"clickable" SBA-15 mesoporous materials: Synthesis, characterization and their reaction with alkynes

Article abstract of DOI:10.1039/b815350g

SBA-15 mesoporous silica has been functionalized with azidopropyl groups through both one-pot co-condensation and post-synthetic grafting. For both these methodologies, azidopropyltriethoxysilane was used to introduce the azidopropyl groups. The azidopropyl modified SBA-15 material synthesized by one-pot co-condensation had hexagonal crystallographic order, pore diameters up of 50 A, and the content of azidopropyl groups was found to be 1.3 mmol g ^-1. The presence of the azidopropyl group was confirmed by multinuclear (¹³C, ²⁹Si) solid state NMR and IR spectroscopy. Both these materials underwent very efficient Cu(I)-catalyzed azide alkyne "click" reaction (CuAAC) with a variety of alkynes. Nearly 85% of the azide present in the SBA-15 material was converted to the corresponding triazole when propargyl alcohol was used as the substrate. This methodology was also used to incorporate mannose into SBA-15. Incubation of this mannose labeled SBA-15 with fluorescein labeled Concanavalin-A led to the formation of a fluorescent silica-protein hybrid material. The ease of synthesis for the azide labeled SBA-15 material together with its ability to undergo very efficient chemoselective CuAAC in water would make it a very attractive platform for the development of covalently anchored catalysts, enzymes and sensors.

Full text of DOI:10.1039/b815350g

www.rsc.org/materials
Volume 19 | Number 10 | 14 March 2009 | Pages 1341–1504
ISSN 0959-9428
PAPER
Sayam Sen Gupta et al.
“Clickable”SBA-15 mesoporous
FEATURE ARTICLE
materials: synthesis, characterization Jung-Il Jin et al.
and their reaction with alkynes Materials science of DNA

PAPER
www.rsc.org/materials | Journal of Materials Chemistry
‘‘Clickable’’ SBA-15 mesoporous materials: synthesis, characterization
and their reaction with alkynes†
Bharmana Malvi,a Bibhas R. Sarkar,a Debasis Pati,a Renny Mathew,b T. G. Ajithkumarb and
a
*
Sayam Sen Gupta
Received 3rd September 2008, Accepted 24th November 2008
First published as an Advance Article on the web 16th January 2009
DOI: 10.1039/b815350g
SBA-15 mesoporous silica has been functionalized with azidopropyl groups through both one-pot
co-condensation and post-synthetic grafting. For both these methodologies,
azidopropyltriethoxysilane was used to introduce the azidopropyl groups. The azidopropyl modified
SBA-15 material synthesized by one-pot co-condensation had hexagonal crystallographic order, pore
diameters up of 50 A, and the content of azidopropyl groups was found to be 1.3 mmol gꢀ1. The
ꢀ
presence of the azidopropyl group was confirmed by multinuclear (13C, 29Si) solid state NMR and IR
spectroscopy. Both these materials underwent very efficient Cu(I)-catalyzed azide alkyne ‘‘click’’
reaction (CuAAC) with a variety of alkynes. Nearly 85% of the azide present in the SBA-15 material
was converted to the corresponding triazole when propargyl alcohol was used as the substrate. This
methodology was also used to incorporate mannose into SBA-15. Incubation of this mannose labeled
SBA-15 with fluorescein labeled Concanavalin-A led to the formation of a fluorescent silica-protein
hybrid material. The ease of synthesis for the azide labeled SBA-15 material together with its ability to
undergo very efficient chemoselective CuAAC in water would make it a very attractive platform for the
development of covalently anchored catalysts, enzymes and sensors.
in a single step, by co-condensation of organosilane with the
Introduction
silica precursor in the presence of a liquid crystalline template.
Both methods have their respective advantages and drawbacks.
The most significant advantage of the co-condensation method
over the grafting method is the uniform distribution of organic
groups on the surface of SBA-15, and higher loading of organic
functionalities without reducing the pore size of SBA-15. The
functional groups installed by such modifications can be used to
anchor various synthetic catalysts, biomolecules and polymers to
generate novel functional materials.
