Adding spatial control to click chemistry: Phototriggered diels-alder surface (Bio)functionalization at ambient temperature

Article abstract of DOI:10.1002/anie.201107095

A photoconjugation strategy based on light-triggered Diels-Alder addition of o-quinodimethanes is compatible with biomolecules and proceeds rapidly at ambient temperature without the need of a catalyst. Spatial control was confirmed by photopatterning of

Full text of DOI:10.1002/anie.201107095

Angewandte
Chemie
DOI: 10.1002/anie.201107095
Clickable Surfaces
Adding Spatial Control to Click Chemistry: Phototriggered Diels–
Alder Surface (Bio)functionalization at Ambient Temperature**
Thomas Pauloehrl, Guillaume Delaittre, Volker Winkler, Alexander Welle, Michael Bruns,
Hans G. Bçrner, Alexandra M. Greiner, Martin Bastmeyer, and Christopher Barner-Kowollik*
The recent years have witnessed a substantial increase of
attention in the modification of materials by the highly
efficient chemical methods often referred to as click chemis-
try.[1] The set of reactions belonging to this class exhibit
specific features such as fast reaction kinetics, high yields,
orthogonal reactivity, and tolerance to a wide range of
solvents. However, in some applications featuring surface
patterns or three-dimensional scaffolds, these characteristics
are not sufficient, because spatial control is also required.
Furthermore, temporal control of chemical reactions could
offer the significant advantage of triggering reactions at any
time, which would be particularly interesting when a specific
sequence of events is required. The use of light as a temporal
and spatial trigger has very recently been applied to click
methods.[2] For instance, Bowman and co-workers reported
patterns of hydrogels fabricated by azide–alkyne cycloaddi-
tion cross-linking initiated by the phototriggered reduction of
CuII into CuI, the latter being the catalyst for this reaction.[3]
However, their method presents the disadvantage of involv-
ing many species, such as the different copper species and the
radical photoinitiator required to generate CuI. Although no
such effect was reported, these species are able to diffuse in
the medium and could hinder the spatially resolved character.
The same applies to the widely used photoinitiated radical
thiol–ene and thiol–yne reactions.[4]
The key to achieve full spatial control is to immobilize one
of the two components and to directly activate it. Popik,
Locklin, and co-workers followed such a path when they used
immobilized cyclopropenone-masked cyclooctynes.[5] Under
UV light, decarbonylation occurs, releasing strained alkynes
reacting rapidly with azides in solution and producing
fluorophore patterns. Following the inspiring work of Lin
and co-workers,[6] who resurrected the seminal work of
Huisgen and Sustmann,[7] we recently explored the photo-
generation of nitrile imines from immobilized diaryl tetra-
zoles to pattern different polymers on cellulose by 1,3-dipolar
cycloaddition with maleimide-functionalized macromole-
cules.[8] Importantly, we have additionally introduced a
novel procedure for click conjugations based on Diels–
Alder addition of hydroxy-o-quinodimethanes (photoenols)
generated by photoisomerization of o-methylphenyl ketones
or aldehydes and demonstrated its ease and efficiency in light-
induced conjugations of polymeric building blocks.[9] The
latter strategy fulfills the harsh set of click conditions required
for polymer–polymer ligation.[1b] Upon performing a screen-
ing study of potential photoenol candidates, we have now
identified the 2-formyl-3-methylphenoxy (FMP) moiety to be
an even more efficient precursor compared to our previously
reported 2-methylbenzophenone derivative. Promotion of the
Diels–Alder endo addition occurs based on hydrogen bond
formation in the photoenol intermediate by increasing both
its lifetime and the amount of formed Z isomer (Scheme 1),
which is, in contrast to the E isomer,[10] highly reactive
towards dienophiles (for example, a maleimide derivative).
Initial model reactions were performed in solution, as
mass spectrometry of samples in solution is an efficient
method for identifying potential side-product formation with
much higher sensitivity and specificity than for example
1H NMR spectroscopy.[11] A 36 W compact fluorescent lamp
at the absorbance maximum of FMP (lmax = 320 nm; Sup-
porting Information, Figure S1) was employed as UV source.
The outcome of such an investigation is shown in Figure 1a,
which depicts the mass spectra of starting FMP-capped
poly(ethylene glycol) methyl ether before irradiation 2
alongside the photostable Diels–Alder cycloadduct 3. Full
conversion with maleimides is typically achieved in less than
15 minutes at ambient temperature (for a kinetic investiga-
[*] T. Pauloehrl, Dr. G. Delaittre, V. Winkler, Prof. Dr. C. Barner-Kowollik
Preparative Macromolecular Chemistry, Institut fꢀr Technische
Chemie und Polymerchemie and Centre for Functional Nano-
structures (CFN), Karlsruhe Institute of Technology (KIT), Enges-
serstrasse 18, 76128 Karlsruhe (Germany)
E-mail: christopher.barner-kowollik@kit.edu
Homepage: http://www.macroarc.de
Dr. G. Delaittre, Dr. A. M. Greiner, Prof. Dr. M. Bastmeyer
Zoologisches Institut, Zell- und Neurobiologie and Centre for
Functional Nanostructures (CFN), Karlsruhe Institute of Technology
(KIT), 76131 Karlsruhe (Germany)
Dr. A. Welle
Institute for Biological Interfaces (IBG I), Karlsruhe Institute of
Technology (KIT), 76344 Eggenstein-Leopoldshafen (Germany)
V. Winkler, Dr. M. Bruns
Institute for Applied Materials (IAM-WPT), Karlsruhe Institute of
Technology (KIT), 76344 Eggenstein-Leopoldshafen (Germany)
Prof. Dr. H. G. Bçrner
Laboratory for Organic Synthesis of Functional Systems, Depart-
ment of Chemistry, Humboldt-Universitꢁt zu Berlin, 12489 Berlin
(Germany)
[**] C.B.K. acknowledges continued funding from the Karlsruhe Institute
of Technology (KIT), the German Research Council (DFG), and the
Ministry of Science and Arts of the state of Baden-Wꢀrttemberg
supporting the current project. T.P.’s PhD studies are funded by the
Fonds der Chemischen Industrie. G.D. thanks the Alexander von
Humboldt Foundation for financial support via a Humboldt
Research Fellowship for Postdoctoral Researchers. The authors are
indebted to Dr. T. Gruendling, K. Oehlenschlaeger, and M. Glassner
for their initial studies on o-quinodimethanes.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201107095.
Angew. Chem. Int. Ed. 2012, 51, 1071 –1074
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1071

