrrolo[2,3-d]pyrimidine derivatives under study. Our results on
the density functional theory calculations, which are provided in
the supplementary data, indicated that the phenyl group linked to
the 2nd position of the pyrimidine ring is coplanar with the
pyrrolo[2,3-d]pyrimidine core in the ground state. Conversely,
the second phenyl moiety at the 4th position of the pyrimidine
ring is twisted in respect to the core in the ground state (due to a
steric hindrance). Note that the pyrrolo[2,3-d]pyrimidine
compounds 2-5 contain different (diphenylamino, dimethylamino
or carbazolyl) end-groups, which most likely cause different
molecular conformations thereby influencing packing
morphology of the formed nanoaggregates. Moreover, the end-
groups can also exhibit intramolecular torsions/vibrations, and
thus, greatly affect photophysical properties of the compounds in
dilute solutions.
In order to evaluate the significance of intramolecular twisting
on the photophysical properties of the derivatives 2-5, they were
incorporated in PS matrix at very low concentration (0.6 wt %).
As in the case of dilute solutions, at such low concentration no
intermolecular interactions are possible, however, oppositely to
the dilute solutions, intramolecular motions of the molecules in
the rigid PS matrix are severely suppressed.35 A comparison of
the fluorescence quantum yield and transient data obtained in
liquid and rigid media thus is expected to provide valuable
information regarding fast intramolecular motions, which
crucially affect radiative relaxation properties of the compounds.
The details of the photophysical properties of pyrrolo[2,3-
d]pyrimidines 2-5 in the dilute solutions and PS matrices are
summarized in Table 1.
2
3
4
5
Table 1. Photophysical properties of the pyrrolo[2,3-d]pyrimidine derivatives in dilute (10-5 M) THF solutions, 0.6 wt % PS matrix
and FONs (formed at THF/water ratio of 1/9).
Dilute solution
PS matrix
λPL max
(nm)
FON
λPL max
(nm)
[b]
Compd.
QY
(%)
λPL max
(nm)
τ (ns)
τr
τnr [c] (ns)
QY
(%)
τ [d] (ns)
QY
(%)
τ [d] (ns)
(ns)
3.7 (7%)
7.7 (59%)
13.4
6.8 (15%)
22.4 (86%)
2
4
2
528
549
6.5
163.5
190.5
6.8
20
24
448
459
20
20
491
(33%)
1.5 (3%)
7.0 (47%)
14.7
1.8 (3%)
9.7 (24%)
22.5 (73%)
3
3.8
3.9
504
(50%)
1.2 (2%)
13.1
(14%)
20.8
(85%)
1.1 (4%)
2.5 (80%)
4.7 (15%)
2.2 (7%)
9.4 (68%)
19.6 (25%)
4
5
1
429
436
<0.8 a)
8.9
<80
<0.8
13.6
28
39
487
7
533
452
1.1 (4%)
4.3 (65%)
10.4 (31%)
402,
415
35
25.3
34

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a) Fluorescence lifetime is limited by the instrument response function of the setup.
b) Radiative lifetime calculated as
.
c) Nonradiative lifetime calculated as
.
d) Fractional intensities of each fluorescent decay component are indicated in brackets.
Compounds 2, 3 and 4 having flexible phenyl end-moieties
(attached directly to the core or as the components of
diphenylamino or dimethylamino groups) demonstrated low PL
QY (1 – 4 %) in the dilute THF solutions accompanied by typical
nanosecond (compounds 2 and 3) or subnanosecond (compound
4) excited state lifetimes, τ. Estimation of radiative and
nonradiative decay time constants, τr and τnr, for the compounds
indicated the presence of an efficient nonradiative relaxation
pathway with up to two orders of magnitude larger nonradiative
decay rate (1/τnr) as compared to the radiative one (1/τr).
