(Bio)molecular surface patterning by phototriggered oxime ligation

Article abstract of DOI:10.1002/anie.201202684

Making light work of ligation: A novel method utilizes light for oxime ligation chemistry. A quantitative, low-energy photodeprotection generates aldehyde, which subsequently reacts with aminooxy moieties. The spatial control allows patterning on surfaces (see scheme) with a fluoro marker and GRGSGR peptide, and can be imaged by time-of-flight secondary-ion mass spectrometry. Copyright

Full text of DOI:10.1002/anie.201202684

Angewandte
Chemie
DOI: 10.1002/anie.201202684
Photoreactions on Surfaces
(Bio)Molecular Surface Patterning by Phototriggered Oxime
Ligation**
Thomas Pauloehrl, Guillaume Delaittre, Michael Bruns, Maria Meißler, Hans G. Bçrner,
Martin Bastmeyer, and Christopher Barner-Kowollik*
Photolabile moieties are widely employed in organic chemis-
try and beyond as a powerful tool for breaking bonds
smoothly without the need for any additional reagents.[1]
Such orthogonal and mild photolytic cleavage is particularly
attractive for solid-supported organic synthesis,[2] in the field
of combinatorial library screening,[3] and for the tracking of
molecular dynamics in biological systems.[4] Of the myriad of
photocleavable protecting groups which have been studied,
the o-nitrobenzyl group is certainly the predominant one
since it enables the caging of a wide range of functionalities,
such as carboxy,[5] amine,[6] hydroxy,[7] and thiol.[8] In addition,
the use of light as a trigger provides a straightforward means
to obtain spatial and temporal control over a desired molec-
ular cleavage,[9] which has found significant attention in
constructing patterns of multiple cell lines,[10] the production
of 3D structured materials for tissue scaffolds,[11] and the
photo-caging of active compounds[12] for instance. Further-
more, the precise positioning of o-nitrobenzyl groups in
polymer chains has allowed for the controlled alteration of
polymer properties upon light stimulus, as demonstrated by
the fabrication of cytocompatible 3D hydrogels that can
photodegrade[13] or photorelease peptides,[14] amphiphilic
dendrimer-based photodestructible micelles,[15] or photo-
cleavable block copolymers to produce functionalized nano-
porous films,[16] to name but a few.[17]
The underlying mechanism for the photocleavage of o-
nitrobenzyl derivatives is well studied[18] and typically leads to
the release of the protecting group in the form of a nitro-
sobenzaldehyde derivative as a byproduct.[19] The idea
presented herein, however, is to take advantage of this
photoreleased aldehyde moiety for oxime formation (see
Scheme 1).[20] In other words, to consider the usual byproduct
as the actual product. As alluded to above, the use of light
would also provide a facile means to confer spatial and
temporal control to the increasingly employed oxime-based
click chemistry. To date, only Maynard and co-workers have
reported molecular patterns employing oxime ligation, how-
ever the reaction requires rather harsh conditions, such as
electron-beam or photoacid generator-based photolithogra-
phies.[21] Interestingly, our method presented herein proceeds
at less-energetic wavelengths (UVA) than light-triggered
strategies recently reported by us and others, which are based
on the nitrile imine-ene 1,3-dipolar cycloaddition (UVC)[22] or
on the Diels–Alder cycloaddition of o-quinodimethanes[23] or
3-(hydroxymethyl)naphthalene-2-ol derivatives[24] (UVB).
Altogether, these different techniques form a versatile tool-
box for photopatterning in different contexts, yet all with high
efficiency under ambient conditions. The current approach
proceeds in two steps: a) a fast and mild photodeprotection
and b) the subsequent oxime ligation reaction (see
Scheme 1). The 2-[(4,5-dimethoxy-2-nitrobenzyl)oxy]tetrahy-
dro-2H-pyranyl (NOTP) scaffold in compound 1 was selected
as a novel highly reactive photocleavable moiety based on its
overall kinetics—photocleavage quantum yield of o-nitro-
veratryl ethers is one order of magnitude higher than that of
[*] T. Pauloehrl, Dr. G. Delaittre, 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)
Engesserstrasse 18, 76128 Karlsruhe (Germany)
E-mail: christopher.barner-kowollik@kit.edu
Homepage: http://www.macroarc.de
Dr. G. Delaittre, Prof. Dr. M. Bastmeyer
Zoologisches Institut, Zell- und Neurobiologie and Centre for
Functional Nanostructures (CFN)
Karlsruhe Institute of Technology (KIT)
Haid-und-Neu-Strasse 9, 76131 Karlsruhe (Germany)
Dr. M. Bruns
Institute for Applied Materials (IAM-WPT) and Karlsruhe Nano
Micro Facility (KNMF), Karlsruhe Institute of Technology (KIT)
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen
(Germany)
M. Meißler, Prof. Dr. H. G. Bçrner
Laboratory for Organic Synthesis of Functional Systems, Depart-
ment of Chemistry, Humboldt-Universitꢁt zu Berlin
Brook-Taylor-Strasse 2, 12489 Berlin (Germany)
[**] C.B.K. acknowledges continued funding from the Karlsruhe Institute
of Technology (KIT), the German Research Council (DFG), the DFG
Centre for Functional Nanostructures (CFN), 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 through a Humboldt Research
Fellowship for Postdoctoral Researchers. We thank Peter Gerstel
(KIT) for his help with the silizanation procedure.
Scheme 1. Photoinduced cleavage of a 2-[(4,5-dimethoxy-2-nitro-
benzyl)oxy]tetrahydro-2H-pyranyl (NOTP) derivative and subsequent
oxime ligation with hydroxylamine derivatives. R=C3H6COOH.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202684.
