Chemically orthogonal trifunctional Janus beads by photochemical sandwich microcontact printing

Article abstract of DOI:10.1039/c2cc36483b

The combination of topographic and chemical orthogonality on polymer particles by site selective immobilization of functional thiols via thiol-ene chemistry provides a trifunctional particle surface with azide and acid functionalities on opposing poles and alkenes in the equatorial area. These Janus beads are accessible for site selective orthogonal chemical reactions as well as biomolecular recognition on the same particle.

Full text of DOI:10.1039/c2cc36483b

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Chemically orthogonal trifunctional Janus beads by
photochemical ‘‘sandwich’’ microcontact printing†
Cite this: Chem. Commun.,
2013, 49, 63--65
b
Tobias Kaufmann,a Christian Wendeln,a M. Talha Gokmen,z Stefan Rinnen,c
Received 5th September 2012,
Accepted 5th November 2012
Maria M. Becker,a Heinrich F. Arlinghaus,c Filip Du Prez*b and Bart Jan Ravoo*a
DOI: 10.1039/c2cc36483b
www.rsc.org/chemcomm
The combination of topographic and chemical orthogonality on minimal amounts of the functional material. Furthermore this
polymer particles by site selective immobilization of functional technique is easy and quick in execution, yet versatile with respect
thiols via thiol–ene chemistry provides a trifunctional particle to tolerable functional groups.18,19 We recently introduced the
surface with azide and acid functionalities on opposing poles and method of ‘‘sandwich’’ microcontact printing as a tool for Janus
alkenes in the equatorial area. These Janus beads are accessible for bead preparation20 and demonstrated its broad applicability.21
site selective orthogonal chemical reactions as well as biomolecular
recognition on the same particle.
In this report we show another versatile application of this
technique by adopting the principles of orthogonal chemistry.
The concept of orthogonal chemical modifications is based on
Janus beads are particles that display different properties on the introduction of two or more different functional groups in a
opposing sides.1,2 These particles are often spherical beads, system, allowing further post-functionalization by specific ligation
however the concept is not restricted to centrosymmetric reactions. The reactions have to be selected in such a way that all
geometries. Possible applications of such anisotropic particles functional groups involved tolerate each other and that exclusive
include the catalysis of two distinct chemical reactions in both addressing of every functional group is assured. During the last few
phases of an oil–water emulsion,3 display applications4 or years, orthogonal chemistry has for example been applied to modify
detection of multiple proteins on barcoded ‘‘Janus chips’’.5
polymers,22–24 dendrons and dendrimers25,26 and also surfaces.27–29
The preparation of bifunctional particles can be roughly In a recent paper we described the preparation of multifunctional
categorized into two main approaches: on the one hand, the orthogonal surfaces by the sequential immobilization of hetero-
particle synthesis can be exploited to realize anisotropic products. bifunctional crosslinkers using microcontact printing.30 Trifunc-
Such methods often employ either co-jetting6 or phase separation tional surfaces with alkene, acid and azide groups were prepared
of two building blocks.7,8 On the other hand, numerous strategies by printing an azide-thiol linker and an acid thiol linker on
have been developed to site selectively post-functionalize originally alkene-modified glass and silicon substrates using thiol–ene
isotropic particles. These strategies include vapor deposition of chemistry. In this communication, we use these hetero-bifunctional
metals9,10 or polymers,11 site specific chemical derivatization of linkers for the preparation of unprecedented chemically orthogonal
beads trapped in a protecting matrix12–14 and printing.15–17 trifunctional polymer Janus beads. Opposing sides of these beads
Especially the technique of microcontact chemistry has the with acid and azide groups are suitable for specific ligation with
beneficial features of linking ‘‘ink molecules’’ covalently and amines by peptide chemistry and with alkynes by copper catalyzed
site specifically onto the surface of the particle by using alkyne–azide cycloaddition (CuAAC), respectively. Remaining alkene
groups in unmodified areas of the Janus beads can be reacted with
thiols using thiol–ene chemistry.
a
¨
¨
Organisch-Chemisches Institut and CeNTech, Westfalische Wilhelms-Universitat
¨
¨
Munster, Corrensstraße 40, 48149 Munster, Germany
Trifunctional bead surfaces were prepared by printing func-
tional thiol inks onto isotropic alkene terminated polymer
particles as depicted in Scheme 1. As substrates, monodisperse
epoxy polymer beads of 5 mm or 170 mm diameter were used.
