7-Amino coumarin based fluorescent phototriggers coupled with nano/bio-conjugated bonds: Synthesis, labeling and photorelease

Article abstract of DOI:10.1039/c2jm30357d

By several synthetic pathways, we designed and synthesized a new series of unsymmetrical substituted 7-amino coumarin-based phototriggers with various nano/bio-conjugated bonds. The photophysical properties of most of the substituted coumarin-based phototriggers, except for compound P9 with maleimide group, showed no significant change compared with that of 7-(diethylamino)-4- (hydroxylmethyl) coumarin (DEACM-OH, the reported symmetric substituted 7-amine coumarin based phototrigger). Four compounds (P2, P4, P9, 14) were successfully conjugated with typical carriers such as mesoporous silica nanoparticles, biocompatible polymer PEG and common protein BSA, respectively. The efficient photorelease of ibuprofen (IBU) in the model cargo delivery system of MD4 confirmed that our designed phototriggers could serve well as a photo-cage for bioactive molecules and the release can be regulated precisely by manipulating the external irradiation conditions. All the results hinted at the superiority of using these coumarin functional compounds for photo-regulated release in biotechnology and biomedical areas. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2jm30357d

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PAPER
7-Amino coumarin based fluorescent phototriggers coupled with
nano/bio-conjugated bonds: Synthesis, labeling and photorelease
*
Qiuning Lin, Chunyan Bao, Guanshui Fan, Shuiyu Cheng, Hui Liu, Zhenzhen Liu and Linyong Zhu
Received 18th January 2012, Accepted 10th February 2012
DOI: 10.1039/c2jm30357d
By several synthetic pathways, we designed and synthesized a new series of unsymmetrical substituted
7-amino coumarin-based phototriggers with various nano/bio-conjugated bonds. The photophysical
properties of most of the substituted coumarin-based phototriggers, except for compound P9 with
maleimide group, showed no significant change compared with that of 7-(diethylamino)-4-
(hydroxylmethyl) coumarin (DEACM-OH, the reported symmetric substituted 7-amine coumarin
based phototrigger). Four compounds (P2, P4, P9, 14) were successfully conjugated with typical
carriers such as mesoporous silica nanoparticles, biocompatible polymer PEG and common protein
BSA, respectively. The efficient photorelease of ibuprofen (IBU) in the model cargo delivery system of
MD4 confirmed that our designed phototriggers could serve well as a photo-cage for bioactive
molecules and the release can be regulated precisely by manipulating the external irradiation
conditions. All the results hinted at the superiority of using these coumarin functional compounds for
photo-regulated release in biotechnology and biomedical areas.
that this kind of phototrigger would be a favored candidate in
intelligent biological materials.
Introduction
The term phototrigger has become more broadly accepted as an
accurate photolabile protective group for caging biologically
active molecules, whose removal only requires light and allows
a very mild activation of sensitive molecules.1–4 This feature is
particularly favourable if access to the reaction site is difficult or
if chemical reagents are of restricted use in biological systems.5,6
For biological and medical applications, the phototrigger must
undergo photolysis rapidly, in high yield, and at a wavelength
which is not detrimental to the biological system. Up to now,
many phototriggers have been developed, such as 2-nitro-
benzyl,7,8 phenacyl,9 benzoin,10 7-nitroindoline,11 and so on.
Most of the uncaging for the phototriggers requires UV light due
to the short wavelength absorption, which is a major drawback
and damaging to cells.12 Thus, the development of new photo-
triggers with long wavelength absorption is of crucial impor-
tance. Recently, 7-amino coumarin based phototriggers have
attracted much attention due to their higher photolysis efficien-
cies, long wavelength absorption and efficient fluorescence
visualization. The mechanism for the photocleavage has been
extensively investigated, they are members of arylalkyl-type
photoremovable protecting groups and the photolysis upon
cleavage of a C–O bond produces a leaving group and a solvent-
trapped coumarin as a photo by-product.13–18 All of this indicates
Phototriggers provide a useful tool in the photo-controlled
release system, since they permit accurate control for the release
including site, timing, and dosage, without physically disturbing
organized biological systems.13,19–22 In many cases, the photo-
release of bioactive molecules needs to be incorporated with
carriers, including biocompatible macromolecules, nano-
particles, proteins and nanocomposites. Especially in intracel-
lular drug delivery systems, suitable carriers protect drugs from
premature degradation, and control drug tissue distribution. To
achieve this aim, various methodologies have been developed, in
which the chemical conjugation labeling is more desirable
because it provides more precise control in terms of the density
and orientation of the caged biomolecules and forms a stable
linkage under in vivo conditions. To maintain the bioactivity of
coupled biomolecules, labeling chemistry should be simple, effi-
cient, site-selective, occur under mild biocompatible conditions
and generate a stable and non-toxic chemical bond. Generally,
the labeling reactions with nano/bio-carriers are carried out by
conjugating the biomolecules on the nano/bio-carriers’ surface,
like –NH2, –COOH, –N3, –OH, –SH by the Michael addition
reaction between maleimide and thiol, the coupling reaction
between NHS (N-hydroxylsuccinimide) activated acid and
primary amine, the click reaction between alkyne and azide, and
so on.23,24 Meanwhile, for some inorganic nanoparticles, conju-
gation can also be operated by directly bonding to the corre-
sponding particle surface, for example, the carboxylic acid for
bonding of ferric oxide based magnetic nanoparticles or
lanthanide nanoparticles,25 the thiol for bonding of silver or gold
Shanghai Key Laboratory of Functional Materials Chemistry, Institute of
Fine Chemicals, East China University of Science and Technology, 130#
Meilong Road, Shanghai, 200237, China. E-mail: linyongzhu@ecust.edu.
cn; Fax: (+86)-21-64253742
6680 | J. Mater. Chem., 2012, 22, 6680–6688
This journal is ª The Royal Society of Chemistry 2012

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nanoparticles and semiconductor quantum dots (QD),26 and
siloxane for bonding of silica nanoparticles,27 respectively.
However, limited 7-amino coumarin based phototriggers have
been reported to act as linkers to couple bioactive molecules with
carriers. The only reported one with a bioconjugated bond was {7-
[bis(carboxymethyl)amino] coumarin-4-yl}methyl (BCMACM)
which was designed to increase the water solubility.28 Herein, we
designed and synthesized a series of unsymmetrical substituted
7-amino coumarin-based phototriggers which were expected to
conjugate with nano/bio-carriers, and finally provide a new
promising strategy for designing and preparing a biological
photorelease system. The successful label and light-controlled
release suggested that the prepared phototrigger served well as
a photocage for the bioactive molecules whose release can be
regulated precisely by controlling the irradiation conditions.
Compound 2. To a pyridine solution (32 mL) of 3-aminophenol
(compound 1, 21.8 g, 0.20 mol) was added dropwise a pyridine
solution (34 mL) containing p-toluenesulfonyl chloride (40.0 g,
0.21 mol) over 30 min at 0 ꢀC. The obtained solution was further
ꢀ
stirred at 0 C for 30 min, and then the pyridine was removed
under reduced pressure. The obtained thick oil was diluted in
ethanol (10 mL), followed by pouring into DI water to precipi-
tate large amounts solid. The solid was filtered, washed with DI
water several times, and dried under vacuum overnight. 51.2 g
(0.19 mol, 97% yield) of 2 as a pink solid was obtained without
further purification. TLC Rf 0.38 (EtOAc : Hexane ¼ 1 : 2).1H
NMR (d6-DMSO, 400 MHz): d ¼ 10.08 (s, 1H), 9.42 (s, 1H), 7.64
(d, J ¼ 8 Hz, 2H), 7.34 (d, J ¼ 8 Hz, 2H), 6.97 (t, J ¼ 8 Hz, 1H),
6.57 (s, 1H), 6.51 (d, J ¼ 8 Hz, 1H), 6.38 (d, J ¼ 8 Hz, 1H), 2.33
(s, 3H); 13C NMR (d6-DMSO, 100 MHz): 158.3, 143.6, 139.4,
137.3, 130.2, 130.1, 127.2, 111.5, 110.8, 107.2, 21.4; MS (ESI):
262.0 [M ꢁ H]ꢁ.
