Site-specific immobilization of proteins at zeolite L crystals by nitroxide exchange reactions

Article abstract of DOI:10.1039/c0cc05474g

Site-selective immobilization of dyes and different protein recognizing entities at the surface of zeolite L crystals using mild radical nitroxide exchange reactions is reported. Exposure of these crystals to aqueous protein solutions leads to site-select

Full text of DOI:10.1039/c0cc05474g

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COMMUNICATION
Site-specific immobilization of proteins at zeolite L crystals by nitroxide
exchange reactionsw
Maike Becker,a Luisa De Cola*b and Armido Studer*a
Received 9th December 2010, Accepted 28th January 2011
DOI: 10.1039/c0cc05474g
Site-selective immobilization of dyes and different protein
recognizing entities at the surface of zeolite L crystals using
mild radical nitroxide exchange reactions is reported. Exposure
of these crystals to aqueous protein solutions leads to site-
selective immobilization of proteins onto the crystals.
Scheme 1 Thermal reversible nitroxide exchange.
Nano- or microscopic inorganic particles linked to functional
Zeolite L,10 a crystalline aluminosilicate, has a defined
(bio)molecules have attracted increased attention during the
morphology, is optically transparent, and can be prepared at
a defined size ranging from 30 nm to 10 mm.11 Zeolite L is
functionalized readily at the external and internal surfaces12
and the crystals are biocompatible. Furthermore, it is possible
to conjugate either the channel entrances or the whole crystal
surface selectively with amine functionalities.13
past years. These biohybrids can be used as components in
optoelectronic devices or drug-delivery systems in the fields
of engineering and biosciences.1 Therefore, improvement of
existing methodologies and development of new concepts for
site-selective chemical surface modification are important. As
known procedures often show incomplete surface modification
or require comparatively harsh reaction conditions, there is
still a demand to develop generally applicable, robust and
experimentally easy to conduct surface chemistry.2 Little is
known about reversible covalent bond formation at inorganic
materials which should be useful for the generation of dynamic
adaptable organic/inorganic hybrid materials.3 Self-assembly
and aggregation behaviour of synthetic hybrid materials, for
example surface modified gold nanoparticles,4a dendrimers4b
and more generally inorganic/polymer hybrid nanostructures,4c
have been investigated. Physical properties like fluorescence5
or magnetic behaviour6 can be tuned as a function of the
hybrid constitution. In the present communication we report
mild reversible nitroxide exchange reactions in alkoxyamines
which are site-selective covalently bound to zeolite L crystals.7
Recently, we successfully achieved dynamic assembly of
zeolite L microcrystals by nitroxide exchange reactions.8 In
these processes, transient C-radicals and persistent nitroxide
radicals are generated by reversible thermal C–O bond homolysis
of the alkoxyamines (Scheme 1).9 Alkoxyamine formation by
cross coupling of the nitroxide with the C-radical leads to cross
over of the nitroxide moieties.
In the present study, zeolite L crystals with a length of 3 mm
were site-selectively functionalized with alkoxyamines bearing
a carboxylic acid functionality by amide bond formation with
amine groups selectively attached at the channel entrances or
all over the crystal (for preparation of these zeolites, see
ESIw).8 Scheme 2 shows the general concept of site-selective
nitroxide exchange for the immobilization of various interesting
molecules at the zeolite. These reactions can be performed
either at the pore entrances (hereafter designated as crystals
1a) or at the whole surface (1b) modified zeolite L crystals.
Initial studies were conducted at 125 1C for 2–4 h in
1,2-dichloroethane (DCE) with crystals of type 1a and 1b
using nitroxides 2 and 3 (large excess) bearing a fluorescent
dye (conditions A, see ESIw). The sample was then centrifuged
and the supernatant solvent was removed. A sample was
suspended in toluene, ultrasonicated, and a drop of the
suspension was placed on a microscope slide. The reaction
success was verified using fluorescence and confocal laser
microscopy. The images shown in Fig. 1 clearly prove the
success of the site-selective chemical functionalization of the
zeolites by thermal nitroxide exchange of crystals 1a and 1b
with nitroxides 2 and 3.
a Institute of Organic Chemistry, University of Munster,
¨
Corrensstr. 40, 48149 Munster, Germany.