Development of mesoporous material based biocatalysts21–24
and biosensors11 has attracted a lot of attention currently. Most
of these materials have been prepared by immobilization of the
enzymes into the pores of the mesoporous channels by physical
adsorption.25,26 Although simple and efficient, this process is
severely limited by the fact that with time, the proteins or
enzymes leach out into the solution. To overcome this, enzymes
have been covalently immobilized by chemically attaching them
to an organo-functionalized mesoporous material bearing alde-
hyde or amine functional groups. Most of the effort has been
directed towards cross linking the amine side-chain of the lysine
to the aminopropyl group of modified mesoporous materials via
glutaraldehyde.27–29 For example, Wang et al. have covalently
immobilized trypsin to a propanal modified SBA-15 via the imine
forming reaction.29 Both these methods have severe limitations,
primarily due to limited control of the spatial orientation of the
immobilized enzyme within the pores. For example, the orien-
tation of the immobilized enzyme may be such that the active site
faces the wall of the mesopore. This would make the enzyme
ineffective and indeed be an obstacle in the development of such
materials. Hence, there is a need to generate functional
Surface functionalized mesoporous materials have emerged as
one of the most important research areas in the field of advanced
functional materials.1–3 They have found wide spread applica-
tions in catalysis,4–6 separation,7,8 decontamination,9 drug
delivery10 and sensor design.11 In particular, mesoporous sili-
ceous matrices like SBA-15 are ideal candidates for functionali-
zation due to their high hydrothermal stability, large pore sizes
ꢀ
(ꢁ90–100A) and thick walls that can be easily functionalized
using simple silanol chemistry.12,13 SBA-15 has been functional-
ized with various organic functional groups. These include
amines,14,15 thiols,16 carboxylic acid,17 sulfonic acid,18 vinyl19 and
nitrogen based heterocycles.9 In general, formulation of a sili-
ceous SBA-15 with covalently linked functional groups can be
designed using two different synthetic strategies for surface
modification. These are post-synthesis modification (also known
as grafting), and direct synthesis or co-condensation.20 Grafting
involves covalent attachment of organic functional groups to the
surface of the mesoporous material by the reaction of a suitable
functional group bearing organosilane. In contrast, direct
synthesis or co-condensation involves modification of the surface
aCReST, Chemical Engineering Division, National Chemical Laboratory,
Dr. Homi Bhabha Road, Pune, 411008, India
bCentral NMR Facility, National Chemical Laboratory, Dr. Homi Bhabha
Road, Pune, 411008, India. E-mail: ss.sengupta@ncl.res.in; Fax: +91 20
25902621; Tel: +91 20 25902747
† Electronic supplementary information (ESI) available: Further
synthesis details; BET surface area and pore size analysis; calibration
curve for semi-quantitative determination of azide groups in SBA-15;
29Si MAS spectra of the different functionalized SBA-15 materials. See
DOI: 10.1039/b815350g
This journal is ª The Royal Society of Chemistry 2009
J. Mater. Chem., 2009, 19, 1409–1416 | 1409

mesoporous materials which can provide a platform to perform
bioorthogonal chemoselective ligation.30 This would provide
spatial control over the molecule, such that it may be attached to
the silica support at the desired orientation.
beaker and 60 mL distilled deionized water was added to it. The
mixture was stirred thoroughly using an overhead stirrer (with
teflon blades) for 4 h to dissolve the gel completely. To this
solution, 240 mL of HCl (2 N) were added dropwise, and the
reaction mixture was allowed to stir for another 1 h at ambient
temperature (ꢁ301–303 K). The temperature of the mixture was
then raised to 313 K and it was allowed to proceed for another
hour. To this warm mixture, tetraethylorthosilicate (TEOS, 18 g,
86.4 mmol) was added dropwise with vigorous stirring. Upon the
completion of addition, the mixture was allowed to stir for 24 h
while maintaining the temperature at 313 K. The synthesis gel
thus formed was then loaded in a 500 mL polypropylene bottle,
sealed using teflon tape and kept at 373 K for 48 h under static
conditions. After this, the bottle was removed from the oven, and
was allowed to cool to ambient temperature. The contents were
then filtered and the residue was washed with copious amount of
water until the pH of the water became neutral. The white solid
was allowed to dry in air, and then crushed using a mortar and
pestle. Calcination of the as-synthesized solid material was done
at 773 K for 6 h in air, using a slow heating ramp of 1 degree per
minute. The sample was cooled to ambient temperature and
preserved under argon atmosphere for further use. Yield: ꢁ 4.7 g.
This material will be represented as CAL-SBA-15.
The recent discovery of the Cu(I)-catalyzed 1,3-dipolar
cycloaddition of organic azides to alkynes has provided the most
powerful ‘‘click chemistry’’ tool for conjugation between
appropriately functionalized binding partners via a 1,2,3-triazole
linkage.31 The favorable thermodynamics, exceptionally high
yields, ease and compatibility with a broad repertoire of func-
tional groups has led to numerous applications of Cu(I)-cata-
lyzed azide-alkyne click chemistry (CuAAC) reaction in various
fields of organic, medicinal, polymer, materials and biological
chemistry.32–34 CuAAC has been successfully used to function-
alize metal nanoparticles,35 single-walled carbon nanotubes,35
powdered silica,36,37 and mesoporous silicon rugate filters.38
Importantly, one of the most promising applications of this
reaction has been in bioconjugation reactions where it has been
shown to be much more effective than traditional methods.39
Therefore, synthesis of azide containing SBA-15 materials would
be very useful in the development of functional materials. The
presence of an organic azide on SBA-15 gives a handle to anchor
various moieties such as enzymes and catalysts via this chemo-
selective ligation.
This study reports the synthesis and characterization of an
SBA-15 mesoporous material containing a clickable azide group,
and demonstrates its reactions with a number of functional
organic molecules bearing alkyne functionality using CuAAC.
These materials were characterized by using FT-IR, powder
XRD, SEM, TEM, multinuclear (13C, 29Si) solid state NMR, and
BET analysis. Further, this study discusses the binding of fluo-
rescein-labeled protein Concanavalin-A to SBA-15 conjugated
with mannose.