.
Angewandte
Communications
The model reactions in solution provided a proof-of-
principle that phototriggered (bio)conjugation can be ach-
ieved without catalyst yet with high efficiency. The light-based
nature of the conjugation technique prompted us to translate
it to the spatially constrained grafting of molecules onto
surfaces to obtain molecular patterns. During the writing of
this manuscript, Popik et al. presented surface grafting by
photoinduced hetero-Diels–Alder reaction onto vinyl ether-
coated slides[12] involving a type of chemistry similar to our
proposed system. However, the photoreactive moiety used
was present as a soluble species, which is detrimental with
respect to the spatial resolution. The method also requires
multistep incorporation of the photoactive 3-hydroxymethyl-
2-naphthol moiety for each different coupling. In contrast, our
key idea is the immobilization of the photoactive FMP moiety
directly onto the surface to perform spatially resolved in situ
grafting of maleimide derivatives, for example sensitive
biomolecules. With the existence of a broad spectrum of
readily available maleimido-functionalized (bio)molecules
one also avoids the complicated incorporation of the photo-
active species mentioned above.
The synthetic route to functionalize silicon surfaces is
rather straightforward. The FMP-functionalized silane 6 was
prepared (Scheme 2), dissolved in anhydrous toluene, and
reacted with activated silicon wafers. Upon confirmation of
effective surface silanization by X-ray photoelectron spec-
troscopy (XPS; Supporting Information, Figure S14), we
performed light-directed Diels–Alder cycloaddition on the
surface using different maleimide derivatives. Imaging time-
of-flight secondary-ion mass spectrometry (ToF-SIMS) is a
commonly utilized and sensitive technique that allows the
highly spatially resolved analysis of molecular patterns on
Scheme 1. Photoinduced isomerization of a 2-formyl-3-methylphenoxy
(FMP) derivative 1 and subsequent Diels–Alder [4+2] cycloaddition
with a dienophile.
tion of the photoreaction in acetonitrile and dichloromethane,
see the Supporting Information, Figures S9 and S10). Impor-
tantly, the photoinduced reactions were also compatible with
polar solvents, such as water (Supporting Information, Fig-
ure S11) and DMF (for example, in the phototriggered
reaction with N-(5-fluoresceinyl)maleimide; Supporting
Information, Figure S12). It should however be noted that
longer irradiation times are typically required with these
solvents for quantitative product formation. As water-borne
catalyst-free conjugation reactions are highly interesting for
biological applications, we also placed our attention on the
attachment of biomolecules, for example, peptides. Figure 1b
depicts the mass spectra of a maleimido-GRGSGR peptide 4
before and after 5 conjugation with a low-molecular-weight
FMP derivative 1. In detail, we irradiated 4 in the presence of
1 (1:1.2 mol/mol) in acetonitrile/phosphate-buffered saline
(PBS; 3:1 v/v) and could again observe quantitative formation
of the expected Diels–Alder adduct (for a detailed inves-
tigation, see the Supporting Information, Figure S13).
Figure 1. ESI-MS spectra of a) the photoconjugation of 2-formyl-3-methylphenoxy PEG (with repeating unit n) before (above, 2) and after (below,
3) 15 min photoconjugation with maleimide, and b) maleimido-GRGSGR peptide before (above, 4) and after (below, 5) 2 h photoconjugation with
FMP derivative 1.
1072
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1071 –1074

