Diffusion-based deprotection in mesoporous materials: A strategy for differential functionalization of porous silica particles

Article abstract of DOI:10.1021/ja070598b

A monodisperse, spherical mesoporous silica (Acid-Prepared Mesoporous Spheres, APMS) was prepared and then functionalized with two types of Fmoc (9-fluorenylmethyloxycarbonyl) terminated silanes with variable chain lengths. N₂ physisorption experiments indicated that, under some conditions, the pores of the solid were completely filled by the Fmoc-protected organosilanes. These blocked pores were then "reopened" by the cleavage of Fmoc groups with a piperidine solution. In contrast to the solution reaction, this deprotection reaction was much slower within the pores. The rate of deprotection was followed by UV/visible spectroscopy, and a plot of Fmoc released versus time showed a sigmoidal shape. An empirical model was applied to the data, which indicated that the reaction was influenced by the concentration and temperature of the piperidine solution as well as the number of Fmoc moieties within the pores. Using this information, we show that the location of the deprotection reaction in the pores of the silica can be empirically controlled. Our work provides a method by which the surface of the porous silica can be functionalized in a well-defined manner. This method can be used to produce materials for catalysis or drug delivery.

Full text of DOI:10.1021/ja070598b

Published on Web 07/18/2007
Diffusion-Based Deprotection in Mesoporous Materials: A
Strategy for Differential Functionalization of Porous Silica
Particles
Kai Cheng and Christopher C. Landry*
Contribution from the Department of Chemistry, 82 UniVersity Place, UniVersity of Vermont,
Burlington, Vermont 05405
Received January 26, 2007; E-mail: christopher.landry@uvm.edu
Abstract: A monodisperse, spherical mesoporous silica (Acid-Prepared Mesoporous Spheres, APMS) was
prepared and then functionalized with two types of Fmoc (9-fluorenylmethyloxycarbonyl) terminated silanes
with variable chain lengths. N
of the solid were completely filled by the Fmoc-protected organosilanes. These blocked pores were then
reopened” by the cleavage of Fmoc groups with a piperidine solution. In contrast to the solution reaction,
2
physisorption experiments indicated that, under some conditions, the pores
“
this deprotection reaction was much slower within the pores. The rate of deprotection was followed by
UV/visible spectroscopy, and a plot of Fmoc released versus time showed a sigmoidal shape. An empirical
model was applied to the data, which indicated that the reaction was influenced by the concentration and
temperature of the piperidine solution as well as the number of Fmoc moieties within the pores. Using this
information, we show that the location of the deprotection reaction in the pores of the silica can be empirically
controlled. Our work provides a method by which the surface of the porous silica can be functionalized in
a well-defined manner. This method can be used to produce materials for catalysis or drug delivery.
Introduction
functionalization of the internal pore surfaces with proper
functional groups is one way to trap various molecules with
The synthesis and modification of mesoporous silica have
recently received much attention,1-7 due to its large internal
different sizes and functionalities inside the pores.1
7-19
The
external particle surfaces can then be modified in a different
manner to provide enhanced biocompatibility, dispersion, or
other properties. The key issue is that chemical modification
of the external surface should not only confer the desired
function but also leave the internal pore volume available to
carry exchangeable molecules or perform a separate function.
Mesoporous silica is synthesized by polymerizing a silica
source in the presence of a surfactant template. The pores of
the solid are created by either calcination of the resulting
organic-inorganic composite in air or surfactant extraction.
Postsynthesis grafting and co-condensation by adding an orga-
nosilane to the synthesis mixture are two popular strategies for
immobilization of functional groups onto mesoporous silica via
surface area and pore volume, tunable and narrowly distributed
pore diameter, controllable surface functionalization, chemical
inertness, and biocompatibility, all of which has made it ideal
8
-10
for use in biomedical applications such as biocatalysis,
1
1
12-15
separation and decontamination, affinity chromatography,
and drug delivery.1 The successful use of porous solids in these
6
applications often requires different surface properties of the
internal (pore) and external surfaces. For example, chemical
(
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Nature 1992, 359, 710.
(
2) Corma, A. Chem. ReV. 1997, 97, 2373.
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3) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.;
Schmitt, K. D.; Chu, C. T.-W.; Olson, D. J.; Sheppard, E. W.; McCullen,
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Mater. 1999, 11, 1452.
(
(
(
(
20
covalent bonds. Although co-condensation provides a more
homogeneous distribution of the functional groups within the
silica than postsynthesis grafting, incomplete attachment and
phase separation during the synthetic process can make it more
difficult to quantitatively control the loadings of functional
5) Gallis, K. W.; KEklund, A. G.; Jull, S. T.; Araujo, J. T.; Moore, J. G.;
Landry, C. C. Stud. Surf. Sci. Catal. 2000, 129, 747.
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2
004, 16, 2157.
2
1,22
(
8) Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003, 42, 3336.
groups.
Another method involving simultaneous extraction
(
9) Cetinus, S. A.; Oztop, H. N. Enzyme Microb. Technol. 2003, 32, 889.
of the surfactant (which is used to create the mesopores during
(
10) Lei, C.; Shin, Y.; Liu, J.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124,
1
1242.
(
(
11) Wang, Y.; Caruso, F. Chem. Mater. 2005, 17, 953.
12) Lakhiari, H.; Legendre, E.; Muller, D.; Jozefonvicz, J. J. Chromatogr. B
(17) Stein, A.; Melde, B. J.; Schroden, R. C. AdV. Mater. 2000, 12, 1403.
(18) Hoffmann, F.; Cornelius, M.; Morell, J.; Froba, M. Angew. Chem., Int.
Ed. 2006, 45, 3216.
(19) Descalzo, A. B.; Martinez-Manez, R.; Sancenon, R.; Hoffmann, K.; Rurack,
K. Angew. Chem., Int. Ed. 2006, 45, 5924.
(20) Vinu, A.; Hossain, K. Z.; Ariga, K. J. Nanosci. Nanotechnol. 2005, 5, 347.
(21) Liu, Y.-H.; Lin, H.-P.; Mou, C.-Y. Langmuir 2004, 20, 3231.
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2003, 15, 4247.
1
995, 664, 163.
(
13) Schiel, J. E.; Mallik, R.; Soman, S.; Joseph, K. S.; Hage, D. S. J. Sep. Sci.
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006, 29, 719.
(
(
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9674
9
J. AM. CHEM. SOC. 2007, 129, 9674-9685
10.1021/ja070598b CCC: $37.00 © 2007 American Chemical Society

Differential Functionalization of Porous Silica Particles
A R T I C L E S
synthesis) and grafting of organic functionalities successfully
provided very well-defined surface functionization.21 However,
used to image the samples. Calcinations were carried out in a box
furnace under conditions of flowing air. The following heating profile
was used for calcinations: 2 °C/min ramp to 450°C, 240 min hold at
all of these methods result in the modification of both interior
and exterior surfaces of mesoporous silicas. Grafting of silane
groups before the extraction of the surfactant has been used to
4
50 °C, 10 °C/min ramp to 550 °C, 480 min hold at 550 °C. Parr
autoclaves were purchased from the Parr instrument Corporation.
N-(9-Fluorenylmethyloxycarbonyl)oxysuccinimide (Fmoc-OSu) was
obtained from Advanced Chem Tech. Alexa Fluor 488 carboxylic acid,
succinimidyl ester was obtained from Molecular Probes. Concentrated
HCl was obtained from J. T. Baker. All other chemicals were obtained
23
exclusively modify the exterior particle surface. For mesopo-
rous silicas synthesized under acidic conditions, however, the
surfactant-silane exchange process cannot be ignored since the
surfactants are not strongly attracted to the silica. This results
from Aldrich. Triethylamine (Et
3
N), hexane, toluene, and methylene
21
in unexpected modification within the pores. We noticed that
reactions within the pores are spatially confined and the diffusion
of reactant and product molecules is physically constrained. This
chloride (CH Cl ) were distilled prior to use, and acetonitrile (MeCN)
2
2
was stored over molecular sieves. Other solvents and chemicals were
used as received.