Relatively fast nonradiative relaxation in the compounds is
attributed to the efficient intramolecular motions performed by
the flexible end-moieties. The inhibition of the intramolecular
torsions upon dispersing the compounds in PS matrices resulted
in a large enhancement of the PL QYs up to 20 – 28 % and in a
noticeable elongation of the average τ due to the reduced
nonradiative decay. Since fluorescence transients of the
pyrrolo[2,3-d]pyrimidine compounds dispersed in a PS matrix
exhibited multi-exponential decay profiles, most likely due to the
various conformers present, the average τ was used for the
absorption dynamics of compound 2, the absorption bands of
compounds 3, 4 and 5 considerably broadened and underwent a
slight red shift with a water fraction above 60% suggesting an
emergence of the new state due to molecule aggregation.
Moreover, the slopes of the long wavelength absorption bands
notably flattened. The long-wavelength trailing edges of the
absorption bands in THF/water mixtures have been observed
before and were attributed to the Mie scattering from the
aggregates.14,19 Note that the absorption spectra of the aggregates
formed at the water fraction of 90% are nearly the same as those
of the neat amorphous films (not shown) suggesting the dominant
amorphous phase of the former.
(a)
(b)
v/v % of water
v/v% of water
90%
90%
80%
80%
70%
comparison. In contrast, the compound
5 bearing bulky
carbazolyl end-moieties featured much higher PL QY in solution
(35 %) and only twice as large nonradiative decay rate as
compared to the radiative one. Obviously, bulky end-groups
severely suppressed intramolecular motions of the flexible
phenyl groups already in the solution resulting in nearly the same
PL QY as that obtained in the PS matrix (39 %). Thus indeed, the
examination of the pyrrolo[2,3-d]pyrimidine fluorescence
properties in the liquid and rigid environments proved a key-role
in their intramolecular motions in the excited state deactivation.
70%
60%
60%
50%
40%
50%
40%
20%
0%
20%
400
0%
250
300
350
450
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Figure
2 Absorption spectra of the pyrrolo[2,3-d]pyrimidine
compounds 2 (a) and 4 (b) in THF/water mixtures with increasing
volume percent (v/v%) of water. The spectra are normalized and
vertically shifted for clarity.
Formation of the pyrrolo[2,3-d]pyrimidine nanoaggregates
with increasing water fraction in THF/water mixtures with solute
molecules was optically monitored by measuring absorption and
PL spectra, PL QY and PL transients. Figure 2 shows the
evolution of absorption spectra of compounds 2 and 4 with
increasing water content in the THF/water mixtures. Generally,
all the derivatives exhibited noticeable changes in the absorption
spectra with the water content above 60 - 70%, whereas below
this value, the spectra were dominated by only single molecule
features. For instance, the spectra of compound 2 solution
showed two dominant absorption bands peaking at 270 nm and
320 nm, which were characteristic of a single pyrrolopyrimidine
molecule.33 At a water content above 70%, the spectra underwent
a significant broadening and a slight red shift up to 10 nm.
Additionally, the lower energy absorption band became more
intense. The noticeable broadening of the two spectra could be
attributed to the newly emerged excitonic states, caused by the
intermolecular interaction in the aggregates. Appearance of the
aggregated phase above 70% water fraction is associated with
reduced volume fraction of the solvent (THF) in the mixture,
which forces the hydrophobic solute molecules to squeeze and
clusterize inside the THF droplets.20
Figure 3 depicts PL spectra dynamics of the compounds 2 and
4 with increasing water content in the THF/water mixtures.
Generally, the PL spectra of all the pyrrolo[2,3-d]pyrimidine
derivatives were mainly dominated by one structureless
fluorescence band, which broadened and redshifted with
increasing water fraction up to 60%. The red shift and broadening
can be explained by the solvatochromic effect evoked by
changing the THF and water ratio (and thus polarity) in the
mixture.37
The absorption spectra of the compounds 3, 4 and 5 in the
THF/water mixtures with water content below 60% shared a very
similar shape, which is characteristic of the carbazole fragments
having bands at about 340 nm and 290 nm.36 Similar to the

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Tetrahedron
(a)
(b)
40
(b)
(a)
30
film
20
10
film
v/v% of water
v/v % of water
90%
80%
90%
80%
70%
0
70%
60%
50%
40
(d)
(c)
60%
50%
40%
20%
30
20
10
0
40%
20%
0%
0%
400
500
600
700
400
500
600
700
Wavelength (nm)
Wavelength (nm)
0
20
40
60
80
100
0
20
40
60
80
100
Figure 3 PL spectra of the pyrrolo[2,3-d]pyrimidine compounds
v/v% of water
2 (a) and 4 (b) in THF/water mixtures with increasing volume
percent (v/v%) of water. The solute concentration in the mixture was
kept constant (1 × 10-5 M). Dashed curves mark PL spectra of the
neat films. The spectra are normalized and vertically shifted for
clarity.
