Synthesis and optical properties of covalently bound Nile Red in mesoporous silica hybrids-comparison of dye distribution of materials prepared by facile grafting and by co-condensation routes

Article abstract of DOI:10.1039/c5ra22736d

The fluorescence dye Nile Red (NR) can be covalently ligated to hexagonally ordered, mesoporous silica materials (MCM-41) via co-condensation and post grafting routes in order to investigate possible differences in the dye distributions. The obtained hybr

Full text of DOI:10.1039/c5ra22736d

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DOI: 10.1039/C5RA22736D.
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DOI: 10.1039/C5RA22736D
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Synthesis and optical properties of covalently bound
Nile Red in mesoporous silica hybrids -
ite this: DOI: 10.1039/x0xx00000x
Comparison of dye distribution of materials prepared
by facile grafting and by co-condensation routes.
Received 00th January 2012,
Accepted 00th January 2012
a
b
c
c
Markus Börgardts, Kathrin Verlinden, Manuel Neidhardt, Tobias Wöhrle,
b
c
b
,a
Annika Herbst, Sabine Laschat, Christoph Janiak, and Thomas J. J. Müller*
DOI: 10.1039/x0xx00000x
www.rsc.org/
The fluorescence dye Nile Red (NR) can be covalently ligated to hexagonally ordered,
mesoporous silica materials (MCM-41) via co-condensation and post grafting routes in order to
investigate possible differences in the dye distributions. The obtained hybrid materials display
emission properties similar to free NR but in contrast to free dye molecules even aqueous gels
are luminescent, rendering these materials particularly interesting for biolabeling applications.
All materials were structurally characterized by nitrogen sorption measurements, small angle
X-ray scattering (SAXS), and transmission electron microscopy (TEM). Their optical
properties were characterized by UV/Vis and fluorescence spectroscopy.
1
2
structure in statu nascendi (co-condensation).
Yet, the
Introduction
determination of the homogeneity of the dye distribution,
especially at low dye loading proved to be difficult and only
little research has been dedicated to the comparison of dye
distribution of hybrid materials synthesized by post grafting
methods and one-pot synthesis. Especially there is barely any
comparison between the two most feasible synthesis methods of
hybrid materials with similar structural properties, i.e. grafting
onto commercially available mesoporous silica and co-
After the discovery of pure inorganic mesoporous silica
materials in 1992, research on mesoporous composite materials
1
-3
modified by organic molecules quickly evolved. By chemical
and physical manipulation of their pore systems, the properties
of rigid silica and various functional molecules can be
combined and lead to unique features of the hybrid materials in
comparison to the individual components. As a consequence
these novel composite materials open novel applications in
1
3,14,15
condensation.
4
Taking advantage of the inherent chemical, thermal and
dimensional stability of silica hybrid materials various
fluorescent dyes have already been incorporated into
mesoporous silica by adsorption or covalent ligation, thus
various topical fields of research, such as catalysis, optical
5
,6
7
8-10
sensing, solid state lasers, and drug delivery.
For sensing
applications mesoporous silica hybrids are particularly
advantageous due to their pore structure (e.g. MCM-41)
enabling facile mass transport at high rates, which is essential
for quick response times to environmental changes.
Furthermore, the shape and size of silica particles can be
controlled by varying the synthetic conditions, thus rendering
them also favorable for medicinal applications, such as drug
1
6,17
enhancing the photostability of the organic components.
Most advantageously a co-condensation strategy could give rise
to a homogenous distribution, thereby avoiding aggregation-
1
8,19
induced self-quenching of the dye molecules.
A typical fluorescence dye displaying emission self-quenching
in the solid state is Nile Red (NR). NR possesses high
fluorescence quantum yields in lipids and unpolar solvents and
is widely used in biological applications as a lipophilic stain or
1
1
delivery or biolabeling. Special sensitivity can be additionally
introduced by modification of the silica9 surface with
biochemical functionalities, such as antibodies.
2
0-22
a
laser dye.
Moreover, the pronounced emission
The simplest route of doping organic dyes into sol-gel matrices
is the adsorption and entrapping of the chromophores during
their synthesis. However, plain adsorption inevitably causes
leakage and migration of the dyes, although this problem can be
circumvented by covalent ligation of the dye to free silanol
groups in the silica framework. In this manner, a dye containing
a reactive functionality can be grafted onto the silanol groups of
solvatochromicity allows its use as polarity sensor in cellular
2
3,24
environments.
Unfortunately, NR´s water insolubility
restricts its application to lipids and highly hydrophobic micro-
environments. But still since NR´s fluorescence does not
interfere with the cellular autofluorescence (typically below
5
50 nm), NR could be an ideal probe for biolabeling and thus
recent work is dedicated to water-soluble, but still luminescent
the silica matrix in the sense of
functionalization. Alternatively and quite elegantly in a highly
convergent fashion, dye with pending trialkoxysilyl
a
postsynthetic
2
5-27
NR derivatives.
As a consequence a new class of
luminescent stains for intracellular imaging could evolve.
Upon incorporation of NR into mesoporous silica, aggregation-
induced self-quenching in aqueous media should be supressed,
a
functionality can be covalently anchored in the mesoporous
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Page 2 of 15
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ARTICLE
leading to hybrid materials suitable for biotechnological Synthesis
2
8,29
applications.
In addition, these hybrids could be ligated to
For the covalent ligation of NR to the silica maVteierwiaAlrtsiclae Osnildinee
biomolecules by decorative functionalization of the outer
surface of silica particles.
9
chain with a terminal triethoxysilyl group
Starting from 3-diethylaminophenol ( ), via the formation of
the nitroso derivative the 2-hydroxy substituted NR
derivative was obtained according to a literature procedure
Scheme 1). By Williamson ether synthesis the phenol
transformed into the propargyl ether in 24 % yield. Finally,
by CuAAC (Cu-catalyzed alkyne-azide cycloaddition) of
alkyne with (3-azidopropyl)triethoxysilane the required
terminal triethoxysilyl group was introduced to give the
triethoxysilyl functionalized NR precursor in 45 % yield.
The NR silica hybrid materials were synthesized via
postsynthetic functionalization of commercially available
MCM-41 with the precursor molecule (postsynthetic grafting)
and NR silica hybrid materials were prepared via in situ co-
DOI: 39/C5RA22736D
had10to.10be introduced.
1
Finally, these NR-silica hybrids should be still luminescent in
the solid sol state. Potential applications can be envisioned in
solid state dye lasers where the luminescent properties of the
dye are concatenated with the inherent stability of the silica
host structure, concomitantly avoiding toxic solvents and
ensuring easier handling with potentially adjustable shape, e.g.
2
,
3
3
2
(
3 was
4
3
3
7
,30
4
as fibres. Moreover, solid-state fluorescent NR hybrids could
be employed in security technology as near-infrared solid state
3
4
3
1
5
dyes.
6
Here we report the synthesis of NR-functionalized hybrid silica
by the two most feasible synthetic methods i.e. postsynthetic
grafting of commercial available MCM-41 and in situ co-
condensation. Materials prepared by both routes were studied
with respect to homogeneity as well as the effect of applied
synthesis route and dye loading of the hybrid. Furthermore, the
structural and optical properties of these novel hybrid materials
are thoroughly studied and discussed.
5
7
condensation upon simultaneous formation of the mesoporous
structure (in situ synthesis). To exclude dye leakage from the
hybrid materials, soxhlet extraction was performed until no
more dye could be detected in the supernatant.
Results and discussion
OH
O
OH
N
NaNO
HCl, H
2
2
O
O
HO
Br
N
N
Et N
2
OH
2
Et N
OH
K CO
0
-5 °C, 4 h
DMF, ,
2
3
Et
2
N
O
O
Et
2
N
O
O
4
h
DMF, 80 °C, 16 h
1
2 (81 %)
3 (66 %)
4
(24 %)
N
O
N
N
Si(OEt)
3
4 2
CuSO · 5 H O (20 mol%)
sodium ascorbate (50 mol%)
N
O
4
+
N3
Si(OEt)3
DMF, 40 °C, 13 h
Et N
O
2
5
(45 %)
Scheme 1. Synthesis of the triethoxysilyl functionalized NR precursor 5.
During the postsynthetic grafting, the triethoxysilane terminus
of precursor
5 reacts with the free silanol groups exposed on the
surfaces in the pores. The major advantage of this strategy is
retaining the initial pore structure of the silica material.
However, the major drawback is the potential non-uniform
distribution of the organic molecules and pore clogging at
elevated substrate concentrations as
a consequence of
preferential functionalization at the pore openings.
In contrast, a homogenous distribution can be achieved by the
in situ co-condensation approach where tetraethoxysilane
(
TEOS), the precursor dye molecule, and the templating agent
are present in the same pot. Especially in cases where self-
quenching of the emission of fluorophores can be expected at
higher concentrations a homogeneous distribution and dilution
is indispensable. Furthermore, it is still possible to obtain
materials at higher degrees of loading since pore clogging is not
an issue. However, since the dye molecules are immediate Scheme 2. Synthesis of grafted (6a-i) and co-condensed (7a-h
components of the silica material, an increase in organic hybrid materials with different dye loadings.
functionalization might cause a decrease in mesoporous order,
)
inevitably causing a total collapse of the structural order at high
doping levels.
Structural characterization
Nitrogen sorption isotherms
2
| J. Name., 2012, 00, 1‐3
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Analysis of the hybrid materials by N
which are of type IV in the IUPAC classification. These obtained by DFT calculations from the N
isotherms represent the particular situation of mesoporous S3 and S4 in ESI†). These calculated distributionsVie
materials possessing adsorption inside micropores at low and relatively narrow around diameters of ca. 1.5 and 2.8 nm
2
sorption gives isotherms
The pore size distributions of the hybrid materials were
isotherms (Figure 2,
2
a
w
reArt
b
ic
i
le O
m
odal
nlin
e
DOI: 10.1039/C5RA22736D
relative pressures followed by multilayer adsorption and by for MCM-41 and its co-condensed and grafted hybrids.
capillary condensation in the pressure region
p
/
p
0
between 0.25 Noteworthy, the volume fraction of the smaller pores around
and 0.40 (Figure 1 and Fig. S1, S2 in ESI†). Pure MCM-41 1.5 nm and of pores larger than 3 nm has decreased in and
exhibits a H4 hysteresis loop in the higher pressure region ( especially compared to neat MCM-41. As there is no decrease
0.45 to 1) which can be attributed to slit-shaped pores or of the volume fraction for low and high loaded materials, this
3
5
6
p
/
p
0
7
=
3
5-37
internal voids of irregular shape.
This behavior is also found change is attributed to the dye extraction conditions leading to
for the co-condensed and grafted materials but with a less slightly modified pores.
pronounced hysteresis loop. In addition, these materials show a
H1 hysteresis loop in the region of the capillary condensation Table 1. Pore volume and surface area of pure MCM-41,
step which can be rationalized with a high degree of pore-size grafted (
uniformity.
6
) and co-condensed (
7
) hybrids.
3
8,39
Grafted
Co-condensed
pore
BET
pore
BET
sample
surface
sample
surface
area
volume
cm³g ]
volume
-
1
area
-1
[
-1
[cm³g ]
-1
[
m²g ]
[m²g ]
neat
MCM-41
0
.77
806
6
a
0.66
0.69
0.65
0.71
0.64
0.63
0.69
0.66
0.66
808
868
748
878
781
750
851
821
852
7a
7b
7c
7d
7e
7f
0.93
0.95
0.93
0.94
0.97
0.81
0.77
0.74
1042
1060
1044
1061
1075
995
6b
6
c
6d
6
6
e
f
6g
7g
7h
995
944
6h
6
i
Figure 1. N
condensed (7h) hybrid material and pure MCM-41. See Fig. S1
and S2 in ESI† for isotherms of 6a-h and 7a-g
2
-sorption isotherms of a grafted (6i), a co-
.
A further distinction between the co-condensed (7) and grafted
(
6
) materials can be made by means of their specific surface
-
1
areas of ~1000 and ~800 m²g , respectively. These differences
can be rationalized by the modified synthesis conditions of the
co-condensed material relative to the synthesis of pure MCM-
4
1 material. Within the series of co-condensed materials,
hybrids 7f exhibit slightly lower surface areas and pore