Experimental section
Compound 3. A mixture of 2 (13.0 g, 49.4 mmol), ethyl ace-
toacetate (7.5 mL, 59.3 mmol), and BiCl3 (1.5 g, 4.9 mmol) was
stirred at 75 ꢀC for 6 days. The reaction mixture was then cooled
to room temperature, diluted with ethanol and filtered. The
obtained residue was washed several times with ethanol and
dried under vacuum overnight. 7.3 g (22.2 mmol, 45% yield) of 3
as a brown yellow solid was obtained without further purifica-
tion. TLC Rf 0.31 (EtOAc : Hexane ¼ 1 : 2). 1H NMR (d6-
DMSO, 400 MHz): d ¼ 10.91 (s, 1H), 7.74 (d, J ¼ 8.4 Hz, 2H),
7.64 (d, J ¼ 8.8 Hz, 1H), 7.38 (d, J ¼ 8 Hz, 2H), 7.09 (dd, J ¼ 8.8,
1.6 Hz, 1H), 7.03 (d, J ¼ 1.6 Hz, 1H), 6.24 (s, 1H), 2.33 (s, 6H);
13C NMR (d6-DMSO, 100 MHz): 160.1, 154.1, 153.3, 144.2,
141.8, 136.7, 130.4, 127.2, 126.9, 115.6, 115.0, 112.9, 105.5, 21.4,
18.3; MS (ESI): 328.0 [M ꢁ H]ꢁ.
Materials
All reagents were purchased from commercial available sources
such as Aldrich or Fisher and used without further purification.
Acetonitrile, dichloromethane, THF, acetone, toluene and DMF
were freshly distilled before use.
Characterization
Proton and carbon magnetic resonance spectra (1H, 13C NMR)
were recorded on a Bruker Avance 500 (400 MHz) spectrometer.
Chemical shifts were reported in parts per million (ppm) down-
field from the Me4Si resonance which was used as the internal
1
standard when recording H NMR spectra. Mass spectra were
recorded on a Micromass GCTTM and a Micromass LCTTM.
Absorption spectra were recorded on a Shimadzu UV-2550 UV-
vis spectrometer. Thermogravimetric analysis (TGA) was per-
formed in air at a heating rate of 10 ꢀC minꢁ1 from room
temperature to 800 ꢀC using a TGA/SDTA851e. The steady-state
fluorescence experiments were performed on a Varian Cary
Eclipses fluorescence spectrometer. Samples for absorption and
emission measurements were measured using 1 cm ꢂ 1 cm quartz
cuvettes. The luminescence quantum yields in solution were
measured by using quinine sulfate (FF ¼ 0.53 in 0.1 M H2SO4) as
a reference. The quantum yield F as a function solvent polarity is
calculated using the following equation:
Compound 4. A solution of 3 (6.5 g, 19.8 mmol), bromoethane
(29.8 mL, 0.40 mol), K2CO3 (4.1 g, 29.7 mmol), NaI (0.3 g, 2.0
mmol), and tetrabutylammonium bromide (0.6 g, 2.0 mmol) in
CH3CN (200 mL) was refluxed for 6 h. The reaction mixture was
allowed to cool to room temperature. After filtration, the solvent
was removed under vacuum. The residue was then dissolved in
CH2Cl2 and washed with water (3 ꢂ 100 mL). The organic layer
was then dried over Na2SO4 and evaporated to remove the
solvents. The obtained crude was recrystallized in ethanol to give
6.2 g (17.4 mmol, 88% yield) of 4 as white solid. TLC Rf 0.47
(EtOAc : Hexane ¼ 1 : 2). 1H NMR (CDCl3, 400 MHz): d ¼ 7.57
(d, J ¼ 8.4 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.25 (d, J ¼ 8 Hz,
2H), 7.18 (dd, J ¼ 8.4, 2 Hz, 1H), 6.90 (d, J ¼ 2 Hz, 1H), 6.27 (s,
1H), 3.62(q, J ¼ 7.2 Hz, 2H), 2.43(s, 3H), 2.42(s, 3H), 1.08 (t, J ¼
6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): 160.4, 153.5, 151.9,
144.0, 142.2, 134.7, 129.7, 127.5, 125.2, 125.0, 119.1, 115.6, 115.1,
45.2, 21.6, 18.7, 13.8; MS (ESI): 357.1 [M]+.
ꢀ
ꢁꢀ
ꢁꢀ
ꢁ
2
Isample
Astd
nsample
nstd
Fsample ¼ Fstd
Istd
Asample
where subscript sample and std denote the sample and standard
respectively, F is the quantum yield, I is the integrated emission
intensity, A stands for the absorbance and n is refractive index.
The time-resolved fluorescence decay was recorded using an
Edinburgh Life-Spec-PS F900 instrument with a 372 nm pulsed
LED and the fluorescence detection wavelength was 460 nm.
Compound 5. SeO2 (5.2 g, 34.8 mmol) and 4 (6.2 g, 17.4 mmol)
were suspended in 300 mL p-xylene, the reaction mixture was
refluxed with vigorous stirring under the protection of an argon
atmosphere. After 24 h, the mixture was filtered and concen-
trated under reduced pressure. The dark brown residual oil was
dissolved in methanol (150 mL), then sodium borohydride
(1.3 g, 34.8 mmol) was added, and the solution was stirred for
3 h at rt. Thereafter, the suspension was carefully neutralized
Preparation of the compounds P1–P9
Compound P1. The synthesis of compound P1 has been
reported in our previously published paper.13
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 6680–6688 | 6681

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with 1 M HCl, diluted with water, and partially concentrated
under reduced pressure to remove methanol. The mixture was
extracted with CH2Cl2 and the obtained organic phase was
washed with water and brine, dried over Na2SO4, and concen-
trated in vacuum. The obtained oil was purified by column
chromatography (EtOAc : Hexane ¼ 1 : 1) to yield 4.14 g (11.1
Compound 7. This compound was prepared from compound 6
in acetone by the same method as compound 4. Purification with
column chromatography (Hexane : EtOAc ¼ 1 : 1) gave 7 (66%
yield) as a yellow solid. TLC Rf 0.28 (EtOAc : Hexane ¼ 1 : 1). 1H
NMR (CDCl3, 400 MHz): d ¼ 7.32 (d, J ¼ 8.8 Hz, 1H), 6.60 (dd,
J ¼ 8.8, 2.4 Hz, 1H), 6.55 (d, J ¼ 2.4 Hz, 1H), 6.28 (s, 1H), 5.82 (m,
1H), 5.18 (m, 2H), 4.83 (s, 2H), 3.96 (t, J ¼ 2.4 Hz, 2H), 3.44(q, J ¼
7.2 Hz, 2H), 1.22 (t, J ¼ 7.2 Hz, 3H); 13CNMR (CDCl3, 100MHz):
163.1, 155.8, 155.6, 150.9, 132.7, 124.3, 116.5, 109.1, 106.8, 105.4,
98.1, 60.7, 52.6, 45.2, 12.2. MS (ESI): 260.1287 [M + H]+.
mmol, 64% yield) of
5 as a gray solid. TLC Rf 0.26
1
(EtOAc : Hexane ¼ 1 : 1). H NMR (CDCl3, 400 MHz): d ¼
7.50 (d, J ¼ 8.4 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 7.27 (d, J ¼
7.2 Hz, 2H), 7.17 (dd, J ¼ 8.4, 2 Hz, 1H), 6.96 (d, J ¼ 2 Hz,
1H), 6.63 (s, 1H), 4.90 (d, J ¼ 1.2 Hz, 2H), 3.63(q, J ¼ 7.2 Hz,
2H), 2.44(s, 3H), 1.09 (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3,
100 MHz): 160.9, 154.1, 153.6, 144.1, 142.2, 134.6, 129.8, 127.5,
125.2, 123.8, 116.7, 115.9, 112.1,60.5, 45.3, 21.6, 13.8; MS (ESI):
373.1 [M]+.