Angewandte
Chemie
photochemically inert (in the absence of a dienophile during
irradiation, no chemical changes of the FMP layer were
detectable by ToF-SIMS) and only reacts in presence of a
suitable dienophile, thus simplifying preparation, handling,
and storage. Subsequent ToF-SIMS composition analysis of
the bromine content on the surface readily reproduced the
shadow mask structures (Figure 2b) of irradiated and non-
irradiated areas with good spatial resolution. This type of
pattern could potentially be used to grow polymers by
surface-initiated metal-catalyzed radical polymerization in a
“grafting-from” approach.[14] In fact, the enormous applic-
ability of polymer surface patterns at different length scales is
well-known in research fields including tissue engineering,
cell biology, and medicinal science.[15]
Scheme 2. Synthesis of the FMP-functionalized silane 6. Reagents and
conditions: a) AlCl3, CH2Cl2; b) 4-bromomethylbenzoate, K2CO3, ace-
tone; c) NaOH, CH2Cl2/methanol (9:1 v/v); d) ethyl chloroformate, 3-
triethoxysilylpropan-1-amine, THF.
Apart from the aforementioned “grafting-from”
approach, our phototriggered strategy can also be utilized
to directly functionalize the FMP surface with polymers, for
example maleimido-capped poly(ethylene glycol) methyl
ether (PEG-Mal) 8 by the “grafting-to” approach (Figure 3a).
By utilizing this methodology, we could find characteristic
mass fragments of PEG only in the irradiated square (Fig-
ure 3b, left), thus confirming the site-specific immobilization.
PEG is commonly used to locally reduce non-specific bind-
ing[16] and to spatially control adhesive and non-adhesive
areas on surfaces.
solid substrates.[13] In contrast to traditional fluorescence
imaging, ToF-SIMS data provides detailed information on the
chemical composition essential for analysis of non-fluorescent
(bio)molecules. For instance, bromine compounds with their
inherent isotopic pattern can be unambiguously detected by
ToF-SIMS; therefore, the bromine-containing dienophile 7
(Figure 2) was utilized in the current study as a molecular
marker to spatially map the locally constrained surface
grafting. The photopatterning was achieved by irradiation of
the functionalized silicon wafers immersed in a solution of 7
by utilizing two shadow masks: first a macroscopic structure
with a square cut into a metal plate and then one with a
micropattern (Figure 2).
Figure 3. a) Representation of the phototriggered double patterning of
w-maleimido poly(ethylene glycol) 8 and ATRP initiator 7. b) ToF-SIMS
images demonstrating successful photopatterning of 8 (left). The
initial photoreactivity is retained on non-irradiated parts and can be
reutilized for further Diels–Alder functionalization by global irradiation
with 7 (right).
Figure 2. a) Representation of the phototriggered Diels–Alder surface
grafting of bromine-containing maleimide derivative 7. ATRP=atom-
transfer radical polymerization. b) ToF-SIMS images of silicon wafers
patterned with 7 utilizing two shadow masks.
After the phototriggered reaction, it is not necessary to
deactivate or cap the remaining FMP groups on the surface, as
the formation of reactive diene species formed during
irradiation is fully reversible. The FMP surface is also
Importantly, the initial photoreactivity of non-irradiated
FMP-functionalized areas is retained and can be utilized for
further Diels–Alder functionalization with a second dieno-
phile. In this context, a preformed PEG-patterned surface
Angew. Chem. Int. Ed. 2012, 51, 1071 –1074
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1073