h
volumes than the other samples, as well as higher structural
disorder, as can be seen in the hysteresis loop of the capillary
condensation step. This could be due to a higher dye content
inside the pores as this would cause a decrease in surface area.
But it is more likely that this decrease is dependent on the
modified synthesis conditions as it is known that the structure
of MCM materials can be controlled by the addition of
Figure 2. Pore size distribution of a grafted hybrid (6i), a co-
condensed hybrid (7h) and pure MCM-41.
4
0-42
These results imply that the surface area as well as the pore
size distribution is an inherent feature of the employed silica
material or rather its synthesis conditions. They are not affected
by the loading with varying amounts of dye. The shape of the
sorption isotherms and their capillary condensation steps show
no distinct change or shift at higher loadings of dye as was the
alcohols.
Thus, as in the synthesis of 7fh a higher amount
of methanol was applied due to solubility issues of the
precursor molecule, it is very likely that this caused a decrease
in the surface area rather than a higher dye content in the
micromolar range. For the grafted hybrids 6a-i the surface areas
2
-1
range between 748 and 878 m g , whereas neat MCM-41 has a
-
1
2
-1
case for organic molecules at concentrations up to 0.77 mmolg
incorporated in the MCM-41 type material.
surface area of 806 m g . As the experimental error in BET
4
3
2
-1
surface area determination can be ±50 m g the values overlap
with their error margins. So functionalized materials 6a-i do not
differ much from neat silica, indicating that dye doping with Small angle X-ray scattering (SAXS)
-
1
0
.6-23 µmolg does not lead to a significant variation of the
In order to gain deeper insight into the structure of the hybrid
materials the samples were studied by small angle X-ray
scattering (SAXS) (Table 2, Figure 3). The post-grafted hybrid
surface area and pore volume.
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materials 6a-i exhibit 4 reflections corresponding to the (100), from the experimental data, the characterization by
110), (200) and (210) planes, whose 1/d values match with a transmission electron microscopy (TEM) gives direct evidence
relation of 1:√3:√4:√7 which is typical for two-dimensional for the structural ordering as a direct image of th
(
V
e
iewsa
A
m
rtic
p
lele
O
.
n
As
line
DOI: 10.1039/C5RA22736D
ordered hexagonal mesostructured material possessing P6mm shown in Figure 4, the hexagonal, honeycomb-like ordering of
symmetry.
3
8,44
Thus, no change in the mesostructure can be the pores as well as the two-dimensional organization of
observed in comparison to neat MCM-41. Furthermore, the channels is evident when the sample is viewed in direction of
post-grafted hybrids as well as the neat MCM-41 exhibit lattice the pores and perpendicular to the pore channels, respectively.
planes around 4.0 nm which is almost unchanged compared to The quality of the obtained silica structures are comparable to
the co-condensed hybrid materials. Like in the gas sorption commercial MCM-41 and show no dependence on the amount
analysis, the co-condensed materials 7g
-h show slightly smaller of incorporated dye. The assumption of voids inside the hybrid
lattice planes as a consequence of the adjusted synthesis material, as indicated by the hysteresis in the sorption
conditions.
isotherms, can be confirmed in the TEM images. Thus all
structural analysis are mutually consistent with each other and
) and confirm the assumed hexagonal mesoporous ordering as well as
the absence of an influence of the dye incorporation (in the
micromolar range) on the structural ordering.
Table 2. Lattice parameters and lattice planes of grafted (
co-condensed (7) hybrid materials.
6
Grafted
Co-condensed
a
a
sample
b(
5
)
a
d
100
sample
b(
5
)
a
d
100
[
mol·
[nm] [nm]
[
mol·
[nm] [nm]
-
1
-1
g ]
0.6
1.5
2.9
5.9
8.8
12
12
18
23
g ]
0.04
0.2
0.4
0.9
1.9
7.4
10
6
6
6
a
b
c
4.71
4.61
4.60
4.62
4.63
4.64
4.71
4.62
4.63
4.08
4.00
3.98
4.00
4.01
4.02
4.08
4.00
4.01
7a
7b
7c
7d
7e
7f
4.65
4.63
4.64
4.64
4.64
4.48
4.47
4.51
4.03
4.01
4.02
4.02
4.02
3.88
3.88
3.91
6
d
6
6
e
f
g
6
6
7g
7h
h
15
6
i
a
Loading of hybrid with
5
Figure 4. (a) TEM image of 7h along the channel direction and
b) TEM image of 7h perpendicular to the channel direction as
(
well as (c) TEM image of 6i along the channel direction and (d)
TEM image of 6i perpendicular to the channel direction.
Figure 3. SAXS pattern of 6h and 7h
.
Excitation and emission properties
In a detailed investigation of the lattice parameters for the
differently dye loaded materials, no significant deviation can be
observed. All co-condensed materials possess d100 spacing in a
range from 3.88 to 4.03 nm corresponding to lattice parameters
ranging from 4.47 to 4.65 nm. In addition, no decrease of the
reflection intensity is observed which also suggests that the
hexagonal structure is not influenced by the addition of dye. In
summary it can be concluded that the applied dye
concentrations in a micromolar range do not interfere with the
formation of the mesoporous structure as long as the synthesis
conditions remain constant.
Determination of dye incorporation
As a consequence of low amounts of dye employed in the
hybrid synthesis to avoid self-quenching of fluorescence, only
UV/Vis spectroscopy proved useful for the determination of the
effective dye concentration. Therefore, suspensions of the
hybrid materials in DMSO were analyzed. But as the loading of
dye in the hybrid materials had to be calculated from the
UV/Vis spectra via the Lambert-Beer's law, the molar
extinction coefficient for the silica matrix-embedded dye had to
be determined. However, this cannot be done precisely as there
is no reference substance and thus the molar extinction
coefficient had to be estimated. For a reasonable estimation the
environment of the dye inside the hybrid material was modelled
and the molar extinction coefficient of the precursor molecule
was determined. Although it cannot completely be ruled out
Transmission electron microscopy (TEM)
In contrast to the gas sorption and SAXS analysis of the hybrid
materials where a hexagonal ordering of the silica is deduced
4
| J. Name., 2012, 00, 1‐3
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RSC Advances
ARTICLE
that these calculations are error-prone due to the estimation of The total fluorescence of nine grafted (
6
) and eight co-
the molar extinction coefficient, the determination of