Compound P4. Compound
7 (80 mg, 0.3 mmol) and
triethoxysilane (560 mL, 3.0 mmol) were added into 5 mL dry
toluene under an argon atmosphere. After the addition of Kar-
stedt catalyst (10 mL), the mixture was allowed to react at rt for
4 h. After removing the solvent in vacuum, silica gel chroma-
tography (EtOAc : Hexane ¼ 2 : 1) was used to yield 60 mg
(0.14 mmol, 47% yield) of P4 as a yellow oil. TLC Rf 0.28
(EtOAc : Hexane ¼ 1 : 1). 1H NMR (CDCl3, 400 MHz): d ¼ 7.28
(d, J ¼ 8.8 Hz, 1H), 6.56 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.46 (d, J ¼ 2.8
Hz, 1H), 6.28 (s, 1H), 4.81 (s, 2H), 3.83 (q, J ¼ 7.2 Hz, 6H), 3.40
(q, J ¼ 6.8 Hz, 2H), 3.30 (t, J ¼ 8 Hz, 2H), 1.71 (m, J ¼ 8 Hz, 2H),
1.22 (t, J ¼ 6.8 Hz, 9H), 1.17 (t, J ¼ 6.8 Hz, 3H), 0.63 (t, J ¼ 8.4
Hz, 2H); 13C NMR (CDCl3, 100 MHz): 163.0, 156.0, 155.4,
150.7, 124.3, 108.7, 106.4, 105.1, 97.7, 60.68, 58.5, 52.9, 45.2,
20.7, 18.3, 12.2, 7.5. MS (ESI): 424.2155 [M + H]+.
Compound 6. 5 (2 g, 5.4 mmol) was added to 15 mL conc.
sulfuric acid and the solution was stirred for 1 h at 0 ꢀC. The
reaction mixture was carefully poured into water, neutralized
with saturated aqueous sodium bicarbonate and extracted with
EtOAc (5 ꢂ 50 mL). The organic solution was dried over
Na2SO4 and evaporated. The residue was purified by column
chromatography (EtOAc : Hexane ¼ 1 : 1) to yield 0.9 g
(4.1 mmol, 88% yield) of 6 as a yellowish solid. TLC Rf 0.32
(EtOAc : Hexane ¼ 1 : 1). 1H NMR (d6-DMSO, 400 MHz): d ¼
7.35 (d, J ¼ 8.8 Hz, 1H), 6.57 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.39 (d,
J ¼ 2 Hz, 1H), 6.25 (s, 1H), 4.64 (s, 2H), 3.10(q, J ¼ 7.2 Hz, 2H),
1.17(t, J ¼ 7.2 Hz, 3H); 13C NMR (d6-DMSO, 100 MHz):161.6,
157.5, 156.2, 152.7, 125.2, 110.6, 106.6, 104.1, 96.7, 59.5, 37.4,
14.5; MS (ESI): 220.1 [M + H]+.
Compound P5. A mixture of 7 (150 mg, 0.58 mmol), thioacetic
acid (824 mL, 11.6 mmol) and AIBN (190 mg, 1.16 mꢀmol) was
dissolved into 10 mL toluene and stirred for 5 h at 80 C. After
removing the solvent in vacuum, silica gel chromatography
(EtOAc : Hexane ¼ 2 : 1) was used to obtain 130 mg (0.39 mmol,
Compound P2. This compound was prepared from compound
6 in acetone by the same method as compound 4. The purifica-
tion with column chromatography (EtOAc : Hexane ¼ 1 : 1)
67% yield) of P5 as
a yellow solid. TLC Rf 0.17
(EtOAc : Hexane ¼ 1 : 1). 1H NMR (CDCl3, 400 MHz): d ¼ 7.30
(d, J ¼ 9.2 Hz, 1H), 6.54 (dd, J ¼ 8.8, 2 Hz, 1H), 6.46 (s, 1H), 6.29
(s, 1H), 4.81 (s, 2H), 3.38 (m, J ¼ 6.8 Hz, 4H), 2.90 (t, J ¼ 7.2 Hz,
2H), 2.36 (s, 3H), 1.88 (m, J ¼ 7.2 Hz, 2H), 1.17 (t, J ¼ 7.2 Hz,
3H); 13C NMR (CDCl3, 100 MHz):195.7, 162.9, 155.9, 150.5,
124.5, 108.8, 106.7, 105.5, 97.9, 60.7, 49.2, 45.3, 30.7, 29.7, 27.3,
26.4, 12.2; MS (ESI): 336.1 [M + H]+.
gave P2 (53% yield) as
a yellow solid. TLC Rf 0.22
(EtOAc : Hexane ¼ 1 : 1). 1H NMR (CDCl3, 400 MHz): d ¼ 7.39
(d, J ¼ 8.8 Hz, 1H), 6.72 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.69 (d, J ¼
2.4 Hz, 1H), 6.35 (s, 1H), 4.87 (s, 2H), 4.09 (d, J ¼ 2.4 Hz, 2H),
3.54 (q, J ¼ 6.8 Hz, 2H), 2.25 (t, J ¼ 2.4 Hz, 1H), 1.27 (t, J ¼ 7.2
Hz, 3H); 13C NMR (d6-DMSO, 100 MHz): 161.4, 157.3, 155.6,
150.5, 125.4, 110.1, 107.4, 105.4, 98.9, 80.9, 74.8, 59.5, 45.7, 39.5,
12.4; MS (ESI): 258.1131 [M + H]+.
Compound P6. A solution of the thioacetate P5 (70 mg, 0.21
mmol) and 2,20-dithiodipyridine (69 mg, 0.32 mmol) in MeOH
(10 mL) was degassed by repeatedly subjecting it to vacuum and
argon purges, followed by addition of 1 mL NH4OH aqueous.
The reaction was left stirring at rt for 4 h, then MeOH was
removed by rotary evaporation and the residue was extracted
with EtOAc and dried over Na2SO4. After evaporation of the
solvent, the residue was chromatographed, eluting with
EtOAc : Hexane (2 : 3) to give 81 mg (0.20 mmol, 91% yield) of
Compound P3. To a solution of 6 (0.3 g, 1.4 mmol) in 5 mL
50%AcOH aqueous at 0 ꢀC, ethylene oxide (1.4 mL, 28.2 mmol)
was added slowly. The solution was allowed to warm to rt and
was stirred for 24 h. The reaction mixture was neutralized with
saturated aqueous sodium bicarbonate and extracted with
EtOAc (3 ꢂ 30 mL). The organic solution was dried over
Na2SO4 and evaporated. The residue was purified by column
chromatography (EtOAc : Hexane ¼ 4 : 1) to yield 0.21 g (0.8
mmol, 57% yield) of P3 as a yellowish solid. TLC Rf 0.22
(EtOAc).1H NMR (CD3OH, 400 MHz): d ¼ 7.46 (d, J ¼
8.8 Hz, 1H), 6.78 (dd, J ¼ 9.2, 2.8 Hz, 1H), 6.62 (d, J ¼ 2.8 Hz,
1H), 6.24 (s, 1H), 4.80 (d, J ¼ 1.2 Hz, 2H), 3.75 (t, J ¼ 6.4 Hz,
2H), 3.56 (m, J ¼ 6.8 Hz, 4H), 1.22 (t, J ¼ 6.8 Hz, 3H); 13C
NMR (CD3OH, 100 MHz): 165.0, 158.6, 157.2, 152.7, 125.7,
110.5, 107.8, 105.2, 98.6, 60.8, 60.2, 53.3, 46.6, 12.2. MS (ESI):
264.1239 [M + H]+.
1
P6 as yellow oil. TLC Rf 0.24 (EtOAc : Hexane ¼ 1 : 1). H
NMR (CDCl3, 400 MHz): d ¼ 8.46 (d, J ¼ 4.8 Hz, 1H), 7.67 (m,
2H), 7.31 (d, J ¼ 8.8 Hz, 1H), 7.10 (m, 1H), 6.56 (dd, J ¼ 8.8, 2.4
Hz, 1H), 6.47 (d, J ¼ 2.4 Hz, 1H), 6.29 (s, 1H), 4.83 (s, 2H), 3.44
(t, J ¼ 7.2 Hz, 2H), 3.39 (q, J ¼ 7.2 Hz, 2H), 2.84 (t, J ¼ 6.8 Hz,
2H), 2.03 (m, J ¼ 7.2 Hz, 2H), 1.16 (t, J ¼ 7.2 Hz, 3H); 13C NMR
(CDCl3, 100 MHz):162.6, 159.7, 156.0, 155.0, 150.5, 149.7, 137.2,
124.5, 120.9, 120.0, 108.8, 106.7, 105.7, 98.1, 60.8, 48.8, 45.3,
35.9, 26.4, 12.2. MS (ESI): 403.1152 [M + H]+.