Synthesis of (3-Triethoxysilylpropyl)carbamic Acid 9H-Fluore-
nylmethyl Ester (Fmoc-APTES). A solution of aminopropyltriethox-
6
produces a molecular size-exclusion effect, which can be used
to control chemical reactions. We reasoned that this size-
exclusion effect could be exploited in combination with
established protection/deprotection methods to produce a me-
soporous silica sphere with amine groups exposed solely on its
external surface, which could then be used to covalently bind
peptides or other biomolecules to the external surface. The
internal pore volume could then be opened by complete
deprotection. Protection/deprotection of surface-bound amines
was selected because it is a well-known reaction, and the
resulting materials are potentially useful. Here, we studied the
rates of diffusion and reaction in the modified mesoporous silica
spheres with UV-visible spectroscopy and confocal fluores-
cence microscopy. An empirical equation can be used to model
the experimental data in order to understand the mechanism of
deprotection within the pores.24
2 2
ysilane, APTES (4.68 mL, 20.0 mmol), in 100 mL of anhydrous CH Cl
was prepared and cooled in an ice bath. In a separate flask, N-(9-
fluorenylmethyloxycarbonyl)oxysuccinimide, Fmoc-OSu (6.88 g, 20.4
2 2
mmol), was dissolved in a small amount of anhydrous CH Cl ; this
solution was then slowly added to the APTES solution with stirring.
After 2 h, rotary evaporation of the solvent left an oily white product
which was purified by flash chromatography with ethyl acetate/hexane
(
1:4) to yield the product (7.8 g, 17.6 mmol, 88% yield) as a white
1
solid. H NMR (500 MHz, CDCl ): δ 7.77 (2H, d, J ) 7.5 Hz, Ar-
3
H), 7.60 (2H, d, J ) 7.6 Hz, Ar-H), 7.40 (2H, t, J ) 7.5 Hz, Ar-H),
7.31 (2H, t, J ) 7.6 Hz, Ar-H), 5.03 (1H, broad s, NH), 4.40 (2H, d,
J ) 6.86 Hz, COOCH ), 4.22 (1H, t, J ) 6.18 Hz, Ar-CH), 3.823
2
2
(6H, q, J ) 7.00 Hz, SiOCH ), 3.208 (2H, q, J ) 6.09 Hz, NHCH
2
),
1
0
1
2
4
.65 (2H, quint, J ) 6.90 Hz, CH2), 1.23 (9H, t, J ) 7.00 Hz, CH
.643 (2H, t, J ) 8.05 Hz, SiCH ). C NMR (125 MHz, CDCl ): δ
2 3
3
),
13
56.6, 144.2, 141.5, 127.8, 127.2, 125.2, 120.1, 66.6, 58.6, 47.5, 43.6,
+
3.4, 18.5, 7.8. ES-MS: calcd for (M + H ) C24
5
H34NO Si 444.2, found
Experimental Section
44.3.
Materials and Methods. H and 13C NMR spectra were recorded
1
Synthesis of ω-Undecenamide. A mixture of urea (15 g, 0.25 mol)
on a Bruker ARX-500 NMR spectrometer operating at 500 and 125
and undecylenic acid (23.01 g, 0.125 mol) was placed in a round-
bottomed flask fitted with a condenser. The mixture was heated to 170-
180 °C for 4 h and then allowed to cool. When the temperature had
dropped to 110-120 °C, a Na CO solution (5%, 100-150 mL) was
3
MHz, respectively. CDCl was used as the solvent and also served as
2
9
an internal reference. Si solid-state MAS NMR experiments were
obtained on the same instrument at a frequency of 79.5 MHz, and
chemical shifts were referenced to 3-(trimethylsilyl)-1-propanesulfonic
acid sodium salt. Samples were spun at 4 kHz, and a 30 s recycle delay
was used. Mass spectra were measured using a Finnegan 4610
spectrometer. FTIR spectra were obtained on a Perkin-Elmer 2000 FTIR
spectrometer. Compressed KBr pellets containing about 2 wt % of
sample were used for these studies. Thermogravimetric analysis (TGA)
was performed on a Perkin-Elmer Pyris 1 DSC-TGA. Scans were
performed between 25 and 800 °C at 5 °C/min. UV-visible spectra
between 200 and 850 nm were obtained using an Ocean Optics S2000
multichannel fiber optic spectrophotometer system. Powder X-ray
diffraction (XRD) experiments were performed on a Scintag X1 θ-θ
diffractometer equipped with a Peltier (solid-state thermoelectrically
2
3
carefully added through the condenser to stop the reaction. The mixture
was then cooled in an ice bath, and the product was collected by
filtration. The crude material was recrystallized in 95% EtOH at least
twice. The final product was dried in vacuo to give an almost colorless
1
solid (14.5 g, 79.1 mmol, 63% yield). H NMR (500 MHz, CDCl ): δ
3
5.81 (1H, ddt, J ) 16.9, 10.2, 6.8 Hz, CH dCH), 5.27 (2H, broad,
2
NH ), 4.91-5.01 (2H, m, CH dCH), 2.22 (2H, t, J ) 7.5 Hz, COCH ),
2
2
2
2.04 (2H, q, J ) 7.1 Hz, CH dCHCH ), 1.642 (2H, quint, J ) 7.5 Hz,
2
2
COCH CH ), 1.30-1.39 (12H, m, alkyl-H).
2
2
Synthesis of 11-Undecenylamine. LiAlH
placed in a 250 mL round-bottom flask containing 50 mL of anhydrous
THF, and the mixture was heated at reflux for 30 min under N . After
this time, heating was stopped and a solution of undecenamide (5.0 g,
7.3 mmol) in 100 mL of anhydrous THF was introduced with stirring.
Reflux was continued under N with stirring for an additional 24 h,
and the reaction was then quenched by dropwise addition of 8 mL of
water to destroy excess LiAlH . The resulting white precipitate was
removed by filtration and washed with 3 × 15 mL EtOAc. After the
combined organic phases were dried over anhydrous Na SO , evapora-
tion of the solvent followed by vacuum distillation (40 ∼ 50 °C at
.06 Torr) gave the product as a colorless oil (3.25 g, 19.2 mmol, 70%
4
(2.3 g, 60.6 mmol) was
2
cooled) detector using Cu KR radiation. N
2
physisorption isotherms
were obtained on a Micromeritics ASAP 2010 instrument. Samples
were degassed at 110 °C under vacuum overnight prior to measurement.
Surface areas were measured using the BET method, pore size
2
2
2
5,26
distributions were calculated from the BJH method,
pore volumes were calculated from the nitrogen adsorption/desorption
data (single-point volume at p/p ) 0.995). Fluorescence confocal
and the total
4
0
2
4
scanning laser microscopy (CSLM) was performed using a BioRad
MRC 1024ES confocal laser scanning imaging system. Dye fluores-
cence was measured at 488 nm, and a 60× oil-immersion lens was
0
1
yield). H NMR (500 MHz, CDCl
3
): δ 5.81 (1H, ddt, J ) 16.9, 10.2,
dCH), 4.91-5.01 (2H, m, CH dCH), 2.67 (2H, t, J )
CH ), 2.03 (2H, q, J ) 7.2 Hz, CH dCHCH ), 1.28-1.44
).
6
7
.8 Hz, CH
.0 Hz, NH
2
2
(
23) Mal, N.-K.; Fujiwara, M.; Tanaka, Y. N. Nature 2003, 421, 350.
2
2
2
2
(
24) Giraldo, J.; Vivas, N. M.; Vila, E.; Badia, A. Pharmacol. Ther. 2002, 95,
(14H, m, alkyl-H), 1.02 (2H, broad s, NH
2
2
1.
(25) Brunauer, S. The Adsorption of Gases and Vapors; Princeton University
Press: Princeton, NJ, 1943.
Synthesis of (9H-Fluoren-9-yl)methyl Undec-10-enylcarbamate
Fmoc-11-Aminoundecene). A solution of 11-undecenylamine (0.677
(
(
26) Webb, P. A.; Orr, C. Analytical Methods in Fine Particle Technology;
Micromeritics Instrument Corporation: Norcross, GA, 1997.
g, 4 mmol) in 30 mL of anhydrous CH
2
Cl
2
was prepared and cooled
J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007 9675

A R T I C L E S
Cheng and Landry
in an ice bath. In a separate flask, N-(9-fluorenylmethyloxycarbonyl)-
oxysuccinimide, Fmoc-OSu (1.417 g, 4.2 mmol), was dissolved in a
small amount of anhydrous CH Cl ; this solution was then slowly added
2 2
RMS6) recirculating unit, which provided precise temperature control,
to within 0.01 °C. A solution of piperidine in DMF (2 to 40%, 50 mL
total volume) was thermally equilibrated at a temperature of 25, 30, or
40 °C for 30 min. Organosilane-modified silica (100.0 mg) was quickly
added to this solution, and the deprotection was carried out for 24 h.
Aliquots of the mixture (500 µL) were removed from the deprotection
mixture at predetermined time intervals. The supernatant was obtained
by centrifugation, and the absorption of the collected supernatants at
289 nm was monitored with UV/visible spectroscopy using a quartz
cell with a 10 mm path length. The amount of Fmoc-piperidine adduct
in the solution was obtained from a calibration curve measured with a
series of different, known Fmoc-APTES solutions that had been treated
under the same conditions.