in condensed
(
7
)
hybrid materials in the solid state was
DMSO should give reasonable estimate as the absorption determined using an integrating sphere (Ulbricht setup). The
spectrum of the free precursor molecule is essentially identical quantum yields in dependence on the matrix-embedded dye
View Article Online
DOI: 10.1039/C5RA22736D
to the absorption spectra of the hybrid materials suspended in concentration are shown in Figure 6 and Table 4. Both types of
DMSO. This suggests that the environment of the silica matrix- materials, co-condensed as well as grafted, reveal a similar
embedded dye is comparable to pure DMSO. Otherwise the dependence of the quantum yield on the concentration.
absorption would be significantly shifted caused by changes of These materials display fluorescence even at very low dye
the environment polarity. The regression analysis of determined doping levels, such as in the nanomolar range. With increasing
concentrations vs applied concentrations served for gaining dye concentration a steep increase in quantum yield is detected
-
1
higher accuracy by recalculating the concentrations (Figure 5, up to a maximum concentration around 2
molg . Quantum
Table 3). yields of 18 and 23 % for the co-condensed and grafted
The incorporation of dye into the silica material proceeds more material, respectively, were determined. At higher dye loading
effectively by grafting than by co-condensation. The slopes of the quantum yields decrease even below the quantum yields for
the regression lines indicate an incorporation of approximately the nanomolar dye doping levels. This asymptotic convergence
half of the applied dye concentration in the hybrid material to a completely quenched fluorescence very likely arises from
obtained by grafting, whereas for the co-condensed material the fluorescence quenching of NR at higher concentrations, as
only one third of the applied concentration is incorporated.
reported for free NR in the solid state.
Although a lower concentration of reactive silanol groups can Although a similar dependence of the quantum yield on the dye
be expected due to calcination of the commercial MCM-41 loading can be observed, the maximum quantum yield of the
source a higher degree of incorporation of dye by grafting was co-condensed material
7 is about 4 % less than for the grafted
proven. material . Moreover, the grafted hybrids reach a quantum yield
6
As a consequence the lower applied dye concentration is still of 7 % even at high concentrations, whereas the co-condensed
sufficient for the grafted materials, in comparison with the materials are nearly quenched (2 %). A possible explanation for
equivalent loaded co-condensed hybrids.
this behavior could be the weak acidity of the silanol groups
45
(
p
K
a
≈ 7.1), which can protonate the NR molecules, and
thereby cause fluorescence quenching.
Figure 5. Applied vs determined concentrations of the hybrid
materials
0.585 x, R² = 0.969; and co-condensed: y = 0.371 x, R² = Figure 6. Quantum yields of grafted (
.948). hybrids.
6 and 7 (linear regression analysis gives for grafted: y
=
0
6) and co-condensed (7)
Table 3. Applied and calculated concentrations of the grafted Table 4. Quantum yields of the grafted (6) and co-condensed
(6
) and co-condensed (
7
) hybrid materials.
(7
) hybrid materials.
Grafted
Co-condensed
applied calculated
conc. of conc. of
Grafted
Co-condensed
calculated
applied
conc. of
calculated
conc. of
calculated
quantum
yield
quantum
yield
5
5
5
5
conc. of
5
conc. of 5
-
1
-1
-1
-1
-1
-1
[
mol·g ]
1.0
2.5
5.0
10
[
mol·g ]
0.6
1.5
2.9
5.9
8.8
12
[
mol·g ]
0.1
0.5
[
mol·g ]
0.04
0.2
[
mol·g ]
0.6
1.5
2.9
5.9
8.8
12
[
mol·g ]
0.04
0.2
6
a
7a
7b
7c
7d
7e
7f
6a
6b
6c
6d
6e
6f
6g
6h
6i
14
23
20
20
17
12
11
8.6
6.7
7a
7b
7c
7d
7e
7f
8.0
15
6
b
6
c
1.0
0.4
0.4
0.9
1.9
7.4
11
15
15
6
d
2.5
0.9
16
6
e
15
20
20
30
5.0
1.9
18
6
f
20
7.4
5.9
3.0
2.1
6
g
12
18
23
7g
7h
30
11
12
18
23
7g
7h
6
h
40
15
6
i
40
Fluorescence quantum yields
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If the functionalization of the co-condensed hybrids
7
can be
assumed to take place preferentially inside the pores, the
fluorophore molecules will face an environment with a higher
effective concentration of silanol groups in comparison to
predominantly outside bound dyes as expected for the grafted
View Article Online
DOI: 10.1039/C5RA22736D
case
6. Thus, the dye molecules in co-condensed hybrids 7 will
experience a higher degree of protonation and quenching of the
4
6
NR luminophore compared to the grafted hybrids 6.
Solvatochromism
The solvatochromic properties of the 2-hydroxy-substituted NR
derivative are not significantly affected by functionalization
to give the precursor molecule . The excitation as well as
3
5
emission maxima remain almost unchanged, except for the
measurement in diethyl ether where the maxima of the
precursor molecule
relative to the 2-hydroxy substituted NR
Upon incorporation of the NR derivative
5
are slightly hypsochromically shifted
(Figure 7).
into mesoporous
Figure 8. Solvatochromism of 7e
.
3
5
Upon changing the environment of the dye the spectroscopic
properties are also influenced. The excitation as well as
emission maxima of hybrid material 7e are bathochromically
shifted in comparison to precursor
Most strikingly, both spectra reveal a much smaller range of
emission and excitation maxima for the hybrid material than for
silica materials (as studied for hybrid material 7e) the
solvatochromic behavior is mostly retained (Figure 8).
However, it was found that the hybrid materials show a red-
edge excitation shift (REES) known from polar dyes in viscous
5 (Figures 9 and 10).
4
7-50
solvents.
Thus, since it was not possible to obtain
absorption spectra in the solid state and, except for DMSO, in
suspension, excitation spectra were recorded for the
determination of the absorption maxima. But due to the
previous discussion it was not possible to correct these
excitation spectra for the inner filter effect. It cannot be ruled
out that the measured spectroscopic data are slightly error-
prone. However, these systematic errors become negligible as
emission spectra were always recorded at excitation in the short
wavelength part of the excitation spectra where no shift of the
emission spectra could be observed (vide infra Figure 16).
the precursor molecule
5
. Whereas the emission of precursor
5
-1
shifts by 4000 cm from hexane to water, the shift decreases to
-
1
about 2000 cm for the hybrid material 7e. This can be
explained by the dominance of the siloxy environment in the
pores, which is only affected to a minor extend upon changing
the solvent polarity in the pores. This assumption is additionally
supported by the emission maxima of the hybrid material in the
solid state as indicated by the grey line at 643 nm in Figure 9.
Only the emission of hybrid material 7e suspended in ether or
ester causes a hypsochromic shift of the emission maximum
and the suspension in water leads to a significant bathochromic
shift relative to the emission of 7e in solid state. The redshift
for the hybrid material in water relative to the emission in the
solid state can be rationalized by extensive hydrogen bonding,
leading to a better stabilization of the excited state. The reverse
is true for hydrogen-bond accepting solvents, such as ethers and
esters which induce a blue shift of the emission.
Figure 7. Excitation and emission maxima of 2-hydroxy
substituted NR (3) and precursor molecule 5.
Figure 9. Emission maxima of
grey line at 643 nm represents the emission of the hybrid
material in the solid state).
5 and 7e in different solvents
(
6
| J. Name., 2012, 00, 1‐3
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ARTICLE
comparable for both the free precursor
material 7e
Comparing the excitation spectra of the differ
5
and the hybrid
.
V
e
ie
n
w
tl
A
y
rtic
l
le
o
O
adnl
e
ine
d
DOI: 10.1039/C5RA22736D
hybrid materials the effect of loading levels on the excitation
maxima for the grafted ( ) and co-condensed ( ) hybrids can be
6
7
considered to be only minimal. For instance the excitation
maximum of grafted material 6a at 589 nm is shifted
-
1
bathochromically for material 6c by approximately 100 cm
accompanied by a decrease in intensity. The co-condensed
hybrids show a similar trend by redshifting from 572 (7c) to
79 nm
7
-
1
5
(7e) (Figure 12) by approximately 200 cm
concomitantly showing a decrease in excitation intensity with
increasing concentration. In addition, the shape of the excitation
spectra is altered as at the red-edge a relative sharp signal arises
whereas the intensity at 590 nm (grafted hybrids
6) and 580 nm
(
co-condensed hybrids ) decreases even further. This effect is
7
Figure 10. Excitation maxima of
grey line at 580 nm represents the excitation of the hybrid
material in the solid state).
5 and 7e in various solvents
attributed to the primary inner filter effect leading to an
attenuation of the excitation light and resulting in a virtual
redshift at high dye concentrations.
(
The emission spectra indicate a pronounced red shift by 1000
The effect of the solvent polarity on the excitation spectra is not
strongly pronounced, possibly due to the lower sensitivity of
the electronic ground state to polarity changes. However, slight
deviations due to an inner filter effect have to be taken into