.
Angewandte
Communications
received a subsequent flood exposure in presence of 7. We
could indeed verify successful sequential photopatterning by
bromine and PEG composition analysis (Figure 3b).
functional patterned substrates to control cell behavior by the
introduced technique.
Received: October 6, 2011
Published online: December 9, 2011
To also demonstrate the feasibility of covalent and site-
specific attachment of peptides by this phototriggered
approach (which also avoids protection chemistry), we
irradiated a freshly prepared FMP-functionalized surface
with maleimido-GRGSGR 4 in acetonitrile/PBS (3:1 v/v).
ToF-SIMS again provided evidence that the peptide was
immobilized in a patterned way that corresponded to the
mask features. In that case, composition analysis was based on
the presence of C4H8N+, a secondary-ion characteristic for
peptides (Figure 4).[17] The successful biopatterning was also
confirmed by characteristic mass fragments for the arginine
Keywords: peptides · photoconjugation · quinodimethanes ·
.
surface patterning · ToF-SIMS
[1] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001,
113, 2056 – 2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021;
b) C. Barner-Kowollik, F. E. Du Prez, P. Espeel, C. J. Hawker, T.
Junkers, H. Schlaad, W. Van Camp, Angew. Chem. 2011, 123, 61 –
64; Angew. Chem. Int. Ed. 2011, 50, 60 – 62.
+
groups of 4 for example (C4H11N3+ + C5H8N3+ + C5H11N4 ;
[2] M. Conradi, T. Junkers, Macromolecules 2011, 44, 7969 – 7976.
[3] B. J. Adzima, Y. Tao, C. J. Kloxin, C. A. DeForest, K. S. Anseth,
C. N. Bowman, Nat. Chem. 2011, 3, 256 – 259.
Supporting Information, Figure S15).
[4] a) C. E. Hoyle, C. N. Bowman, Angew. Chem. 2010, 122, 1584 –
1617; Angew. Chem. Int. Ed. 2010, 49, 1540 – 1573; b) R.
Hoogenboom, Angew. Chem. 2010, 122, 3489 – 3491; Angew.
Chem. Int. Ed. 2010, 49, 3415 – 3417.
[5] S. V. Orski, A. A. Poloukhtine, S. Arumugam, L. Mao, V. V.
Popik, J. Locklin, J. Am. Chem. Soc. 2010, 132, 11024 – 11026.
[6] a) W. Song, Y. Wang, J. Qu, M. M. Madden, Q. Lin, Angew.
Chem. 2008, 120, 2874 – 2877; Angew. Chem. Int. Ed. 2008, 47,
2832 – 2835; b) Y. Wang, W. Song, W. J. Hu, Q. Lin, Angew.
Chem. 2009, 121, 5434 – 5437; Angew. Chem. Int. Ed. 2009, 48,
5330 – 5333.
[7] J. S. Clovis, A. Eckell, R. Huisgen, R. Sustmann, Chem. Ber.
1967, 100, 60 – 70.
[8] M. Dietrich, G. Delaittre, J. P. Blinco, A. J. Inglis, M. Bruns, C.
Barner-Kowollik, Adv. Funct. Mater., DOI: 10.1002/
adfm.201102068.
Figure 4. Locally constrained biofunctionalization on a surface. ToF-
SIMS image (on the right) for bioconjugation with maleimido-
GRGSGR 4.
[9] a) T. Gruendling, K. K. Oehlenschlaeger, E. Frick, M. Glassner,
C. Schmid, C. Barner-Kowollik, Macromol. Rapid Commun.
2011, 32, 807 – 812; b) M. Glassner, K. K. Oehlenschlaeger, T.
Gruendling, C. Barner-Kowollik, Macromolecules 2011, 44,
4681 – 4689.
[10] a) P. G. Sammes, Tetrahedron 1976, 32, 405 – 422; b) J. L. Charl-
ton, M. M. Alauddin, Tetrahedron 1987, 43, 2873 – 2889; c) J. L.
Segura, N. Martꢀn, Chem. Rev. 1999, 99, 3199 – 3246; d) S. M.
Mellows, P. G. Sammes, J. Chem. Soc. D 1971, 21 – 22.
[11] L. Nebhani, C. Barner-Kowollik, Macromol. Rapid Commun.
2010, 31, 1298 – 1305.
[12] S. Arumugam, V. V. Popik, J. Am. Chem. Soc. 2011, 133, 15730 –
15736.
[13] C.-Y. Lee, G. M. Harbers, D. W. Grainger, L. J. Gamble, D. G.
Castner, J. Am. Chem. Soc. 2007, 129, 9429 – 9438.
[14] B. Charleux, F. DꢁAgosto, G. Delaittre, Adv. Polym. Sci. 2011,
233, 125 – 183.
The photoenol-mediated conjugation strategy possesses
many interesting features, making it a first-class click reaction.
It can proceed rapidly at ambient temperature in a wide range
of (polar) solvents. No catalyst is required and no by-product
is formed, and thus no purification steps are required. Most
importantly, control over time and space is inherent owing to
its light-induced nature. Herein, we have demonstrated the
high efficiency and speed of the phototriggered Diels–Alder
(bio)conjugation in solution as well as on a surface by means
of the covalent attachment of three dienophiles: a small-
molecule ATRP initiator, a polymer, and a peptide. We
envisage many applications for this procedure ranging from
the photolithographic grafting of conductive polymers for
LED development to the generation of a variety of complex
architectures, including polymer–protein conjugates. Our
efforts are currently directed towards the production of
[15] Z. Nie, E. Kumacheva, Nat. Mater. 2008, 7, 277 – 290.
[16] X. C. Nan Cheng, J. Colloid Interface Sci. 2010, 348, 71 – 79.
[17] H. J. Mathieu, Surf. Interface Anal. 2001, 32, 3 – 9.
1074
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1071 –1074