Attachment of Fluorescent Dye to Free Amines of Deprotected
Silica-Fmoc. Deprotected silica-Fmoc samples (2.0 mg) were suspended
3
in 200 µL of a 100 mM NaHCO solution. The suspensions were
then transferred to vials containing Alexa Fluor 488 carboxylic acid,
succinimidyl ester (10 µg, 0.0155 µmol). After incubation for 4 h
at room temperature and 12 h at 4 °C with continuous shaking, the
samples were recovered by centrifugation, washed successively with a
to the APTES solution with stirring. After 2 h, rotary evaporation of
the solvent left an oily white product which was purified by flash
chromatography on silica gel with ethyl acetate/hexane (1:4) to yield
1
the product as a white solid (1.42 g, 3.63 mmol, 91% yield). H NMR
(500 MHz, CDCl
3
): δ 7.77 (2H, d, J ) 7.5 Hz, ArsH), 7.60 (2H, d,
J ) 7.6 Hz, ArsH), 7.40 (2H, t, J ) 7.5 Hz, ArsH), 7.31 (2H, t, J )
7
4
)
.6 Hz, ArsH), 5.81 (1H, ddt, J ) 16.9, 10.2, 6.8 Hz, CH
.91-5.01 (2H, m, CH dCH), 4.70 (1H, broad s, NH), 4.41 (2H, d, J
6.7 Hz, COOCH ), 4.22 (1H, t, J ) 6.18 Hz, ArsCH), 3.18 (2H,
m, NHsCH ), 2.04 (2H, q, J ) 7.0 Hz, CH dCHCH ), 1.29-1.38
14H, m, alkyl-H).
Synthesis of (9H-Fluoren-9-yl)methyl 11-(Triethoxysilyl)unde-
cylcarbamate (Fmoc-AUTES). After bubbling dry N into 20 mL of
a dry dichloromethane solution of Fmoc-11-aminoundecene (0.714 g,
2
dCH),
2
2
2
2
2
(
2
1
1
.82 mmol) and triethoxysilane (0.3286 g, 0.367 mL, 2.0 mmol) for
0 min, 0.2 µmol of Pt(dvs), platinum(0)-1,3-divinyl-1,1,3,3,-tetra-
methyldisiloxane complex, 3 wt % in xylenes (15 µL) was added and
the resulting mixture was refluxed for 12 h. After removing the solvent
under reduced pressure, an oily white product was obtained which was
further purified by flash chromatography with ethyl acetate/hexane (1:
3
100 mM NaHCO solution, distilled water, and MeOH, and then air-
dried and stored at 4 °C in the dark. The procedure could also be used
at any stage of the silica modification to locate free amines within the
samples.
4
) to yield the product as a white solid (0.85 g, 1.53 mmol, 84% yield).
1
H NMR (500 MHz, CDCl
3
): δ 7.77 (2H, d, J ) 7.5 Hz, ArsH),
.60 (2H, d, J ) 7.6 Hz, ArsH), 7.40 (2H, t, J ) 7.5 Hz, ArsH),
.31 (2H, t, J ) 7.6 Hz, ArsH), 4.74 (1H, broad s, NH), 4.41 (2H, d,
), 4.22 (1H, t, J ) 6.46 Hz, ArsCH), 3.82 (6H,
q, J ) 7.00 Hz, SiOCH ), 3.19 (2H, q, J ) 5.8 Hz, NHCH ), 1.65-
.22 (14H, m, alkyl-H), 0.636 (2H, t, J ) 8.2 Hz, SiCH
125 MHz, CDCl ): δ 156.6, 144.2, 141.5, 127.8, 127.2, 125.2,
Results and Discussion
7
7
Acid-Prepared Mesoporous Spheres (APMS) were used in
this study. They are easily and rapidly synthesized, have pores
J ) 6.6 Hz, COOCH
2
2
2
with an average diameter of 30 Å, and have a 5 µm particle
13
1
(
2
). C NMR
4-7,28
diameter.
They were chosen for this study because of their
3
monodisperse, spherical particle morphology, which was easily
1
1
5
20.1, 66.6, 58.5, 47.5, 41.3, 32.8, 30.2-29.5, 29.3, 26.9, 22.9, 18.5,
4
,5,28
recognized by microscopy.
The postsynthesis grafting
+
4.3, 10.6. ES-MS: calcd for (M + H ) C32
5
H50NO Si 556.34, found
procedure was used to immobilize functionalized amines on
APMS through covalent linkages between organic molecules
57.1.
Synthesis of APMS. Cetyltrimethylammonium bromide (1.20 g, 3.29
29-35
and silanol groups.
The degree of functionalization and the
mmol) and concentrated HCl (4.5 g, 36.5 wt %, 45.0 mmol) were added
to distilled water (55.5 g) and stirred rapidly until the surfactant had
dissolved. Tetraethoxysilane (5.65 g, 27.1 mmol) was then added, and
the mixture was stirred at room temperature for 1 h. The mixture was
then transferred to a Teflon-lined Parr autoclave and heated at 150 °C
for 40 min. The mixture was then cooled to room temperature, and the
resulting white precipitate was collected by filtration, air-dried, and
calcined (1.10 g).
Synthesis of Functionalized Silica. A calcined sample of either
APMS or Nucleosil (0.60 g) was suspended in 20 mL of dry toluene
in a dry, inert atmosphere. A solution of organosilane (0.1-5 mmol)
dissolved in 5 mL of dry toluene was then added, and the mixture was
refluxed at 110 °C for 24 h. After the mixture was cooled to room
temperature, the white powder was recovered by filtration, air-dried,
and then vacuum-dried for 24 h prior to further use.
distribution of functional groups were influenced by the densities
of surface silanol groups (Si-OH) on the mesoporous silica
wall, the reactivities of the organosilanes, and their accessibility
to surface silanols. The physical properties of the surface-
functionalized APMS were characterized using X-ray diffraction
29
(
XRD), Si MAS NMR spectrometry, FT-IR spectroscopy, and
thermogravimetric analysis (TGA).
The removal of the Fmoc group was usually achieved by
treatment with piperidine in N,N′-dimethylformamide (DMF).36
The mechanism of the Fmoc-deprotection reaction involves the
formation of aromatic cyclopentadiene intermediates, which are
rapidly eliminated to form dibenzofulvene, which is further
scavenged by piperidine to form the Fmoc-adduct 1-((9H-
fluoren-9-yl)methyl) piperidine. This product strongly absorbs
UV radiation at 289 nm, offering the potential to monitor the
deprotection reaction by UV/visible spectroscopy. Because the
deprotection reactions occurred within the confined space of
the pore network, the reaction rates were significantly different
from those in solution. The concentration and temperature of
Quantitation of Organic Loading on APMS by UV-visible
Spectrophotometry. In a typical analysis, silica-Fmoc (4.0 mg) and
NH F (15.0 mg) were placed in a plastic vial. After rapid addition of
4
concentrated HCl (120 µL), the mixture was incubated at 50 °C for 30
min with continuous vortexing. Distilled water (0.5 mL) was then added,
and the remaining organic components were extracted with CH
2
Cl
2
(5
×
1 mL). After dilution to 10 mL with additional CH Cl
absorbance of Fmoc-adduct in the extractant was determined at 289
2
2
, the
(
28) Gallis, K. W.; Landry, C. C. U.S. Patent 6,334,988, 2002.
nm using UV-visible spectroscopy, using a calibration curve that was
(29) Zhao, X. S.; Lu, G. Q. J. Phys. Chem. B 1998, 102, 1556.
(30) Jal, P. K.; Patel, S.; Mishra, B. K. Talanta 2004, 62, 1005.
prepared using standard amounts of Fmoc-APTES.27 Samples were
(
31) De Juan, F.; Ruiz-Hitzky, E. AdV. Mater. 2000, 12, 430.
assayed in triplicate.
(32) Feng, X.; Fryxell, G. E.; Wang, L.-Q.; Kim, A. Y.; Liu, J.; Kemner, K. M.
Science 1997, 276, 923.
33) Lim, M. H.; Stein, A. Chem. Mater. 1999, 11, 3285.
Kinetic Study of the Deprotection Reaction of Silica-Fmoc.
Kinetic studies were performed in a 100 mL round-bottom flask with
a double-layer water jacket connected to a Brinkman Lauda (Model
(
(34) Liu, J.; Shin, Y.; Nie, Z.; Chang, J. H.; Wang, L.-Q.; Fryxell, G. E.; Samuels,
W. D.; Exarhos, G. J. J. Phys. Chem. A 2000, 104, 8328.
(35) Lindner, E.; Schmneller, T.; Auer, F.; Mayer, H. A. Angew. Chem., Int.