account.
-1
-7
cm upon increase of the dye concentration from the 10 over
-6
-5
-1
1
0
to 10 molg (Figure 13). This bathochromic shift is
identical for grafted ( ) and co-condensed ( ) materials? as
6
7
indicated by almost superimposable spectra at comparable dye
loading.
Furthermore, the excitation as well as emission spectra of
Thus, this bathochromic shift can be attributed to reabsorption
of the hypsochromic part of the emission at higher
precursor
5 and hybrid material 7e do not change for
measurements in methanol, DMSO and water. In all three cases
the environment created by the silica matrix is comparable to
the environment of the dye in the respective solvent. This is
also reflected in the differences in the Stokes shifts of the
47
concentrations by dyes with small Stokes shifts. Although NR
in general exhibits a large Stokes shift after incorporation in
51
silica such reabsorption events become possible (Figure 14).
precursor molecule
5 and the hybrid material 7e (Figure 11).
Figure 11. Difference between Stokes shifts of precursor Figure 12. Excitation spectra of selected grafted (
molecule and hybrid material 7e
condensed ( ) hybrid materials with variable dye loading
6) and co-
5
.
7
(emission = 700 nm).
Some solvents hardly show any change in the Stokes shift of
the hybrid material 7e compared to precursor molecule
5
,
although their excitation and emission maxima vary
significantly. A striking example is the identical Stokes shift of
-
1
precursor
= 1600 cm ) in hexane, although the emission and
excitation of hybrid 7e is shifted bathochromically by 3100
5 (ṽ = 1600 cm ) and hybrid material 7e
-
1
(
ṽ
-
1
cm in comparison to compound
5 (Figures 9 and 10). Thus, it
can be concluded that the incorporation of the dye into a silica
matrix significantly changes the spectroscopic properties but
the influence of the solvent polarity on these properties is
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Moreover, there is no change in the absorption spectra of the
suspensions of hybrid materials in DMSO, e
V
x
ie
c
w
e
A
p
r
t
ticlefo
O
r
nline
a
DOI: 10.1039/C5RA22736D
shoulder at 640 nm (Figure S 25, S 26, supporting information)
for some hybrid materials which can be attributed to protonated
NR species. This is additionally supported by fluorescence
quenching studies. Therefore, reabsorption effects in the solid
state causing a bathochromic shift are by far more plausible
4
6,57
than aggregate formation.
The effect of REES can be found for the hybrid materials
suspended in different solvents as well as in the solid state,
although less pronounced. For hybrid materials 6f and 7g the
-
1
emission shifts bathochromically by 133 and 88 cm ,
respectively, upon excitation at 546 and 664 nm. This effect is
much more pronounced for the hybrid materials in suspension
as shown in Figure 15, with maximum shifts of about 1000
-
1
cm . It is striking that predominantly the grafted material
shows bigger shifts compared to the co-condensed hybrids and
that nearly no REES can be observed in water.
Figure 13. Emission spectra of selected differently loaded A thorough inspection of the REES of the grafted (6b) and co-
grafted ( ) and co-condensed (
) hybrid materials (shoulder at condensed (7e) hybrid materials suspended in acetone clearly
95 nm is a measurement caused artefact).
6
7
6
shows that at excitation wavelengths lower than 540 nm both
materials do not distinctly shift the emission in dependence of
There is no evidence for the formation of aggregates although the excitation wavelength (Figure 16). Only upon excitation in
the excitation spectra display a different shape and a slight red the red-edge a shift in the emission maxima can be observed.
shift at higher dye concentrations. These findings could imply As all grafted materials show a more pronounced REES it can
the forming of aggregates, but considering the spectroscopic be assumed that the distribution of dye molecules is more
data from reports on NR aggregation this scenario seems inhomogeneous. While some quite unrestricted molecules are
2
8,51-54
unlikely in our case.
If the alteration of shape in the located on the outside surface, some relatively restricted
excitation spectra was caused by aggregation, it would be quite fluorophores reside inside the pore channels, and a fraction of
unlikely that for the grafted and co-condensed materials, molecules which is bound to the pore openings, where high dye
despite their very different synthesis conditions, nearly identical concentrations are present, enhancing their restrain. In contrast,
aggregates would be formed. Furthermore, literature reported the relatively homogenous distribution inside the pores with
aggregates of NR adsorbed in MCM-41 materials and only slight changes in the restriction of the molecules inside the
suspended in dichloromethane possess full width at half channels could give rise to a less pronounced REES for the co-
maximum (fwhm) values of their emission band around 1000 condensed materials.
-
1 54
cm . Even though these values are quite high, they become
possible due to higher disorder in a restricted environment
1
200
000
800
5
5,56
leading to a broadening of the emission band.
But
1
comparing the fwhm values of the synthesized materials which
-
1
are around 1500 cm for hybrids suspended in dichloromethane
-
1
and even 2000 cm in solid state, aggregation seems even more
unlikely. In addition there is no reduction of fwhm at high dye
loading levels, relative to the low loaded hybrid material.
6
4
2
00
00
00
0
6b
7e
Figure 15. REES of 6b and 7e suspended in different solvents
-
1
(
range of excitation
n
-hexane: 3333 cm , 1,4-dioxane: 2389
-
1
-1
-1
cm , ethylacetate: 1374 cm , CH
3
2
Cl
2
: 3607 cm , acetone:
O: 2309 cm ).
-
1
-1
-1
871 cm , MeOH: 4127 cm , H
2
Figure 14. Excitation and emission spectra of 6c (in the solid
state).
8
| J. Name., 2012, 00, 1‐3
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Journal Name
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ARTICLE
View Article Online
DOI: 10.1039/C5RA22736D
Figure 17. Fluorescence spectra of water suspended hybrid
materials 7e (solid lines) and 6b (dashed lines) upon addition of
HCl at the indicated concentrations.
Figure 16. REES of 6b and 7e in acetone.
Fluorescence quenching
For further investigation of the different hybrid materials
fluorescence quenching experiments were conducted. Upon
addition of hydrochloric acid to the water suspended hybrid
materials 7e and 6b, a decrease in fluorescence intensity can be
monitored (Figure 17).
Both materials show an emission maximum at 664 nm with a
small shoulder at 720–750 nm in the emissive state. Upon
addition of an acid to the samples the emission maximum at
6
64 nm decreases and slightly shifts bathochromically to 670
nm for high acid concentrations. For the two materials with
-
1
comparable dye loading levels of 1.5 and 1.9
molg the
emission spectra at low acid concentrations (up to 6.00
mmolL ) look very similar. But upon further increase of the
-
1
acid concentration, the co-condensed material 7e is more
Figure 18. Stern-Volmer plots of water suspended hybrid
materials 6b (blue circles) and 7e (red squares) upon addition of
HCl at different concentrations.
efficiently quenched than the grafted material 6b
.
For the
identical relative fluorescence intensity for the co-condensed
hybrid 7e a smaller acid concentration is need than for the
grafted hybrid 6b (Figure 18). This indicates the presence of
unequally distributed dye loading inside the different materials,
which can also be observed in the plot of fluorescence
intensities against acid concentrations.
In this Stern-Volmer plot the curves deviate from linearity with
increasing acid concentrations towards the x-axis which is
indicative for the presence of more than one species of
fluorophores with variable accessibility to the quencher. These
different classes of fluorophores can also be deduced from the
excitation spectra of the quenched fluorophores (Figure 19).
In the emissive state the different materials show slightly
variable excitation maxima. For the co-condensed material 7e
the maximum lies at 602 nm, whereas the maximum of the
grafted hybrid 6b is slightly shifted bathochromically to 608
-
1
nm. Upon addition of 200 mmolL of acid both materials show
the same two excitation maxima at 588 and 640 nm, but in the
case of the grafted material 6b the signal at 640 nm is
significantly more intense than in the co-condensed case 7e
.
This newly arising redshifted excitation maximum at 640 nm
corresponds to the literature known absorption maximum of
+
46,57
NR-H .
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alleys to applications of NR-based hybrid materials. Further
studies directed to access novel functional organic hybrid
materials by this approach are currently underway.View Article Online
DOI: 10.1039/C5RA22736D
Experimental
General Considerations
Reagents, catalyst and solvents were purchased reagent grade
and used without further purification. Products were purified
with column chromatography on silica gel 60 (0.040-0.063
mm) from M&N using flash technique.
1
13
H, C and 135-DEPT NMR spectra were recorded on a
Bruker AVIII-300 and the resonance of the particular solvent
1
13
was locked as internal standard (CDCl
7
3
: H