Figure 4. Fluorescence quantum yield of the compounds 2 (a), 3
(b), 4 (c) and 5 (d) in THF/water mixtures with increasing volume
percent (v/v%) of water. Lines serve as guides for the eye.
Further evidence for the newly arisen phase is the
enhancement of the PL QYs just above the critical water fraction
60 - 70% (see Fig. 4). As discussed above, the compounds 2, 3
and 4 were found to possess very low PL QYs in pure THF
solutions due to efficient intramolecular torsions resulting in high
nonradiative deactivation rates. The addition of water up to 60%
induced no changes in the PL QY of the compounds indicating
the governing role of single molecule species. The slight decrease
of PL QY from 4% to 1% observed for the compound 2 with the
addition of water content up to 50% could be ascribed to the
solvation effect.37 The observed enhancement of the PL QY just
above 60 % of water fraction occurring concomitantly with the
drastic changes in the absorption and PL spectra dynamics is
attributed to the suppression of intramolecular torsions due to
molecule aggregation. The PL QYs continued growing in the
cases of compounds 2, 3 and 4 until 90% of the solvent in the
The red shift of the PL spectrum from 527 nm to 550 nm,
from 445 nm to 495 nm and from 440 nm to 475 nm were
observed for the compounds 2, 4 and 5 respectively, with the
increasing water fraction from 0 % to 60 %. Interestingly, the PL
spectrum of the dilute THF solution of compound 3 contained
two fluorescence bands, the first peaking at 440 nm and the
second, far more intense, at 550 nm. With only slight addition of
water (up to 20%), the short-wavelength band became the
dominant, while the second one almost disappeared.
Nevertheless, an increase of the water fraction up to 60 %
resulted in a similar red shift of the short-wavelength band to 474
nm. Since there were no drastic changes in the shape of the PL
spectra of all the derivatives 2-5 as compared to that observed in
pure THF, it is safe to assume that the pyrrolo[2,3-d]pyrimidines
retained their molecular characteristics in the mixtures up to
60 % water. When water content exceeded the critical value of
60 - 70% the sudden changes in the dynamics of the PL spectra
appeared, which manifested an emergence of the new aggregated
phase. In particular, the PL spectra of the compounds 2 and 5
exhibited a sudden blue shift, whereas a sharp red shift occurred
for the compounds 3 and 4. However, after the appearance of the
new phase, the PL spectra of all the derivatives began shifting in
the opposite direction, i.e. to shorter wavelengths with increasing
water content in the mixture. It is worth noting that similar to the
absorption spectra, the peak positions and shapes of the PL
spectra of the THF/water mixtures with the largest water fraction
of 90% were found to be very similar to those of the neat
amorphous films (see Fig. 3). The spectra obviously lacked
distinct vibronic peaks. This again confirmed our assumption on
the amorphous nature of the pyrrolo[2,3-d]pyrimidine aggregates
formed by reprecipitation method.
mixtures was replaced by the water. The compound
2
demonstrated a steep increase of the PL QY from 1% up to 20%
with increasing water fraction up to 90%, thus delivering the
maximal QY turn-on ratio of 20. In analogy, the compound 3
exhibited a 10-fold increase in the PL QY (from 2% to 20%),
whereas this increase for the compound 4 was only 7-fold, i.e.
from 1% to 7%. The PL QY data of the pyrrolo[2,3-d]pyrimidine
derivatives 2-5 in the THF/water (1/9) mixtures are summarized
in Table 1. Although PL QYs of the derivatives 2-4 are low and
rather similar at the water fraction of ~50% (before the onset of
AIE effect), they diverge at the water fraction of 90% when the
fluorescent aggregates are formed. Particularly, the QYs of the
fluorescent aggregates of compounds 2 and 3 are nearly the same
as those of their dilute dispersions in PS matrices (see Table 1)
signifying an absence of special molecule arrangements in the
aggregates. Most likely, the molecules in these aggregates pack
in a random way with a relatively large average intermolecular
distance, and thus, relatively weak intermolecular interactions.