Synthesis of azide functionalized SBA-15 (one-pot co-conden-
sation technique). For the functionalization of SBA-15 using the
one-pot co-condensation technique, the typical synthesis of SBA-
15 as described previously was modified following the strategies
described by Wang et al.15 The composition of the synthesis gel
was 0.9 TEOS : 0.1 AzPTES : 6.1 HCl : 0.017 P123 : 165 H2O. In
a typical synthesis batch, 4 g of Pluronic P123 was dissolved in
30 mL H2O for 4 h, to this 120 mL of 2N HCl was added and
stirred for another 1 h at ambient temperature. The temperature
was raised to 313 K and the mixture was stirred at 313 K for
a further 1 h. Then 8.1 g TEOS were added dropwise and the
mixture was allowed to prehydrolyze for 3 h before the addition
of 1.1 g of AzPTES, after which it was allowed to stir for a total
of 24h (including prehydrolysis) at 313 K. The mixture was
transferred to a 250 mL polypropylene vessel and heated at
373 K for 48 h under static conditions. The contents were cooled,
filtered, washed with water and dried. The white solid was then
refluxed in ethanol for 24 h (200 mL ethanol per 1.5 g of solid),
filtered and dried to get the azide-functionalized SBA-15. Yield:
ꢁ2.9 g. This material will be referred as AZP-SBA-C.
Experimental
Synthesis
Synthesis of 3-azidopropyltriethoxysilane, AzPTES. 3-Chlor-
opropyltriethoxysilane (abbreviated as Cl-PTES; 2 g, 8.3 mmol)
was added to a solution of sodium azide (1.08 g, 16.6 mmol) and
tetrabutylammonium bromide (0.644 g, 2 mmol) in dry aceto-
nitrile (50 mL), under nitrogen atmosphere. The reaction mixture
was stirred under reflux for 18 h. After completion of the reac-
tion, the solvent was removed under reduced pressure. The crude
mixture was diluted in n-pentane and the suspension was filtered
over Celite. Solvent was removed from the resulting filtrate and
the crude oil obtained was distilled under reduced pressure of
0.025 mbar at 62 ꢂC to give AzPTES (3-azidopropyltriethoxy-
silane) as a colorless liquid. Yield: 1.52 g, 74%. 1H NMR
(500 MHz, CDCl3): d 0.66 (t, 2H, J ¼ 8.25 Hz), 1.21 (t, 3H,
J ¼ 6.88 Hz), 1.66–1.73 (m, 2H), 3.25 (t, 2H, J ¼ 7.16 Hz), 3.80
(q, 2H, J ¼ 6.88 Hz). 13C NMR (50 MHz, CDCl3) d 7.59, 18.23,
22.64, 53.8, 58.41. FT-IR (NaCl, cmꢀ1): 2098 (-N]N+]Nꢀ, s).
Synthesis of azide functionalized SBA-15 (post-synthesis
grafting technique). To a suspension of 1 g of CAL-SBA-15 in
50 mL of toluene, 2 mL AzPTES were added, and the mixture
ꢂ
was stirred for 16 h at 80 C under nitrogen atmosphere. After
the completion of reaction, the contents were cooled, filtered and
washed with toluene until it became free from AzPTES. The
sample was then dried at 80 ꢂC for 8h in a vacuum oven and
preserved under argon atmosphere for further use. Yield: ꢁ1.2 g.
This material will be referred as AZP-SBA-G.
Synthesis of SBA-15. SBA-15 material was synthesized
following the procedure reported by Zhao et al.,12 with slight
modifications. In a typical batch synthesis, 8 g of the nonionic
block copolymer Pluronic P123 was taken in a polypropylene
Modification of AZP-SBA-C and AZP-SBA-G by Cu(I) cata-
lyzed azide-alkyne cycloaddition reaction (CuAAC). For CuAAC,
the azide functionalized SBA-15 (AZP-SBA-C or AZP-SBA-G)
was incubated with 3 equivalents of the corresponding alkyne
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ꢂ
(1–4, see Scheme 2) in a 1:1 water/t-butanol mixture containing
CuSO4 (1 equivalent) and sodium ascorbate (4 equivalent). In
a typical click reaction, AZP-SBA-C (300 mg, 0.395 mmol of
azide) was incubated with propargyl alcohol (70.07 mL, 1.185
mmol) in 10 mL of t-BuOH/water (1:1) containing sodium
ascorbate (156.42 mg, 0.790 mmol) and copper sulfate (98.75 mg,
0.395 mmol). The mixture was stirred for 12 h and another
156.42 mg (2 equivalents) of sodium ascorbate was added and the
reaction mixture was incubated for a further 12 h. After
completion of reaction, the reaction mixture was centrifuged and
the residue was first washed with water twice and then sequen-
tially washed with 0.1 M N,N-diethyldithiocarbamate sodium in
methanol (15 mL), methanol (15 mL), and acetone (15 mL)
respectively. The last three washings were repeated thrice.
Finally, the faint yellowish white powder obtained was dried at
80 ꢂC in vacuum oven for 6–8 h. Yield: ꢁ250 mg.