= 7.26,
C  =
1
11 3
7.0, DMSO-d
6
: H

= 2.50,
C  = 39.52). MALDI mass
spectra were measured on a Bruker Daltronics Ultraflex I
spectrometer, HR-ESI mass spectra were measured on a UHR-
QTOF maXis 4G Bruker Daltronics and GC mass spectra were
obtained on a GCMS-QP2010 S spectrometer from Shimadzu.
Combustion analyses were carried out on Elementar vario
MICRO CUBE in the microanalytical laboratory of the Institut
für Pharmazeutische und Medizinische Chemie at the Heinrich-
Heine-Universität Düsseldorf. IR spectra were recorded on
Shimadzu IRAffinity-1. Melting points were measured on a
Figure 19. Excitation spectra of water suspended hybrid
materials 6b (dashed lines) and 7e (solid lines) upon addition of
HCl at the indicated concentrations (emission = 720 nm).
As the grafted materials show a more intense signal of the
+
protonated NR species NR-H than in the co-condensed case it
+
can be assumed that the degree of NR-H is higher in the
grafted hybrid 6b than in the co-condensed 7e. This can be
rationalized by preferred functionalization with the dye
molecules on the outside surface and the pore openings of
grafted materials enabling an easier protonation. In turn, in the
co-condensed material the dye molecules are incorporated
within the pores or even within the silica walls to a larger
extent. In addition the surrounding silica matrix can act as a
proton buffer.
Büchi Melting Point B-540.
isotherms were determined at 77 K by a Quantachrome Nova
200e sorption analyser. Prior to the measurements the samples
N -adsorption-desorption
2
4
were degassed at 80.0 °C for 20 h. Specific surface areas were
calculated using the Brunauer-Emmett-Teller (BET) equation in
the low pressure interval p/p < 0.3. Pore size distributions were
0
calculated from the adsorption branch using the non-local
density function theory based on a slit-pore model. The total
pore volume was calculated at the point of p/p = 0.95. X-ray
diffraction data were collected on a Bruker AXS Nanostar C.
The radiation source was a Siemens X-ray canal with a power
0
Conclusions
The fluorescence dye 2-hydroxy NR was functionalized via
propargylation and CuAAC reaction with an azido substituted
triethoxysilane derivative to furnish a luminescent precursor for
covalent ligation. The formation of mesoporous hybrid
materials by incorporation into silica matrices with variable dye
loading was accomplished by two synthetic approaches, i. e. by
grafting onto a mesoporous MCM-41 host or by in situ co-
condensation with concomitant formation of the mesoporous
silica-dye hybrid. The structural and optical properties of these
novel hybrids were thoroughly studied. Analysis of the
materials by nitrogen sorption measurements in combination
with SAXS and TEM confirmed a two-dimensional hexagonal
columnar ordered mesoporous structure which is not affected
by functionalization with the NR dye in the applied micromolar
concentrations. Investigation of the optical properties revealed a
red-edge excitation shift (REES) and fluorescence quenching,
which were utilized to analyze the dye distribution inside the
different materials. Therefore it can be qualitatively assumed
that different dye species are present inside the grafted and co-
condensed materials. While grafted materials are predominantly
functionalized on the outside surface and in pore openings at
high dye concentrations, the co-condensed materials obviously
possess a more homogenous dye distribution and predominant
functionalization inside the pores or silica walls. The hybrid
materials retain, although much less pronounced, some optical
K1
of 1500 W. The nickel-filtered monochromatic Cu radiation
wavelength of 1.5405 nm was maintained by using crossed
Göbel mirrors as monochromator. Real-time detection was
enabled using a Bruker HI-Star detector. Samples were
measured in Hilgenberg glass capillaries with an outer diameter
of 0.7 nm. Pre-measurement calibration was carried out at
2
98 K with silver behenate as a standard reference. Data
analysis was carried out using the following software: SAXS
from Bruker, Datasqueeze (v. 2.2.8) from Heiney, QTIPlot
(
v.0.9.8) from Ion Vasilief and LCDiXray (v.1.0) from Golbert.
Mesoporous structures were probed by HR-TEM at the institute
of microstructure research in Jülich using a FEI TECNAI G²
operated at 200 kV. All HR-TEM samples were prepared by
placing
a drop of the hybrid material suspension in
dichloromethane onto
temperature.
a
carbon-coated grid at room
Excitation and emission spectra were recorded on a Hitachi F-
000 fluorescence spectrophotometer at T = 293 K. Excitation
7
of fluorescence was always carried out at the excitation
maximum. Quantum yield determinations of the hybrid
powders were obtained with an integrating sphere. Data
analysis and quantum yield calculations were performed with
the software FL Solutions Version 4.0 by Hitachi.
characteristics of the NR, such as the solvatochromism. Synthetic procedures
However, in contrast to the native dye, these synthesized
hybrids possess quantum yields of about 20 % in the solid state
and even fluorescence in aqueous media which opens new
5
8
(
3-Azidopropyl)triethoxysilane (1)
1
0 | J. Name., 2012, 00, 1‐3
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+
Sodium azide (708 mg, 10.9 mmol) and sodium iodide (154 (w), 3046 (br). HR-MS (ESI) calcd. for (C20
H
18
N
2
O
3
+ H) :
m
/
z
= 335.13902 (100%), 336.14237 (22%); Found: 335.13893
mg, 1.03 mmol) were placed in a Schlenk flask under nitrogen
atmosphere and dissolved in DMSO (10 mL). Then (3-
chloropropyl)triethoxysilane (1.35 g, 5.61 mmol) was added
(
(
100%), 336.14204 (15%). Anal. calcd. for Vie 20
C
w Ar
H
ticle
N
Online
O
18
2 3
DOI: 10.1039/C5RA22736D
334.4): C 71.84, H 5.43, N 8.38; Found: C 71.66, H 5.52,
N 8.09.
and the solution was stirred at 60 °C for 18 h. n-Hexane (10
mL) was added and the reaction mixture was stirred at room 9-(Diethylamino)-2-(prop-2-yn-1-yloxy)-5
temp for 3 h. The solution was washed with deionized water (3 benzo[a]phenoxazin-5-one (4)
H-
9
-(Diethylamino)-2-hydroxy-5
H-benz[a]phenoxazin-5-one (3)
x 5 mL) and brine (1 x 5 mL), and the organic layer was dried
with anhydrous magnesium sulfate. The solvents were removed
(
334 mg, 1.00 mmol) and potassium carbonate (280 mg, 2.00
mmol) were dissolved in DMF (5 mL). To this solution
propargyl bromide (145 mg, 1.22 mmol) was added and the
mixture was stirred at 80 °C for 16 h. The reaction mixture was
diluted with diethyl ether (25 mL) and brine (25 mL). The
organic layer was separated and the aqueous layer washed with
diethyl ether (3 x 25 mL). The combined organic layers were
dried with anhydrous magnesium sulfate and the solvents were
-
3
in vacuo and the residue was dried at 10 mbar for 4 d to give
3-azidopropyl)triethoxysilane (1.03 g, 74%) as a colorless oil.
(
1
H NMR (300 MHz, CDCl
3
):
2
 = 0.64-0.71 (m, 2 H, -SiCH -),
3
1
-
3
.23 (t, JHH = 7.0 Hz, 9 H, Si(OCH CH
3)3), 1.65-1.77 (m, 2 H,
2
3
SiCH
2
C
H
2
-), 3.26 (t,
J
HH = 7.0 Hz, 2 H, -SiCH
2
CH
2
C
H
2
-),
3
13
.82 (q, JHH = 7.0 Hz, 6 H, Si(OCH2CH ) ). C NMR (75
3 3
MHz, CDCl3):
 = 7.6 (CH ), 18.3 (CH ), 22.7 (CH ), 53.8 removed in vacuo. The residue was adsorbed on celite and
2
3
2
+
(
[
CH ), 58.5 (CH ). GC-MS:
m
/
z
= 163 [(C H N O Si) ], 119 purified by chromatography on silica gel (
n
-hexane/ethyl
as a red
2
2
3
9
3
3
+
+
(C
3
H
7
O
3
Si) ], 79 [(HO)
3
Si ].
acetate 2:1, with 5% triethylamine) to give compound
solid (89.0 mg, 24%), Mp 192-194 °C.
4
3
2
1
3
5
-(Diethylamino)-2-nitrosophenol (2)
H NMR (300 MHz, CDCl
3
):