Ed. 1999, 38, 2154.
(27) See the Supporting Information.
(36) Atherton, E.; Sheppard, R. C. The Peptides 1987, 9, 1.
9
676 J. AM. CHEM. SOC. VOL. 129, NO. 31, 2007
9

Differential Functionalization of Porous Silica Particles
A R T I C L E S
Scheme 1. Syntheses of Organosilanes Used in This Studya
a
Abbreviations: Fmoc, N-(9-fluorenylmethyloxycarbonyl); LiAlH4, lithium aluminum hydride; HSi(OEt)3, triethoxysilane; Pt(dvs), Platinum(0)-1,3-
divinyl-1,1,3,3-tetramedisiloxane complex, 3 wt % in xylenes.
piperidine solutions could therefore be varied to study the effects
on reaction rate by measuring the absorbance of the released
Fmoc adducts.
Fluorescence confocal scanning laser microscopy (CSLM),
in combination with fluorescent labeling, was used to study the
locations of various molecules within APMS. The ability of the
confocal microscope to create a thin focal plane allows an optical
In order to study the density and distribution of the incor-
porated functional groups, we initially prepared a series of silica
samples in which different amounts of organosilanes were
immobilized on the surface by varying the concentrations of
3
0
the silanes during the modifications. The organic moieties on
the silica surface were characterized by TGA, which gave the
2
1,37
organic contents.
TGA data for APMS-Fmoc are sum-
“
slice” to be taken through the center of the APMS particle;
marized in Table 1 (actual TGA traces can be found in the
Supporting Information). Additional confirmation of the organic
loadings was obtained by UV/visible spectrophotometry of the
Fmoc groups. In this analysis, the Fmoc groups were cleaved
from the silica framework by etching the silica in aqueous HF,
and the Fmoc groups were recovered from the mixture by
extraction. Absorbance of the extractant at 289 nm was then
regions of the particle outside the focal plane do not appear
in the final image. This technique allows fluorescence origi-
nating from within the pores of APMS to be distinguished
from fluorescence on the external surface. Using dyes that can
form peptide bonds allows free amines bound to the internal
pore surfaces to be distinguished from those on the external
surface.
Surface Functionalization of APMS. 9-Fluorenylmethy-
loxycarbonyl propylcarbamate (Fmoc-propylamine) was intro-
duced by direct reaction of (9H-fluorenyl)methyl 3-(triethoxysilyl)-
propylcarbamate (Fmoc-APTES, Scheme 1) to silica surfaces.
Other organosilanes, including (9H-fluorenyl)methyl 11-(tri-
ethoxysilyl)undecylcarbamate (Fmoc-AUTES), 3-aminopropy-
ltriethoxysilane (APTES), were also studied for the comparisons.
Fmoc-AUTES was selected as an immobilized functional linker
on the silica due to its longer alkyl chain;27 therefore, the
incorporation of the long alkyl moiety of Fmoc-AUTES was
expected to fill the pores of the silica completely. Fmoc-APTES
was obtained by the reaction of APTES with N-(9-fluorenyl-
methyloxycarbonyl)oxysuccinimide (Fmoc-OSu) in high yield
27
correlated with a calibration curve. The results of both methods
correlated closely. The slight difference between the two
methods may be derived from either incomplete hydrolysis of
ethoxy groups, leading to higher organosilane loadings in TGA,
or incomplete recovery of Fmoc in the extraction step, leading
to lower organosilane loading in UV/visible spectrophotometry.
Since TGA could be accomplished more readily, it was used
for the remaining calculations. There were three or four distinct
weight losses in the TGA profiles that were due to loss of
MeOH, organic functional groups, and dehydration of the
3
7
surface hydroxyl groups. It has been reported that the loading
of organosilanes on the silica surface by condensation of Si-
OR groups can be regarded as a Langmuir adsorption process.21
Because the temperature was maintained at the boiling point of
toluene (110 °C), the degree of surface functionalization was
determined by the concentration of silanes in solution (Csilane)
during the functionalization. The organic loadings were calcu-
lated from the TGA data using units of mole per gram of pure
silica (mol/g of SiO2). As the concentration of the organosilane
increased, the loading also increased until it reached the
maximum possible loading. The curves of surface loadings were
(90%). In contrast, Fmoc-AUTES was prepared through a series
of reactions since the triethoxysilane was unavailable. Undey-
lenic acid was reacted with urea to give ω-undecenamide, which
was further converted to 11-undecenylamine by reduction with
LiAlH4. Before the hydrosilation, the amine groups were
protected with Fmoc. The resulting amide derivative was then
hydrosilated with triethoxysilane in the presence of the Karstedt
catalyst (platinum(0)-1,3-divinyl-1,1,3,3-tetramethyldisiloxane
complex, {Pt2((CH2dCH)Me2Si)2O}3).
(
37) Zhao, X. S.; Lu, G. Q.; Whittaker, A. K.; Millar, G. J.; Zhu, H. Y. J. Phys.
Chem. B 1997, 101, 6525.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007 9677

A R T I C L E S
Cheng and Landry
Table 1. Organic Loading and Surface Coverage of Organosilanes on APMS and Nucleosil
organosilane
organosilane loading, Γb
surface density,
R
surface coverage,
(%)
θ
samplea
concentration (mM)
(mmol/g SiO
(molecule/nm-2)
2
)
APMS-3-NH2-4
APMS-3-NH2-8
4.2
8.3
21
42
63
0.730
1.28
2.01
2.31
2.40
2.57
2.58
0.400
0.701
1.10
1.26
1.31
1.41
1.42
25.26
44.15
69.55
79.93
83.04
88.93
89.27
APMS-3-NH2-21
APMS-3-NH2-42
APMS-3-NH2-63
APMS-3-NH2-83
APMS-3-NH2-150
83
150
APMS-3-Fmoc-20
APMS-3-Fmoc-33
APMS-3-Fmoc-45
APMS-3-Fmoc-60
APMS-3-Fmoc-100
APMS-3-Fmoc-135
APMS-3-Fmoc-200
20
33
45
0.445
0.554
0.645
0.737
0.891
0.988
1.11
0.244
0.303
0.353
0.403
0.488
0.541
0.608
33.46
41.65
48.50
55.41
66.99
74.29
83.53
60
100
135
200
APMS-11-Fmoc-10
APMS-11-Fmoc-20
APMS-11-Fmoc-50
APMS-11-Fmoc-83
APMS-11-Fmoc-100
APMS-11-Fmoc-200
10
20
50
83
100
200
0.128
0.203
0.422
0.523
0.549
0.617
0.0701
0.111
0.231
0.286
0.300
0.338
14.71
23.33
48.51
60.11
63.10
70.92
Nucleosil-3-Fmoc-200
Nucleosil-11-Fmoc-200
200
200
0.553
0.392
0.772
0.548
-
-
deprotected APMS-3-Fmoc-135 (6 h)
deprotected APMS-3-Fmoc-135 (24 h)
-
-
0.395 Fmoc- (40%) + 0.593 -NH2 (60%)
0.119 Fmoc- (12%) + 0.869 -NH2 (88%)
a
Nomenclature: “APMS-n-R-m”, where n ) carbon atoms in organic “linker”, R ) functionalization, and m ) organosilane concentration in loading
b
solution. Calculated from TGA data.
Table 2. Parameters Obtained from Langmuir Fitting of Loading Data for Fmoc-APTES, Fmoc-AUTES, and APTES
maximum
R2
intercept
(1/
slope
equilibrium constant,
K (M-1)
maximum loading,
density, R
max
silanes
Γ
max
)
(1/
Γ
maxK)
Γ
max (mmol/g)
(nm-2)
APTES
Fmoc-APTES
Fmoc-AUTES
0.351
0.750
1.16
0.003 52
0.0355
0.0675
100
21.1
17.0
2.85
1.33
0.862
1.56
0.728
0.472
0.99
0.99
0.99
fit by the Langmuir adsorption equation (eq 1).21
lower (less than 25 M ), consistent with the fact that both
organosilanes contain hydrophobic groups as the terminal group,
which were not favorably adsorbed onto the hydrophilic silica
walls. Moreover, Fmoc-AUTES has a longer alkyl chain,
causing more steric hindrance to accessing the pores and
resulting in a lower equilibrium constant.
