Photolabile protecting group bonded to aminopropyl silica-gel beads

Article abstract of DOI:10.1246/bcsj.82.997

4, 5-Dimethoxy-2-nitrobenzyloxycarbonyl group acting as a photolabile protecting group was bonded on 3-amino- propyl silica gels through a covalent bond ina 46% yield. The deprotection was performed under UV-irradiation. The resulting amino group was anal

Full text of DOI:10.1246/bcsj.82.997

Short Articles
Bull. Chem. Soc. Jpn. Vol. 82, No. 8, 997–999 (2009)
997
SiO2-NH
PPG
Photolabile Protecting Group
Bonded to Aminopropyl
Silica-Gel Beads
Si
O
NO2
O
O
O
O
Si
O
O
Si
N
H
N
H
O
OMe
Si
OMe
1a
Scheme 1. 3-Aminopropyl silica gel 1a protected by 4,5-
dimethoxy-2-nitrobenzyloxycarbonyl group acting as PPG.
Jin Matsumoto,1 Yoshiya Senda,2 Hiroki Masuda,1
Tomokazu Fuchikawa,1 Tsutomu Shiragami,1
and Masahide Yasuda*1
OH
O
NO
O
O
O
NO2
N
R
h
ν
R
NH CO2
R
H
1Department of Applied Chemistry, Faculty of Engineering,
University of Miyazaki, Gakuen-Kibanadai,
Miyazaki 889-2192
2
O
O
N
H
N
H
OMe
OMe
OMe
OMe
OMe
OMe
Scheme 2. Deprotection scheme of PPG.
2Asahi Organic Chemicals Co., Ltd.,
2-5955 Nakanose, Nobeoka 882-8688
H2tpp
NH
O
H2tpp
O
C9H19
O
Received December 24, 2008; E-mail: yasuda@cc.miyazaki-
u.ac.jp
SiO2-NH
SiO2-NH
O
1b
1c (57%)
SiO2 NH
2d, i
2c, i
2d, i
NH2
C9H19
NH
O
PPG
3b
4,5-Dimethoxy-2-nitrobenzyloxycarbonyl group acting
as a photolabile protecting group was bonded on 3-amino-
propyl silica gels through a covalent bond in a 46% yield.
The deprotection was performed under UV-irradiation. The
resulting amino group was analyzed through the use of a
confocal laser-scanning microscope using 4-(10,15,20-
triphenylporphyrinyl)phenyl fluorophore.
NH
2a, i
O
2c, i
ii
O
O
SiO2-NH2
x0= 0.833 mmol g-1
SiO2
SiO2
NH
SiO2 NH
NH
1a (46%)
3a (quant.)
xP= 0.383 mmol g-1 xA= 0.383 mmol g-1
3c (60%)
xR= 0.231 mmol g-1
Ph
O
N O C
O
O
O
O
O
NH
N
N
HN
PPG
Ph
N O C (CH2)9-H
N O
N
H
O
O
O
Ph
2c
2a
Photolabile protecting groups (PPG) have been widely used
in organic synthesis in solutions in order to be transformed to a
variety of functional group after deprotection under irradia-
tion.1 Also PPG is often bonded to a solid matrix such as
polystyrene beads2 or a glass surface3 to prepare functional
micro-beads by controlling the site and amount. Functional
micro-beads have been used as catalysts and scavengers in
organic synthesis, carriers of solid-phase synthesis, and
selective sensors for biomolecules. However, the introduction
of PPG on to silica gel (SiO2) has been scarcely reported.4 In
order to facilitate the site-controlled functionalization of silica
gel, 3-aminopropyl silica gel (SiO2-NH2) was N-protected with
PPG (Scheme 1).
2d
H2tpp
Scheme 3. The reaction routes of 1a and its transformation.
