Functionalization of fluorous thin films via"click" chemistry

Article abstract of DOI:10.1039/b821148e

The first covalent modification of thin films non-covalently immobilized via fluorous interactions was demonstrated with "click" reactions in 70-80% yields.

Full text of DOI:10.1039/b821148e

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Functionalization of fluorous thin films via ‘‘click’’ chemistryw
Catherine M. Santos, Amit Kumar, Wen Zhang and Chengzhi Cai*
Received (in Berkeley, CA, USA) 26th November 2008, Accepted 16th March 2009
First published as an Advance Article on the web 9th April 2009
DOI: 10.1039/b821148e
The first covalent modification of thin films non-covalently
immobilized via fluorous interactions was demonstrated with
‘‘click’’ reactions in 70–80% yields.
Surface functionalization based on ‘‘click’’ chemistry1 with
copper-catalyzed azide–alkyne cycloaddition (CuAAC)
reactions has attracted great interest.1–3 Although CuAAC
on a variety of substrates has previously been demonstrated,2,4–11
introducing suitable handles to the substrate surface to render
it ‘‘clickable’’ has been achieved through covalent immobiliza-
tion, which can be cumbersome. For example, glass surfaces
presenting ethynyl or azido groups were prepared by lengthy
reactions.5,7 Since many applications involving optical
detection are performed on glass substrates, a practical
method for generating ‘‘clickable’’ glass surfaces is valuable.
Herein, we show such a method based on the fluorous
immobilization of ‘‘clickable’’ thin films on commercially
available fluorous glass slides. We demonstrate that the
fluorous immobilized films are sufficiently stable to allow
functionalization via CuAAC in aqueous or suitable organic
solvents, and in a microarray format. This method is
complimentary to the direct fluorous immobilization of tagged
molecules,12–17 and extends the application of fluorous
thin films.
The efficiency of direct fluorous immobilization has been
demonstrated by the preparation of high quality microarrays
Scheme 1 The preparation of ‘‘clickable’’ fluorous films and demon-
of fluorous-tagged carbohydrates and small molecules on
fluorous surfaces.12–16 Despite the exciting development of
this practical immobilization method, its scope and limitations
await to be established. For example, for systems where
non-fluorous interactions are strong, a sufficiently large
perfluorocarbon tag, e.g., C8F17, is required to immobilize
the molecules.13 However, large fluorous tags greatly decrease
the solubility of molecules in aqueous solution. In fact, to
date all fluorous thin films were deposited from a solution
containing organic solvents that may possibly denature the
molecules. These concerns can be circumvented by our
click chemistry-based approach on fluorous thin films.
Furthermore, click chemistry is specific and compatible with
a wide variety of functional groups, and can be performed in
both aqueous and organic solvents.1,2
stration of their covalent functionalization via the CuAAC reaction.
prepared simply by immersion of a fluorous glass slide
(Fluorous Tech., Inc.) in a 1 mM solution of the fluorous
azide 1 or alkyne 2 in methanol, followed by washing with
methanol. The CuAAC reaction on the surfaces were
catalyzed by a complex of Cu+ with ligand 3 containing a
secondary amine and a triazole ring. The amine is as an
electron donor to Cu+, which accelerates the reaction, while
the triazole ring readily dissociates to allow formation of the
Cu(I)-acetylide–ligand complex.18 The Cu+–ligand 3 complex
is similar to those reported by Fokin and coworkers for
CuAAC,18 but is modified with oligo(ethylene glycol) chains
to increase water solubility. Thus, using a general procedure,19
the sulfur-containing alkyne 4, the azido-tagged biotin
derivative 5, or the FITC-labeled azide 6 was readily ‘‘clicked’’
onto surface A or B, leading to surface C, D or E.
As shown in Scheme 1, ‘‘clickable’’ surfaces A and B,
presenting azido and ethynyl groups, respectively, were readily
All films were characterized by X-ray photoelectron spectro-
scopy (XPS), and selected results are presented in Fig. 1. An
XPS survey of azido-terminated surface A (Fig. 1(a)) shows
the presence of the expected C, F, N and O signals. The broad
N 1s signal in the high resolution scan (Fig. 1(b)) was fitted
and deconvoluted into three peaks: 400.1 eV and 403.2 eV
attributed to the azido group (NQNQN and NQNQN,
Department of Chemistry & Center for Materials Chemistry,
University of Houston, Houston, Texas 77204, USA.