6682 | J. Mater. Chem., 2012, 22, 6680–6688
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1
Compound 8. A mixture of compounds 3 (5.0 g, 15.2 mmol)
and 1,2-dibromoethane (13.2 mL, 152 mmol) was dissolved in
250 mL distilled acetonitrile. Then cesium carbonate (7.4 g,
22.8 mmol) was added to the solution and the obtained mixture
was refluxed for 6 h. After filtration, the solution was evaporated
and the obtained crude was recrystallized with ethanol to give
6.1 g (14.0 mmol, 92% yield) of 8 as a gray solid. TLC Rf 0.28
(EtOAc : Hexane ¼ 1 : 1). 1H NMR (CDCl3, 400 MHz): d ¼ 7.59
(d, J ¼ 8.4 Hz, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.28 (d, J ¼ 8.4 Hz,
2H), 7.25 (dd, J ¼ 6.4, 2Hz, 1H), 6.89 (d, J ¼ 2 Hz, 1H), 6.31 (d,
J ¼ 1.2 Hz, 1H), 7.67 (m, 2H), 7.31 (d, J ¼ 8.8 Hz, 1H), 7.10 (m,
1H), 6.56 (dd, J ¼ 8.8, 1.2 Hz, 1H), 3.92 (t, J ¼ 7.2 Hz, 2H), 3.42
(t, J ¼ 7.2 Hz, 1H), 2.45 (s, 6H); 13C NMR (CDCl3, 100 MHz):
160.1, 153.6, 151.8, 144.4, 142.2, 134.5, 129.9, 127.5, 125.4, 125.2,
119.7, 116.1, 115.5, 52.3, 28.9, 21.6, 18.7; MS (EI): 437.1[M]+.
TLC Rf 0.17 (EtOAc : Hexane ¼ 1 : 1). H NMR (CDCl3, 400
MHz): d ¼ 7.23 (d, J ¼ 9.2 Hz, 1H), 6.51 (dd, J ¼ 8.8, 2.4 Hz,
1H), 6.40 (d, J ¼ 2.8 Hz, 1H), 6.24 (s, 1H), 4.73 (s, 2H), 3.35–3.46
(m, 6H), 1.11 (t, J ¼ 2.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz):
161.9, 154.8, 154.7, 149.2, 123.6, 107.9, 106.3, 104.8, 97.2, 59.5,
48.4, 47.9, 44.7, 11.0. MS (ESI): 311.1114 [M + Na]+.
Compound P8. A mixture of P7 (100 mg, 0.35 mmol) and PPh3
(144 mg, 0.55 mmol) was dissolved in 5 mL THF (containing one
drop of water) under argon atmosphere. After stirring for 24 h at
RT, the solvent was removed under vacuum. The residue was
purified by column chromatography (CH2Cl2 : MeOH ¼
100 : 5–100 : 10) to give 70 mg (0.27 mmol, 76% yield) of P8 as an
orange-yellow solid. TLC Rf 0.20 (CH2Cl2 : MeOH ¼ 100 : 10).
1H NMR (CD3OH, 400 MHz): d ¼ 7.43 (d, J ¼ 9.2 Hz, 1H), 6.76
(dd, J ¼ 8.8, 2.4 Hz, 1H), 6.60 (d, J ¼ 2.4 Hz, 1H), 6.23 (s, 1H),
4.78 (s, 2H), 3.47–3.54 (m, 4H), 2.89 (t, J ¼ 6.8 Hz, 2H), 1.21 (t,
J ¼ 6.8 Hz, 3H); 13C NMR (CD3OH, 100 MHz): 164.9, 158.5,
157.1, 152.4, 125.9, 110.5, 108.0, 105.5, 98.8, 60.8, 52.2, 46.4,
39.5, 12.4. MS (ESI): 263.1397 [M + H]+.
Compound 9. This compound was prepared from compound 8
using the same method as compound 6. Purification with column
chromatography (EtOAc : Hexane ¼ 1 : 2) was carried out to
give
9 (91% yield) as a yellow solid. TLC Rf 0.38
(EtOAc : Hexane ¼ 1 : 2). 1H NMR (CDCl3, 400 MHz): d ¼ 7.38
(d, J ¼ 8.4 Hz, 1H), 6.57 (dd, J ¼ 8.8, 2 Hz, 1H), 6.50 (d, J ¼ 2
Hz, 1H), 6.00 (s, 1H), 3.65 (t, J ¼ 5.2 Hz, 2H), 3.60 (t, J ¼ 6 Hz,
2H), 2.35 (s, 3H); 13C NMR (CDCl3, 100 MHz): 161.9, 155.8,
153.1, 150.5, 125.8, 111.1, 110.5, 109.8, 98.5, 44.8, 31.0, 18.6. MS
(ESI): 304.0 [M + Na]+.
Compound 12. To a solution of 11 (200 mg, 0.62 mmol) in 5 mL
anhydrous DMF was added to furan-protected maleimide29
(217 mg, 1.24 mmol) and K2CO3 (257 mg, 1.86 mmol). The
mixture was heated to 50 ꢀC for 2 h, and then cooled to rt. The
solvent was removed in a vacuum and the residue was extracted
with Et2O (3 ꢂ 50 mL), dried over Na2SO4, and evaporated
under vacuum. Purification with column chromatography
(EtOAc : Hexane ¼ 4 : 1) gave 135 mg (0.33 mmol, 53% yield) of
12 as a yellow solid. TLC Rf 0.26 (EtOAc). 1H NMR (d6-DMSO,
400 MHz): d ¼ 7.46 (d, J ¼ 9.2 Hz, 1H), 6.72 (dd, J ¼ 8.8, 2.4 Hz,
1H), 6.62 (d, J ¼ 2.4 Hz, 1H), 6.55 (s, 2H), 6.11 (s, 1H), 5.15 (s,
2H), 4.68 (s, 2H), 3.55 (t, J ¼ 6.8 Hz, 2H), 3.40 (m, 4H), 2.93 (s,
2H), 1.09 (t, J ¼ 7.2 Hz, 3H); 13C NMR (d6-DMSO,
100 MHz):176.4, 161.0, 156.8, 155.4, 150.1, 136.4, 125.1, 108.6,
106.4, 104.5, 97.3, 80.3, 59.0, 47.2, 46.5, 44.7, 35.2, 11.9. MS
(ESI): 433.1 [M + Na]+.
Compound 10. This compound was prepared from compound
9 using the same method as compound 4. The obtained crude was
purified by column chromatography (EtOAc : Hexane ¼ 1 : 2) to
give 10 (92% yield) as
a yellow solid. TLC Rf 0.62
(EtOAc : Hexane ¼ 1 : 2). 1H NMR (CDCl3, 400 MHz): d ¼ 7.42
(d, J ¼ 8.8 Hz, 1H), 6.61 (dd, J ¼ 8.8, 2.8 Hz, 1H), 6.54 (d, J ¼ 2.4
Hz, 1H), 5.99 (s, 1H), 3.75 (t, J ¼ 7.2 Hz, 2H), 3.50 (q, J ¼ 7.2 Hz,
2H), 3.46 (t, J ¼ 8 Hz, 2H), 2.35 (s, 3H), 1.23 (t, J ¼ 7.2 Hz, 3H);
13C NMR (CDCl3, 100 MHz): 161.8, 155.9, 152.7, 149.7, 125.8,
110.2, 109.8, 108.5, 98.3, 52.3, 45.7, 27.6, 18.4, 12.4. MS (EI):
309.1[M]+.
Compound P9. A solution of 12 (135 mg, 0.33 mmol) in 5 mL
anisole was heated to 140 ꢀC for 2 h. Then the mixture was
allowed to cool down to rt and evaporated in vacuum to give
a pale brown solid. Purification with column chromatography
(EtOAc : Hexane ¼ 2 : 1) was carried out to give 85 mg (0.25
mmol, 76% yield) of P9 as a brown-yellow oil. TLC Rf 0.22
(EtOAc : Hexane ¼ 2 : 1). 1H NMR (CDCl3, 400 MHz): d ¼ 7.36
(d, J ¼ 8.8 Hz, 1H), 6.72 (s, 2H), 6.70 (dd, J ¼ 9.2, 2 Hz, 1H), 6.60
(d, J ¼ 2.4 Hz, 1H), 6.32 (s, 1H), 4.85 (s, 2H), 3.75 (t, J ¼ 6.8 Hz,
2H), 3.55 (t, J ¼ 6.8 Hz, 2H), 3.44 (q, J ¼ 7.2 Hz, 2H), 1.22 (t, J ¼
6.8 Hz, 3H); 13C NMR (d6-DMSO, 100 MHz):171.4, 161.5,
157.3, 155.9, 150.7, 135.1, 125.6, 108.9, 106.8, 104.9, 97.7, 59.5,
47.6, 44.9, 34.9, 12.4. MS (ESI): 343.1281 [M + H]+.