Reagent: i) Im, CH2Cl2, room temp, 3 days and ii) h¯,
CHCl3-TFA, 2 h.
a1 a2 a3
a4
a5
1a (4.0)
1a (2.0)
1a (1.0)
1a (0.5)
The 4,5-dimethoxy-2-nitrobenzyloxycarbonyl group was
selected as it is a PPG which can generate an amino group
under UV irradiation (Scheme 2).5 Using succinimidyl ester
of N-PPG-3-aminopropanoic acid 2a, the PPG was bonded
on micrometer SiO2-NH2 (average diameter: 95.4 ¯m, area:
229 m2 g¹1, pore volume: 1.08 cm3 g¹1, quantity of NH2 (x0):
0.833 mmol g¹1) (Scheme 3). N-PPG-protected SiO2-NH2 1a
was prepared by the reaction of SiO2-NH2 (1 g) with 2a
(1 mmol) in CH2Cl2 (8 mL) for 3 days under a consistent gentle
shaking in the presence of imidazole (Im; 1.67 mmol) acting as
base to give free amino group as SiO2-NH2. The beads were
separated by filtration and washed with CHCl3/MeOH (5:1) to
obtain 1a. IR spectra of 1a is shown in Figure 1. N-PPG
protection of SiO2-NH2 was confirmed by the appearance of
1a (0)
2b
2000
1500
Wavenumber/cm-1
1000
Figure 1. IR spectral changes of 1a during UV-irradiation
for a given time (t/h) shown in parenthesis along with IR
spectra of 2b. After irradiation for 4 h, 1a was completely
transformed to 3a. The assignment of a1 through a5 are
shown in text.
¹1
characteristic absorption of the PPG at 1709 cm for C=O
Published on the web August 7, 2009; doi:10.1246/bcsj.82.997

998
Bull. Chem. Soc. Jpn. Vol. 82, No. 8 (2009)
© 2009 The Chemical Society of Japan
0.4
0.3
0.2
0.1
0
-50
0
50
0
1
2
3
Distance/µm
Amount of 2a/mmol against unit gram
of SiO2-NH2
Figure 3. Distribution of the fluorescence intensity of 1b
(dotted line) and 3b (solid line) at cross section located at
the 25 ¯m depth from the surface. Inset: the cross sectional
fluorescence image of 3b at the 25 ¯m depth from surface.
Measurement was performed by a CLSM under irradiation
at 543 nm.
Figure 2. Dependence of the PPG amount (xP) in 1a on the
amounts of 2a used.
¹1
stretching of the urethane bond (a1), 1582 cm for the C=C
stretching of the benzene ring (a2), 1523 cm
¹1
for NO
¹1
stretching of the nitro group (a3), and 1279 cm for the C-O
stretching of the MeO group (a4) in addition to the absorption
of the Si-O stretching (a5) at 800 cm¹1. These assignments
were performed by comparison with the IR spectrum of a
model compound, N-propyl-3-[(4,5-dimethoxy-2-nitrobenzyl-
oxycarbonyl)amino]propanamide (2b). The amounts of the
PPG (xP/mmol g¹1) in 1a were determined by IR absorption
spectrophotometry. The xP was determined for 1a which was
prepared by the reaction of SiO2-NH2 with a specific amount of
2a. In Figure 2, xP was plotted against the amounts of 2a
added. As the amount of 2a increased, the xP also gradually
(H2tpp)-bonded silica gel 3b was examined through use of
a fluorescence image measured by a confocal laser-scanning
microscope (CLSM) under excitation at 543 nm. As shown in
Figure 3, the fluorescence of 3b was coming from the area
between the surface and a 10 ¯m depth. It showed that the
formed amino groups located at shallow site in the silica gel
beads. In the fluorescence image of H2tpp-incorporated SiO2-
NH2 1b which was prepared by the reaction of SiO2-NH2 with
2d (Figure 3), the fluorescence was coming from the entire
inside of the SiO2-NH2 beads. These results demonstrate that
2a cannot reach the deeper parts of the SiO2-NH2 beads
possibly due to the adsorption of the polar nitro group in shal-
low sites, as has been reported for o-hydroxybenzophenone.7
The reactivity of the resulting amino group on 3a was
checked by decanoylation with 2c which was performed by the
reaction of 3a with 2c in the presence of Im. The decanolyated
silica gel 3c was subjected to IR absorption spectrophotometry
in order to determine the amount of the decanoyl group (xR).