¨
We then analyzed the influence of the structure of the
alkoxyamine moiety on the exchange reaction. To this end,
we prepared two additional types of alkoxyamine zeolite
conjugates 7a, 8a (Scheme 2) and compared their reactivities
with the reactivity of the parent 1a bearing the 2,2,6,6-tetra-
methylpiperidin-N-oxyl radical (TEMPO) as the nitroxide
moiety. 7a bears a sterically more hindered 1-tert-butyl-
3,3,5,5-tetraethyl-2-piperazinone group14 and in 8a the TEMPO
E-mail: studer@uni-muenster.de; Fax: +49 (0)251-83-36523;
Tel: +49 (0)251-83-33291
b Department of Physics and Center for Nanotechnology, CeNTech,
University of Munster, Mendelstr. 7, 48149 Munster, Germany.
¨
¨
E-mail: decola@uni-muenster.de; Fax: +49 (0)251-980-2834;
Tel: +49 (0)251-980-2873
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c0cc05474g
c
3392 Chem. Commun., 2011, 47, 3392–3394
This journal is The Royal Society of Chemistry 2011

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Scheme 2 (i) Nitroxide exchange reaction on entrance functionalized zeolite L crystals 1a for immobilization of various biomolecules at the
crystal pore entrances under mild conditions. (ii) Schematic representation of an alkoxyamine modified crystal of type 1b for all over chemical
surface modification. (iii) Zeolite conjugates 7a and 8a.
Na,Na-bis(carboxymethyl)-L-lysine hydrate (NTA) linker
(Fig. 2). Nitroxide exchange was performed for 2–4 h at
125 1C or rt in DCE or water. Unprotected mannose 9 was
immobilized site-specifically at the zeolite L surface. The success
was proven by subsequent complexation of the crystals with
rhodamine labelled concanavalin A (ConA)16,17a (Fig. 2: 10a,
10b). For complexation, the crystals were treated with an excess
of protein in diluted buffer at rt for 1–4 h (conditions B, ESIw).
Fluorescence images show a specific red fluorescence either at
the channel entrances or at the whole surface depending on the
crystal type 1a or 1b used for the initial exchange.17b
Fig. 1 Red (2, rhodamine) and yellow (3, nitrobenzooxadiazole
(NBD)) fluorescent nitroxides. Fluorescence images 4a, 4b (with 3 and
1a, 1b), 6a, 6b (with 2 and 1a, 1b) and confocal laser microscopy images
5a, 5b (with 2 and 1a, 1b) recorded after nitroxide exchange reactions.
To exclude non-specific binding of ConA onto the surface,
we performed control experiments of non-functionalized as
well as alkoxyamine modified zeolites. After extensive washing
only little fluorescence was observed (ESIw).18 We then
decided to apply the extraordinarily high and robust
biotin–streptavidin binding for selective protein immobilization.19
Nitroxide exchange with biotin derivative 11, followed by
alkoxyamine is connected to the surface via a tetraethylene
glycol linker (see ESIw).
We found that in comparison to zeolite 1a, neither immobi-
lization of the sterically hindered alkoxyamine nor the tetra-
ethylene glycol linker resulted in a measurable difference in the
nitroxide exchange with 2 at 125 1C.
We also studied the effect of the reaction temperature on the
surface exchange reactions on crystals 1a and 7a, 7b at 125 1C,
90 1C, 70 1C and at room temperature (rt) with nitroxide 2.
Pleasingly, exchange at the zeolite L surface was also possible
at rt for both systems 1 and 7. This is in agreement with our
previously reported results.8,15 As expected, reactions occurred
faster at higher temperature and we noted only a small difference
for the rt experiments in going from system 1a to 7a (see ESIw).