Compound 11. This compound was prepared from compound
10 using the same method as compound 5. Purification with
column chromatography (EtOAc: Hexane ¼ 1 : 1) was done to
give 11 (55% yield) as a yellow solid. TLC Rf 0.18 (EtOAc:
1
Hexane ¼ 1 : 1). H NMR (CDCl3, 400 MHz): d ¼ 7.37 (d, J ¼
8.8 Hz, 1H), 6.66 (dd, J ¼ 8.8, 2.4 Hz, 1H), 6.61 (d, J ¼ 2.4 Hz,
1H), 6.34 (s, 1H), 4.85 (d, J ¼ 1.2 Hz, 2H), 3.75 (t, J ¼ 8.4 Hz,
2H), 3.50 (q, J ¼ 7.2 Hz, 2H), 3.46 (t, J ¼ 7.2 Hz, 2H), 1.23 (t, J ¼
7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz): 162.5, 155.9, 155.1,
149.8, 124.7, 108.7, 107.4, 106.3, 98.3, 60.7, 52.2, 45.6, 27.7, 12.4.
MS (ESI): 325.9[M]+.
Compound P7. The solution of compound 11 (220 mg, 0.68
mmol) in 5 mL anhydrous DMF was stirred with the presence of
NaN3 (132 mg, 1.36 mmol) and NaI (102 mg, 0.68 mmol) at 90
ꢀC for 4 h. The solvent was removed in vacuum. The residue was
extracted with Et2O (3 ꢂ 50 mL), dried over Na2SO4, and
evaporated under vacuum. Purification with column chroma-
tography (EtOAc : Hexane ¼ 1 : 1) was carried out to give
159 mg (0.55 mmol, 81% yield) of P7 as an orange-yellow oil.
Preparation of the labeled composites M2, M4, M9, and MD4
Compound 13. Under dark conditions, a mixture of 7 (20 mg,
0.08 mmol), ibuprofen (20 mg, 0.096 mmol), EDC (31 mg, 0.16
mmol), and DMAP (2 mg, 0.016 mmol) was dissolved in 10 mL
anhydrous CH2Cl2 under an argon atmosphere. After stirring at rt for
4 h, the solvent was removed under vacuum. The obtained crude
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productwaspurified bycolumnchromatography(EtOAc : Hexane¼
1 : 5) to give 35 mg (0.078 mmol, 98% yield) of 13 as a yellowish oil.
TLC Rf 0.28 (EtOAc : Hexane ¼ 1 : 5). 1HNMR(CDCl3, 400MHz):
d ¼ 7.24 (d, J ¼ 8.4 Hz, 2H), 7.17 (d, J ¼ 9.6 Hz, 1H), 7.12 (d, J ¼ 8.0
Hz, 2H), 6.52 (m, 2H), 6.04 (s, 1H), 5.83 (m, 1H), 5.14–5.24 (m, 4H),
3.97 (t, J ¼ 2.4 Hz, 2H), 3.84 (q, J ¼ 6.8 Hz, 1H), 3.44 (q, J ¼ 6.8 Hz,
2H), 2.47 (d, J¼ 7.2 Hz, 2H), 1.87 (m, J¼ 6.8 Hz, 1H), 1.57 (d, J¼ 6.8
Hz, 3H), 1.23 (t, J ¼ 6.8 Hz, 3H), 0.91 (d, J ¼ 6.8 Hz, 6H); 13C NMR
(CDCl3, 100 MHz):174.0, 161.7, 156.0, 151.1, 149.3, 140.9, 136.9,
132.6, 129.5, 127.2, 124.3, 116.6, 108.9, 106.9, 106.4, 98.3, 61.7, 52.6,
45.3, 45.1, 45.0, 30.1, 22.4, 18.3, 12.2. MS (EI): 447.3 [M]+.
MD4. This material was prepared from MSN-OH material
and compound 14 by the same method as M4.
General methods of the photolysis and photorelease
The photolytic reaction of the MD4 based drug delivery system
was performed by irradiating the MD4 suspension (2 mg mLꢁ1 in
an acetonitrile : water solution (9 : 1, v/v)) with a CHF-XM-
500w lamp (l ¼ 450 nm, 25 mW cmꢁ2). Between certain time
intervals, a small aliquot (100 mL) of the suspension was taken
out and centrifuged (8000 r minꢁ1) for 10 min, the obtained
transparent solution was analyzed by reversed-phase HPLC
using a BetaBasic-18 column eluted with a mixture of 90%
methanol and 10% water at a flow rate of 0.5 mL minꢁ1. The
chromatogram was plotted by using absorbance detection at
230 nm.
Compound 14. Under dark conditions, this compound was
prepared from compound 13 by the same method as
compound P4. Purification with column chromatography
(EtOAc : Hexane ¼ 1 : 5) gave 14 (86% yield) as a yellowish oil.
TLC Rf 0.28 (EtOAc : Hexane ¼ 1 : 5). 1H NMR (CDCl3,
400 MHz): d ¼ 7.22 (d, J ¼ 8.0 Hz, 2H), 7.15 (d, J ¼ 8.8 Hz, 1H),
7.11 (d, J ¼ 8.0 Hz, 2H), 6.50 (m, 2H), 6.00 (s, 1H), 5.18 (m, 2H),
3.84 (q, J ¼ 6.8 Hz, 6H), 3.78 (q, J ¼ 6.8 Hz, 1H), 3.40 (q, J ¼ 7.2
Hz, 2H), 3.30 (t, J ¼ 8.0 Hz, 2H), 2.45 (d, J ¼ 7.2 Hz, 2H), 1.86
(m, J ¼ 6.8 Hz, 1H), 1.72 (m, J ¼ 8 Hz, 2H), 1.55 (d, J ¼ 7.2 Hz,
3H), 1.24 (t, J ¼ 6.8 Hz, 9H), 1.18 (t, J ¼ 6.8 Hz, 3H), 0.89 (d, J ¼
6.8 Hz, 6H), 0.63 (t, J ¼ 8.4 Hz, 2H); 13C NMR (CDCl3, 100
MHz):174.0, 161.8, 156.2, 150.8, 149.3, 140.9, 136.9, 129.5, 127.2,
124.3, 108.6, 106.4, 106.0, 97.8, 61.7, 58.5, 52.9, 45.2, 45.1, 45.0,
30.2, 29.7, 22.4, 20.7, 18.3, 12.2, 7.5. MS (EI): 611.3280 [M]+.
Results and discussion
Synthesis
In principle, the introduction of a substituent with labeling
activity on the 7-amino coumarin should be based on main-
taining the inherent photocleavable properties and long wave-
length absorption of phototriggers. The chemical modification of
the 7-position amino group of coumarin provided chances to
introduce nano/bio-conjugated bonds, which was easier than
modifying those directly on the heterocyclic backbone. N-alkyl-
ation on the 7-position amino was proven to be the most effective
method to keep the long-wavelength absorption associated with
the np electron structure between the nitrogen atom and
aromatic ring.
Synthesis of MSN. The MSN–OH material was synthesized as
described in the reported literature.30 TEM, powder X-ray
diffraction (XRD), and nitrogen sorption isotherms analysis
confirmed that the 120 nm particles had a honeycomb-like
structure with a 2.7 nm average pore diameter and a surface area
Although amide bond substitution was more facile than the N-
alkylation, it was limited due to the blue-shift of absorption.32
The easily achieved symmetric N-bisalkylation such as bis-car-
boxylmethyl16,28 substitution might cause undesirable cross-
linking during the labeling process. Especially in the bio-labeling
reaction, the cross-linking might change the conformation of
biomacromolecules such as proteins. Therefore, based on the
requirements of conjugation with various carriers, we designed
nine of the unsymmetrical N-alkylation substituted phototriggers
with an essential structure of 7-(ethylamino)-4-(hydroxymethyl)
coumarin (shown in section A of Scheme 1), where R represents
the other substituted groups that were used to generate different
nano/bio-conjugated bonds, and the 4-hydroxymethyl was
reserved to cage bioactive molecules by generating photo-
cleavable bonds of carboxylates, carbonates or carbamates.
In general, there are two methods to obtain the 4-hydroxy-
methyl coumarin phototriggers: hydrolysis from the corre-
sponding Cl or Br33 and oxidation by SeO2, followed by
subsequent reduction by NaBH4 from 4-methyl.28 Here, the
latter is more desirable because the introduction of Cl or Br may
cause side reactions during the following N-alkylation process.