Their preparation by, respectively, precipitation polymerization
and microfluidics can be found elsewhere.21 The epoxy beads
were perfunctionalized with alkene groups by reaction with
allyl mercaptane (see ESI†). The steps for successful surface
modification with functional thiols include the following: a
first flat stamp made of poly(dimethyl siloxane) (PDMS) is
E-mail: b.j.ravoo@uni-muenster.de; Fax: +49 251 8336557; Tel: +49 251 8333287
b Department of Organic Chemistry, Ghent University, Krijgslaan 281 S4, 9000
Gent, Belgium. E-mail: Filip.DuPrez@UGent.be; Fax: +32 9 2644972;
Tel: +32 9 2644503
c
¨
¨
¨
Physikalisches Institut, Westfalische Wilhelms-Universitat Munster, Wilhelm-
¨
Klemm-Straße 10, 48149 Munster, Germany. E-mail: arlinghaus@wwu.de;
Fax: +49 251 8333682; Tel: +49 251 8339064
† Electronic supplementary information (ESI) available: Synthesis, general
procedures and surface analytical data. See DOI: 10.1039/c2cc36483b
‡ Currently staff scientist at Life Technologies, Svelleveien 29, 2004 Lillestrom,
Norway.
c
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Chem. Commun., 2013, 49, 63--65 63

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Scheme 1 Schematic illustration of the preparation of chemically orthogonal Janus
beads by ‘‘sandwich’’ microcontact printing of functional thiols on alkene bead
surfaces; (i) 80 mM thiol 1 and 2, 40 mM DMPA in ethanol, UV-LED (365 nm), 5 min.
Fig. 1 Fluorescent Janus particles obtained by coupling dansyl cadaverine and
rhodamine alkyne onto chemically orthogonal beads. Fluorescence microscopy
images (a, b), light microscopy image (c) and CLSM 3D volume plot (d). The
diameter of the particle in (d) is 5.6 mm.
covered with azido-thiol ink 1 solution (80 mM of 1 in ethanol
including 40 mM dimethoxyphenyl acetophenone (DMPA), see
ESI†), subsequently dried in a stream of argon and fixed into a
UV-transparent object carrier connected to a mechanical press.
A second flat stamp is loaded with the acid-thiol 2 (see ESI†) as
outlined above and a monolayer of alkene functionalized poly- the polymer beads, ethynyl trifluorotoluene (see ESI†) was coupled
mer beads is applied. The first stamp is brought into conformal to the azide-decorated side of the beads via CuAAC and the
contact with the polymer beads from the top and the stamp– Janus beads were imaged by Time-of-Flight Secondary Ion Mass
bead–stamp sandwich is irradiated with light of 365 nm using Spectrometry (ToF-SIMS). We emphasize that ToF-SIMS imaging
a
UV-LED for 5 min. The trifunctional polymer beads of the beads is highly challenging due to the curvature and poor
were cleaned by washing with solvents of different polarity conductance of the polymer microparticles. ToF-SIMS indicated
(water, dimethyl formamide, dichloromethane, diethyl ether) the formation of Fꢀ and of CNꢀ fragments selectively on one side
and subsequent extraction in hot ethanol for 9 h.
of the beads (see Fig. S2, ESI†). The fluoride signal indicates the
As a proof of concept experiment, acid and azide terminated successful regiospecific immobilization of the fluorine containing
poles of the beads were specifically brought to reaction with alkyne, whereas the CNꢀ fragment can be attributed to the
dansyl cadaverine via a peptide coupling reaction or rhodamine immobilization of the nitrogen containing azide-thiol linker in
alkyne using CuAAC chemistry, respectively. After detection of the same area. These measurements confirm the results obtained
each immobilized fluorophore individually (see Fig. S1, ESI†), by fluorescence microscopy.
both poles were derivatized in sequential reactions (peptide
In a more sophisticated experiment, protein-modified Janus
coupling followed by CuAAC) (Fig. 1). As can be observed from beads were prepared. Three different biomolecules were used
these images, both fluorophores were exclusively immobilized as protein binding sites on polymer beads: a biotin alkyne
on opposing poles of the beads due to selective reaction of derivative, an amino-a-mannoside and an amino-b-lactoside
dansyl cadaverine with surface bound carboxylic acid moieties (see ESI† for chemical structures). These ligands are known to
and rhodamine alkyne with surface azide groups. Large poly- bind specifically to different proteins, namely streptavidin,
mer beads of 170 mm diameter (a, c) are selectively modified as concanavalin A (ConA) and peanut agglutinin (PNA) respec-
well as small polymer beads of 5 mm diameter (b, d). The tively. Binding one of these ligands to one pole of the chemi-
constructed 3D-volume plot of stacked confocal laser scanning cally orthogonal Janus beads resulted in highly preferential
microscopy (CLSM) images (d) highlights the exclusiveness of adsorption of the respective proteins on one side of the beads
modification on opposing poles and a 3601 rotation (see video, (see Fig. S3, ESI†). In a following step, two of the reactive
ESI†) reveals the size of the printed patches on the small beads. bioligands were combined on one bead by sequential reaction
Furthermore, it should be noted that the ‘‘yield’’ of the two of biotin alkyne (by CuAAC) followed by either the amino-
sequential reactions of surface bound alkenes with azide thiol a-mannoside or the amino-b-lactoside (by peptide coupling).
