Synthesis, characterization and protein binding properties of supported dendrons

Article abstract of DOI:10.1039/b908014g

Novel benzylether type aldehyde and acetal terminated dendrons were synthesized and attached to a silica gel support; a linear spacer was also introduced as a control material. The supported dendritic compounds were mainly characterized by solid state ¹³C CPMAS NMR, elemental analysis and X-ray photoelectron spectroscopy (XPS) and the presence of free aldehydes was determined by the purpald test. Bovine serum albumin (BSA) protein was coupled to the dendronized support by imine bond formation, followed by irreversible reduction of the carbon-nitrogen double bond. A significant positive dendritic effect was observed on the antibody binding capacity of immobilised BSA as measured by fluorescence immunoassay (FIA). The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b908014g

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Synthesis, characterization and protein binding properties of supported
dendrons†
a
b
a
Olga Iliashevsky,a Liron Amir,ab Robert Glaser, Robert S. Marks and N. Gabriel Lemcoff
*
Received 22nd April 2009, Accepted 11th June 2009
First published as an Advance Article on the web 10th July 2009
DOI: 10.1039/b908014g
Novel benzylether type aldehyde and acetal terminated dendrons were synthesized and attached to
a silica gel support; a linear spacer was also introduced as a control material. The supported dendritic
compounds were mainly characterized by solid state 13C CPMAS NMR, elemental analysis and X-ray
photoelectron spectroscopy (XPS) and the presence of free aldehydes was determined by the purpald
test. Bovine serum albumin (BSA) protein was coupled to the dendronized support by imine bond
formation, followed by irreversible reduction of the carbon–nitrogen double bond. A significant
positive dendritic effect was observed on the antibody binding capacity of immobilised BSA as
measured by fluorescence immunoassay (FIA).
In this study, the first results on BSA immobilization on silica
ꢀ
gel modified with aldehyde terminal group Frechet type den-
Introduction
Dendrimers are highly branched monodispersed macromolecules
possessing a large number of symmetrical terminal groups. The
unique fractal-like dendritic geometry brings about many potential
applications.1–7 Notably, one of the most exploited properties of
dendrimers is their multivalency. Additive effects, an increase in
efficiency ofbinding; as well as dendritic effects, asynergisticincrease
in affinity, may be observed in dendrimer–substrate associations.8,9
Furthermore, the attachment of dendrimers to solid supports,10,11
taking advantage of the aforementioned effects and the unique
dendritic architecture, has been reported in catalytic reactions,12–14
separation processes15,16 and biological applications.17–22
drons are presented. To determine whether dendronized sup-
ported proteins could be used for different applications and what
effect the dendritic support may have; the immunoactivity of the
immobilized protein was evaluated on different dendron gener-
ations (Gn) and on a linear spacer (Ln) that reproduced the length
parameters of the second generation dendron.
Results and discusion
Characterization of dendron grafting by elemental analysis
All elemental analyses were repeated in triplicate. The amount (in
mmol) of grafted dendron per gram of material (dendron
modified silica gel), defined as n* [mmol gꢀ1], was calculated by
the following equations:
Aldehyde terminated dendrimers can be utilized for the
conjugation of biologically active molecules.17 Aldehyde groups
are widely used as an immobilization entity due to covalent bond
formation with amino side groups, primarily lysines, on the
protein surface.23–27
%CGn ꢀ %Csilica
XGn
n*Gn
¼
The immobilization of proteins and peptides onto solid
supports22,28–30 has proved to be important in many areas such as
enzymatic catalysis,31 biochips32 and biosensors.33,34 In recent
years, various chemical methods for the immobilization of
proteins have been developed, besides the aforementioned alde-
hyde based covalent linking, ranging from the use of a thiol-ene
reaction, through Diels–Alder, Staudinger ligation and ‘click’
methodologies.35–41 Two main strategies are widely used: the first
based on physical adsorption, while the second employs the
formation of covalent bonds.28 In the physical adsorption
method, proteins usually retain high activities but leaching may
be significant due to weak interactions.42 On the other hand,
covalent bonds are more difficult to disrupt, but may lead to
conformational changes and as a result hinder activity.31
%CðMwꢀ2Þ ꢀ %Csilica
XGn
¼
ꢁ 1000
Mw ꢀ 2
where XGn is the relative mass ratio of the dendron in the dendron
modified silica gel, %CGn is the percentage of carbon in the
dendron modified silica gel according to elemental (carbon)
analysis, %Csilica is the percentage of carbon in the amine
modified silica gel according to elemental (carbon) analysis and
%C(Mwꢀ2) is the theoretical percentage of carbon in the den-
dron’s molecular weight minus 2 (Mwꢀ2), where 2 is the weight
of the two hydrogen atoms lost during the grafting reaction.