Samples were preheated at 100 C for 12 hours in the vacuum
line. Single point BET surface area was obtained from the
nitrogen adsorption-desorption data at P/P0 ꢁ 0.249. Pore size
distributions were calculated using the BJH method. Semi-
quantitative FT-IR spectra were recorded on a Perkin Elmer FT-
IR spectrum GX instrument by making KBr pellets. Pellets were
prepared by mixing 3 mg of sample with 97 mg of KBr. Yields for
AAC reactions were calculated from corrected area under the
curve characteristic for the azide stretch at ꢁ2100 cmꢀ1. The
average of three values was used for all calculations. Qualitative
FT-IR spectra were recorded on a Perkin-Elmer spectrometer in
the diffuse reflectance mode operating at a resolution of 4 cmꢀ1
.
Elemental analyses were carried out on a Thermo Finnigan
FLASH EA 1112 series instrument.
Solid-state NMR. 29Si and 13C cross polarization magic angle
spinning (CPMAS) NMR experiments were carried out on
a Bruker AVANCE 300 wide bore spectrometer equipped with
a superconducting magnet with a field of 7.1 Tesla. The operating
frequencies for 13C and 29Si were 75.4MHz and 59.6MHz
respectively. The samples were packed into a 4mm zirconia rotor
and loaded into a 4mm BL MAS probe and spun about the magic
angle (54.74) at 10kHz using a standard ramp-CP pulse sequence
was used for both the experiments. The RF powers were 50kHz
and 60kHz for the 29Si and 13C CPMAS experiments. The contact
times were 6ms and 3ms for the 29Si and the 13C CPMAS
experiments. All the chemical shifts were referenced to TMS.
Typically 10000 to 25000 scans with a recycle delay of 3s were
collected depending on the sensitivity of the sample.
Note: Click reaction with a-D-propargyl mannopyranoside
was carried out in water.
Labeling of SBA-C-4 with fluorescein labeled Concanavalin A.
10 mg of mannose labeled SBA-15 (SBA-C-4, see Table 1) or
alcohol labeled SBA-15 (SBA-C-1, see Table 1) was incubated
with fluorescently labeled Concanavalin-A (300mL, 1 mg/mL) in
20 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM MnCl2,
0.1 mM CaCl2 and 0.15 M NaCl. After incubation for 30 min
both samples were washed extensively (3 times) with the above
mentioned buffer. The samples were dispersed in the buffer, and
put under a microscope under both bright-field and epi-fluores-
cence modes (with a 470–490 nm bandpass filter).
Epi-fluorescence microscopy. Bright-field transmittance and epi-
fluorescence microscopy was performed on samples that were
drop-cast on glass cover slips. A 150mW Xe lamp and a 100mW
halogen lamp attached to an inverted microscope (Nikon 2000U)
were used as light sources to excite the samples for bright-field and
epi-fluorescence modes, respectively, in order to show that the
concentrations of the samples near the surface were approximately
the same and the same areas were probed in both the imaging
methods. For epi-fluorescence microscopy, proper excitation filters
(470–490 nm bandpass filter, Chroma) were chosen to excite fluo-
rescein. A Plan Apo 0.6 NA 40X air-objective was used to collect
the fluorescence (500–580 nm emission filter, Chroma), which was
subsequently imaged using a color digital camera (Nikon 4500)
equipped with a 10ꢃ eyepiece and a home-built microscope
adapter. For bright-field images, the exposure time was set at 30 ms,
while the epi-fluorescence images were captured at lower excitation
powers at 250 ms exposure to avoid photobleaching of samples.
The images were processed using NIH ImageJ image analyses
software and the raw data for both the sample and the control were
adjusted to the same brightness and contrast levels to ensure that
the stark contrast was clearly visible in the electronic format.
Characterization
Powder X-ray diffraction of all the samples was carried out in
a PANalytical X’pert Pro dual goniometer diffractometer. A
proportional counter detector was used for low angle experi-
ments and an X’celerator solid state detector was employed in the
ꢀ
low angle experiments. The radiation used was Cu Ka (1.5418 A)
with a Ni filter and the data collection was carried out using a flat
holder in Bragg-Brentano geometry (0.5 to 5ꢂ; 0.2ꢂ minꢀ1). Care
was taken to avoid sample displacement effects. SEM images
were obtained on Leica Stereoscan 440 microscope. HR-TEM
images were taken on a FEI Technai F30 operating at 300 kV
with FEG. The samples were prepared by dispersing a large
number of solid particles in acetone by sonication, and dropping
the resulting suspension on a copper grid of 400 mesh and
allowed to dry in air. Nitrogen adsorption and desorption studies
were carried out at 100 ꢂC using a Micromeritics instrument.