The yield (100xR/xA) for the formation of 3c from 3a was
determined to be 60%. This value is similar to the value of the
yield (57%) for 1c which was prepared by the reaction of SiO2-
NH2 with 2c.
increased until reaching a maximum level at approximately
¹1
0.8 mmol g
of 2a, nearly equal to the amounts of x0
(0.833 mmol g¹1). At the maximum point, 0.383 mmol g xP
was formed in 1a. This value corresponds to 46% of the
protection yield (=100xP/x0) based on x0.
¹1
¹1
As a consequence, 0.450 mmol g of the amino groups
remained in 1a, however, the residual amino groups of 1a did
not react with acylation reagents such as Ac2O and succin-
imidyl decanoate (2c). It was suggested that the residual
amino groups remained deep within narrow channels of the
silica gel to prevent the approach of any of the reagents.
Therefore, the following deprotection was performed without
the protection of the residual amino groups. The cleavage of the
PPG from 1a was carried out under 366 nm irradiation in
CHCl3-trifluoroacetic acid (TFA; 50 mM). The addition of TFA
was effective in the decomposition of the acetal intermediate
generated by the irradiation (Scheme 2). Removal of PPG was
confirmed by the disappearance of a3 in the IR spectra
(Figure 1). The absorption of a3 disappeared entirely upon
irradiation for 4 h, resulting in complete transformation from 1a
to the PPG-deprotected SiO2-NH2 3a. Therefore, the quantity
(xA) of the free amino groups formed was assumed to be equal
to the quantity of NO2 consumed. i.e., xP = xA.
In conclusion, easy deprotection of PPG from 1a was
achieved, since SiO2 is highly transport to visible light.
Therefore, 1a will be extensively applicable to the preparation
of micro-beads with multifunctionalities such as catalysts,
scavengers, and biosensors because of large surface area and
high adsorption ability.8
Experimental
Instruments. 1H (400 MHz) and 13C NMR (100 MHz) spectra
were taken on a Bruker AV 400M spectrometer for CDCl3
solutions with tetramethylsilane used as an internal standard.
Matrix-assisted laser desorption/ionization mass spectra (MALDI-
MS) were measured on a Bruker Daltonics Autoflex III TOF/TOF
in the positive ion mode. FT-IR was measured on a JASCO Herscel
FT/IR-300 with a Micro-20 spectrometer. UV-vis spectra of the
solution were obtained with a JASCO V-550 spectrophotometer.
In order to analyze the distribution of the formed amino
groups, 3a were treated with 5-[4-(succinimidyloxycarbonyl)-
phenyl]-10,15,20-triphenylporphyrin (2d).6 The distribution
of 4-(10,15,20-triphenylporphyrinyl)phenyl chromophore

J. Matsumoto et al.
Bull. Chem. Soc. Jpn. Vol. 82, No. 8 (2009)
999
Absorption and fluorescence microscopic spectroscopy of silica
gel beads was performed on an Olympus FV-300 confocal laser
scanning microscope (CLSM) equipped with spectrophotometer
(STFL 250, Seki Technotron) linked to CLSM using an optical
fiber.
(2e; 5.0 mmol) was reacted with HOSu (6.0 mmol) in CH2Cl2
(20 mL) in the presence of DMAP (5.0 mmol) and DCC
(7.5 mmol) for 2 days at room temperature. After the filtration of
the dicyclohexylurea, the CH2Cl2 solution was evaporated and then
the residual oil was purified by column chromatography in silica
gel to give 2c.
Preparation of Succinimidyl 3-[(4,5-Dimethoxy-2-nitro-
benzyloxycarbonyl)amino]propanoate (2a). 4,5-Dimethoxy-2-
nitrobenzyl alcohol (5 mmol) was reacted with p-nitrophenyl
chloroformate (6.0 mol) in dimethylacetamide (DMA, 20 mL) in
the presence of Et3N (10 mmol) at room temperature for 2 days to
give p-nitrophenyloxycarbonyl 4,5-dimethoxy-2-nitrobenzyl car-
bonate (4a) in a 71% yield.9 The reaction of 4a (1.0 mmol) with
ethyl 3-aminopropanoate (2.0 mol) was performed in the presence
of 4-(dimethylamino)pyridine (DMAP, 1.0 mmol) in DMA
(20 mL) at room temperature for 2 days. p-Nitrophenol was
removed from the reaction mixture by extraction with an aqueous
solution of NaHCO3 and CHCl3. The CHCl3 solution was
evaporated and then the residual oil was poured into hexane
(200 mL) to give ethyl 3-[(4,5-dimethoxy-2-nitrobenzyloxycar-
bonyl)amino]propanoate (4b) as a yellow precipitate in 83% yield.