For example, in the calculation of G2p, elemental analysis
afforded %Csilica ¼ 7.78%, %CG2 ¼ 25.02% and according to
calculations %C(Mwꢀ2) ¼ 72.11% and Mwꢀ2 ¼ 1015.12 g$molꢀ1
.
Thus,
aDepartment of Chemistry, Ben-Gurion University of the Negev, P.O.Box
653, Beer-Sheva, 84105, Israel. E-mail: lemcoff@bgu.ac.il; Fax: (+97)-8-
6461740; Tel: (+97)-8-6461196
bDepartment of Biochemical Engineering, Ben-Gurion University of the
Negev, P.O.Box 653, Beer-Sheva, 84105, Israel
25:02 ꢀ 7:78
XG2p
n*G2p
¼
¼ 0:268
72:11 ꢀ 7:78
0:268
ꢁ 1000 ¼ 0:26mmol,gꢀ1
† Electronic supplementary information (ESI) available: Experimental
and characterization details. See DOI: 10.1039/b908014g
¼
1015g,molꢀ1
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Table 1 Calculated amount of grafted dendron based on elemental analysis results
Compound
Gn (termini)
Mwꢀ2 (g$molꢀ1
)
%CGn
%C(Mwꢀ2)
XGn
n* (mmol$gꢀ1
)
23
24
14
15
17
18
20
21
L2p (1)
L2 (1)
G0p (1)
G0 (1)
G1p (2)
G1 (2)
G2p (4)
G2 (4)
374.4
330.4
162.2
118.2
446.5
358.4
1015.1
838.9
21.61
20.89
19.11
17.34
21.18
18.95
25.02
22.46
76.91
79.91
73.98
81.25
72.56
77.00
72.11
75.81
0.200
0.182
0.171
0.130
0.207
0.161
0.268
0.216
0.54
0.55
1.05
1.10
0.46
0.45
0.26
0.26
The parameter n* allows one to compare the different materials
at the same level of binder molecule loading. n* multiplied by the
number of dendron termini provides the number of aldehyde
groups (in mmol) per gram of modified silica material. Calcula-
tions of grafted dendron after hydrolysis were performed by the
same method. According to elemental analysis, the protecting
groups were nearly quantitatively removed (Table 1) (NMR
evidence supports this observation, vide infra). Additionally,
elemental analysis indicates that higher generation dendrons
afford lower molar loadings on the silica, probably due to steric
hindrance effects. This is quite fortuitous since a very similar
number of aldehyde groups per gram of dendronized material are
obtained in all cases, except for the linear spacer modified silica
where about half the number of aldehydes per gram of material
were obtained (the molar loading of the L2 molecules were
similar to the G1 molecules).
Characterization of dendron grafting by XPS
Although XPS is considered to be quantitative, it is a surface
chemical analysis technique and for correct results it requires
samples with a homogeneous surface. Since our dendronized
silica gels have a microporous heterogeneous surface, we could
only extract qualitative data from the XPS measurements. After
the deprotection of the aldehyde by acidic hydrolysis, the amount
of carbon atoms per nitrogen and silicon atoms should be
reduced and this relative difference may be detected by XPS. As
shown in Table 2, XPS analysis indicates a decrease of C/N and
C/Si ratios after acetal hydrolysis, as expected.