during mCP and subsequent CuAAC with rhodamine alkyne from Subsequent incubation of the biofunctionalized Janus beads in
solution is obviously very high, as the rhodamine modified a mixed solution of fluorescently labeled proteins (DyLight 405-
patches can even be detected by light microscopy (c).
labeled streptavidin and rhodamine-labeled PNA or DyLight
In order to apply an additional analytical method to verify the 405-labeled streptavidin and fluorescein-labeled ConA) resulted
site selective orthogonal chemical attachment of ink molecules to in simultaneous yet site specific protein immobilization on the
c
64 Chem. Commun., 2013, 49, 63--65
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B.J.R. is grateful to the Deutsche Forschungsgemeinschaft
for financial support. F.D.P. acknowledges the Belgian Program
on Interuniversity Attraction Poles initiated by the Belgian
State, Prime Minister’s Office (Program P7/05) for financial
¨
support. Prof. H. J. Galla and P. Seelheim (Westfalische
¨
Wilhelms-Universitat Mu¨nster) are acknowledged for access
to and help with CLSM.
Notes and references
1 J. Du and R. K. O’Reilly, Chem. Soc. Rev., 2011, 40, 2402–2416.
2 J. Yoon, K. J. Lee and J. Lahann, J. Mater. Chem., 2011, 21,
8502–8510.
3 S. Crossley, J. Faria, M. Shen and D. E. Resasco, Science, 2010, 327,
68–72.
4 T. Nisisako, T. Torii, T. Takahashi and Y. Takizawa, Adv. Mater.,
2006, 18, 1152–1156.
Fig. 2 Schematic illustration of protein Janus beads. Overlay of fluorescence
images of rhodamine-labeled PNA bound to amino-b-lactoside and DyLight
405-labeled streptavidin bound to biotin alkyne (a) and fluorescein-labeled ConA
bound to amino-a-mannoside and DyLight 405-labeled streptavidin bound to
biotin alkyne (b).
5 D. Dendukuri, D. C. Pregibon, J. Collins, T. A. Hatton and
P. S. Doyle, Nat. Mater., 2006, 5, 365–369.
6 K.-H. Roh, D. C. Martin and J. Lahann, Nat. Mater., 2005, 4,
759–763.
beads (Fig. 2). Fig. 2 clearly demonstrates the site specific
immobilization of the ligands as well as their selectivity
towards proteins. We note that amino-functionalized carbo-
hydrates were immobilized on acid patches by two different
strategies. Whereas the amino-a-mannoside was bound to the
acid patch of the beads via peptide coupling reaction using
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoro-
borate (TBTU), the amino-b-lactoside was coupled to the beads
¨
7 R. Erhardt, A. Boker, H. Zettl, H. Kaya, W. Pyckhout-Hintzen,
G. Krausch, V. Abetz and A. H. E. Mu¨ller, Macromolecules, 2001,
34, 1069–1075.
8 I. K. Voets, A. de Keizer, P. de Waard, P. M. Frederik, P. H. H.
Bomans, H. Schmalz, A. Walther, S. M. King, F. A. M. Leermakers
and M. A. Cohen Stuart, Angew. Chem., Int. Ed., 2006, 45, 6673–6676.
9 Q. Chen, S. C. Bae and S. Granick, Nature, 2011, 469, 381–384.
10 Q. Chen, J. K. Whitmer, S. Jiang, S. C. Bae, E. Luijten and S. Granick,
Science, 2011, 331, 199–202.
in a two step procedure: acid functionalized beads were first 11 R. T. Chen, B. W. Muir, G. K. Such, A. Postma, K. M. McLean and
F. Caruso, Chem. Commun., 2010, 46, 5121–5123.
activated by reaction with dicyclohexyl carbodiimide and
N-hydroxysuccinimide and subsequently exposed to a solution
´
12 C. Casagrande and M. Veyssie, C. R. Acad. Sci., Ser. II, 1988, 306,
1423–1425.
of the b-lactoside in dimethyl formamide. As can be seen from 13 X. Y. Ling, I. Y. Phang, C. Acikgoz, M. D. Yilmaz, M. A. Hempenius,
G. J. Vancso and J. Huskens, Angew. Chem., Int. Ed., 2009, 48,
7677–7682.