-1
Γ
KCsilane
θ )
)
(1)
Γmax
1 + KCsilane
The equilibrium constant (K) and the maximum loading in mmol
of organosilane/g of SiO2 (Γmax) were obtained from the
parameters of the fitting data of surface loading by the Langmuir
adsorption model. Strict application of this model would assume
complete independence of adsorbate molecules, and the orga-
nosilanes in our experiments could interact with the surface
and with each other. Although a more detailed description is
not appropriate, use of the Langmuir model allowed quantitative
comparison of data sets to be made. The loading curves of
these silanes are shown in Figure 1, and the parameters
calculated from the Langmuir equation are listed in
Table 2. APTES achieved a saturated monolayer on the
silica surface below 0.075 M, while Fmoc-APTES and
Fmoc-AUTES require significantly higher concentrations (above
The surface density of the organosilanes (R) was calculated
from eq 2, where NA is Avogadro's number and SBET is the BET
surface area of the porous silica in m /g :
2
21
2
ΓNA
mol ‚ m
R )
×
(2)
(
3
18
2
)
SBET
10 mmol ‚ 10 nm
When the coverage of APTES on APMS was 83.0% (Table 1),
2
its surface density was 1.31 molecules/nm . Consistent with the
results shown above, the surface densities of Fmoc-APTES and
2
Fmoc-AUTES were lower than this value (1.11 molecules/nm
2
at 83.5%, 0.34 molecules/nm at 70.9%, respectively), due to
the steric hindrance and hydrophobicities of those molecules.
Finally, the theoretical maximum densities of the attached silanes
0
.15 M). APTES also showed a much higher equilibrium
-
1
21
constant (about 100 M ). It has been reported that
there are strong interactions between hydrophilic silanols on
the silica surface and APTES which contributes to the
higher equilibrium constants.21 In contrast, the equilibrium
constants of Fmoc-APTES and Fmoc-AUTES were much
were calculated from the Langmuir fitting parameter (Table 2).
The functional silanes can be ranked in order of increasing
maximum surface densities as follows: Fmoc-AUTES (0.48
2
2
molecules/nm ) < Fmoc-APTES (0.73 molecules/nm ) <
2
APTES (1.58 molecules/nm ).
9678 J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007

Differential Functionalization of Porous Silica Particles
A R T I C L E S
Table 3. Physical Properties of Selected Samples from Table 1
surface
area
pore
avg pore
diameter
(Å)
volume
2
3
sample
(m /g)
(cm /g)
unmodified APMS
1180
686
624
519
444
331
88.3
5.72
500
32.7
0.625
0.315
0.274
0.248
0.209
0.144
0.0489
0.0118
30
23
23
24
22
22
21
<20
APMS-3-Fmoc-20
APMS-3-Fmoc-33
APMS-3-Fmoc-45
APMS-3-Fmoc-60
APMS-3-Fmoc-100
APMS-3-Fmoc-135
APMS-3-Fmoc-200
APMS-11-Fmoc-50
APMS-11-Fmoc-200
0.231
0.061
23
<20
unmodified Nucleosil
Nucleosil-3-Fmoc-200
Nucleosil-11-Fmoc-200
431
332
315
0.911
0.641
0.671
80
66
67
deprotected APMS-3-Fmoc-135 (6 h)
deprotected APMS-3-Fmoc-135 (24 h)
691
764
0.300
0.378
25
26
introduction of the organic moieties led to a decrease in pore
diameter, surface area, and pore volume. The functionalized
APMS samples increasingly showed characteristics of Type I
isotherms as a function of the degree of silation, indicating that
the pore diameter had decreased. Calculation of the average pore
diameters of the samples indicated a decrease from 30 Å for
unmodified APMS to 21 Å for APMS-3-Fmoc-135. APMS-3-
Fmoc-200 did not even show the presence of any pores, since
they were completely filled with organic moieties. The changes
in measured pore diameter were consistent with the surface area
2
2
(
5.72 m /g) and pore volume (0.0118 m /g) for this sample. It
Figure 1. (Top panel) Isotherms of organosilanes loaded into APMS.
is also worth noting that the sharpness of the pore size
distribution decreased as the degree of silation increased, which
(Bottom panel) Linear fits of the organosilane loadings derived from the
Langmuir equation: (a) APTES; (b) Fmoc-APTES; (c) Fmoc-AUTES.
4
0,41
is indicative of inhomogeneous pore filling.
A similar
Physical Characterization of Materials. 29Si MAS NMR
spectra of calcined APMS before and after silation are shown
in Figure 2. For the calcined APMS, the resonance centered at
δ ) -111 ppm with two shoulders at -103 and -93 ppm was
decrease in the porosity of APMS was observed upon binding
of Fmoc-AUTES, although the decreases occurred at different
organic loadings. An examination of the data in Table 1 shows
that APMS-3-Fmoc-45 and APMS-11-Fmoc-50 have approxi-
4
3
2
assigned to siloxane (Q , Si(OSi)4), single silanol (Q , Si(OSi)3-
mately the same surface areas (519 and 500 m /g, respectively),
2
29,37,38
OH), and geminal silanol (Q , Si(OSi)2(OH)2) sites.
The
while their organic loadings are different (0.645 vs 0.422 mmol
spectrum of functionalized APMS showed two additional
resonances at approximately δ ) -58 and -67 ppm, which
represented T2 (RSi(OSi)2(OH)) and T3 (RSi(OSi)3) sites,
respectively. The relative amounts of each type of Si atom were
calculated from the deconvolution of the spectra. The slight
of organosilane/g of SiO ). A more dramatic comparison is
2
APMS-3-Fmoc-200 and APMS-11-Fmoc-200, which have
2
surface areas of 5.72 and 32.7 m /g but loadings of 1.11 and
0.617 mmol/g. These differences can be attributed to the longer
chain of Fmoc-AUTES (23 Å, almost twice that of Fmoc-
APTES), which can occupy the available pore space more
completely. Finally, a commercially available material, Nucleo-
sil-50, was used for comparison. This material had a very broad
pore size distribution with an average pore diameter of ap-
proximately 80 Å. Nucleosil could be easily modified by
reaction with either Fmoc-APTES or Fmoc-AUTES, which is
indicated by the fact that Nucleosil-3-Fmoc-200 and Nucleosil-
11-Fmoc-200 showed higher surface densities than their APMS-
based counterparts at similar organosilane loadings. Addition-
ally, at a concentration of organosilane in solution that
essentially resulted in pore closure in APMS (200 mM),
Nucleosil still retained 75% of its surface area and pore volume
3
2
decreases in the number of Si atoms in Q and Q environments
were due to the reaction of silanol groups with the organosilanes,
3
2
and the presence of Si atoms in T and T environments in the
functionalized APMS samples further indicated the existence
of the covalent linkage between the organic moieties and silica
walls.
Data from the characterization of the porous samples by
N2 physisorption are shown in Table 3. The isotherm of
unmodified APMS exhibited a Type IV isotherm4
,5,39
with a
point of inflection at approximately p/p0 ) 0.2-0.4, consistent
with the presence of mesopores. This sample had the large
2
3
surface area (1177 m /g) and pore volume (0.625 cm /g)
characteristic of mesoporous materials.4 As expected, the
(
40) Luan, Z.; Xu, J.; He, H.; Klinowski, J.; Kevan, L. J. Phys. Chem. B 1996,
(
38) Chong, A. S. M.; Zhao, X. S. J. Phys. Chem. B 2003, 107, 12650.
39) Sing, K. S. W.; Everett, D. H.; Haul, R. A. W.; Moscou, L.; Pierotti, R.
A.; Rouquerol, J.; Siemieniewska, T. Pure Appl. Chem. 1985, 57, 603.
100, 19595.
(
(41) Luan, Z.; He, H.; Zhou, W.; Cheng, C. F.; Klinowski, J. J. Chem. Soc.,
Faraday Trans. 1995, 91, 2955.
J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007 9679

A R T I C L E S
Cheng and Landry
Scheme 2. Fmoc Deprotection Reaction, Synthesis of Fmoc-Functionalized Silicas, and Deprotection Strategy
for both Fmoc-APTES and Fmoc-AUTES, which is consistent
with the broad pore size distribution and therefore the number
of large pores within this material.
The mechanism of the Fmoc deprotection reaction is shown
3
6,43
in Scheme 2.