E-mail: cai@uh.edu; Fax: +1 713 743 2709; Tel: +1 713 743 2710
w Electronic supplementary information (ESI) available: Experimental
procedures for the surface modification and characterizations, and the
synthesis and characterization of all compounds used. See DOI:
10.1039/b821148e
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169.8 eV, attributable to the sulfonic acid group22 (Fig. 1(c)).
The broad N 1s signal was centered at 400.4 eV (Fig. 1(d)).
The S : N ratio provides a rough estimation of the reaction
yield of B80%.23 The broad N 1s peak can be fitted and
deconvoluted into five peaks with the following tentative
assignments: 398.7 eV (N–NQN),6 400.0 (NQN),6 400.2
(NQNQN), 401.2 (OQC–N) and 403.2 eV (NQNQN).
The ratio of the peak areas is 0.7 : 1.5 : 0.5 : 2.5 : 0.2, consistent
with the resulting surface upon the CuAAC reaction in B80%
yield. In a control experiment, surface A was subjected to the
same reaction conditions but in the absence of copper. No
peak was observed in the high resolution scan of the S 2p
region (Fig. 1(c), red curve), thus, no attachment of alkyne 4 to
azido surface A was observed in the absence of the catalyst for
the CuAAC reaction.
CuAAC could also be performed on ethynyl-terminated
surface B (Scheme 1) with azido-labeled biotin 5 following
the general procedure.19 The XPS narrow scans of the S 2p
and N 1s regions for the resulting biotinylated surface, D, are
shown in Fig. 1(e) and (f), respectively. Based on the S : N
ratio, the yield of the reaction was estimated to be B70%.23
This yield is among the highest of those reported for click
reactions of azides with surfaces presenting ethynyl groups.2
The close proximity of the ethynyl groups may lead to side
reactions, such as copper-catalyzed homocoupling in the
presence of adventitious O2.24 A broad peak at 162.7 eV in
the S 2p region was attributed to the biotin.25 The broad N 1s
signal was fitted and deconvoluted into three peaks with the
following tentative assignments: 398.7 (N–NQN), 400.0
(NQN) and 401.2 eV (OQC–N). The ratio of the peak areas
is 0.7 : 1.4 : 3.2, which correlates well with the calculated ratio
for a 70% yield reaction (0.7 : 1.4 : 3.1). Unreacted azides were
not present after the CuAAC reaction, as no peak was
observed near 403 eV.
The presence of biotin covalently attached to the surface
upon CuAAC was confirmed by its specific binding with
FITC-labeled avidin. Specifically, a solution of FITC–avidin
(0.5 mg mLÀ1) in PBS buffer was spotted on a biotinylated
surface, D. As a control, a solution of biotin-saturated
FITC–avidin (1 mg mLÀ1) in PBS buffer was spotted on the
Fig. 1 XPS spectra of the fluorous thin films before and after the
CuAAC reactions. (a) Survey and (b) 1s narrow scan of
N
azido-modified surface A. The inset of (b) is the overlay of the spectra
obtained before and after 4 h immersion of surface A in methanol.
(c) S 2p and (d) N 1s narrow scan of surface C upon CuAAC reaction
of surface A and 4. The red curve in (c) is the negative control without
adjacent area. The spotting was performed using a SpotBots
2
microarrayer (Telechem Int. Inc., CA). The samples were
incubated at 58% relative humidity for 30 minutes, and then
washed twice with PBS buffer. Fluorescence images show that
the FITC–avidin bound to surface D (Fig. 2(a)), while biotin-
saturated FITC–avidin did not bind to the surface (Fig. 2(b)),
thus proving that the surface was biotinylated and specifically
bound to avidin.
the copper catalyst. (e)
S 2p and (f) N 1s narrow scan of
biotin-presenting surface D. The dotted lines under the deconvoluted
N 1s curves correspond to the difference between the original and
fitted curves.
respectively),4,20 and 401.3 eV assigned to the amide
nitrogen.6,21 The calculated ratio of the areas under the three
peaks is 1.9 : 1 : 1.1, close to the expected ratio (2 : 1 : 1) for
film A. To address the stability of the films during the reaction,
a sample of A was immersed in methanol for 4 h. XPS showed
that the intensities of the N signal remained unchanged
(inset of Fig. 1(b)). Furthermore, only a small change in the
F : C ratio (1.19 vs. 1.21) before and after the immersion was
observed. The films were also stable in PBS buffer for 4 h
(data not shown).