Conversely, the reduced QY of compound 4 aggregates as
compared to that of its dilute PS dispersion suggest the presence
of the special molecule packing in the aggregates facilitated by
the electron-donating dimethylamino moiety. The less sterically-
hindered dimethylamino group as compared to the
diphenylamino moiety of compound 3 can more easily approach
the electron-withdrawing pyrrolopyrimidine core in the

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5
neighboring molecule by forming intermolecular charge transfer
state, which in-turn can reduce the QY of the aggregates.38
water fraction above 50% mainly due to the presence of different
phases and different conformers in the mixture, but nevertheless
the tendencies towards the shortening or lengthening of their
average decay time could be clearly observed. The average τ of
the compound 2 exhibited shortening with increasing water
fraction from 0% to 50% and then gradual lengthening of τ with
further increased water content up to 90%. The initial slight
shortening of τ occurring simultaneously with steady PL
redshifting and decreasing of PL QY was explained by the
solvatochromic effect, whereas the subsequent rapid lengthening
of τ occurring concomitantly with the PL blueshifting and
increasing of the PL QY obviously signified a restriction of
intramolecular motions in the aggregated phase. Similar PL
decay behaviors with increasing water content above 50% were
also observed for compounds 3 and 4 in THF/water mixtures,
although the increase in average τ was rather moderate (Fig. 5b).
An increase in the average τ of the aggregates with increasing
water content can be explained by the continuous reduction of
intermolecular distance in the reduced-size THF droplets, thereby
causing enhanced intermolecular interaction and stronger
suppression of intramolecular torsions. However, just a slight
excess of water (up to 50%) in the mixture is insufficient to
obstruct torsional motions of the single molecules.
The constant blue shifting of the PL spectrum accompanied by
continuously growing PL QY observed with an increasing water
fraction above 60 - 70% (after the emergence of the aggregated
phase) most probably reflects the denser packing of the
molecules in the aggregates. Greater spatial restrictions imposed
by the continuously reduced volume fraction of the solvent not
only impedes the intramolecular torsions (and so the nonradiative
decay rate), but also the planarization of the molecules in the
excited state. The restricted planarization thus reduces the extent
of the molecule conjugation resulting in the blue shifting of the
PL bands. An increasing number of fluorescent aggregates versus
weakly fluorescent single molecules might also contribute to the
effects discussed.
As opposed to the PL QY behaviors of compounds 2-4,
compound 5 bearing bulky carbazolyl end-groups exhibited
negligible dependence of the QY on the water fraction in the
mixture, and retained the QY value (34%) in aggregates similar
to that estimated in dilute solution or PS matrix (see Table 1).
The spatial restrictions occurring in the aggregated phase had no
effect on the PL efficiency, since fast intramolecular motions
were already suppressed by the bulky carbazolyl moieties. On the
other hand, highly twisted carbazolylphenyl side-arms prevented
close packing of the compound 5 molecules implying more
disordered or amorphous-like aggregates.
In contrast, the average τ of compound 5 possessing bulky
carbazolyl end-groups was found to be roughly insensitive to the
amount of water (in the range of 0 – 90%) in the THF/water
mixture. This is consistent with inhibition of fast intramolecular
motions by the bulky end-groups already in the dilute solution
and also with a highly twisted molecular structure of the
compound 5 preventing closer packing and stronger squeezing of
the molecules in the aggregates.
(a)
103
90%
80%
102
70%
0%
50%
(b)
103
90%
80%
70%
102
20%
50%
0
20
40
60
Time (ns)
Figure 5 Fluorescence transients of the compounds 2 (a) and 4 (b) in
THF/water mixtures with increasing volume percent (v/v%) of water.