Table 1 Yield of CuAAC reaction with different substrates
Mesoporous Alkyne
Material substrate
CuAAC
reaction yield (%)
Product
AZP-SBA-C Propargyl alcohol (1)
Phenylacetylene (3)
a-D(+)-propargyl
SBA-C-1 85
SBA-C-3 63
SBA-C-4 55
Results and discussion
Synthesis strategy of azide functionalized SBA-15
mannopyranoside (4)
AZP-SBA-G Propargyl alcohol (1)
a-D(+)-propargyl
mannopyranoside (4)
SBA-G-1 90
SBA-G-4 79
The azide functionalized SBA-15 was synthesized by both one-
pot co-condensation and post-synthetic grafting methods as
presented in Scheme 1. During the synthesis of AZP-SBA-C via
This journal is ª The Royal Society of Chemistry 2009
J. Mater. Chem., 2009, 19, 1409–1416 | 1411

Scheme 1 Synthesis of azide functionalized SBA-15 materials.
one-pot co-condensation, the ratio of TEOS and AzPTES was
kept as 9:1 since the usage of higher amounts of organosilica
precursor may lead to functionalized materials with reduced long
range order.15 The organosilica precursor AzPTES was obtained
by the displacement of the chloro group in 3-chloropropyl-
triethoxysilane with an azido group using sodium azide. In the
post-synthetic grafting method, 1 g of calcined SBA-15 (CAL-
SBA-15) was reacted with 8.1 mmol of AzPTES in toluene at
80 ꢂC under an inert atmosphere to synthesize AZP-SBA-G. The
amount of azidopropyl group that was incorporated into
the SBA-15 framework was determined by elemental analysis.
The concentration of azide was found to be 1.9 mmol/g for AZP-
SBA-G while that for AZP-SBA-C was found to be 1.3 mmol/g.
Therefore, during co-condensation, approximately 85% of
AzPTES added was incorporated into the silica framework of
AZP-SBA-C. The amount of azide incorporated in these mate-
rials was estimated to be doubled when HPLC grade silica
beads36 were used as the support matrix. The structure and
morphology of AZP-SBA-C and AZP-SBA-G was established
unambiguously using XRD, TEM, SEM, NMR and N2
adsorption-desorption isotherms, as discussed below.
Fig. 1 Powder XRD patterns of different SBA-15 samples via post-
synthesis grafting: [A] CAL-SBA-15; [B] AZP-SBA-G; [C] SBA-G-1.
Characterizations
The powder XRD patterns of grafted AZP-SBA-G and co-
condensed AZP-SBA-C are shown in Fig. 1 and 2 respectively.
All of the materials showed the characteristic high intensity 100
peak near the 2q value of 1ꢂ. The other significant peaks corre-
sponding to 110 and 200 diffractions were also observed indi-
cating that well-ordered one-dimensional hexagonal mesoporous
channels of SBA-15 were formed and remained intact under the
functionalization environment. However, the higher order (110)
and (200) diffractions become significantly less resolved for the
azide-functionalized samples AZP-SBA-G (Fig. 1, curve B) and
AZP-SBA-C (Fig. 2, curve A), showing that the long-range order
decreases slightly upon incorporation of azidopropyl groups into
the SBA-15 matrix. A similar phenomenon has been observed in
aminopropyl-functionalized SBA-15 by Wang and co-workers.15
The SEM images displayed highly dispersed wormlike external
morphology while the TEM micrographs showed the long
parallel channel-structure with thick walls of the mesoporous
materials AZP-SBA-G and AZP-SBA-C respectively (Fig. 3).
The TEM and SEM pictures indicate that the mesoporosity was
retained in both the azide grafted SBA-15 materials. Hence both
Fig. 2 Powder XRD patterns of different SBA-15 samples via co-
condensation: [A] AZP-SBA-C; [B] SBA-C-1.
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Fig. 4 Comparative FT-IR spectra of the different SBA-15 materials.
Fig.
3 SEM images and TEM micrographs of different azido-
functionalized SBA-15 materials. A-1 and A-2 for AZP-SBA-G; B-1 and
B-2 for AZP-SBA-C.
the grafting and co-condensation techniques did not interfere
with the structural properties of SBA-15. N2 adsorption-
desorption isotherms of AZP-SBA-C exhibit the characteristic
type IV isotherm with steep increase in adsorption at P/P0
¼
0.45–0.7 due to capillary condensation of the nitrogen in the
mesopores.† The BJH pore-size distribution (PSD) analysis
shows very narrow PSD values in the range 4–6 nm. The pore
diameter, BET surface, micropore surface area and the wall
thickness were determined to be 5 nm, 503 m2/g, 92.3 m2/g and
3.7 nm respectively.† These values are consistent with other
organo-functionalized SBA-15 materials that have been reported
before.15–17,19
The FT-IR spectra of the various functionalized SBA-15
materials are presented in Fig. 4. The spectra of both the azido-
functionalized SBA-15 materials (AZP-SBA-C and AZP-SBA-G)
display an absorbance at ꢁ2100 cmꢀ1 which is a characteristic
stretching vibration of any organic azide (N3). The absence of this
peak in CAL-SBA-15 showed that the azidopropyl group has
been successfully incorporated into both AZP-SBA-C and
AZP-SBA-G sample. The samples showed absorbance peaks at
1220, 1070, 800 and 466 cmꢀ1 respectively. These peaks are typical
of Si–O–Si bands that are associated with the formation of the
silica networks. Weak peaks associated with Si–OH groups in the
940–960 cmꢀ1 range were also observed for the functionalized
mesoporous materials. The strong peak around 1630 cmꢀ1 might
be attributed to the bending vibration of H2O. Therefore, the
FT-IR spectra indicate that the azido group was efficiently
incorporated in the matrix of the mesoporous SBA-15 both by
post-synthesis grafting and one-pot co-condensation techniques.