4b (1 mmol) was hydrolyzed in CHCl3-EtOH (18 mL v/v 1:2) in
the presence of an aqueous solution of Et4NOH (10%, 3 mL)
to give 3-[(4,5-dimethoxy-2-nitrobenzyloxycarbonyl)amino]pro-
panoic acid (4c) in 81% yield. The reaction of 4c (1.0 mmol) with
N-hydroxysuccinimide (HOSu; 1.20 mol) was performed in
CH2Cl2 (15 mL) in the presence of DMAP (1.0 mmol) and N,N¤-
dicyclohexylcarbodiimide (DCC, 1.44 mmol) for 3 days at room
temperature. After the filtration of the dicyclohexylurea, the
CH2Cl2 solution was evaporated and then the residual oil was
poured into hexane (200 mL) to give 2a as a pale yellow
precipitate in 79% Yield.
1
2c: Yield 79%. H NMR: ¤ 0.88 (t, J = 6.8 Hz, 3H), 1.27 (m,
12H), 1.74 (quint, J = 7.4 Hz, 2H), 2.60 (t, J = 7.4 Hz, 2H), 2.84
(s, 4H); 13C NMR: ¤ 14.06, 22.64, 24.54, 25.54, 28.75, 29.04,
29.30, 29.50, 29.58, 30.90, 31.86, 168.64, 169.14. HRMS
(MALDI-TOF) Found: m/z 292.1446. Calcd for C14H23NO4Na:
[M + Na]+, 292.1519.
Quantitative Analysis of Micro-Beads by IR Absorption
Spectrophotometry. In order to determine xP, a mixture of 2b
with SiO2-NH2 was prepared in a given molar ratio (m2b/mS)
where m2b and mS denoted the molar amounts of 2b and SiO2-
NH2. In the IR spectra of the mixed samples, the area ratios of a3 of
2b to a5 of SiO2-NH2 were plotted against m2b/mS to form
calibration curves. In order to determine xR, a mixture of decanoic
acid (2e) with SiO2-NH2 was prepared in a specified molar ratio
(m2e/mS) where m2e and mS represented the molar amounts of 2e
and SiO2-NH2. In the IR spectra of the mixed samples, the area
¹1
ratio of the C-H stretching (a6) at 3000-2830 cm of 2e to a5
absorption of SiO2-NH2 were plotted against m2e/mS to make
calibration curves.
Deprotection of 1a. The irradiation of 1a (10 mg) was carried
out in a CHCl3-TFA (50 mM) solution (8 mL) by an Eikosha high-
pressure mercury lamp through a Pyrex filter. The beads were then
separated by filtration and washed with CHCl3-MeOH (5:1) to
produce 3a.
Supporting Information
2a: 1H NMR: ¤ 2.88 (t, J = 6.2 Hz, 2H), 2.89 (s, 4H), 3.64 (q,
J = 6.2 Hz, 2H), 3.95 (s, 3H), 3.98 (s, 3H), 5.52 (s, 2H), 5.71 (br t,
J = 6.2 Hz, 1H), 7.03 (s, 1H), 7.71 (s, 1H); 13C NMR: ¤ 25.53,
32.17, 36.73, 56.35, 56.41, 63.69, 108.09, 109.79, 128.45, 139.54,
147.97, 153.59, 155.72, 167.29, 169.04. HRMS (MALDI-TOF)
Found: m/z 448.1061. Calcd for C17H19N3O10Na: [M + Na]+,
448.0963.
The spectral data of 4a, 4b, and 4c are available free of charge
on the web at http://www.csj.jp/journals/bcsj/.
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8
Preparation of Succinimidyl Decanoate (2c). Decanoic acid
9