Lines mark multi-exponential fits to the experimental data.
Figure 5 shows PL transients of the pyrrolo[2,3-d]pyrimidines
2 and 4 in THF/water mixtures measured at different water
fractions. The transients of all the compounds generally showed
non-exponential decay profiles (on a nanosecond time scale) with
Figure 6. SEM images of the compound 3 nanoaggregates
formed in THF/water mixtures with 70 (a), 80 (b) and 90 (c) volume
percent of water. The solute concentration in the mixture was kept
constant (1 × 10-5 M).

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Tetrahedron
A scanning electron microscopy aided in visualizing the
molecule aggregation resulted in effective fluorescence turn-on.
The aggregation induced emission with a maximal 20-fold
emission efficiency enhancement (from 1% up to 20%) was
obtained for the pyrrolo[2,3-d]pyrimidine possessing a highly-
twisted 4-(diphenylamino)phenyl end-moiety at position 7 of the
heterocycle. The size tuning of the FONs in the range of 50 –
600 nm was accomplished by the controlling solvent/non-solvent
ratio in the THF/water mixtures while maintaining solute
concentration constant (1 × 10-5 M). Spherical shape of the FONs
revealed by SEM accompanied by featureless PL bands indicated
the dominant amorphous nature of the nanoparticles. Intense
fluorescence of pyrrolo[2,3-d]pyrimidine-based FONs and the
structural similarity to biogenic purines could be exploited for
their application in biosensing.
pyrrolo[2,3-d]pyrimidine-based molecular aggregates casted on
crystalline silicon substrates. The typical SEM images of the
compound 3 nanoaggregates formed in THF/water mixtures with
70, 80 and 90 volume percent of water are displayed in Fig. 6.
The similar images were also acquired for the rest of the
compounds revealing an almost perfectly round shape formed at
the different THF/water ratio, and thus verifying their amorphous
nature.20 A clear tendency of the reduction of mean particle size
with the increasing water fraction was observed. The mean
diameters of the nanoaggregates of all the pyrrolo[2,3-
d]pyrimidine compounds varied from about 600 nm (at 70% of
water) to less than 50 nm (at 90% of water). Although the size of
the compound 2, 3 and 5 nanoparticles formed at the water
fraction of 80% and 90% was similar 50 – 100 nm, the
nanoaggregates of compound 4 at the same water content were
found to be noticeably smaller, i.e. less than 50 nm in diameter.
The decreasing nanoaggregate size with increasing water fraction
in the mixture for the pyrrolo[2,3-d]pyrimidines 2-5 justified the
blueshifting of the PL spectra and constantly increasing PL QYs
to be caused by the more dense packing of the molecules in the
nanoparticles.
4. Experimental
4.1. Instrumentation
1H and 13C NMR spectra were recorded using Varian INOVA
spectrometer (300 MHz and 75 MHz, respectively) at room
temperature. High resolution mass spectra were carried out on a
quadrupole, time-of-flight mass spectrometer (microTOF-Q II,
Bruker Daltonik GmbH, Bremen, Germany) or on Dual-ESI Q-
TOF 6520 (Agilent Technologies) mass spectrometer. Column
chromatography was performed using Silica gel 60 (0.040-
0.063 mm, Merck). The course and purity of the reaction
products were monitored by TLC using Silica gel 60 F254
aluminum plates (Merck). Visualization was accomplished by
UV light. Melting points were determined in open capillaries
with
a
digital melting point IA9100 series apparatus
(ThermoFischer Scientific) and are uncorrected.
Formation of organic nanoparticles in aqueous solutions was
accomplished by the reprecipitation method. Purified water at
certain volume percentage was added to the dilute THF solutions
of pyrrolo[2,3-d]pyrimidine derivatives under vigorous stirring,
while retaining their constant molar concentration (1 × 10-5 M) in
the mixture. Neat amorphous films of the studied compounds
were prepared by spin-coating 1 × 10-3 M THF solutions on glass
substrates. The spin-coating technique was also employed to
form polystyrene (PS) films with molecularly dispersed
compounds at a mass ratio of 0.6%.