The Fmoc-piperidine adduct strongly absorbs
in the UV region (λmax ) 273, 289, and 300 nm) and has been
used as a marker of amine deprotection in other studies.36 To
study the kinetics of amine deprotection in porous solids, the
reaction was monitored by taking solution aliquots and measur-
ing the change in absorbance at 289 nm. The release rates of
the Fmoc-adducts were expressed as the percentage of Fmoc
groups released, determined from the Fmoc-organosilane load-
ings in Table 1, as a function of time. An example of this plot
is shown in Figure 3. Several empirical models were examined
Nitrogen physisorption data on the deprotected materials
were also obtained. It can be clearly observed that exposure of
the Fmoc-organosilane modified solids to a solution of 5%
piperidine in DMF led to a significant increase in pore diameter,
surface area, and pore volume. Based on TGA experiments,
6
0% of the Fmoc groups of APMS-3-Fmoc-135 were re-
moved after 6 h, and 88% were removed after 24 h. A
2
drastic increase in surface area (from 88.3 to 764 m /g) and
3
24
pore volume (from 0.0489 to 0.378 cm /g) indicated the
for their ability to fit the sigmoidal curves shown in this figure.
blocked pores were “reopened” by the cleavage of bulky
Fmoc groups from Fmoc-modified silica. Importantly, the
isotherm of deprotected APMS-3-Fmoc-135 reverted to Type
IV from Type I, indicating that the mesoporous properties of
the solid had been regenerated, with a measured pore diameter
of 26 Å. The increase of 5 Å in pore diameter after the
deprotection was consistent with the difference in molecular
diameter between the Fmoc-aminopropyl and free-aminopropyl
moieties.
The best results were obtained by using the Hill equation, which
is also known as the variable slope sigmoid and is frequently
2
4,44
used to model pharmacological phenomena.
This equation
is used for many physical, chemical, biological, and environ-
mental processes that are driven by two different mechanisms:
4
4,45
diffusion and chemical or biological reaction.
equation is shown below (eq 3):
The Hill
Ymax
Y(t) ) Y +
(3)
0
H
Deprotection Studies. The deprotection reaction of Fmoc
1
+ (t /t)
1/2
groups confined in pores with diameters approaching molecular
6
diameters, as in APMS, is a diffusion-controlled process. In
where Y(t) is the cumulative amount of the Fmoc-adduct at time
t, Y0 is the initial amount of Fmoc-adduct, Ymax is the maximum
amount of Fmoc-adduct released, t1/2 is the time at which Y(t)
reaches Ymax/2, and H is the Hill coefficient, which gives the
slope of the sigmoidal curve at t1/2. The plots of Fmoc-adduct
release for modified APMS can be divided into three partss
this case, piperidine first diffuses into the pores to reach the
Fmoc-aminopropyl moieties, where it reacts quickly to form
an Fmoc-piperidine adduct, 1-(9H-fluoren-9-ylmethyl)piperidine,
which then diffuses out of the pores. The reaction rate is thus
determined by several factors including the chemical kinetics
of surface deprotecting reaction, the diffusivity of the reagent
or product in the solvent, the tortuosity factor, and porosity of
(
43) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis: A Practical
Approach; Oxford University Press: Oxford, 2000.
4
2
the silica particles.
(44) Lombardo, T.; Ionescu, A.; Lefevre, R. A.; Chabas, A.; Ausset, P.; Cachier,
H. Atmos. EnViron. 2005, 39, 989.
(
42) Andrade, J., J. S.; Street, D. A.; Shibusa, Y.; Havlin, S.; Stanley, H. E.
(45) Calace, N.; Nardi, E.; Petronio, B. M.; Pietroletti, M. EnViron. Pollut. 2002,
118, 315.
Phys. ReV. E 1997, 55, 772.
9680 J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007

Differential Functionalization of Porous Silica Particles
A R T I C L E S
Figure 3. Cumulative amount of Fmoc-piperidine adduct released from
APMS-3-Fmoc-135 in a DMF solution containing 5% piperidine at room
temperature. The three phases of the process (lag, burst, and saturation)
were characterized as described in the text. The first derivative of the curve
is shown in (b).
Figure 2. 29Si MAS NMR of (a) unmodified APMS and (b) APMS-3-
The length of the lag phase (tlag) can be obtained from eq 5.
Ym - Y0
Fmoc-135. Chemical shifts from deconvolution of the spectra are marked,
4
and relative peak areas normalized to the area of the Q peak in (a) are
given in parentheses.
tlag ) t -
(5)
m
Km
the lag phase, the burst phase, and the saturated phase (Figure
3
)sand described by the parameters in eq 3.2 In the lag phase,
4
Table 4 shows the values of selected Hill parameters, all of
which were obtained by fitting Fmoc-adduct release curves for
various samples to eq 3. The correlation coefficients (R ) were
the release curve starts with a small value (Y0) and a slope of
almost zero, slowly increasing at the beginning (t < tlag). The
length of the lag phase (tlag) depends on the intrinsic charac-
teristics of the heterogeneous reaction on the exterior surface
of the silica beads. In the burst phase, the slope of the release
curve increases to a maximum value (Km) and then slowly
decreases again until it is effectively horizontal. The saturated
2
in all cases greater than 0.995.
In organic synthesis, N-Fmoc groups are typically removed
by treatment with 20-50% v/v piperidine in DMF. Under these
conditions and in solution, the Fmoc groups can be quickly
cleaved to afford the free amine and dibenzofulvene in less than
1
min. Lower concentrations of piperidine in DMF (2%, 5%,
phase was expected to appear when Y(t) reaches more than 90%
of Ymax.24 The first derivative of the release curve (eq 4, Figure
and 10%) have been studied in an attempt to kinetically control
3
6
the deprotection reaction. Although t1/2 for deprotection of
Fmoc-amino derivatives in solution varied with the structure
of each compound, the approximate value of t1/2 for the
deprotection of Fmoc-amino acid in 20% piperidine DMF
solution at room temperature was only 6 s. Even in 5%
3b) illustrates the rate of release; the time at maximum release
rate (tm) can be found from this equation, and it can also be
used to determine the rate at tlag (Klag).
tH-1
H
t
H
1/2
∂
Y(t)
3
6
K )
) Ymax
H
(
(4)
piperidine, t1/2 was only 20 s. However, the half-lives for the
heterogeneous system are very different. In this case, t1/2 in 20%
H 2
∂
t
t
+ t )
1/2
J. AM. CHEM. SOC.
9
VOL. 129, NO. 31, 2007 9681

A R T I C L E S
Cheng and Landry
Table 4. Parameters from Eqs 3 and 5 for the Release of Fmoc-Piperidine Adducts under Various Conditionsa
temp
C)
piperidine
(vol %)
Y
max
T
1
/
2
T
m
T
lag
Y
lag
sample
(
°
(%)
(min)
K
m
(min)
K
lag
(min)
(%)
APMS-3-Fmoc-100
25
2
5
10
20
40
2
5
2
5
89.96 ( 1.29
82.41 ( 2.07
81.57 ( 1.50
73.93 ( 1.02
67.01 ( 1.17
89.01 ( 1.48
87.54 ( 1.60
94.19 ( 0.77
90.03 ( 1.54
233.8 ( 4.4
129.1 ( 5.6
105.7 ( 2.8
95.42 ( 1.92
94.93 ( 1.83
106.9 ( 2.3
72.94 ( 2.01
39.18 ( 0.46
24.35 ( 0.61
0.302
0.420
0.500
0.509
0.460
0.658
0.783
1.90
175.6
76.38
59.90
56.57
57.24
81.53
42.62
30.01
17.16
0.0766
0.176
0.278
0.359
0.420
0.184
0.416
0.576
0.984
81.74
26.29
19.92
19.53
19.22
38.16
14.39
14.00
7.093
6.26
4.63
5.54
7.02
8.07
7.01
5.99
8.06
6.98
25
25
25
25
30
30
40
40
2.67
APMS-3-Fmoc-33
APMS-3-Fmoc-45
APMS-3-Fmoc-60
APMS-3-Fmoc-100
APMS-3-Fmoc-135
APMS-3-Fmoc-200
25
25
25
25
25
25
5
5
5
5
5
5
-
1.92b
-
-
-
-
-
-
4.03b
-
-
-
-
-
82.16 ( 1.55
81.03 ( 2.70
90.96 ( 2.25
94.30 ( 6.21
68.78 ( 2.04
129.1 ( 5.6
312.0 ( 9.8
591.1 ( 35.5
0.800
0.412
0.198
0.133
42.65
75.81
198.1
465.6
0.708
0.199
0.0946
0.0431
15.37
26.18
73.65
231.9
10.9
5.22
6.97
9.99
APMS-11-Fmoc-50
APMS-11-Fmoc-200
25
25
5
5
-
16.18b
128.3 ( 5.5
-
0.392
-
43.75
-
1.47
-
8.293
-
12.2
82.50 ( 2.11
Nucleosil-3-Fmoc-200
Nucleosil-11-Fmoc-200
25
25
5
5
82.60 ( 3.81
87.54 ( 2.33
33.28 ( 2.49
45.45 ( 1.82
1.62
1.17
19.95
19.09
2.77
2.48
6.629
4.405
18.4
10.9
a
A complete list of parameters may be found in the Supporting Information. b T1/2 values for samples with low organosilane loadings were estimated
directly from the release curves.
piperidine solution at room temperature is 95.4 min and is 234
min in 2% piperidine solution. These significant differences are
evidence of the presence of diffusion constraints during the
deprotection reaction. In the absence of piperidine, APMS-Fmoc
exhibited less than 3% Fmoc release in DMF solution over a
period of 24 h (Figure 4a), indicating that Fmoc-groups are
chemically stable. As expected, higher concentrations of pip-
eridine resulted in lower values of t1/2. Even in 40% piperidine
solution, however, t1/2 is still 94.9 min. The effects of three
different temperatures (25, 30, and 40 °C) on t1/2 were also
studied. As shown in Figure 4b and Table 4, t1/2 decreased
remarkably as temperature increased for piperidine concentra-
tions of both 2 and 5%. This can be explained by the accelerated
diffusion of the piperidine and Fmoc-piperidine adduct through
the pores of silica at higher temperatures. Higher temperatures
also increased the rate of the Fmoc-deprotection reaction itself,
resulting in a smaller value of t1/2.