14 K. Kamalasanan, S. Jhunjhunwala, J. Wu, A. Swanson, D. Gao and
the protein binding experiments, both chemical reactions
worked equally well.
A third functionality is present in the form of non-contacted
alkenes on the bead surface as well as in the bead interior.
These were addressed by thiol–ene reaction of a mannoside
thiol on large beads (170 nm diameter) that were previously
coupled with dansyl cadaverine by peptide coupling and
rhodamine alkyne by CuAAC. The surface bound mannoside
S. R. Little, Angew. Chem., Int. Ed., 2011, 50, 8706–8708.
15 O. Cayre, V. N. Paunov and O. D. Velev, Chem. Commun., 2003,
2296–2297.
16 Z. Li, D. Lee, M. F. Rubner and R. E. Cohen, Macromolecules, 2005,
38, 7876–7879.
17 S. Jiang and S. Granick, Langmuir, 2009, 25, 8915–8918.
18 B. J. Ravoo, J. Mater. Chem., 2009, 19, 8902–8906.
19 C. Wendeln and B. J. Ravoo, Langmuir, 2012, 28, 5527–5538.
was incubated with fluorescein-labeled ConA to visualize the 20 T. Kaufmann, M. T. Gokmen, C. Wendeln, M. Schneiders, S. Rinnen,
H. F. Arlinghaus, S. A. F. Bon, F. E. Du Prez and B. J. Ravoo, Adv.
Mater., 2011, 23, 79–83.
21 T. Kaufmann, M. T. Gokmen, S. Rinnen, H. F. Arlinghaus, F. E. Du
carbohydrate functionalization. However, fluorescence micro-
scopy (see Fig. S4, ESI†) does not show selective protein
attachment in the form of a ‘‘belt’’ structure in non-contacted
regions, but rather a homogeneous distribution of ConA in the
beads. This is ascribed to the porous nature of the employed
Prez and B. J. Ravoo, J. Mater. Chem., 2012, 20, 6190–6199.
22 M. Malkoch, R. J. Thibault, E. Drockenmuller, M. Messerschmidt,
B. Voit, T. P. Russell and C. J. Hawker, J. Am. Chem. Soc., 2005, 127,
14942–14949.
polymer particles. Although no site selective attachment can 23 S. K. Yang and M. Weck, Macromolecules, 2007, 41, 346–351.
¨
24 R. K. Iha, K. L. Wooley, A. M. Nystrom, D. J. Burke, M. J. Kade and
be observed, this result may be valuable when considering
applications in heterogeneous catalysis of chemical reactions,
where a high surface area equipped with a catalyst would be
desirable. Successful protein attachment clearly demonstrates
the accessibility of remaining alkenes on the surface and in the
interior of the beads.
In summary, we could demonstrate that three different
chemical functionalities can be introduced selectively in different
surface regions of small and large polymer microbeads. The
combination of topographic and chemical orthogonality on
polymer particles is unprecedented and provides access to a
wide range of Janus particles.
C. J. Hawker, Chem. Rev., 2009, 109, 5620–5686.
25 M. B. Steffensen and E. E. Simanek, Angew. Chem., Int. Ed., 2004, 43,
5178–5180.
¨
26 L. Roglin, E. H. M. Lempens and E. W. Meijer, Angew. Chem., Int.
Ed., 2011, 50, 102–112.
27 S. G. Im, K. W. Bong, B.-S. Kim, S. H. Baxamusa, P. T. Hammond,
P. S. Doyle and K. K. Gleason, J. Am. Chem. Soc., 2008, 130,
14424–14425.
28 N. Gupta, B. F. Lin, L. M. Campos, M. D. Dimitriou, S. T. Hikita,
N. D. Treat, M. V. Tirrell, D. O. Clegg, E. J. Kramer and C. J. Hawker,
Nat. Chem., 2010, 2, 138–145.
29 A. Pulsipher, N. P. Westcott, W. Luo and M. N. Yousaf, J. Am. Chem.
Soc., 2009, 131, 7626–7632.
30 C. Wendeln, S. Rinnen, C. Schulz, T. Kaufmann, H. F. Arlinghaus
and B. J. Ravoo, Chem.–Eur. J., 2012, 18, 5880–5888.
c
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Chem. Commun., 2013, 49, 63--65 65