Figure 7 Fluorescence microscopy image of the compound 3
fluorescent nanoaggregates formed in THF/water (1/9) mixture.
Fluorescence microscopy was employed for imaging the large
ensembles of pyrrolo[2,3-d]pyrimidine nanoaggregates formed in
THF/water (1/9) mixtures and casted on glass substrates. Figure 7
shows the fluorescence microscopy image of compound 3
fluorescent nanoparticles, which were easily discerned against
the “dark” background owing to their high PL efficiency. The
compounds 2, 4 and 5 demonstrated similar homogenously
distributed and well separated fluorescent nanoaggregates formed
at the water fraction of 90% in the water/THF mixtures.
Dilute solutions of the nanoparticles as well as molecularly
dispersed compounds were placed in 10 mm quartz cuvettes for
optical measurements. Absorption spectra of the dilute solutions
and neat films were registered using UV-Vis-NIR
spectrophotometer
Lambda
950
(Perkin-Elmer).
Photoluminescence spectra of the samples were recorded on a
back-thinned CCD spectrometer (Hamamatsu PMA-11) using a
narrow band of a Xe lamp spectrum (FWHM = 15 nm) peaking
at 340 nm as an excitation source. Photoluminescence quantum
yields (QY) of the samples were estimated utilizing the
integrating sphere (Sphere Optics) coupled to the CCD
spectrometer. Fluorescence transients were measured using a
time-correlated single photon counting technique on a PicoHarp
3. Conclusions
Formation of water-stable FONs from sterically-hindered
pyrrolo[2,3-d]pyrimidine compounds by the reprecipitation
technique
was
demonstrated.
Bearing
different
4-
(diphenylamino)-, 4-(dimethylamino)- or 4-(9-carbazolyl)phenyl
end-moieties, the compounds were compared for their influence
on nanoparticle formation dynamics, nanoparticle size tunability,
spectral features and emission efficiency enhancement upon
aggregation. Formation dynamics of the pyrrolo[2,3-
d]pyrimidine FONs was optically assessed by examining changes
in absorption and PL spectra, PL lifetime and quantum yield
occurring with continuously varied THF/water ratio in the
mixture. Rapid intramolecular torsions of the flexible phenyl
end-moieties in the pyrrolo[2,3-d]pyrimidines were proved to
cause efficient nonradiative relaxation of the single molecules in
solution, whereas the suppression of these motions upon
300 system (PicoQuant GmbH) equipped with
a 330-nm
wavelength light-emitting diode as a pulsed excitation source.
Field-emission scanning electron microscopy (FE-SEM) of the
FONs casted on crystalline silicon plates was performed by SU-
70 microscope (Hitachi) at 2 kV. The contrast of the SEM images
was enhanced by depositing a thin layer of chromium on the
samples. Fluorescence microscopy images of the casted FONs
were obtained using fluorescence microscope BX51 (Olympus).
4.2. Materials

ACCEPTED MANUSCRIPT
7
All the pyrrolo[2,3-d]pyrimidine derivatives 2-5 were
synthesized by a combination of Pd(0) catalyzed Suzuki cross-
coupling reaction followed by Cu(I) catalyzed N-arylation
reaction34, 39, 40 starting from 2,4-dichloropyrolo[2,3-d]pyrimidine
(1) in accordance with the scheme 1.
anhydrous dioxane (3 ml) was flushed with argon and 5.0 mol%
CuI, anhydrous K3PO4 (0.062 g, 0.29 mmol), 9-(4-bromophenyl)-
9H-carbazole (0.045 g, 0.14 mmol), 10.0 mol% trans-1,2-
diaminocyclohexane were added under stirring and argon flow.