Data from the lag and burst phases of the release curves
(Table 4 and Figure 5) provide interesting information about
the deprotection reaction on this porous substrate. On flat
surfaces, heterogeneous deprotection reactions are said to
involve sequential steps: the reagent diffuses to the surface and
4
6,47
quickly reacts to form products, which then diffuse away.
Thus, as long as the concentration of piperidine is sufficiently
high, the surface reaction should take place quickly and tlag
should be short or nonexistent. Indeed, APMS-3-Fmoc-33 and
-45, with relatively low organosilane loadings and open pores,
showed hyperbolic rather than sigmoidal Fmoc-adduct release
curves, and eqs 3-5 could not be applied effectively. At
intermediate organosilane loadings, the fact that tlag exists and
increases with loading indicates that diffusion of piperidine and
the Fmoc-piperidine adduct within the pores is hindered. This
must be due to inhomogeneity in the distribution of Fmoc within
the pores, consistent with other reports indicating that higher
Figure 4. Cumulative release of the Fmoc-piperidine adduct from APMS-
-Fmoc-100 in (a) DMF solutions containing piperidine concentrations
between 0 and 40% at room temperature and in (b) DMF solutions
3
containing 2 or 5% piperidine at temperatures between 25 and 40 °C.
(
46) Aris, R. The Mathematical Theory of Diffusion and Reaction in Permeable
Catalysts; Clarendon: Oxford, 1975.
densities of functional group are located on the exterior surfaces
and near the openings of mesoporous channels rather than in
(47) Cussler, E. L. Diffusion: Mass Transfer in Fluid Systems, 2nd ed.;
Cambridge University Press: Cambridge, 1997.
9682 J. AM. CHEM. SOC.
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Differential Functionalization of Porous Silica Particles
A R T I C L E S
Figure 6. Fluorescence CSLM images of deprotected APMS-3-Fmoc-n
samples with Alexa 488 (a fluorescent green dye) covalently bound to the
free amine, where n ) (a) 33, (b) 45, (c) 60, (d) 100, (e) 135, and (f) 200.
Samples were deprotected in 5% piperidine in DMF at room temperature
for 40 min. The tlag for each sample (see Table 4) is listed in the top right
corner of each image (bar ) 5 µm).
Figure 5. Cumulative release of the Fmoc-piperidine adduct from various
APMS-3-Fmoc samples in a solution of DMF containing 5% piperidine at
room temperature.
the porous interior.21,33,34 One might expect that APMS-3-Fmoc-
are all important to the overall rate of release. Significantly,
2
00, which contained pores that were essentially completely
m
K was approximately 5 times larger at 40 °C than at 25 °C.
filled, would have both an unusually large tlag and a relatively
short burst phase, since penetration of piperidine to the porous
interior of the particles is continually hindered by organosilane
groups. As anticipated, these features are found for the release
curve of APMS-3-Fmoc-200. After the pore entrances are
opened during the lag phase, the piperidine is able to access
the Fmoc-organosilane groups within the pores. Since the
majority (over 99.9%) of the total surface area of APMS is
located within the pores, as long as the concentration of
piperidine is sufficiently high the release rate should increase
dramatically after the pore entrances are cleared. It is also found
that increased organosilane surface densities should produce
longer burst phases, reflected in a release curve with a broader
shape and a longer tm. The value of the maximum rate, Km,
also decreases as the organosilane surface increases, due to
hindered molecular diffusion within pores of materials with
higher organosilane loadings.
Visual Analysis of Deprotection by Confocal Microscopy.
Effective use of the data above requires experimental demon-
stration that the deprotected amines can be refunctionalized, for
example, through peptide bond formation, to allow APMS to
be differentially modified. Based on the above results, we
theorized that tlag could be a controlling factor in this differential
modification. Fluorescence confocal scanning laser microscopy
(
CSLM), combined with fluorescent labeling, was used to locate
deprotected propylamine moieties with APMS-Fmoc samples.
Our initial studies used a constant concentration of 5% piperidine
in DMF for 40 min for the deprotection of various APMS-3-
Fmoc-modified materials. The fluorescent dye Alexa Fluor 488
(
λmax ) 488 nm), as the carboxylate succinimidyl ester, was
bound to any free amines present after the deprotection step,
providing the location of free amines. Although the molecular
diameter of the dye is larger than piperidine or its derivative,
the labeling time (16 h) was long enough to allow the dye to
penetrate the pores of the solid. CSLM was then used to select
an optical slice located at the center of a particle. As shown in
Figure 6, the interiors of most of the particles appeared darker
than the exteriors, and the particles appeared as rings, indicating
that there were fewer labeled propylamine moieties within the
pores than on the external particle surface. The brightness and
thickness of the ring corresponded to the number of deprotected
amine groups and the penetrating depth of the piperidine during
the deprotection, respectively. APMS-3-Fmoc-135 and APMS-
We also performed studies varying the concentration of
piperidine and the temperature for a given substrate with an
intermediate organosilane loading (APMS-3-Fmoc-100). De-
creasing the piperidine concentration from 5 to 2% caused a
dramatic increase in tlag and tm, with a decrease in the rate of
the Fmoc-piperidine adduct released (Km). Conversely, increas-
ing the concentration of piperidine from 5 to 10% had the
opposite effects; increasing the concentration further did not
produce a dramatically different result. This provides support
for the results described above, since it appears that as long as
the concentration of piperidine in the solution is high enough
that diffusion to the surface is not hindered (i.e., there is always
piperidine available for deprotection), the limiting factor in the
rate of deprotection is the access of the piperidine to the internal
pore surface. Thus, Km values are similar at piperidine concen-
trations of 5-40%. In order to have the organosilane loading
be the rate-limiting effect, concentrations of at least 5% should
be used. Similarly, increasing the temperature of the system
had a significant effect on tlag, tm, and Km, indicating that
molecular diffusion of piperidine both to the solid surface and
through the pores and the rate of the deprotection reaction itself
3
-Fmoc-200 showed thin, well-defined rings, indicating that the
deprotection reaction was restricted mainly to the external
particle surface even after 40 min. As demonstrated above, the
tlag for these materials (74 and 232 min, respectively) is longer
than the deprotection time (40 min), and less than 5% of the
total number of Fmoc groups (those located on the external
surface) were deprotected. The lack of homogeneity in the
CSLM image of APMS-3-Fmoc-200 lends support to the idea
that the groups are not distributed homogeneously on the
external surface. APMS-3-Fmoc-100 exhibited a clearly defined
but slightly wider ring. For this material, tlag is approximately
26 min but t is 76 min, so that although the groups near the
m
pore entrances have been deprotected, the rate of Fmoc release
J. AM. CHEM. SOC.
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VOL. 129, NO. 31, 2007 9683

A R T I C L E S
Cheng and Landry
Figure 8. Cumulative release of the Fmoc-piperidine adduct from
Nucleosil-3-Fmoc-200, Nucleosil-11-Fmoc-200, APMS-3-Fmoc-135, and
APMS-11-Fmoc-200 in DMF solution containing 5% piperidine at room
temperature.
increased almost linearly as the function of deprotection time
in the burst phase. After APMS-3-Fmoc-135 was exposed to
5
% piperidine for 24 h, all of the internal and external Fmoc-
propylamine moieties were completely deprotected.