= 1.25 (t,
JHH
HH = 7.1 Hz, 4 H, -NC
HH
-), 6.28 (s, 1 H, 6-H), 6.42 (d, J =
J
HH = 7.1 Hz, 6 H, -
2.4 Hz, H,
CH ), 4.88 (t,
4
3
-Diethylaminophenol ( ) (6.00 g, 36.1 mmol) was dissolved in
1
NCH CH3),
2.58
), 3.45 (q,
(t,
J
=
2
2
3
a mixture of deionized water (8 mL) and concentrated
hydrochloric acid (14 mL). The solution was cooled to 0 °C and
a solution of sodium nitrite (2.49 g, 36.2 mmol) in deionized
water (20 mL) was added dropwise over a period of 30 min and
the stirring was continued at 0 °C for 4 h. The solution was
filtered and the residue recrystallized from ethanol (100 mL).
After adding diethyl ether (80 mL) a brown solid precipitated
-
OCH
2
CC
H
H
2
3
4
4
J
HH = 2.4 Hz, 1 H, -OC
H
2
3
4
2
1
(
.6 Hz, 1 H, 8-H), 6.62 (dd,
J
HH = 9.1 Hz,
J
HH = 2.6 Hz, 1 H,
3
4
0-H), 7.22 (dd, JHH = 8.7 Hz, JHH = 2.6 Hz, 1 H, 3-H), 7.57
3
4
d, JHH = 9.1 Hz, 1 H, 11-H), 8.10 (d, JHH = 2.6 Hz, 1 H,
3
13
1
-H), 8.23 (d, JHH = 8.7 Hz, 1 H, 4-H). C NMR (75 MHz,
): = 12.6 (CH ), 45.1 (CH ), 56.1 (CH ), 96.3 (CH),
05.3 (CH), 107.2 (CH), 109.6 (CH), 118.4 (CH), 124.8 (Cquat),
26.4 (Cquat), 127.9 (CH), 131.2 (CH), 134.0 (Cquat), 139.8
CDCl
1
1
(
1
3

3
2
2
-
3
and was filtered off. Drying at 50 °C and 10 mbar gave 5-
(
diethylamino)-2-nitrosophenol (
2
) (5.72 g, 81%) as a brown
solid.
Cquat), 146.8 (Cquat), 150.8 (Cquat), 152.1 (Cquat), 160.1 (Cquat),
1
3
-
1
H NMR (300 MHz, DMSO-d
H, -CH ), 3.82 (m, 4 H, -CH -), 6.75 (d, JHH = 2.2 Hz, 1 H, 6-
6
):

= 1.27 (t,
J
HH = 7.1 Hz, 6
83.1 (Cquat). IR: ṽ / cm = 679 (m), 591 (m), 704 (m), 743
3
3
2
(m), 756 (m), 789 (m), 807 (m), 820 (s), 845 (m), 864 (m), 880
m), 893 (m), 924 (m), 1005 (s), 1034 (m), 1080 (s), 1115 (s),
3
H), 7.21 (dd,
J
HH = 10.4 Hz, J = 2.2 Hz, 1 H, 4-H), 7.55 (d,
(
3
J
HH = 10.4 Hz, 1 H, 3-H).
1
1
1
1
2
150 (m), 1179 (m), 1194 (m), 1207 (m), 1250 (s), 1267 (s),
292 (m), 1315 (m), 1335 (m), 1373 (m), 1404 (s), 1454 (m),
468 (m), 1491 (m), 1518 (w), 1530 (w), 1555 (m), 1572 (m),
589 (s), 1638 (w), 1687 (w), 1726 (m), 2106 (w), 2332 (w),
9
-(Diethylamino)-2-hydroxy-5H-benz[a]phenoxazin-5-one
3
1
(
3)
A solution of 5-(diethylamino)-2-nitrosophenol (
.60 mmol) and 1,6-dihydroxynaphthalene (0.75 g, 4.70 mmol)
2
) (1.00 g,
359 (w), 2872 (w), 2928 (m), 2959 (m), 3194 (m). MALDI-
+
5
TOF:
m
/
z
= 373.4 (C23
H
20
N
2
O
3
+H) .
in DMF (90 mL) was heated to 155 °C for 4 h. The solvent was
removed in vacuo and the residue was purified by
9
-(Diethylamino)-2-((1-(3-(triethoxysilyl)propyl)-1H-1,2,3-
triazol-4-yl)methoxy)-5 -benzo[a]phenoxazin-5-one (5)
-(Diethylamino)-2-(prop-2-yn-1-yloxy)-5 -benzo[ ]phenoxa-
zin-5-one (80.0 mg, 0.210 mmol), copper sulfate
HH = 7.0 Hz, 6 pentahydrate (12.0 mg, 0.0500 mmol) and sodium ascorbate
7.1 Hz, H, (20.0 mg, 0.100 mmol) were dissolved in DMF (1 mL). Then a
HH = 2.6 Hz, 1 H, 8-H), solution of (3-azidopropyl)triethoxysilane (270 mg, 1.90 mmol)
.74 (dd, JHH 4= 9.1 Hz, JHH = 2.7 Hz, 1 H, 10-H), 7.07 (dd, in DMF (2 mL) was added and the mixture was stirred at 40 °C
chromatography on silica gel (
give 9-(diethylamino)-2-hydroxy-5H-benz[a]phenoxazin-5-one
) (8.66 g, 66%) as a violet solid, Mp 298 °C.
n-hexane/ethyl acetate 1:1) to
H
9
H
a
(
3
(4)
1
3
6
H NMR (300 MHz, DMSO-d ):  = 1.14 (t, J
3
H, -CH3), 3.45 (q,
-
JHH
=
4
4
CH
6
2
-), 6.11 (s, 1 H, 6-H), 6.57 (d, J
3
4
3
3
J
HH = 8.6 Hz,
JHH = 2.5 Hz, 1 H, 3-H), 7.52 (d, JHH = 9.1 Hz, for 13 h. Then diethyl ether (50 mL) and brine (20 mL) were
3
4
1
8
H, 11-H), 7.85 (d, JHH = 2.4 Hz, 1 H, 1-H), 7.95 (d, JHH
=
added to the solution and the aqueous layer was washed with
.6 Hz, 1 H, 4-H), 10.41 (s, 1 H, -OH). C NMR (75 MHz, diethyl ether (4 x 50 mL). The combined organic layers were
): = 12.4 (CH ), 44.4 (CH ), 96.0 (CH), 104.0 (CH), dried with anhydrous magnesium sulfate and the solvent was
08.1 (CH), 109.8 (CH), 118.3 (CH), 123.8 (Cquat), 127.4 (CH), removed in vacuo. The residue was purified by chromatography
1
3
DMSO-d
6

3
2
1
1
(
30.7 (CH), 133.7 (Cquat), 138.6 (Cquat), 146.3 (Cquat), 150.6 on silica gel (
n
-hexane/ethyl acetate 1:1) to give compound
5
as
-
1
Cquat), 151.5 (Cquat), 160.5 (Cquat), 181.5 (Cquat). IR:
ṽ
/ cm = a red solid (60.0 mg, 45%).
1
6
8
38 (m), 671 (m), 687 (m), 702 (m), 745 (m), 783 (m), 797 (m),
18 (m), 847 (m), 887 (m), 910 (m), 953 (m), 1013 (m), 1028 -NCH CH CH2Si-), 1.24
H
NMR (300 MHz, CDCl3):
(m, 15

= 0.61 (m,
H,
2
H,
2
2
Si(OCH CH3) ,
2
3
3
(
(
(
(
m), 1028 (m), 1045 (m), 1074 (m), 1112 (vs), 1150 (m), 1159 -NCH CH3), 2.05 (m, 2 H, -NCH CH2CH Si-), 3.47 (q, JHH
m), 1180 (m), 1221 (m), 1258 (vs), 1288 (m), 1317 (vs), 1377 7.1 Hz, 4 H, -NCH2CH ), 3.80 (q, 6 H, Si(OCH2CH ) , 4.39 (t,
=
2
2
2
3
3 3
3
m), 1406 (m), 1439(m), 1474 (m), 1485 (m), 1505 (m), 1520
JHH = 7.2 Hz, 2 H, -NCH2CH CH Si-), 5.42 (s, 2 H,
m), 1537 (m), 1562 (m), 2625 (br), 2870 (w), 2924 (w), 2963 -OCH2C-), 6.30 (s, 1 H, 6-H), 6.45 (d, JHH = 2.6 Hz, 1 H, 8-H),
2 2
4
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1‐3 | 11