Characterization of dendron grafting by the purpald test
The reaction of purpald43 (4-amino-3-hydrazino-5-mercapto-
1,2,4-triazole) in 1 N NaOH with aldehydes has been used in
solution to detect and quantify aldehyde presence taking
advantage of the deeply coloured reaction product. This test has
also been found to be suitable for the qualitative determination
of aldehyde groups grafted on solid supports.43b However,
specific conditions should be employed and calibrated according
to the type of support materials.
Characterization of dendron grafting by NMR
The modified silica gels were characterized by solid state 13C
CPMAS NMR. The disappearance of the acetal signals at ca. 100
ppm, the ethylene signals at 65 ppm and the appearance of an
aldehyde peak around 190 ppm in the spectrum of 21 are
indicative of a fruitful deprotection of the acetal groups (Fig. 1).
See the ESI† for the other materials.
Dendron/linear spacer modified silica gels before and after
hydrolysis were analyzed for the presence of aldehyde groups
using purpald. Indeed, orange/brown colours were developed in
the cases of the unprotected dendron/linear spacer modified silica
gel (Table 3) exclusively.
Protein binding
Once aldehyde terminated dendronized silica materials of three
generations and the related supported linear spacer were secured
and characterized, (corresponding to 1, 2 and 4 free aldehyde
groups per dendron for the different generations and one alde-
hyde per linear spacer molecule) BSA was immobilized on the
Table 2 C/N and C/Si ratios obtained from XPS
Compound
Gn
C/N
C/Si
23
24
14
15
17
18
20
21
L2p
L2
G0p
G0
G1p
G1
G2p
G2
20.3
15.0
11.1
9.9
19.9
11.8
17.0
13.4
1.7
1.3
1.5
1.1
1.8
1.3
2.3
2.2
Fig. 1 13C CPMAS NMR of 20 (top spectrum) and 21 (bottom spec-
trum).
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Table 3 Purpald test
lysine cross-linking in BSA with small molecule cross-linkers
support the assumption that a multivalent dendrimer may bind
multiple lysines on the BSA surface.44 It is also possible that the
observed improvement may be due to reduced antibody access in
proteins bound to lower generation dendrons due to protein
conformational changes. Further experiments are ongoing to try
to elucidate these assumptions.
Compound
Gn
Colour change
23
24
14
15
17
18
20
21
L2p
L2
G0p
G0
G1p
G1
G2p
G2
Not observed
Observed
Not observed
Observed
Not observed
Observed
Not observed
Observed
Conclusions
In conclusion, three generations of acetal terminated benzylether
dendrons were synthesized and characterized, together with
a linear spacer that emulates the distance of the termini to the
focal group in the second generation dendron. These dendrons
were efficiently attached to a silica gel support and deprotected.
The new materials were characterized by NMR, XPS and
elemental analysis. BSA protein could be covalently bound to the
dendronized silica. A noticeable dendritic effect on protein
binding could be assessed by FIA analyses; higher generation
dendrimers led to more efficient protein binding on the silica
surface. Distancing the protein from the silica surface using
a linear spacer also showed activity enhancement. However, the
use of a multivalent molecule, able to simultaneously bind several
neighbouring lysine groups, was more than two times more
efficient than the monovalent analogue evidencing a significant
positive dendritic effect. Additionally, the ability to judiciously
deprotect supported dendrons (by acidic hydrolysis) may allow
for future site specific protein binding for advanced materials.
silica support by incubation of a solution of protein (10% THF in
PBS) with the supported dendrons. The imines obtained were
then reduced with NaCNBH3 to irreversibly immobilize the
protein on the support.