Fig. 5 represents the solid state 13C CP-MAS NMR spectra of
the functionalized SBA-15 materials. The spectrum of the as-
synthesized azido-functionalized SBA-15 material synthesized
using the co-condensation technique (as-synthesized AZP-
SBA-C, Fig. 5a) displays signals for both the surfactant
Fig. 5 13C CP MAS NMR spectra of the different functionalized SBA-
15 materials.
EO20PO70EO20 and azidopropyl group. The peaks at ꢁ21 and
78 ppm correspond to the peaks for the surfactant while the three
peaks C1 (8.68 ppm), C2 (22 ppm), C3 (53 ppm) represent the
three C-atoms of the azido-propyl chain in the order as pre-
sented. The relative intensities of the C1, C2, C3 peaks appear to
be less in comparison to the surfactant peaks because of the
higher concentration of the latter in the as-synthesized matrix.
After the as-synthesized AZP-SBA-C was extracted with ethanol
to remove surfactant, the peaks characteristic of the surfactant
disappeared, while the C1, C2, and C3 peaks of the azido-propyl
group remained (Fig. 5b). The 13C CP MAS NMR spectrum of
AZP-SBA-G also showed three distinct peaks corresponding to
the C1, C2. C3 carbon atoms (Fig. 5c). The 13C CP-MAS NMR
spectra of both AZP-SBA-C and AZP-SBA-G were similar
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J. Mater. Chem., 2009, 19, 1409–1416 | 1413

washing was the use of dithiocarbamate to remove the Cu(I), as
reported earlier.40 Attempts to remove copper by using other
chelating reagents like pH 8 Na2EDTA led to the loss of
a significant amount of the SBA-15 material indicating that
a part of the silica had leached out during this process. Use of
higher amounts of catalyst and longer reaction times did not
improve the yield of the reaction. The extent of the reaction was
estimated using IR spectroscopy, by monitoring the decrease in
the integrated intensity of nas(N3) at 2100 cmꢀ1 (Fig. 4D, SBA-C-
1). To get a semi-quantitative estimate of the conversion of the
azide upon CuAAC, a calibration curve was made using quan-
titative amounts of AZP-SBA-C or AZP-SBA-G in KBr and
plotting the integrated intensities of the azide IR peak at 2100
cmꢀ1 to the concentration of the azidopropyl labeled silica in the
KBr pellet.† We were, however, unable to observe the appear-
ance of a 1,2,3-triazole stretch since it interfered with a strong
peak at 1650 cmꢀ1 from the bending vibration of water adsorbed
to SBA-15. Since we were unable to observe the appearance of
the triazole peak in the IR, we turned our attention to finding
substrates that have a characteristic IR peak that does not
interfere with the azide functionalized SBA-15 materials. For
such substrates, their successful attachment would allow us to
follow the reaction both by the appearance of their signature IR
peak and with the disappearance of the azide stretch of the
starting SBA-15 material. The methyl ester of 2-pentynoic acid
(2) was thus chosen as the substrate for CuAAC since it had
a characteristic carbonyl stretch at 1727 cmꢀ1, a region that
displayed no peaks for the azide labeled SBA-15 materials. The
successful attachment of 2 was confirmed from the IR spectra in
which concomitant disappearance of nas(N3) at 2100 cmꢀ1 and
the appearance of n(C]O) at 1727 cmꢀ1 was observed (Fig. 4E,
SBA-C-2).
Fig. 6 29Si CP MAS NMR spectra of the different functionalized SBA-
15 materials.
which indicated that the same material was produced by co-
condensation and grafting.
The 29Si CP MAS NMR spectra of the different azide-func-
tionalized SBA-15 materials are presented in Fig. 6. All three
spectra display prominent peaks at around ꢀ93, ꢀ102 and ꢀ112
ppm respectively. These may be assigned to the different types
of Si sites namely Q2 [(SiO)2Si(OH)2], Q3 [(SiO)3Si(OH)] and Q4
[(SiO)4Si] that are classified based on the total number of silanol
groups (Si–OH) at the Si centre (i.e. 2, 1, 0 respectively). The 29Si
MAS spectra were obtained to evaluate the relative intensities of
Q3 and Q4 for both AZP-SBA-C and AZP-SBA-G.† It was
observed that in both the samples that the intensity of the Q4
signal was found to be higher than the Q3 signal. This is
expected since SBA-15 is a thick-walled material in which much
of the Si exists as (SiO)4Si. Two other distinct peaks that are
observed at ꢀ59.9 and ꢀ68 ppm may be attributed to the
functionalized sites of the Si framework namely T2 [R(SiO)2-
Si(OH)] and T3 [R(SiO)3Si] respectively, where R is the azido-
propyl group.