Then after 10 hours 1.0 mol% CuI and 10.0 mol% trans-1,2-
diaminocyclohexane were added. Addition of CuI was continued
every two hours in portions of 1.0 mol% until the amount of CuI
reached 10.0 mol%. Then after 2 hours 1.0 mol% CuI and 10.0
mol% trans-1,2-diaminocyclohexane were added. Addition of
CuI was continued every two hours in portions of 1.0 mol% until
the amount of CuI reached 15.0 mol%. Total reaction time was
35 hours. After cooling the reaction mixture to room temperature
ethyl acetate (5 mL) was added, resulting solution was filtered
through a layer of silica gel eluting with ethyl acetate. The filtrate
was concentrated under reduced pressure, the residue was
dissolved in a minimal amount of chloroform and purified by
column chromatography using hexane→hexane:ethyl acetate as
an eluent to give target compound (0.042 g, 59%) as a colourless
solid, mp 326.8-327.7 °C; UV (THF), λ, nm (ε, l·mol-1·cm-1): 245
(5.3×104), 282 (2.7×104), 292 (3.3×104), 329 (2.1×104); δH
(CDCl3): 7.26 [1H, d, J = 3.9 Hz, 5-H (pp)], 7.35-7.64 [18H, m,
3×7-9,12-14-H (2-carb., 4-carb., N7-carb.), 7.80-7.94 [7H, m, 6-
H (pp), 3×10,11 (2-carb., 4-carb., N7-carb.)], 8.19-8.28 [8H, m,
2,6-H (N7-Ph), 3×3,5-H (2-Ph, 4-Ph, N7-Ph)], 8.65 [2H, d, J = 8.4
Hz, 2,6-H (4-Ph)], 9.00 [2H, d, J = 8.4 Hz, 2,6-H (2-Ph)]; δC
(CDCl3): 103.1, 109.9, 110.1, 110.2, 115.4, 120.4, 120.5, 120.61,
120.62, 120.73, 120.74, 123.82, 123.83, 123.9, 125.3, 126.3,
126.4, 126.43, 127.1, 127.5, 128.4, 129.4, 130.1, 130.9, 136.6,
136.8, 137.1, 137.5, 139.6, 140.0, 140.8, 140.9, 141.1, 153.1,
157.3, 158.2. HRMS (ESI): MH+, found 843.3189. C60H38N6
requires 843.3230.
R
Cl
2 R = H; R' = Ph2N
(i)
N
N
3 R = 9-carbazolyl, R' = Ph2N;
4 R = 9-carbazolyl, R' = Me2N;
5 R = R' = 9-carbazolyl
(ii)
N
Cl
N
N
N
H
1
2-5
R
R'
Scheme 1. Reagents and conditions: (i) 2 mol% Pd(OAc)2, 4
mol% (2-biphenyl)dicyclohexylphosphine, 2.4 equiv.
RC6H4B(OH)2, 4.8 equiv. K3PO4, 1,4-dioxane, ꢀ, Ar; (ii) 4-
R'C6H4-Br, CuI, trans-1,2-diaminocyclohexane, K3PO4, 1,4-
dioxane, ꢀ, Ar.
Synthesis of 2,4-diphenyl-7-[4-(diphenylamino)phenyl]-7H-
pyrrolo[2,3-d]pyrimidine (2) and 2,4-di[4-(9H-carbazol-9-
yl)phenyl]-7-[4-(diphenylamino)phenyl]-7H-pyrrolo[2,3-
d]pyrimidine (3) were reported by us earlier.34
2,4-Di[4-(9H-carbazol-9-yl)phenyl]-7-[4-
(dimethylamino)phenyl]-7H-pyrrolo[2,3-d]pyrimidine (4).