We could also show that the nitroxide exchange is a reversible
process. To this end, we treated rhodamine conjugated zeolite L
crystals (images 5a, 6a in Fig. 1) with an excess of TEMPO for
4 h in DCE at 125 1C and found that the red fluorescence of the
dye could no longer be observed at the pore entrances (see ESIw).
Exchange reactions worked equally well in H2O.
As radical chemistry tolerates various functional groups,
our approach should allow for site-selective immobilization of
different biomolecules and proteins at the crystal. We synthe-
sized nitroxides 9, 11 and 13 bearing mannose, biotin and the
Fig. 2 Nitroxides 9, 11, 13 and 15 (left). Fluorescence microscopy
images of site-selective protein immobilization (a) with crystals 1a and (b)
with crystals 1b: ConA (10), streptavidin (12), eGFP (14) and BSA (16).
c
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Chem. Commun., 2011, 47, 3392–3394 3393

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interaction with green fluorescence labelled streptavidin
(Oysters-488) allowed for site-specific protein immobilization
(Fig. 2; 12a and 12b). We further extended this concept using
the well established complexation of his-tagged eGFP to nickel
bound NTA. After rt nitroxide exchange on 1a and 1b with
NTA nitroxide 13, subsequent complexation of Ni2+ and
addition of his-tagged eGFP, site-selective protein immobili-
zation was achieved (Fig. 2, 14a and 14b).
3 Dynamic covalent bonds: P. T. Corbett, J. Leclaire, L. Vial,
K. R. West, J.-L. Wietor, J. K. M. Sanders and S. Otto, Chem.
Rev., 2006, 106, 3652; J.-M. Lehn, Chem. Soc. Rev., 2007, 36, 151.
4 (a) L. R. Hirsch, R. J. Stafford, J. A. Bankson, S. R. Sershen,
B. Rivera, R. E. Price, J. D. Hazle, N. J. Halas and J. L. West,
Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 13549; (b) E. C. Wiener,
M. W. Brechbiel, H. Brothers, R. L. Magin, O. A. Gansow,
D. A. Tomalia and P. C. Lauterbur, Magn. Reson. Med., 1994,
31, 1; (c) Y. Wang, A. Yu and F. Caruso, Angew. Chem., Int. Ed.,
2005, 44, 2888.
5 M. Bruchez Jr, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos,
Science, 1998, 281, 2013.
6 S. Angelos, E. Choi, F. Vogtle, L. De Cola and J. I. Zink, J. Phys.
¨
Chem. C, 2007, 111, 6589.
We also focused on the direct covalent attachment of
a protein to a zeolite crystal and chose the reliable reaction
of succinimido maleimides with thiols for covalent attachment
of a nitroxide to a protein.20 The TEMPO-maleimide derivative
1521 was reacted with free thiols of unfunctionalized
b-lactoglobulin A (LGA) or of rhodamine tagged bovine
serum albumin (BSA). Mass spectrometry analysis revealed
successful conjugation of the proteins with 15 (see ESIw). For
LGA a single product with the correct mass (LGA + 15) was
identified. However, for the heterogeneous BSA, as expected,
several product peaks were observed. Both nitroxide modified
proteins were then reacted with crystals 1a and 1b at rt.