The dependence of the substrate concentration for the
conversion of the available azide groups on AZP-SBA-C or AZP-
SBA-G by CuAAC is shown in Fig. 7. Treatment of AZP-SBA-G
(100 mg) with 1.5 equivalents of propargyl alcohol led to the
formation of SBA-C-1 in which 90% of all available azides were
converted to the triazole (Table 1). Similar results were obtained
with AZP-SBA-C although 3 equivalents of propargyl alcohol
were needed to convert 85% of the available azides. The very high
extent of the reaction leads us to conclude that in AZP-SBA-C,
most azides are present in the surface of SBA-15. These data are in
contrast to amino grafted SBA-15 material that was synthesized
by co-condensation with aminopropyltriethoxysilane.41 In this
CuAAC of N3-SBA-15 with various alkyne containing substrates
The azidopropyl labeled silicas AZP-SBA-C and AZP-SBA-G
were subjected to Cu(I) catalyzed azide alkyne cycloaddition
reaction (CuAAC) with various organic substrates (Scheme 2).
Propargyl alcohol (1) was used as the substrate during the initial
experiments to optimize the reaction conditions. The CuAAC
reaction was attempted using CuSO4/ascorbate in an equimolar
mixture of t-BuOH and water. For example, treatment of AZP-
SBA-C (25 mg) with 1 (0.1 mmol, 3-fold molar excess with
respect to azide groups on azidopropyl grafted silica) using
CuSO4/ascorbate in 1:1 H2O/t-BuOH for 24 h converted 85% of
the available azide groups into the corresponding triazole
product. Control reactions performed without the use of CuSO4
showed no conversion of the azide. After the reaction, an
extensive washing protocol was followed to remove the Cu(I),
ascorbate and any unreacted 1. One of the key steps in the
Scheme
2 CuAAC reaction of azide-functionalized SBA-15 with
various substrates.
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This journal is ª The Royal Society of Chemistry 2009

phenylacytelene (3), and a-D(+)-propargyl mannopyranoside (4)
as substrates. The extent of the click reaction as observed using
IR as described before is shown in Table 1. Both the substrates
were successfully incorporated into the azido functionalized
SBA-15 by CuAAC. The conversion of available azides was less
for both phenylacytelene and a-D(+)-propargyl mannopyrano-
side than for propargyl alcohol. There may be several factors
responsible for this observation. One of them is that propargyl
alcohol, a much smaller molecule, is accessible to more azides
during the click reaction.
The presence of mannose in the a-D(+)-propargyl man-
nopyranoside labeled SBA-15, SBA-C-4, was confirmed by
probing its affinity for the protein Concanavalin-A (Con-A). The
plant lectin Con-A exists as a tetramer in Tris-HCl buffer (pH
7.4) and binds up to four a-D(+)-mannopyranoside units in the
presence of Na+, Ca2+ and Mn2+ ions. Commercially available
fluorescein labeled Con-A (300 mg) was incubated with both
SBA-C-4 and SBA-C-1 (10 mg) in Tris-HCl buffer containing
Na+, Ca2+ and Mn2+ ions. After incubation for 30 min both the
samples were extensively washed with Tris-HCl buffer contain-
ing Na+, Ca2+ and Mn2+ ions. The samples were dispersed in the
above mentioned buffer and were then put under a microscope
under both bright-field and epi-fluorescence modes (with a 470–
490 nm bandpass filter). The fluorescence observed from SBA-C-
4 incubated with Con-A is much more intense than that of the
corresponding SBA-C-1 that was incubated with Con-A (Fig. 8).
The intense fluorescence is observed since the fluorescently
labeled Con-A will bind to the mannose on the exterior surface of
SBA-C-4. The material SBA-C-1 was used as a control to elim-
inate the effect of triazole on the binding on Con-A. The diam-
eter of tetrameric Con-A (each subunit is around
4.2 nm ꢃ 3.9 nm ꢃ 2 nm)42 is larger than the pore size of SBA-C-4
(5 nm) and this would probably attach to the mannose on the
external surface of SBA-C-4, rather than inside the channels. The
observance of intense fluorescence in Fig 8D indicates that
mannose successfully conjugates to AZP-SBA-C to form SBA-C-
4 via CuAAC. The weak fluorescence observed in SBA-C-1 is
Fig. 7 Dependence of the percentage of azide converted in AZP-SBA-C
or AZP-SBA-G on propargyl alcohol concentration. Reactions were
performed with 100 mg of the respective azide functionalized SBA-15
material in 3 ml H2O/t-BuOH (1:1) containing CuSO4 (0.13 mmol) and
sodium ascorbate (0.26 mmol).
material it was observed that only 25% of the available amino
groups are on the surface. Since the azidopropyl group is more
hydrophobic than the protonated aminopropyl group, it can act
as a co-surfactant and be solubilized by the Pluronic micelles
leading to its presence in the micelle-silica interface. The presence
of the azides at this interface during synthesis is probably the
reason for its high density at the surface of AZP-SBA-C. The
presence of the azides mostly on the surface of AZP-SBA-C
coupled with the very high efficiency of the CuAAC reaction
makes AZP-SBA-C a very attractive material for attachment of
alkyne containing functional molecules. It should also be noted
that the percentage of available azide for click reaction is more in
AZP-SBA-G than in AZP-SBA-C. This can be explained by the
fact that during the direct synthesis of AZP-SBA-C by co-
condensation, some of the azides may be trapped in the wall and
therefore be unavailable for conjugation.