Comparative Release Studies on Related Materials. As
discussed earlier, several factors, such as the pore diameter and
organosilane loading of the solids, significantly influenced the
release rate of the Fmoc-adduct. To clearly understand these
factors, we compared several Nucleosil and APMS samples
modified with 3-carbon and 11-carbon alkyl chains. Not
included in the figure are the data for APMS-3-Fmoc-200 due
to concerns with the inhomogeneous distribution of amines in
this sample (Figure 6) and the fact that it was less effectively
fitted to the Hill equation (Figure 5). For comparison, APMS-
3-Fmoc-135 was used instead. The release curves of the Fmoc-
piperidine adduct from these samples in DMF containing 5%
piperidine at room temperature are shown in Figure 8. It is
apparent that the release curves of samples other than APMS-
3-Fmoc-135 are more like hyperbolic plots, although they still
fit well to the Hill equation (correlation coefficients in all cases
were greater than 0.99), and had extremely small values of tlag.
For a given organosilane loading, Nucleosil and APMS can be
ranked in order of increasing t1/2 as Nucleosil-3-Fmoc-200 <
Nucleosil-11-Fmoc-200 < APMS-11-Fmoc-200 < APMS-3-
Fmoc-135, which is consistent with the relative pore sizes of
these materials. It was previously shown that, for higher
organosilane loadings, the diffusion of the reagents within the
pores is the rate-determining step; therefore, materials with larger
pore diameters and surface areas show faster diffusion rates and
faster rates of adduct release. Finally, the fluorescence CSLM
images of these samples after deprotection with 5% piperidine
at room temperature for a moderate time (40 min) as well as
after exhaustive deprotection (24 h) are shown in Figure 9. As
expected, Nucleosil-3-Fmoc-200 and Nucleosil-11-Fmoc-200
(tlag ) 6.6 and 4.4 min, respectively) did not show a clearly
defined ring after 40 min deprotection time, since the percentage
of free amines in each material is 64 and 48%. APMS-3-Fmoc-
135 (tlag ) 74 min), with only 4.3% free amine after 40 min,
shows only a thin, dim ring. APMS-11-Fmoc-200 might be
expected to show a more homogeneous dye distribution, since
tlag (8.3 min) is significantly lower than the deprotection time.
Figure 7. Fluorescence CSLM images of deprotected APMS-3-Fmoc-135.
Samples were deprotected in 5% piperidine in DMF at room temperature
for various times: (a) 5, (b) 10, (c) 20, (d) 40, (e) 60, (f) 90, (g) 120, (h)
2
40, (i) 360, (j) 540, (k) 900, and (l) 1400 min. For this sample, tlag ) 74
min, tm ) 198 min (bar ) 5 µm).
has just begun to increase. Since the tlag has been exceeded, the
groups within the pores have begun to be deprotected. As
expected the samples with smaller values of tlag and tm show
distinct penetration of the dye to the interior of the particle,
with APMS-3-Fmoc-45 and APMS-3-Fmoc-33 showing es-
sentially complete deprotection and dye penetration. Interest-
ingly, these samples also showed the dimmest images, since
the total number of free amines and therefore dye molecules
within the sample was low.
We then performed experiments on a single sample with a
relatively large value of tlag (APMS-3-Fmoc-135), attempting
to control its degree of deprotection by varying its time of
exposure to 5% piperidine (Figure 7). For this sample, tlag is 74
min, and the deprotection time was varied from 5 to 1440 min.
Most of the images showed a clearly defined ring. When the
deprotection time was less than tlag, the images consistently
showed a more clearly defined ring, and the brightness of the
ring increased with time. When the deprotection time became
longer than tlag, diffusion of the piperidine within the pores led
to the broadening and brightening of the ring, although a rapid
increase in dye penetration within the interior did not occur until
after the deprotection time exceeded tm (198 min). Even at tm,
however, a calculation using eq 3 and data from Table 4
indicated that only 36% of the amines were deprotected by the
time tm was reached; at 360 min, 56% of the amines were
deprotected, reflected by the brightness of the images. Figure 5
indicates that the cumulative amount of released Fmoc-adduct
9684 J. AM. CHEM. SOC.
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VOL. 129, NO. 31, 2007

Differential Functionalization of Porous Silica Particles
A R T I C L E S
ments in which TEG-modified APMS was loaded with a
chemotherapeutic agent, thus using APMS as a drug delivery
5
1
vehicle. We are currently performing additional studies using
TEG-modified APMS to enhance transfection of DNA plasmids
and shRNA fragments. Although the modification methods are
slightly different, the studies described in this article provide
the quantitative basis for subsequent use of selectively modified
APMS.
Conclusions
This study has illustrated a new and simple strategy by which
the functional groups within the pores or on the surfaces of the
mesoporous silica can be selectively activated for subsequent
modification. The surface functionalization of APMS and
Nucleosil with two Fmoc-modified organosilanes has been
studied in detail. The number of organosilane groups that could
be loaded onto the mesoporous surface was mainly determined
by the concentration of the organosilane solution, the size and
properties of the organosilane, and the physical properties of
the mesoporous silica (density of free silanol groups, surface
area, pore volume, and pore diameter). Under some conditions,
the pores of the solid were completely filled by the Fmoc-
protected organosilanes. These blocked pores could be “re-
opened” by the cleavage of sterically hindered Fmoc groups
from Fmoc-amino by using a solution of piperidine in DMF.
UV/visible spectroscopy was used to study the release rate of
an the Fmoc-piperidine adduct following organosilane depro-
tection, and the data were fit to the Hill equation. The rate of
deprotection was influenced by the concentration and temper-
ature of the piperidine solution, the organosilane loading and
distribution within the solid, and the physical properties of the
solid. The parameters from the Hill equation, particularly tlag,
tm, and Km, were used to design a process by which the internal
and external organosilanes could be differentially modified.
Fluorescence CSLM was used to visualize this process by
labeling deprotected amines with fluorescent dyes. For example,
when tlag was greater than the deprotection time, the external
organosilanes were selectively deprotected, allowing the location
of surface functionalization to be precisely controlled. Current
applications of this research in our laboratory include the
selective attachment of peptides and polymers to the external
surface of APMS, along with the modification of the pores for
exchange of other molecules.
Figure 9. Fluorescence CSLM of deprotected samples with Alexa-488
bound to the free amine: (1) Nucleosil-3-Fmoc-200, (2) Nucleosil-11-Fmoc-
2
00, (3) APMS-11-Fmoc-200, and (4) APMS-3-Fmoc-135. Samples were
deprotected in 5% piperidine in DMF at room temperature for (a, left
column) 40 min and (b, right column) 24 h. The tlag for each sample is
listed in the top right corner of the images in column a (bar ) 5 µm). .
However, the ring is thicker and brighter than that for APMS-
3-Fmoc-135, indicating the internal deprotection has begun, and
eq 3 shows that in fact only 23% of the total amines are
deprotected by this time.
Examples of Applications of Selectively Modified APMS.
Based on these experiments it is possible to envision the use of
selectively modified APMS in several applications, particularly
drug delivery and biomolecular affinity binding. For example,
in other studies we have modified the external surface of APMS
with tetraethylene glycol (TEG), which enhanced the ability of
APMS to penetrate the cell membranes of malignant mesothe-
liomas in vitro and epithelial cells in vivo.48 This is consistent
with many other studies showing that polyethylene glycol
Acknowledgment. The authors wish to thank Prof. Doug
Taatjes for assistance in obtaining confocal images and Profes-
sors Jose Madalengoitia and Martin Case for useful discussions.
This work was funded in part by J. M. Huber, Inc.
4
9,50
Supporting Information Available: Tabulated TGA, and UV/
modification enhances uptake of chemotherapeutic agents.
The rapid uptake of the APMS particles led to further experi-
2
9
visible data and associated spectra, Si MAS NMR, FTIR, and
powder XRD spectra, N2 physisorption isotherms, and pore size
distributions. This material is available free of charge via the
Internet at http://pubs.acs.org.
(
48) Blumen, S. R.; Cheng, K.; Ramos-Nino, M.; Taatjes, D.; Weiss, D.; Landry,
C. C.; Mossman, B. T. Am. J. Resp. Cell Mol. Biol 2007, 36, 333.
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A.; Shatos, M.; Doherty, J.; Weller, A.; Bergeron, M. J. Free Rad. Biol.
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(
JA070598B
(
50) Veronese, F. M.; Schiavon, O.; Pasut, G.; Mendichi, R.; Andersson, L.;
Tsirk, A.; Ford, J.; Wu, G.; Kneller, S.; Davies, J.; Duncan, R. Bioconj.
Chem. 2005, 16, 775.
(51) Blumen, S. R.; Cheng, K.; MacPherson, M.; Ramos-Nino, M. E.; James,
T. A.; Taatjes, D. J.; Landry, C. C.; Mossman, B. T. Cancer Res., submitted.
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