Besides, with phototrigger P1 which had been synthesized in our
previous report,13 the other N-alkylation substitutions were
carried out after the oxidation and reduction processes of
4-methyl to avoid inactivation of the substituted group.
of 1081 m2 gꢁ1
.
M2. The N3-PEG1000-N3 was obtained as described in the
reported literature.31 To a solution of N3-PEG1000-N3 (0.8 g,
0.74 mmol) and P2 (0.4 g, 1.56 mmol) in 20 mL THF : water
(4 : 1), were added, respectively, the copper sulfate (80 mg,
0.5 mmol) and sodium ascorbate (180 mg, 0.91 mmol). The
mixture was stirred at rt for 3 h under an argon atmosphere.
After filtration, the solution was evaporated, and the residue was
chromatographed eluting with CH2Cl2 : CH3OH (100 : 5) to give
1.08 g (0.66 mmol, 90% yield) product as a brown-yellow oil.
M4. The MSN-OH material (50 mg) and P4 (20 mg,
0.047 mmol) were dispersed in 10 mL anhydrous toluene under
argon atmosphere. The mixture was stirred at rt for another 36 h,
followed by centrifuging (8000 r minꢁ1, 10 min) and washing with
toluene and ethanol for several times, respectively. The obtained
nanoparticles were finally dried under high vacuum.
M9. Compound P9 (10 mg, 0.029 mmol) and 0.1 g bovine
serum albumin (BSA) were dissolved in DMSO : distilled water
(1 : 1, 3 mL). The resulting solution was stirred at rt for 4 h.
Upon completion, the solution was transferred to an 8000 Da
dialysis membrane and dialyzed against DMSO : distilled water
(1 : 1) for 48 h, then against distilled water for 48 h. Afterwards,
the solution was lyophilized to afford a white solid.
As shown in Scheme 2, phototriggers P2–P6 were synthesized
starting from the key building block 7-(ethylamino)-4-(hydroxyl-
methyl) coumarin (6) which was prepared from n-amino
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Scheme 2 Synthesis of P1–P9: a) 4-toluene sulfonyl chloride, C5H5N,
ꢀC, 30 min, 97%; b) CH3COCH2COOEt/BiCl3, 75 ꢀC, 6 days, 45%; c)
0
BrCH2CH3/K2CO3/NaI/TBAB, CH3CN, reflux, 6 h, 88% for 4, 92% for
10; d) SeO2, p-xylene, reflux, 24 h; then NaBH4, MeOH, rt, 3 h, 64% for 5,
55% for 11; e) conc. H2SO4, 0 ꢀC, 1 h, 88% for 6, 91% for 9; f)
BrCH2CH^CH/K2CO3/NaI/TBAB, acetone, reflux 6 h, 53% for P2, and
ethylene oxide, 50% AcOH aq, 0 ꢀC, 24 h, 57% for P3, BrCH2CH]CH2/
K2CO3/NaI/TBAB, acetone, reflux 6 h, 66% for 7; g) triethoxysilane/
Karstedt catalyst, toluene, rt, 4 h, 47%; h) AcSH/AIBN, toluene, 80 ꢀC,
5 h, 67%; i) 2,20-dithiodipyridine/NH4OH, MeOH, rt, 4 h, 91%; j)
BrCH2CH2Br/Cs2CO3, CH3CN, reflux, 6 h, 92%; k) NaN3/NaI, DMF,
90 ꢀC, 4 h, 81%; l) PPh3, THF/H2O, rt, 76%; m) furan-protected mal-
eimide/K2CO3, DMF, 50 ꢀC, 2 h, 53%; n) anisole, 140 ꢀC, 2 h, 76%.
Scheme 1 Structures and schematic representation of A) modification of
7-amino coumarin-based phototriggers, B) labelled composites with
phototriggers, C) typical photo-controlled drug delivery system, and D)
photolysis mechanism for the photo-controlled drug release system.
The latter was obtained from 7-(tosylamino)-4-methyl coumarin
(3) in four steps similar to the preparation procedures of inter-
mediate 6. The Ts protecting group in compound 3 enhanced the
activity of the N–H bond which made it easy to be further
substituted by 1,2-dibromoethane. Compound P7 was synthe-
sized by nucleophilic displacement with sodium azide in 81%
yield, which was further reduced with PPh3 to obtain compound
P8 in 76% yield. Compound P9 was obtained in two steps from
compound 11 by 1) coupling with furan-protected maleimide in
53%, and 2) deprotection in 76% yield.
phenol (1) in five steps by 1) protection of amino with 4-toluene
sulfonyl chloride in 97% yield; 2) cyclization with ethyl acetoa-
cetate catalyzed by BiCl3 in 45% yield; 3) alkylation with bro-
moethane in 88% yield; 4) oxidation by SeO2 and subsequent
reduction by NaBH4 in 64% yield; 5) deprotection of tosyl in 88%
yield.34,35 As depicted in Scheme 2, compound P2 and 7 were
prepared according to the slightly modified procedure reported
by Hagen and coworkers,28 in which compound 6 was treated
with an excess of propargyl bromide or allyl bromide in the
presence of potassium carbonate, and a catalytic amount of
sodium iodide and tetra-n-butylammonium bromide in acetone
in 53% and 66% yield respectively. N-ethoxylation of compound
6 with ethylene oxide in aqueous acetic acid led to compound P3
in 57% yield. The compounds P4 and P5 were obtained from
compound 7 by a hydrosilylation reaction in 47% yield and
a typical thiol-ene addition in 67% yield, respectively. The further
reaction in situ for compound P5 with 2,20-dithiodipyridine in
basic conditions gave the pyridyl disulfide functional compound
P6 in 91% yield.
Nano/bio-labeling application
In these synthesized phototriggers, compound P1 and compound
P8 were designed to activate the corresponding amino or
carboxylic acid-containing carriers by 1-ethyl-3-(3-dimethylami-
nopropyl) carbodiimide hydrochloride (EDC) in the presence of
N-hydroxylsuccinimide (NHS). Compound P2 and P7 were
designed to activate the corresponding alkyne or azide-containing
carriers by simple, efficient ‘‘click chemistry’’. The introduction of
hydroxyl substitution in compound P3 was used to link with
hydroxyl or amino contained carriers by the activation of N,N0-
disuccinimidyl carbonate (DSC), N,N0-carbonyldiimidazole
Since the substitution reaction to compound 6 did not work for
1,2-dibromoethane, phototriggers P7–P9 were prepared as shown
in Scheme 2, starting from another key building block 7-(N-(2-
bromoethyl)-N-ethylamino)-4-(hydroxylmethyl) coumarin (11).
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(CDI), or p-nitrophenyl chloroformate. The introduction of
functionalized triethoxy silane in compound P4 was used for
bonding with silica-based carriers directly by simple alcohol-
based silanization. Compound P5 with a thioacetate group was
used directly to bond on the surface of silver or gold nanoparticles
and semiconductor quantum dots (QDs) after the deprotection of
thiol. Compound P6 and P9 were synthesized to conjugate thiol-
containing carriers by disulfide exchange reactions in reduction
conditions and thiol Michael addition reactions, respectively.
In this paper, as shown in Scheme 3, compounds P2, P4 and
P9 were chosen as the representative phototriggers to label with
inorganic nanoparticles, biocompatible polymer, and protein,
respectively. Among numerous inorganic nanoparticles, meso-
porous silica nanoparticles (MSN) have recently attracted much
attention due to its superior properties of mesoporous structure
and good biocompatibility.36–38 The synthesis and characteriza-
tion of MCM-41-type MSN material was represented in the
experimental section. In our previous work,13 we have detailed
the amide coupling for the preparation of M1 between the
carboxylic acid of compound P1 with the aminopropyl-func-
tionalized MSN material (shown in section B of Scheme 1). Here,
we exhibited another conjugation method through simple sila-
nization between the silane precursor P4 and MSN material at rt
condition. Poly(ethylene glycol) (PEG) is a biocompatible arti-
ficial synthetic linear polyether with a wide range of sizes and
terminal functional groups, which has been widely used in bio-
logical fields.39 In this article, bis-azide terminal PEG1000 was
used to couple the alkyne of compound P2 via ‘‘click chemistry’’.