The product SBA-C-1, formed upon reaction of AZP-SBA-C
and 1, was further characterized by XRD, 13C NMR and 29Si
NMR. The XRD showed one intense (100) peak diffraction in
the proximity of 2q ¼ 1ꢂ, indicating well-ordered hexagonal
arrays and showing that the mesoporosity of the material does
not change after undergoing CuAAC reaction (Fig. 1C and 2B).
The 13C NMR shows extra peaks at 122 ppm and 146.7 ppm
which correspond to the C4 and C5 atoms of the triazole as
shown in Fig. 5d. The extra peak due to the C6 carbon is not
observed as it is masked by the C3 peak from the linker. The 29Si
CP MAS spectrum of the SBA-C-1 material shows little or no
change from the starting AZP-SBA-C indicating that the Si sites
does not undergo any chemical change during the reaction and
its work-up (Fig. 6C). This is expected since the T1 and T2 Si sites
are from the azido-propyl modified silica and should not undergo
any change during the course of the click reaction.
Fig. 8 A) SBA-C-1 treated with fluorescent labeled Con-A in bright field
mode. B) SBA-C-1 treated with fluorescent labeled Con-A in epi-fluo-
rescence mode. C) SBA-C-4 treated with fluorescent labeled Con-A in
bright field mode. D) SBA-C-4 treated with fluorescent labeled Con-A in
epi-fluorescence mode. The scale bar shown is equivalent to 10 microns.
We then extended the CuAAC to other substrates to explore
the efficiency of this reaction on azidopropyl functionalized
SBA-15. The reaction was carried out with a five-fold excess of
This journal is ª The Royal Society of Chemistry 2009
J. Mater. Chem., 2009, 19, 1409–1416 | 1415

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probably due to the non-specific interaction between fluo-
rescently labeled Con-A with the hydroxyl groups of silica or the
1,2,3-triazole functional group of SBA-C-1. This specificity of
Con-A for mannose-labeled SBA-15 can be used in the devel-
opment of sensors. Similar sensors have been developed for
sensing ion transport in ion channels using the interaction of
biotin-labeled MCM-41 with the protein avidin.11
Conclusion
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21 M. Hartmann, Chemistry of Materials, 2005, 17, 4577–4593.
22 H. H. P. Yiu, P. A. Wright and N. P. Botting, Journal of Molecular
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23 Y. Zhu, S. Kaskel, J. Shi, T. Wage and K.-H. van Pee, Chemistry of
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24 Y. Zhu, W. Shen, X. Dong and J. Shi, Journal of Materials Research,
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25 J. Kim, J. W. Grate and P. Wang, Chemical Engineering Science, 2006,
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26 H. H. P. Yiu, P. A. Wright and N. P. Botting, Microporous and
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The synthesis of azide labeled SBA-15 has been successfully
achieved using both the one-pot co-condensation and post-
synthetic grafting methods. The azide labeled SBA-15 materials
retains their mesoporosity after synthesis and the incorporation
of the organic functionality was confirmed by IR and solid state
NMR. These materials undergo very efficient CuAAC with
a variety of alkynes. When AZP-SBA-G was used, only 1.5 eq of
alkyne was required to covert 90% of the available azides to
triazole. Similar efficiency is very difficult to attain in aqueous
solution by using amine and carboxylic labeled SBA-15, which
are materials typically used to anchor various functional mole-
cules. The azide labeled SBA-15 was further used as a platform
to synthesize a silica-protein hybrid material composing of
mannose labeled SBA-15 and Con-A. Such hybrid materials may
be important in the development of biosensors. The ease of
synthesis for the azide labeled SBA-15 material together with its
ability to undergo very efficient chemoselective CuAAC in water
would make it a very attractive platform for the development of
covalently anchored catalysts, enzymes and sensors. Such efforts
are under way in our laboratory.
29 P. Wang, S. Dai, S. D. Waezsada, A. Y. Tsao and B. H. Davison,
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30 I. S. Carrico, Chem. Soc. Rev., 2008, 37, 1423–1431.
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Acknowledgements
BM, BRS and DP acknowledge CSIR, New Delhi, India for
fellowships. SSG acknowledges Professor G. Ranga Rao, IIT-
Madras, for help regarding BET analysis and Professor Arindam
Chowdhury, IIT-Bombay, for help regarding fluorescence
microscopy. SSG also acknowledges Dr CVV Satyanaryana,
NCL for helpful discussions and NCL start-up grant and DST,
New Delhi (Grant No: SR/S1/PC-56/2008) for funding.
34 L. Jean-Franc¸ois, Angewandte Chemie International Edition, 2007, 46,
1018–1025.
35 D. A. Fleming, C. J. Thode and M. E. Williams, Chemistry of
Materials, 2006, 18, 2327–2334.
36 Z. Guo, A. Lei, X. Liang and Q. Xu, Chem. Commun., 2006, 4512–
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37 K. M. Kacprzak, N. M. Maier and W. Lindner, Tetrahedron Letters,
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38 S. Ciampi, T. Bocking, K. A. Kilian, J. B. Harper and J. J. Gooding,
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39 Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless and
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