Site-specific red fluorescence was detected in the reaction with
the BSA-nitroxide (Fig. 2, 16a and 16b). For the non-fluorescent
LGA nitroxide, we conducted z potential measurements and
compared the results with the z potentials of the fluorescent
7 Polymers and polymer brushes by radical nitroxide exchange:
C. J. Hawker, G. G. Barclay and J. Dao, J. Am. Chem. Soc., 1996,
118, 11467; N. J. Turro, G. Lem and I. S. Zavarine, Macromolecules,
2000, 33, 9782; O. G. Ballesteros, L. Maretti, R. Sastre and
J. C. Scaiano, Macromolecules, 2001, 34, 6184; Y. Higaki,
H. Otsuka and A. Takahara, Macromolecules, 2004, 37, 1696;
G. Yamaguchi, Y. Higaki, H. Otsuka and A. Takahara, Macro-
molecules, 2005, 38, 6316; S. B. Jhaveri, M. Beinhoff, C. J. Hawker,
K. R. Carter and D. Y. Sogah, ACS Nano, 2008, 2, 719; M. K. Brinks
and A. Studer, Macromol. Rapid Commun., 2009, 30, 1043; Nitroxides
in synthesis see: T. Vogler and A. Studer, Synthesis, 2008, 1979.
8 B. Schulte, M. Tsotsalas, M. Becker, A. Studer and L. De Cola,
Angew. Chem., Int. Ed., 2010, 49, 6881.
9 H. Fischer, Chem. Rev., 2001, 101, 3581; A. Studer, Chem.–Eur. J.,
2001, 7, 1159; A. Studer, Chem. Soc. Rev., 2004, 33, 267; A. Studer
and T. Schulte, Chem. Rec., 2005, 5, 27.
10 G. Calzaferri and A. Devaux, in Supramolecular Effects in Photo-
chemical and Photophysical Processes, ed. V. Ramamurthy and
Y. Inoue, Wiley, New York, 2010.
BSA–zeolite conjugates.
z potential measurements were
performed at rt at pH 7.4 in NH4HCO2-buffer. z (mV) were
similar (pore entrance modified BSA–zeolite: À31.32;
LGA–zeolite: À23.7; all over modified BSA: À2.89; LGA:
À5.42). Based on these results and the fluorescence images
shown in Fig. 2, we believe that both BSA and LGA were
immobilized successfully by nitroxide exchange reactions.
We reported that surface nitroxide exchange reactions
are well suited for a site-selective modification of zeolite L
microcrystals. We have shown that the nitroxide exchange is a
reversible process that can be conducted under mild, physio-
logical conditions at the zeolite surface. Proteins can be
immobilized site-specifically to zeolite L crystals by covalent
bond formation or by using his-tag, streptavidin tag or
concanavalin A–mannose interactions.
11 S. Megelski and G. Calzaferri, Adv. Funct. Mater., 2001, 11, 277;
A. Zabala Ruı
Chem., 2005, 136, 77; A. Zabala Ruı
and G. Calzaferri, in Materials Syntheses, A Practical Guide, ed.
´
z, D. Bruhwiler, T. Ban and G. Calzaferri, Monatsh.
¨
´
z, D. Bruhwiler, L.-Q. Dieu
¨
U. Schubert, N. Husing and R. Laine, Springer, Wien, 2008.
¨
12 A. Devaux, Z. Popovic, O. Bossart, L. De Cola, A. Kunzmann and
G. Calzaferri, Microporous Mesoporous Mater., 2006, 90, 69;
A. Zabala Ruız, H. Li and G. Calzaferri, Angew. Chem., Int.
´
Ed., 2006, 45, 5282; Z. Popovic, M. Otter, G. Calzaferri and L. De
Cola, Angew. Chem., Int. Ed., 2007, 46, 6188; G. Calzaferri,
´
A. Zabala Ruız, H. Li and S. Huber, Patent WO, 2007/012216.
13 S. Huber and G. Calzaferri, Angew. Chem., Int. Ed., 2004, 43, 6738;
M. Busby, H. Kerschbaumer, G. Calzaferri and L. De Cola,
Adv. Mater., 2008, 20, 1614.
14 S. Miele, P. Nesvadba and A. Studer, Macromolecules, 2009, 42,
2419; I. C. Wienhofer, A. Studer, Md. T. Rahman, T. Fukuyama
¨
and I. Ryu, Org. Lett., 2009, 11, 2457.