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Page 12 of 15
Journal Name
ARTICLE
3
4
6
J
1
1
.66 (dd,
J
HH = 9.1 Hz,
J
HH = 2.7 Hz, 1 H, 10-H), 7.23 (dd, Grafted NR MCM hybrids (6a-f)
3
4
3
HH = 8.7 Hz,
H, 11-H), 7.69 (s, 1 H, -CC
J
HH = 2.5 Hz, 1 H, 3-H), 7.60 (d,
J
HH = 9.1 Hz,
Six NR-functionalized silica hybrid materials were synthesized
by postsynthetic grafting. For employing small amounts of the
4
H
N-), 8.18 (d, JHH = 2.5 Hz, 1 H,
View Article Online
3
13
DOI: 10.1039/C5RA22736D
-H), 8.23 (d,
): = 7.47 (CH
5.1 (CH ), 52.6 (CH ), 58.5 (CH ), 62.5 (CH ), 96.3 (CH),
J
HH = 8.7 Hz, 1 H, 4-H). C NMR (75 MHz,
precursor
) = 0.610 m
ethanol were prepared. The solution of dye
5
four different stock solutions of
c
5
5
1
(
5
) = 49.0
M,
CDCl
3

2
2
), 12.6 (CH ), 18.3 (CH ), 24.2 (CH ),
3
3
2
c
2
(5
M
and
c
3
(5
) = 5.00 m
M
,
C
4
(
) = 5.72 m
M
in
4
1
1
2
2
2
, MCM-41, and
05.3 (CH), 106.9 (CH), 109.6 (CH), 118.4 (CH), 122.8 (CH),
24.8 (Cquat), 126.1 (Cquat), 127.9 (CH), 131.2 (CH), 134.1
quat), 139.8 (Cquat), 143.5 (Cquat), 146.9 (Cquat), 150.8 (Cquat),
ethanol were subsequently added to the reaction vessel and
stirred at room temp for 20 h, followed by stirring at 80 °C for
(
C
2
4 h (for experimental details, see Table 5). The obtained
suspensions were centrifuged (10 min, 4000 rpm), decanted and
resuspended in ethanol (20 mL) and 2 aqueous hydrochloric
-
1
1
6
9
1
1
1
1
3
6
52.1 (Cquat), 160.9 (Cquat), 183.2 (Cquat). IR: ṽ / cm = 638 (m),
69 (m), 683 (m), 716 (m), 789 (s), 818 (s), 881 (m), 910 (m),
53 (m), 1011 (m), 1030 (m), 1072 (vs), 1111 (vs), 1161 (m),
182 (m), 1194 (m), 1223 (m), 1256 (s), 1271 (s), 1314 (s),
346 (m), 1406 (s), 1439 (m), 1458 (m), 1470 (m), 1479 (m),
497 (m), 1520 (m), 1558 (m), 1582 (vs), 1620 (m), 1641 (m),
670 (w), 1680 (w), 2891 (w), 2928 (w), 2972 (w), 3084 (w),
134 (w). HR-MS (ESI) calcd. for (C H N O Si+H) :
20.28989; Found: 620.28975; calcd. for (C26
= 536.19599; Found: 536.19595.
M
acid solution (1 mL), upon which the red suspensions turned
blue. After heating to 80 °C for 24 h the reaction mixtures were
centrifuged, the solids transferred into a Soxhlet extraction
thimble and extracted with ethanol over a period of 48 h. The
obtained powders were washed with triethylamine (2 mL) and
ethanol (20 mL), which led to a color change back to red. The
solids were washed with ethanol (3 x 20 mL) and centrifuged
each time as described until the supernatant reached pH 7. The
+
3
2
41
5
6
m/z =
+
29 5 6
H N O Si+H) :
m/z
-
3
obtained violet powders were dried at 60 °C and 10 mbar for 3
d to mass constancy.
Synthesis of Hybrid Materials
Table 5. Experimental details of the synthesis of NR grafted MCM-41 hybrid materials 6.
Applied loading
of hybrid with
Determined loading of
hybrid with
Yield of NR grafted
MCM-41 hybrid
Volume of stock
solution [mL]
Mass of
MCM-41 [g] ethanol [mL]
Volume of
Sample
5
5
-
1
-1
[
mol·g ]
1.0
2.5
5.0
10
[
mol·g ]
0.6
materials
0.408
0.560
1.64
6 [g]
6
a
8.91 of c
0.50 of c
14.3 of c
2.00 of c
3.00 of c
3.50 of c
14.3 of c
5.24 of c
6.99 of c
1
3
2
3
3
4
2
4
4
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
0.437
1.00
1.75
1.00
1.00
1.00
0.437
1.00
1.00
6.10
14.5
0.70
13.0
12.0
11.5
0.70
9.76
8.01
6
b
1.5
2.9
5.9
8.8
12
12
18
23
6
c
6
d
0.624
0.752
0.802
0.396
0.804
0.892
6
e
15
20
20
30
6
f
6
g
6
h
6
i
40
4
3
Co-condensed NR MCM-hybrids (7a-h)
suspensions were centrifuged (10 min, 4000 rpm), decanted and
washed with ethanol before they were centrifuged again. Then
the residues were suspended in ethanol (80 mL) and
concentrated aqueous hydrochloric acid solution (2 mL) and
stirred at 80 °C for 24 h. The mixtures were centrifuged, and
the solids were transferred into a Soxhlet extraction thimble and
extracted with ethanol over a period of 48 h. The obtained
powders were washed once with triethylamine (2 mL) and
ethanol (80 mL) upon which they turned red again. The solids
were washed with ethanol (3 x 80 mL) and centrifuged as
described above till the supernatant reached pH 7. The obtained
For the synthesis of the co-condensed NR-functionalized silica
hybrids a solution of tetraethyl orthosilicate (TEOS), variable
hexadecyl-trimethyl-ammonium
bromide (C16TMABr), ethylamine, methanol, and deionized
water was prepared with molar ratios of 1.00 : x : 0.140 : 2.40 :
amounts of precursor
5
,
2
.00 : 100 / (molar ratios x of precursor
5 are given in Table 6).
First the template C TMABr was dissolved in deionized water
1
6
and ethylamine, which was employed as a 70 wt % aqueous
solution before TEOS and the molar amount x of precursor in
methanol were added. For the syntheses of materials 7f-h 10
molar equivalents of methanol were used. The mixtures were
stirred at room temperature for 24 h with a speed of 750 rpm
before they were heated to 100 °C for 24 h. The obtained
5
-
3
violet powders were dried at 60 °C and 10 mbar for 3 d to
mass constancy.
Table 6. Experimental details of the synthesis of co-condensed NR hybrid materials
Applied loading of hybrid Determined loading of hybrid
Molar ratio x of
precursor
7.
Yield of co-condensed NR
hybrid materials 7 [g]
Sample
-
1
-1
with
5
[
mol·g ]
with
5
[
mol·g ]
5
5
-
-
-
-
-
-
-
-
7
a
0.1
0.5
1.0
2.5
5.0
20
0.04
0.2
0.4
0.9
1.9
7.4
11
2.08 · 10
1.04 · 10
2.08 · 10
5.20 · 10
1.04 · 10
4.16 · 10
6.24 · 10
8.32 · 10
2.23
2.01
2.86
2.83
2.87
1.23
1.47
1.41
4
4
4
3
3
3
3
7
b
7
c
7
d
7
7
7
e
f
g
40
30
7
h
15
1
2 | J. Name., 2012, 00, 1‐3
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Page 13 of 15
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ARTICLE
Acknowledgments
18 D. Aiello, A.M. Talarico, F. Teocoli, E.I. Szerb, I. Aiello, F. Testa
The authors cordially thank the Strategic Research Fund of the
and M. Ghedini, New J. Chem., 2011, 35, 141.
View Article Online
Heinrich
Heine
University,
the
Deutsche 19 Q. He, J. Shi, X. Cui, J. Zhao, Y. Chen and J. Zhou, J. Mater. Chem.,
DOI: 10.1039/C5RA22736D
Forschungsgemeinschaft DFG (Mu 1088/9-1), and the Fonds
der Chemischen Industrie for financial support.
2009, 19, 3395.
20 P. Greenspan, E. P. Mayer and S. D. Fowler, J. Cell Biol., 1985, 100
65.
21 N. N. Alekseev, SU 1 109 393, 1983.
,
9
Notes and references
a
Institut für Organische Chemie und Makromolekulare Chemie, 22 H. Tajalli, A. G. Gilani, M. S. Zakerhamidi and P. Tajalli, Dyes
Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225
Pigm., 2008, 78, 15.
Düsseldorf, Germany.
23 J. L. Meinershagen and T. Bein, J. Am. Chem. Soc., 1999, 121, 448.
b
Institut für Anorganische Chemie und Strukturchemie, Abteilung für 24 G. Hungerford and J. A. Ferreira, J. Lumin., 2001, 93, 155.
Metallorganische Chemie, Heinrich-Heine-Universität Düsseldorf, 25 J. Jose and K. Burgess, J. Org. Chem., 2006, 71, 7835.
Universitätsstraße 1, D-40225 Düsseldorf, Germany.
26 J. Jose, A. Loudet, Y. Ueno, R. Barhoumi, R. C. Burghardt and K.
Burgess, Org. Biomol. Chem., 2010, , 2052.
c
Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring
8
5
5, D-70569 Stuttgart, Germany.
27 T. Behnke, C. Würth, K. Hoffmann, M. Hübner, U. Panne and U.
Resch-Genger, J. Fluoresc., 2011, 21, 937.
†
Electronic Supplementary Information (ESI) available: structural 28 T. Felbeck, T. Behnke, K. Hoffmann, M. Grabolle, M. M. Lezhina,
characterization of hybrid materials
and TEM), spectroscopic characterization of precursor
materials and
hybrid materials 6b and 7e, fluorescence quenching of hybrid materials 30 R. Sastre and A. Costela, Adv. Mater., 1995,
6
, and
7
(nitrogen sorption, SAXS,
U. H. Kynast and U. Resch-Genger, Langmuir, 2013, 29, 11489.
5
and hybrid 29 J. Qian, X. Li, M. Wie, X. Gao, Z. Xu and S. He, Opt. Express, 2008,
6
7
(UV/Vis and fluorescence), red-edge excitation shift of
16, 19568.
7
, 198.
6
b
and 7e. See DOI: 10.1039/b000000x/
31 S.-Y. Park, Y. Kubota, K. Funabiki, M. Shiro and M. Matsui,
Tetrahedron Lett., 2009, 50, 1131.
3
2 S. A. Martin-Brown, Y. Fu, G. Saroja, M. M. Collinson and D. A.
1
2
C. T. Kresge, M. E. Lenowicz, W. J. Roth, J. C. Vartuli and J. S.
Higgins, Anal. Chem., 2005, 77, 486.
Beck, Nature, 1992, 359, 710.
33 M. Meldal and C. W. Tornoe, Chem. Rev., 2008, 108, 2952.
C. T. Kresge, J. C. Vartuli, W. J. Roth and M. E. Leonowicz, Stud. 34 M. Berchel, J.-P. Haelters, D. Afonso, A. Maroto, L. Deschaps, P.
Surf. Sci. Catal., 2004, 148, 53.
Giamarchi and P.-A. Jaffrès, Eur. J. Org. Chem., 2014, 1076.
3
4
5
C. T. Kresge and W. J. Roth, Chem. Soc. Rev., 2013, 42, 3663.
A. Taguchi and F. Schüth, Micropor. Mesopor. Mat., 2005, 77, 1.
A. B. Descalzo, M. D. Marcos, C. Monte, R. Martínez-Máñez and K.
Rurack, J. Mater. Chem., 2007, 17, 4716.
35 K. S. W. Sing, D. H. Everett, R. A. W. Haul, L. Moscou, R. A.
Pierotti, J. Rouquérol and T. Siemieniewska, Pure Appl. Chem.
1985, 57, 603.
,
36 M. Kruk and M. Jaroniec, Chem. Mater., 2001, 13, 3169.
6
7
G. Wirnsberger, B. J. Scott and G. D. Stucky, Chem. Commun., 2001, 37 H.-P. Lin, S.-T. Wong, C.-Y. Mou and C.-Y. Tang, J. Phys. Chem. B
19.
2000, 104, 8967.
F. Marlow, M. D. McGehee, D. Zhao, B. F. Chmelka and G. D. 38 D. Zhao, Y. Wan and W. Zhou, Ordered Mesoporous Materials
,
1
,
Stucky, Adv. Mater., 1999, 11, 632.
Wiley-VCH GmbH & Co. KGaA, Weinheim, 2013.
8
9
N. Vadia and S. Rajput, Asian J. Pharm. Clin. Res., 2011,
4
, 44.
39 G. A. Tompsett, L. Krogh, D. W. Griffin and W. C. Conner,
Langmuir, 2005, 21, 8214.
V. Mameava, C. Sahlgren and M. Lindén, Adv. Drug Deliv. Rev.
013, 65, 689.
0 J. M. Rosenholm, C. Sahlgren and M. Lindén, Nanoscale, 2010,
870.
,
,
2
40 P. Cool, E. F. Vasant and O. Collart, in Inorganic Chemistry in
Focus II, G. Meyer, D. Naumann and L. Wesemann (eds.), Wiley,
Weinheim, 2005, 319.
1
1
1
1
1
1
1
1
2
1
1 Y.-S. Lin, C.-P. Tsai, H.-Y. Huang, C.-T. Kuo, Y. Hung, D.-M. 41 M. T. Anderson, J. E. Martin, J. G. Odinek and P. P. Newcomer,
Huang, Y.-C. Chen and C.-Y. Mou, Chem. Mater., 2005, 17, 4570.
Chem. Mater., 1998, 10, 1490.
2 F. Hoffmann, M. Cornelius, J. Morell and M. Fröba, Angew. Chem. 42 S. Liu, P. Cool, O. Collart, P. van der Voort, E. F. Vasant, O. I.
Int. Ed., 2006, 45, 3216.
Lebedev, G. van Tendeloo and M. Jiang, J. Phys. Chem. B, 2003,
107, 10405.
3 M. Sharifi, R. Marschall, M. Wilhelm, D. Wallacher and M. Wark,
Langmuir, 2011, 27, 5516.
43 Z. Zhou, A. W. Franz, S. Bay, B. Sarkar, A. Seifert, P. Yang, A.
Wagener, S. Ernst, M. Pagels, T. J. J. Müller and W. R. Thiel, Chem.
4 N. Gartmann and D. Brühwiler, Angew. Chem. Int. Ed., 2009, 45
354.
5 D. Aiello, R. Aiello, F. Testa, T. Martino, I. Aiello, M. La Deda and 44 K. J. Edler, in Porous Materials, D. W. Bruce, D. O´Hare and R. I.
M. Ghedini, J. Photochem. Photobiol.,A, 2009, 201, 81.
Walton (eds.), John Wiley & Sons, Ldt, Chinchester, 2011, 108.
6 C. M. Carbonaro, R. Corpino, P. C. Ricci, D. Chiriu and C. Cannas, 45 M. L. Hair and W. Hertl, J. Phys. Chem., 1970, 74, 91.
AIP Conference Proceedings, 2014, 1624, 23.
46 E. M. Moreno and D. Levy, Chem. Mater., 2000, 12, 2334.
7 G. Schulz-Ekloff, D. Wöhrle, B. van Duffel and R. A. Schoonheydt, 47 J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Third
,
6
Asian J., 2010, 5, 2001.
Micropor. Mesopor. Mat., 2002, 51, 91.
Edition, Springer, Baltimore, 2006.
4
8 A. P. Demchenko, Luminescence, 2002, 17, 19.
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1‐3 | 13