As is well known, bovine serum albumin (BSA) is a favourite
protein model in natural macromolecules. It has an exposed thiol
group at the position of the cysteine-34 residue which can
effectively react with the maleimide group of compound P9 in
aqueous environments.40 All the successful labels for M2, M4,
and M9 were confirmed by UV and fluorescence spectra after
complete purification. As shown in Fig. 1, the appearance of the
absorption around 370 nm and luminescence around 460 nm
suggested the successful labeling of the coumarin phototriggers
P2, P4, and P9. The labelings were quantified at 13.2 mmol gꢁ1
for M9 by UV calculation. The labeling quantifications of
insoluble M4 and PEG polymer M2 were further proved by TGA
analysis at 266 mmol gꢁ1 as reported in our previous paper13 (not
shown) and NMR comparison at 497 mmol gꢁ1 (as shown in
Fig. 1C), respectively.
Photophysical properties
The photophysical properties of compounds P2-P9, composites
M2, M4, and M9 were summarized in Table 1. Similar to the
reported symmetric substituted phototrigger of 7-(diethy-
lamino)-4-(hydroxylmethyl) coumarin (DEACM-OH), the
Scheme 3 Synthesis of labelled composites M2, M4, M9, and the photo-
controlled drug release system MD4: a) P2/CuSO4/sodium ascorbate,
THF/H2O (4/1), rt, 3 h; b) P4 or MSN-OH material (in dark), toluene, Ar
gas, rt, 36 h; c) P9, DMSO/H2O (1/1), rt, 4 h; d) ibuprofen/EDC/DMAP,
CH2Cl2, rt, in dark, 4 h, 98%; e) triethoxysilane/Karstedt catalyst,
toluene, rt in dark, 4 h, 86%.
Fig. 1 a) Absorption spectra; b) Fluorescence spectra of labelled
1
composites M2, M4, M9, and MD4 in ethanol solutions; c) H NMR
spectra of composites M2.
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absorption spectra for compounds P2-P9 showed maxima at
around 370 nm with large molar extinction coefficients (3max
(bis-exponential decays). Its S1 state was significantly quenched
by the maleimide ring based on the photoinduced electron
transfer (PET) mechanism.43 Nevertheless, the photophysical
properties, either fluorescence quantum yield or S1 state life time
would be recovered once it was labelled to BSA, named M9, via
an SH Michael addition reaction destroying the strong electron
deficiency of maleimide ring (Table 1).
z
20 000 Mꢁ1 cmꢁ1). The fluorescence spectra of the compounds
corresponded to the respective absorption spectra with an
average Stokes shift of 90 nm. Except for compound P9, the
fluorescence quantum yields FF of all other compounds were
0.4–0.75. There were no significant changes for their fluorescence
lifetimes (c.a. 3–4 ns) following the mono-exponential decay law.
According to the photo-SN1 cleavage mechanism reported by
Bendig and Furuta,33,41 the photolysis of caged compound was
closely correlated with the S1 excited state. This indicated that
there was no significant interaction between the S1 state of the
coumarin moiety and the various substitutions except maleimide,
which hinted that the photocleavable properties would not
be affected and would be similar to that for the reported
DEACM-OH.
Photo-controlled release
To validate the application of our N-unsymmetrical substituted
coumarin phototriggers in a biological photo-controlled release
system, MD4 was designed and fabricated as a model, in which
compound P4 acted as linker to conjugate MSN material with
a
leaving cargo, nonsteroidal anti-inflammatory drug of
ibuprofen (IBU). As shown in Scheme 3, compound 14 was
prepared firstly by the esterification between compound 7 and
IBU, and then hydrosilylation. The labeling of compound 14 to
MSN material was similar to that for composite M4. The
successful labeling was also validated via UV-vis spectroscopy,
and quantified at 179 mmol gꢁ1 by TGA analysis. As shown in
Fig. 1 and Table 1, the absorption of MD4 showed further
hyperchromic effects from 380 nm to 392 nm and extended up
450 nm, which was more favorable for biological applications.
The reduced fluorescence quantum yields, FF, for MD4
compared with M4 can be explained by the competition between
the photochemical reaction (ester cleavage) and radioactive
action.41
After conjugating with carriers, the maximum absorption of
the composites showed was red-shift. Especially for M4, the
maximum shifted from 375 nm to 387 nm and extended up
450 nm. It suggested that our designed phototriggers provided
the possibility to allow photouncaging under visible light irra-
diation (l < 400 nm) without any biological damage. For M2,
PEG polymer conjugated by click chemistry, there was no
significant change for both fluorescence lifetime and quantum
yield, except for 6 nm redshift of the emission peak compared
with the parent compound P2. However, composite M4, derived
from the incorporation of phototrigger P4 into mesoporous
silica nanoparticles, exhibited a slight difference of photophysical
data. As shown in Table 1, no significant change of fluorescence
quantum yields but bis-exponential fluorescence decays (a
shorter component of 0.36 ns and a normal component of
3.24 ns) indicated a heterogeneous microenvironment of photo-
trigger molecules within the nanoparticles.42 Compound P9 was
exceptional due to its much lower fluorescence quantum
yield and an additional shorter emission lifetime value
To check the stability of MD4, the solution of MD4 in ace-
tonitrile : water solution (9 : 1, v/v) was allowed to stand over-
night at 37 ꢀC in the dark, and no release of IBU could be
detected by analytical HPLC. The visible light irradiation at
450 nm (a xenon lamp) resulted in IBU release. The time course
of the drug release under photolysis was monitored by HPLC. As
shown in Fig. 2, the photolytic processes progressed effectively,
and the maximum release had reached 95% after 2 h irradiation
(25 mW cmꢁ2). The incomplete photolysis may be attributed to
an internal filtering effect from the photo by-product.44 The
efficient photorelease confirmed that the substitution on the
7-amine group of coumarin did not affect the photocleavable
property of the phototrigger. Precise control of the photolytic
release was demonstrated by monitoring the progress of IBU
release after periods of exposure to light and dark conditions, as
shown in inset (a) of Fig. 2. The distinctive ‘‘stepped’’ profile
revealed that the cargo release only proceeded under light
conditions, thus demonstrating ‘‘light-regulated precise release’’.
In addition, there were no detectable side-products in the
photolysis for MD4 (inset (b) of Fig. 2). All the results indicated
that this kind of coumarin-based phototrigger could act well as
the photo-responsive linker between the cargo and carrier, and
the cargo release could be precisely controlled by manipulating
external light conditions.
Table 1 Absorption maximun l , emission maximun lmf ax, fluorescent
max
abs
quantum yields FF, and fluorescence lifetime sf of the designed photo-
triggers P2–P9, prodrug 14 and labelled composites M2, M4, M9, and
MD4. Solvent: ethanol in all casesa
max
labs
(nm)
lfmax
(nm)
Compounds
FF
sf (ns)
Ref
P2
P3
P4
P5
P6
P7
P8
P9
378
362
373
375
371
373
368
369
368
468
456
468
467
466
465
463
462
460
0.44
0.75
0.57
0.40
0.58
0.60
0.64
0.46
0.03
3.31
3.62
3.30
3.49
3.48
3.35
3.53
3.04
3.24(0.91)
0.36(0.09)
2.77
14
M2
M4
380
367
387
477
462
476
0.33
0.72
0.45
3.62
3.48(0.89)
1.20(0.11)
4.24
2.64(0.91)
0.39(0.09)
Conclusions
M9b
MD4
376
392
467
482
0.45
0.35
We synthesized a new series of unsymmetrical substituted
7-amino coumarin-based phototriggers coupled with various
nano/bio-conjugated bonds. The photophysical properties of
most of the substituted coumarin-based phototriggers, except for
a
Ref: DEACM-OH. b values obtained in DMSO : H2O (1 : 4).
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 6680–6688 | 6687

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11 G. Papageorgiou, D. C. Ogden, A. Barth and J. E. T. Corrie, J. Am.
Chem. Soc., 1999, 121, 6503.
12 G. C. R. Ellis-Davies, Nat. Methods, 2007, 4, 619.
13 Q. Lin, Q. Huang, C. Li, C. Bao, Z. Liu, F. Li and L. Zhu, J. Am.
Chem. Soc., 2010, 132, 10645.
14 V. Hagen, B. Dekowski, N. Kotzur, R. Lechler, B. Wiesner, B. Briand
and M. Beyermanm, Chem.–Eur. J., 2008, 4, 1621.