We thank the DFG (TRR61) for funding and Dr. Manuel
Tsotsalas for the preparation of zeolite L crystals.
15 S. Marque, H. Fischer, E. Baier and A. Studer, J. Org. Chem.,
2001, 66, 1146; A. L. J. Beckwith, V. W. Bowry and K. U. Ingold,
J. Am. Chem. Soc., 1992, 114, 4983; A. Studer, Angew. Chem.,
Int. Ed., 2000, 39, 1108.
16 J. W. Becker, G. N. Reeke Jr, B. A. Cunningham and
G. M. Edelman, Nature, 1976, 259, 406; A. J. Kalb (Gilboa) and
J. R. Helliwell, in Handbook of Metalloproteins, ed. A. Messerschmidt,
R. Huber, T. Poulos and K. Weghardt, Wiley, New York, 2001.
17 (a) D. K. Mandal, N. Kishore and C. F. Brewer, Biochemistry,
1994, 33, 1149; (b) Binding of ConA indicates a high mannose
concentration at the surface. Binding of ConA to single mannose is
weak (glycocluster effect): J. J. Lundquist and E. J. Toone, Chem.
Rev., 2002, 102, 555.
18 Adsorption of proteins to zeolites is comparatively weak at pH
values around 7 and stronger for small nanozeolites at higher
temperatures: Y.-Y. Hu, Y.-H. Zhang, N. Ren and Y. Tang,
J. Phys. Chem. C, 2009, 113, 18040; A. Tavolaro, P. Tavolaro
and E. Drioli, Colloids Surf., B, 2007, 55, 67.
Notes and references
1 G. Schmid, Nanoparticles, from Theory to Application, Wiley-
VCH, Weinheim, Germany, 2003; L. Nebhani and C. Barner-
Kowollik, Adv. Mater., 2009, 21, 3442; G. Whitesides, Nat.
Biotechnol., 2003, 21, 1161; M. Ferrari, Nat. Nanotechnol., 2008,
3, 131; J. Schulte, Nanotechnology: Global Strategies, Industry
Trends and Applications, Wiley, Portland, OR, 2005; V. Wagner,
A. Dullaart, A. Bock and A. Zweck, Nat. Biotechnol., 2006, 24,
1211; M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161; K. Park, S. Lee,
E. Kang, K. Kim, K. Choi and C. Kwon, Adv. Funct. Mater., 2009,
19, 1553; D. A. LaVan, T. McGuire and R. Langer, Nat. Biotechnol.,
2003, 21, 1184.
2 M. J. Shenton and G. C. Stevens, J. Phys. D: Appl. Phys., 2001, 34,
2761; C. Oehr, M. Muller, B. Elkin, D. Hegemann and U. Vohrer,
¨
19 N. M. Green, Adv. Protein Chem., 1975, 29, 85.
Surf. Coat. Technol., 1999, 116, 25; P. Favia and R. d’Agostino,
Surf. Coat. Technol., 1998, 98, 1102; C.-M. Chan, T.-M. Ko and
H. Hiraoka, Surf. Sci. Rep., 1996, 24, 1; L. J. Gerenser, J. Adhes.
Sci. Technol., 1993, 7, 1019; K.-K. Iu, X. Liu and J. K. Thomas,
J. Photochem. Photobiol., A, 1994, 79, 103; M. Yamaguchi,
A. Tanaka and T. Suzuki, J. Electron Microsc., 1992, 41, 7.
20 H. Y. Song, M. H. Ngai, Z. Y. Song, P. A. MacAry, J. Hobley and
M. J. Lear, Org. Biomol. Chem., 2009, 7, 3400; S.-J. Xiao,
M. Wieland and S. Brunner, J. Colloid Interface Sci., 2005, 290,
172; M. D. Partis, D. G. Griffiths, G. C. Roberts and
R. B. Beechey, J. Protein Chem., 1983, 2, 263.
21 G. Wu and W. L. Hubbell, Biochemistry, 1993, 32, 879.
c
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