The antibody binding capacity of the BSA bound on different
generations of dendronized silica was measured by a fluorescence
immunoassay (FIA), with fluorescein isothiocyanate (FITC)
conjugated chicken anti-BSA antibody (Fig. 2).
To our satisfaction, a more than three fold increase in
immunoactivity was observed for the higher generation den-
drons. Even though the calculated number of aldehyde groups
according to elemental analyses in 15, 18 and 21 was approxi-
mately the same; the higher generation supported dendron
scaffold 21 afforded a significantly larger fluorescence signal
when the protein was bound. The results suggest that either more
protein is bound with the same amount of aldehyde in the higher
generation dendrons, or that the protein bound by higher
generation dendrons is more efficient at binding its antibody. The
introduction of a non-dendritic spacer also elicited more efficient
binding when compared to the G0 support; however, the use of
a dendritic scaffold of the same spacer length provided a signifi-
cantly superior binding (even when the four fold ratio of alde-
hydes per molecule of dendron to linear spacer is taken into
account).
Experimental
Materials and reagents
All reactions were run under a dry nitrogen atmosphere using
standard Schlenck techniques. All solvents and reagents were of
reagent grade quality, purchased commercially, and used
without further purification, except for THF and toluene which
were freshly distilled from sodium benzophenone, acetone which
was dried under CaSO4 and freshly distilled, and dichloro-
methane which was dried under activated molecular sieves 4A
The enhancement of binding capacity with the generation
increase might be explained by superior protein binding through
a multivalency effect. The results by Kim and coworkers on
˚
(pore size 4A) prior to use. Flash chromatography was per-
formed on Fluka 0.05–0.15 mm basic alumina (pH ¼ 9.5 ꢂ 0.5).
˚
Silica gel with a 0.04–0.063 mm particle size and 60 A pore
diameter was obtained from Merck; and was dried in vacuo at
110 ꢃC overnight before use. Antibodies were purchased from
ICN biomedicals, Inc. (#654381 chicken anti-BSA affinity
purified FITC conjugated).
General
Nuclear magnetic resonance (NMR) spectra were acquired on
a Bruker DMX 500 spectrometer; coupling constants are
reported in Hertz (Hz). The 1H NMR chemical shifts were
referenced to the residual protio solvent peak at 7.26 ppm in
neutralized chloroform-d (CDCl3). Solid-state 13C CPMAS
NMR was acquired on a Bruker DMX 500 spectrometer and
chemical shifts were referenced to a glycine external standard at
176.0 ppm. LC-MS data was acquired with a Bruker esquire 3000
(Bruker Daltonics). GC-MS data was acquired with an Agilent
6850 GC-MS apparatus. Determination of C and N by elemental
Fig. 2 Function experiment of the post immobilization of BSA. Fluo-
rescence measurements determined the amount of fluorescein iso-
thiocyanate conjugated antibody to BSA, immobilized on the
dendronized silica, per one gram of silica, normalized by the number of
aldehyde groups obtained from elemental analysis results.
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analysis was obtained with a Perkin-Elmer 2400 series II
Analyzer. XPS surface analysis was performed with a Thermo-
Electron Escalab-250 instrument including a hemispherical
analyzer (energy resolution 0.45 eV or better) and microfocused
monochromatic X-ray excitation (Al source). General survey
spectra were recorded for quantitative and qualitative analysis.
Fluorescence in the FIA was read using Synergyꢀ HT Multi-
Mode Microplate Reader (Biotek) in wavelengths of 528/485 nm.
a solution of diisopropyl azodicarboxylate (DIAD) (0.75 cm3, 3.8
mmol) in THF (50 cm3). The reaction was warmed to room
temperature and stirred for 24 h. The reaction was stopped by
adding water and evaporated to remove THF. The aqueous layer
was extracted twice with ethyl acetate, each organic layer was
washed with 1 M aqueous KOH and an equal volume of water.