Upon CuAAC reaction between surface A and alkyne 4 to
form surface C, XPS showed the presence of an S 2p peak at
Fig. 2 Fluorescence image of biotinylated surface D after spotting a
solution of (a) FITC–avidin and (b) biotin-saturated FITC–avidin.
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Notes and references
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Fig. 3 Functional microarrays on fluorous slides via click chemistry.
(a) Fluorescence image after incubation of arrays of azide 6 in the
presence of the Cu+ catalyst on ethynyl-terminated surface B.
(b) Fluorescence image of the negative control with 6 on B without
the Cu+ catalyst. (c) Fluorescence image after incubation of arrays of
FITC–BSA–azido on B. (d) Fluorescence image of the negative
control with FITC–BSA–azido on B without the Cu+ catalyst.
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19 General procedure for the surface CuCAAC reaction: to a reaction
vial containing the azido- or alkynyl-terminated surfaces A or B
was added a solution of Cu(MeCN)4PF6 (2.5 mM), ascorbic acid
(50 mM), and 3 (25 mM) in degassed methanol, or a solution of
CuSO4 (5 mM), ascorbic acid (50 mM), and 3 (25 mM) in water.
After 10 min, a solution of alkyne 4, or azide 5 or 6 (10 mM) in
methanol–water (1 : 1 v/v) or water was added. After incubation
for at least 4 h under N2, the film was taken out and immersed in
EDTA (10 mM) for 5 min followed by washing with methanol
and water, and drying under a stream of argon.
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The surface CuAAC reaction on the fluorous immobilized
thin films could also be performed in microarray format. Thus,
a mixture of the azide-tagged FITC dye 6 (10 mM) with
Cu(MeCN)4PF6 (2.5 mM), ligand 3 (25 mM) and ascorbic
acid (50 mM) in a methanol–water (1 : 9 v/v) mixture was
spotted on the ethynyl-terminated fluorous thin film B
(Scheme 1). Similarly, click reactions on a microarray format
were also performed to attach proteins, such as BSA
(bovine serum albumin) modified with both FITC and azido
groups (see the ESIw). The samples were incubated at 58%
relative humidity for 6 h, and washed with water and methanol
(see the ESIw). The fluorescence images in Fig. 3(a) and (c)
show the intense green spots corresponding to the immobilized
FITC dye in the resulting surface, E, and FITC-labelled BSA,
respectively. As a negative control, the same reaction mixtures
in the absence of the copper catalyst were spotted on adjacent
areas. No fluorescence was observed on these areas (Fig. 3(b)
and (d)) upon otherwise the same treatment, thus establishing
that the molecules cannot be immobilized in the absence of the
Cu+ catalyst for the click reaction.
In conclusion, we have developed a practical method for
surface immobilization on fluorous substrates via click
chemistry. The thin films with ethynyl or azido handles are
readily prepared on commercial fluorous glass slides, and such
non-covalently immobilized thin films are surprisingly stable,
allowing covalent functionalization in good yields (70–80%)
via CuAAC reactions in aqueous or suitable organic solvents,
and in microarray format. This method is complementary to
the direct deposition of fluorous-tagged compounds. Further-
more, the same approach can be used for other ‘‘click’’
reactions on fluorous surfaces.
23 The percentage yield was calculated using the following formulae:
for the click reaction of sulfonic acid alkyne 4 on azide-modified
fluorous film A; yield (%) ={1/((N/S) À 6)} Â 100, and for the
reaction of the biotin-functionalized azide 5 on the alkyne modified
fluorous film B; yield (%) = {4/((N/S) À 2)Â 100.
This work was supported by the Welch Foundation, the
National Science Foundation grant DMR-0706627, the
Institute of Biomedical Imaging Science and the Texas Center
for Superconductivity at the University of Houston.
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2856 | Chem. Commun., 2009, 2854–2856