15 P. Neveu, I. Aujard, C. Benbrahim, T. L. Saux, J.-F. Allemand,
S. Vriz, D. Bensimon and L. Jullien, Angew. Chem., Int. Ed., 2008,
47, 3744.
16 M. Noguchi, M. Skwarczynski, H. Prakash, S. Hirota, T. Kimura,
Y. Hayashi and Y. Kiso, Bioorg. Med. Chem., 2008, 16, 5389.
17 J. Babin, M. Pelletier, M. Lepage, J. Allard, D. Morris and Y. Zhao,
Angew. Chem., Int. Ed., 2009, 48, 3329.
€
18 S. Hartner, H.-C. Kim and N. Hampp, J. Polym. Sci., Part A: Polym.
Chem., 2007, 45, 2443.
19 S. S. Agasti, A. Chompoosor, C.-C. You, P. Ghosh, C. K. Kim and
V. M. Rotello, J. Am. Chem. Soc., 2009, 131, 5728.
20 J. A. Johnson, Y. Y. Lu, A. O. Burts, Y. Xia, A. C. Durrell,
D. A. Tirrell and R. H. Grubbs, Macromolecules, 2010, 43, 10326.
21 S. K. Choi, T. Thomas, M.-H. Li, A. Kotlyar, A. Desai and
J. R. Baker Jr, Chem. Commun., 2010, 46, 2632.
22 S. S. Banerjee and D.-H. Chen, Nanotechnology, 2009, 20, 185103.
23 M. Shi, J. Lu and M. S. Shoichet, J. Mater. Chem., 2009, 19, 5485.
24 O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010,
62, 284.
Fig. 2 a) The time course of drug release for the MD4 drug delivery
system under 450 nm visible light irradiation (25 mW cmꢁ2) determined
by HPLC analysis. Inset (a) is the partial progress for the release of IBU
from MD4 in light and dark conditions. ‘‘ON’’ indicates the beginning of
light irradiation; ‘‘OFF’’ indicates the end of light irradiation; (b) is the
time course of HPLC profiles for released IBU from 0–120 min under
irradiation of 450 nm visible light (25 mW cmꢁ2).
25 C.-J. Carling, F. Nourmohammadian, J.-C. Boyer and N. R. Branda,
Angew. Chem., Int. Ed., 2010, 49, 3782.
26 M. Barczewski, S. Walheim, T. Heiler, A. B1aszczyk, M. Mayor and
T. Schimmel, Langmuir, 2010, 26, 3623–3628.
27 H. Langhals and A. J. Esterbauer, Chem.–Eur. J., 2009, 15, 4793–
4796.
28 V. Hagen, B. Dekowski, V. Nache, R. Schmidt, D. Geibler,
D. Lorenz, J. Eichhorst, S. Keller, H. Kaneko, K. Benndorf and
B. Wiesner, Angew. Chem., Int. Ed., 2005, 44, 7887.
compound P9 with a maleimide group, showed no significant
change compared with DEACM-OH, the reported symmetric
substituted 7-amino coumarin-based phototrigger. Four repre-
sentative compounds P2, P4, P9 and 14 were successfully
labelled with typical carriers of polyethylene glycol (PEG)
polymer (M2), mesoporous silica nanoparticles (MSN) (M4 and
MD4), and bovine serum albumin (BSA) (M9). The efficient
photo-regulated release of the drug IBU in the model MD4
system displayed a new promising platform for designing and
preparing photocontrollable release systems with a precise
modulation by manipulating external light conditions in bio-
logical areas.
29 M. T. Reetz, M. Rentzsch, A. Pletsch, A. Taglieber, F. Hollmann,
ꢀ
€
R. J. G. Mondiere, N. Dickmann, B. Hocker, S. Cerrone,
M. C. Haeger and R. Sterner, ChemBioChem, 2008, 9, 552.
30 C.-Y. Lai, B. G. Trewyn, D. M. Jeftinija, K. Jeftinija, S. Xu,
S. Jeftinija and V. S.-Y. Lin, J. Am. Chem. Soc., 2003, 125,
4451.
31 S. S. Iyer, A. S. Anderson, S. Reed, B. Swanson and J. G. Schmidt,
Tetrahedron Lett., 2004, 45, 4285.
32 B. H. Ruan, D. C. Cole, P. Wu, A. Quazi, K. Page, J. F. Wright,
N. Huang, J. R. Stock, K. Nocka, A. Aulabaugh, R. Krykbaev,
L. J. Fitz, N. M. Wolfman and M. L. Fleming, Anal. Biochem.,
2010, 399, 284.
Acknowledgements
33 T. Furuta, S. S.-H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee,
E. M. Callaway, W. Denk and R. Y. Tsien, Proc. Natl. Acad. Sci.
U. S. A., 1999, 96, 1193.
34 S. Mizukami, T. Nagano, Y. Urano, A. Odani and K. Kikuchi, J. Am.
Chem. Soc., 2002, 124, 3920.
35 K. C. Majumdar, S. Chakravorty and A. Taher, Synth. Commun.,
2008, 38, 3159.
36 Q. He, Z. Zhang, Y. Gao, J. Shi and Y. Li, Small, 2009, 5, 2722.
ꢁ
37 M. Vallet-Regı, F. Balas and D. Arcos, Angew. Chem., Int. Ed., 2007,
46, 7548.
38 D. P. Ferris1, J. Lu, C. Gothard, R. Yanes, C. R. Thomas, J. Olsen,
J. F. Stoddart, F. Tamanoi and J. I. Zink, Small, 2011, 7,
1816.
39 K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert, Angew.
Chem., Int. Ed., 2010, 49, 6288.
This work was supported by NSFC (2117308), ‘‘Chen Guang’’
project (09CG25), Shanghai Sci. Tech. Comm. (11JC1403300),
Shanghai Rising-star program ‘‘10QA1401600’’ and Funda-
mental Research Funds for the Central University.
Notes and references
1 M. Goeldner, R. S. Givens, Dynamic Studies in Biology:
Phototriggers, Photoswitches and Caged Biomolecules, Wiley-VCH,
Weinheim, 2005.
2 P. Rai, S. Mallide, X. Zheng, R. Rahmanzadeh, Y. Mir, S. Elrington,
A. Khurshid and T. Hasan, Adv. Drug Delivery Rev., 2010, 62, 1094.
3 S. Sortino, J. Mater. Chem., 2012, 22, 301.
40 A. J. (T.) Dirks, S. S. Berkel, N. S. Hatzakis, J. A. Opsteen,
F. L. Delft, J. J. L. M. Cornelissen, A. E. Rowan, J. C. M. Hest,
F. P. J. T. Rutjes and R. J. M. Nolte, Chem. Commun., 2005, 4172.
41 T. Eckardt, V. Hagen, B. Schade, R. Schmidt, C. Schweitzer and
J. Bendig, J. Org. Chem., 2002, 67, 703.
4 M. Wirkner, J. M. Alonso, V. Mans, M. Salierno, T. T. Lee,
A. J. Garcia and A. Del Campo, Adv. Mater., 2011, 23, 3907.
5 A. P. Pelliccioli and J. Wirz, Photochem. Photobiol. Sci., 2002, 1, 441.
6 J.-P. Pellois and T. M. Muir, Angew. Chem., Int. Ed., 2005, 44, 5713.
7 S. Petersen, J. M. Alonso, A. Specht, P. Duodu, M. Goeldner and
A. Campo, Angew. Chem., Int. Ed., 2008, 47, 3192.
€
ꢁ
42 V. Lebret, L. Raehm, J.-O. Durand, M. Smaıhi, C. Gerardin,
N. Nerambourg, M. H. V. Werts and M. Blanchard-Desce, Chem.
Mater., 2008, 20, 2174.
8 L. Yu, C. Lv, L. Wu, C. Tung, W. Lv, Z. Li and X. Tang, Photochem.
Photobiol., 2011, 87, 646.
43 S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and
S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559.
44 C. Bao, G. Fan, Q. Lin, B. Li, S. Cheng, Q. Huang and L. Zhu, Org.
Lett., 2012, 14, 572.
9 R. S. Givens, A. Jung, C.-H. Park, J. Weber and W. Bartlett, J. Am.
Chem. Soc., 1997, 119, 8369.
10 C. P. McCoy, C. Rooney, C. R. Edwards, D. S. Jones and
S. P. Gorman, J. Am. Chem. Soc., 2007, 129, 9572.
6688 | J. Mater. Chem., 2012, 22, 6680–6688
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