The combined organic layers were dried over sodium sulfate,
filtered and evaporated. The resultant solid was washed with
methanol, filtered and then was further purified by column
chromatography on basic alumina using (20/80 petroleum ether/
ethyl acetate) as eluent to give the desired product 10 (0.56 g,
42%) as a white powder: dH(500 MHz; CDCl3) 3.91 (3 H, s,
CO2CH3), 4.14–4.03 (4 H, m, 2 ꢁ CHOCH2), 5.02 (2 H, s,
PhOCH2), 5.10 (2 H, s, PhOCH2), 5.83 (1 H, s, CHOCH2), 6.98
(2 H, d, J 8.7, 2 ꢁ H-Ph), 7.16 (1 H, br d, H-Ph), 7.34 (1 H, t,
J 7.8, H-Ph), 7.36 (2 H, d, J 8.7, 2 ꢁ H-Ph), 7.45 (2 H, d, J 8.3, 2
ꢁ H-Ph), 7.50 (2 H, d, J 8.3, 2 ꢁ H-Ph), 7.65–7.64 (2 H, m, H-
Ph); dC(125 MHz; CDCl3) 52.2, 65.3, 69.7, 69.9, 103.5, 115.0,
120.4, 122.1, 126.7, 127.3, 128.9, 129.3, 129.3, 131.4, 137.7, 137.9,
158.6, 158.7, 166.9; m/z (EI) 269 (51, M-C15H13O4), 163 (100, M-
C8H7O3), 91 (47).
Synthesis
Acetal protected compounds 1–7 (Scheme 1) were prepared by
a convergent repetitive methodology according to literature
procedures.45
Methyl 4-(4-(1,3-dioxolan-2-yl)benzyloxy)benzoate (8)
A solution of p-hydroxybenzoic acid methyl ester (6.8 g,
28 mmol), 1 (4.2 g, 28 mmol), K2CO3 (4.4 g, 33 mmol) and
18-crown-6 (0.37 g, 1.40 mmol) in acetone (150 cm3) was refluxed
for 24 h. Then, acetone was removed under reduced pressure. To
the resultant solid, water was added and the aqueous layer was
extracted twice with ethyl acetate. The organic layer was dried
over magnesium sulfate, filtered, and evaporated. The resultant
solid was washed with methanol and filtered to afford the desired
product 8 (7.6 g, 86%) as a white powder: dH(500 MHz; CDCl3)
3.88 (3 H, s, CO2CH3), 4.14–4.03 (4 H, m, 2 ꢁ CHOCH2), 5.13
(2 H, s, PhOCH2), 5.82 (1 H, s, CHOCH2), 6.97 (2 H, d, J 9.0, 2
ꢁ H-Ph), 7.44 (2 H, d, J 7.9, 2 ꢁ H-Ph), 7.51 (2 H, d, J 7.9, 2 ꢁ
H-Ph), 7.99 (2 H, d, J 9.0, 2 ꢁ H-Ph); dC(125 MHz; CDCl3) 51.9,
65.3, 69.7, 103.4, 114.5, 122.9, 126.8, 127.3, 131.6, 137.3, 137.9,
162.3, 166.8; m/z (EI) 314 (M+, 20%), 283 (11, M-CH3O), 163
(100, M-C8H7O3), 91 (59).
(3-(4-(4-(1,3-dioxolan-2-yl)benzyloxy)benzyloxy)phenyl)
methanol (11)
To a suspension of LAH (0.90 g, 24 mmol) in THF (50 cm3)
ꢃ
cooled to 0 C was added dropwise a solution of 10 (3.3 g, 7.9
mmol) in THF (50 cm3). Then, the solution was allowed to warm
to room temperature and stirred for 24 h. Excess LAH was
quenched by slow addition of a semi-saturated solution of
sodium sulfate. THF was removed under reduced pressure. The
resultant aqueous solution was extracted twice with ethyl acetate.