RSC Advances
Page 14 of 15
Journal Name
ARTICLE
4
5
5
5
9 S. Mukherjee, H. Raghuraman and A. Chattopadhyay, Biochim.
Biophys. Acta, 2007, 59.
View Article Online
DOI: 10.1039/C5RA22736D
0 R. Saxena, S. Shrivastava, S. Haldar, A. S. Klymchenko and A.
Chattopadhyay, Chem. Phys. Lipids, 2014, 183, 1.
1 E. Fleige, B. Ziem, M. Grabolle, R. Haag and U. Resch-Genger,
Macromolecules, 2012, 45, 9452.
2 T. Behnke, C. Würth, E.-M. Laux, K. Hoffmann and U. Resch-
Genger, Dyes Pigm., 2012, 94, 247.
5
5
3 R. Varghese and H.-A. Wagenknecht, Chem. Eur. J., 2010, 16, 9040.
4 C. Martín, P. Piatowski, B. Cohen, M. Cil, M. T. Navarro, A. Corma
and A. Douhal, J. Phys. Chem. C, 2015, 119, 13283.
5 F. Würthner, T. E. Kaiser and C. R. Saha-Möller, Angew. Chem. Int.
Ed., 2011, 50, 3376.
5
5
5
5
6 J. Mei, Y. Hong, J. W. Y. Lam, A. Qin, Y. Tang and B. Z. Tang, Adv.
Mater., 2014, 26, 5429.
7 N. L. Selivanov, L. G. Samsonova, V. Y. Artyukhov and T. N.
Kopylova, Russ. Phys. J., 2011, 54, 601.
8 G. Dördelmann, H. Pfeiffer, A. Birkner and U. Schatzschneider,
Inorg. Chem., 2011, 50, 4362.
1
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1
33x100mm (300 x 300 DPI)