A
solution of 2,4-di(4-(9H-carbazol-9-yl)phenyl)-7H-pyrrolo[2,3-
d]pyrimidine33 (0.1 g, 0.17 mmol) in anhydrous dioxane (3 ml)
was flushed with argon and 1.0 mol% CuI, anhydrous K3PO4
(0.062 g, 0.29 mmol), 4-bromo-N,N-dimethylbenzeneamine
(0.028 g, 0.14 mmol), 10.0 mol% trans-1,2-diaminocyclohexane
were added under stirring and argon flow. Addition of portions of
1.0 mol% CuI to the reaction mixture was continued every two
hours until the amount of CuI reached 5.0 mol%. Then after 2
hours 1.0 mol% CuI and 10.0 mol% trans-1,2-
diaminocyclohexane were added. Addition of CuI was continued
every two hours in portions of 1.0 mol% until the amount of CuI
reached 10.0 mol%. Then after 2 hours 1.0 mol% CuI and 10.0
mol% trans-1,2-diaminocyclohexane were added. Addition of
CuI was continued every two hours in portions of 1.0 mol% until
the amount of CuI reached 14.0 mol%. Total reaction time was
32 hours. After cooling the reaction mixture to room temperature
ethyl acetate (5 mL) was added, resulting solution was filtered
through a layer of silica gel eluting with ethyl acetate. The filtrate
was concentrated under reduced pressure, the residue was
dissolved in a minimal amount of chloroform and purified by
column chromatography using hexane:ethyl acetate (16:1) as an
eluent to give target compound (0.065 g, 65%) as a yellow solid,
mp 281.4-282.6 °C; UV (THF), λ, nm (ε, l·mol-1·cm-1): 239
(4.8×104), 257 (3.9×104), 293 (2.7×104), 342 (2.4×104); δH
(CDCl3): 3.11 (6H, s, N(CH3)2), 6.98 [2H, d, J = 9.3 Hz, 3,5-H
(N7-Ph)], 7.12 [1H, d, J = 3.6 Hz, 5-H (pp)], 7.35-7.66 [13H, m,
6-H (pp), 2×7-9,12-14-H (2-carb., 4-carb.)], 7.75-7.79 [4H, m,
2×3,5-H (2-Ph, 4-Ph)], 7.89 [2H, d, J = 8.7 Hz, 2,6-H (N7-Ph)],
8.21 [2H, d, J = 6.3 Hz, 10,11-H (4-carb.)], 8.23 [2H, d, J = 6.6
Hz, 10,11-H (2-carb.)], 8.63 [2H, d, J = 8.7 Hz, 2,6-H (4-Ph)],
8.95 [2H, d, J = 9 Hz, 2,6-H (2-Ph)]; δC (CDCl3): 40.9, 101.4,
110.2, 110.3, 113.0, 114.9, 120.3, 120.5, 120.6, 120.7, 123.8,
123.9, 125.5, 126.3, 126.4, 127.0, 127.3, 127.4, 129.9, 130.2,
130.8, 137.9, 138.3, 139.1, 139.6, 140.9, 141.0, 149.9, 153.0,
156.8, 157.7. HRMS (ESI): MH+, found 721.3053. C50H36N6
requires 721.3074.
Acknowledgments
The research was funded by a grant (No. MIP-073/2011) from
the Research Council of Lithuania. Dr. S. Šakirzanovas is
acknowledged for performing SEM measurements.
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ACCEPTED MANUSCRIPT
SUPPLEMENTARY DATA
Optical study of the formation of pyrrolo[2,3-d]pyrimidine-based
fluorescent nanoaggregates
Lina Skardžiūt÷a*, Karolis Kazlauskasa, Jelena Dodonovab, Jonas Bucevičiusb, Sigitas
Tumkevičiusb, and Saulius Jurš÷nasa
a Institute of Applied Research, Vilnius University, Saul÷tekio 9-III, LT-10222 Vilnius, Lithuania
b Department of Organic Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania
Results of the density functional theory calculations
Figure 1s shows the optimized ground state geometries of the pyrrolo[2,3-d]pyrimidine
compounds. The values of the dihedral angles are provided in Table 1s. All calculations were
carried out using density functional theory (DFT) as implemented in Gaussian03 software package.1
Molecular structures of compounds 2-5 were optimised using B3LYP hybrid functional and 6-
311G** basis set.
Figure 1s. Optimized ground state geometries of compound 2 (a), compound 3 (b), compound 4 (c)
and compound 5 (d). The values of the dihedral angles are summarized in Table 1s.
Table 1s. Dihedral angles in the optimized ground state geometries of the pyrrolo[2,3-d]pyrimidine
compounds.

ACCEPTED MANUSCRIPT
a
b
c
a1
-
55°
57°
55°
b1
-
56°
58°
55°
c1
-
-
Compound 2
Compound 3
Compound 4
Compound 5
33°
33°
32°
33°
0°
0°
0°
0°
38°
41°
45°
38°
-
58°
References
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Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.;
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X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.;
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