The combined organic layers were dried over sodium sulfate,
filtered and evaporated to afford the desired product 11 (2.8 g,
91%) as a white powder: dH(500 MHz; CDCl3) 4.14–4.03 (4 H, m,
2 ꢁ CHOCH2), 4.64 (2 H, s, OHCH2), 4.99 (2 H, s, PhOCH2),
5.09 (2 H, s, PhOCH2),5.83 (1 H, s, CHOCH2), 6.93 (1 H, br d,
H-Ph), 6.94 (1 H, br d, H-Ph), 6.96 (2 H, d, J 8.7, 2x H-Ph), 7.00
(1 H, br s, H-Ph), 7.27 (1 H, t, J 7.0, H-Ph), 7.35 (2 H, d, J 8.7, 2
ꢁ H-Ph), 7.44 (2 H, d, J 8.1, 2 ꢁ H-Ph), 7.50 (2 H, d, J 8.1, 2 ꢁ
H-Ph); dC(125 MHz; CDCl3) 65.1, 65.2, 69.6, 103.4, 113.1, 114.1,
114.9, 119.2, 126.7, 127.2, 129.3, 129.1, 129.5, 137.6, 138.0, 142.5,
158.5, 159.0; m/z (EI) 269 (48, M-C7H7O2), 163 (100, M-
C14H13O3), 91 (57).
(4-(4-(1,3-Dioxolan-2-yl)benzyloxy)phenyl)methanol (9)
To a suspension of lithium aluminium hydride (LAH) (2.7 g, 72
mmol) in THF (100 cm3) cooled to 0 ꢃC was added dropwise
a solution of 8 (7.6 g, 24 mmol) in THF (100 cm3). Then, the
solution was allowed to warm to room temperature and stirred
for 24 h. Excess LAH was quenched by slow addition of a semi-
saturated solution of sodium sulfate. THF was removed under
reduced pressure. The resultant aqueous solution was extracted
twice with ethyl acetate. The combined organic layers were dried
over sodium sulfate, filtered and evaporated to acquire the
desired product 9 (6.4 g, 93%) as a white powder: dH(500 MHz;
CDCl3) 4.14–4.02 (4 H, m, 2 ꢁ CHOCH2), 4.59 (2 H, s,
OHCH2), 5.08 (2 H, s, PhOCH2), 5.82 (1 H, s, CHOCH2), 6.94
(2 H, d, J 8.3, 2 ꢁ H-Ph), 7.27 (2 H, d, J 8.3, 2 ꢁ H-Ph), 7.43
(2 H, d, J 8.3, 2 ꢁ H-Ph), 7.49 (2 H, d, J 8.3, 2 ꢁ H-Ph); dC(125
MHz; CDCl3) 64.9, 65.3, 69.7, 103.4, 114.9, 126.7, 127.3, 128.6,
133.4, 137.6, 138.0, 158.2; m/z (EI) 286 (M+, 21%), 163 (100, M-
C7H7O2), 91 (88).
3-(4-(4-(1,3-dioxolan-2-yl)benzyloxy)benzyloxy)benzyl
methanesulfonate (12)
To a stirred solution of 11 (0.98 g, 2.5 mmol) in dichloromethane
(15 cm3), were added triethylamine (0.53 cm3, 3.8 mmol) and
methanesulfonyl chloride (0.23 cm3, 3.0 mmol) at 0 ꢃC. The
reaction was stirred for 2 h. Then, the solution was washed with
water and the organic layer was dried under sodium sulfate,
filtered and evaporated to afford the desired product 12 (1.2 g,
92%) as a white powder: dH(500 MHz; CDCl3) 2.87 (3 H, s,
SO3CH3), 4.14–4.024 (4 H, m, 2 ꢁ CHOCH2), 4.99 (2 H, s,
SO3CH2), 5.09 (2 H, s, PhOCH2), 5.20 (2 H, s, PhOCH2), 5.83
(1 H, s, CHOCH2), 6.98 (2 H, d, J 8.6, 2 ꢁ H-Ph), 6.99 (2 H, m,
2 ꢁ H-Ph), 7.01 (1 H, br d, H-Ph), 7.30 (1 H, t, J 7.8, H-Ph), 7.34
Methyl 3-(4-(4-(1,3-dioxolan-2-yl)benzyloxy)benzyloxy)
benzoate (10)
To a mixture of 9 (0.93 g, 3.25 mmol), m-hydroxybenzoic acid
methyl ester (0.55 g, 3.62 mmol) and triphenylphosphine (PPh3)
(0.95 g, 3.62 mmol) in THF (20 cm3) at 0 ꢃC was added dropwise
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Scheme 1 Synthesis of materials. a: p-hydroxybenzoic acid methyl ester, 18-C-6, K2CO3 acetone, b: LAH, THF, c: m-hydroxybenzoic acid methyl ester,
PPh3, DIAD,THF. d: MsCl, DCM, Et3N.
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(2 H, d, J 8.6, 2 ꢁ H-Ph), 7.45 (2 H, d, J 8.3, 2 ꢁ H-Ph), 7.50
(2 H, d, J 8.3, 2 ꢁ H-Ph); dC(125 MHz; CDCl3) 38.3, 65.2, 69.6,
69.7, 71.3, 103.4, 115.0, 114.9, 115.0, 115.9, 121.1, 126.7, 127.2,
128.9, 129.2, 130.0, 134.7, 137.7, 137.9, 158.5, 159.0; m/z (EI) 269
(43, M-C8H9O4S), 163 (100, M-C15H15O5S), 91 (61).
Protein immobilization and measurement of the protein binding
capacity by FIA
Five milligrams of dendronized silica were poured into 6 tubes,
and then incubated for 7 h. with 200 ml of 400 mg/cm3 BSA
solution in 10% THF in PBS. The imines were reduced by
a solution of 0.02 M NaCNBH3. The tubes were centrifuged, and
the precipitate was washed 6 times with 10% THF in PBS. Non-
dendronized silica amine was used as the control. Blocking of
non-specific binding sites was done by incubating the samples
with 200 ml of 1 M glycine in 10% THF in PBS for 2 h. The tubes
were centrifuged and the supernatant removed and washed twice
with 10% THF in PBS. The antibody binding reaction was per-
formed by incubation of the samples with FITC conjugated anti
BSA, 100 ml of 1/100 diluted commercial solution to each tube for
8 h. Four washing steps followed.
Modification of the silica gel surface
Modification of the silica gel surface was carried out by standard
silanization procedures using 3-aminopropyltrimethoxysilane.
To a stirred suspension of silica gel (10 g) in dry toluene (120
cm3) under nitrogen was added
a solution of 3-amino-
propyltrimethoxysilane (3.6 cm3) and the mixture was refluxed
for 80 h. After cooling to room temperature it was filtered by
suction and washed with toluene, dichloromethane, and meth-
anol and dried in vacuo to afford 13.
The modified silica materials were mixed with 150 ml of 10%
THF in PBS and moved to a fluorescence 96-well black plate.
The fluorescence was read in wavelengths of 528/485 nm.
The amount of amino groups according to elemental
(nitrogen) analysis introduced onto silica gel was 1.6 mmol gꢀ1
(amino functionalized silica gel).
Acknowledgements
Dendron immobilization on 3-aminopropyltrimethoxysilane
modified silica 13
We thank Prof. Micha Polak for useful discussions regarding the
XPS analyses. This research was supported by the Israel Science
Foundation (227/05) and The Edmond J. Safra Foundation.
Modified silica gels 14, 17, 20, 23 (Scheme 1) were prepared by
post-synthetic immobilization of protected compounds 1, 4, 7
and 12 through the reactive functional group (either the bromide
or mesylate) at the focal point. Aldehyde terminated materials
were obtained by acetal deprotection by acidic hydrolysis. The
References
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€
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