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most other o-nitro-veratryl derivatives[4]—as well as for the
ease of incorporation of the (tetrahydropyran-2-yl)oxy group
through dihydropyran (see the Supporting Information for
full synthetic procedure).
Initial model reactions were performed in solution on
a poly(ethylene glycol) methyl ether (PEG) functionalized
with 1, a system amenable to mass spectrometry, a method
which allows the detection of potential side-product forma-
tion with much higher sensitivity and specificity than
1H NMR spectroscopy.[25] A low-cost compact fluorescent
lamp (lmax = 370 nm, 14 mWcmÀ2, 18 W) was employed as the
UVA source for the photodeprotection. The outcome of the
deprotection is shown in Figure 1, which depicts the mass
spectrum of the fully photodeprotected nitrosobenzaldehyde-
capped PEG 3. Complete cleavage upon mild irradiation was
typically achieved within only 3 min at ambient temperature
(see also Supporting Information Figure S29 for UV spectra).
Subsequent overnight reaction of 3 with hydroxylamine
hydrochloride resulted in the desired aldoxime 4 (Figure 2).
The presence of a small quantity of impurities may also be
observed, which can all be assigned to the multiple side-
reactions undergone by the nitroso moiety,[26] for example,
dimerization yielding 5, two- and four-electron reduction
leading to 6 and 8, or subsequent condensation generating 7.
Owing to the undesirable nitroso-centered reactions this
strategy is not suitable for polymer–polymer conjugation in
solution, as such reactions are subject to a stringent set of click
requirements.[27] However, it is noteworthy that all the
products derived from the reaction between 3 and hydroxyl-
amine contain the desired oxime bond. Consequently, when
the reaction sequence is performed on a surface, no influence
on the grafting density or the spatial control is to be expected.
These findings, as well as the light-based nature of the
conjugation technique, prompted us to translate it to the
spatially constrained grafting of molecules onto surfaces to
produce molecular patterns.
Figure 1. ESI-MS spectra of NOTP-capped poly(ethylene glycol) methyl
ether before (top) and after irradiation (middle; lmax =370 nm,
14 mWcm2, 18 W, 3 min). Bottom: Structural formulae for starting
compound 2 and photoproduct 3.
The synthetic route to functionalize silicon surfaces is
straightforward. A NOTP-functionalized silane was prepared
(Scheme 2), dissolved in anhydrous toluene, and used to treat
activated silicon wafers. Upon successful silanization—as
demonstrated by X-ray photoelectron spectroscopy (XPS, see
Figure 2. ESI-MS spectrum of nitrosobenzaldehyde-capped poly(ethylene glycol) methyl ether 3 after reaction with hydroxylamine hydrochloride to
form 4. Species 5, 6+8, and 7 can be assigned to dimerization, reduction, and condensation of the nitroso moiety, respectively. See the
Supporting Information for details.
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À
the presence of nitrite (NO2 ) and tetrahydro-2H-pyranyl
(C5H9O+) ions (Figure S37)—fragments initially present in
the protected molecule—while only the irradiated part
exhibited fluorine functionalization, as demonstrated by the
detection of FÀ and C6F5À ions, after immersion of the wafer in
the solution of O-[(perfluorophenyl)methyl] hydroxylamine
hydrochloride (Figure 3b).
Scheme 2. Synthesis of the NOTP-functionalized silane. Reagents:
ethyl chloroformate, triethylamine, 3-(triethoxysilyl)propan-1-amine,
THF.
To also demonstrate the feasibility of a covalent and site-
specific attachment of biomolecules by this phototriggered
approach, we irradiated a freshly prepared NOTP-function-
alized wafer and treated it with a solution of (2-amino-
Figure S35)—the photopatterning was achieved by irradia-
tion of the silicon wafer covered with a shadow mask for 3 min
(2.5 JcmÀ2 UV dose, lower or comparable to that of the most
efficient soft photopatterning techniques[28]). Subsequently,
the mask was removed and the silicon wafer was immersed in
a solution of O-[(perfluorophenyl)methyl] hydroxylamine
hydrochloride, which was utilized as a molecular marker to
spatially map the locally constrained surface grafting (Fig-
ure 3a). Analysis of the photopatterning was by time-of-flight
oxy)acetamido
Gly-Arg-Gly-Ser-Gly-Arg
peptide
(GRGSGR). ToF-SIMS again provided evidence that the
peptide was immobilized in a pattern corresponding to the
mask features (Figure 3c). In that case, composition analysis
was based on the presence of CH3N2 and C4H8N+ ions,
+
characteristic secondary ions for arginine-containing pep-
tides.[30]
In summary, we have presented the highly efficient
phototriggered deprotection of a novel o-nitrobenzyl acetal
at 370 nm. The strategy allows not only the quantitative and
extremely rapid photorelease of aldehyde functionalities but
also for the first time the spatial control over the click-type
oxime ligation using mild conditions. Indeed, we translated
the two-step procedure to the fabrication of patterned silicon
surfaces and confirmed the site-specific attachment of amino-
oxy-functionalized (bio)molecules, that is, a small marker
molecule and a peptide, by ToF-SIMS. We envisage that the
introduced technique can be extended to the production of
functional patterned substrates to study/control cell behavior.
However, the potentially cell-toxic nitroso group should be
first neutralized, for example, with glutathione.[31]
Received: April 6, 2012
Revised: June 8, 2012
Published online: && &&, &&&&
Keywords: click chemistry · oxime ligation · patterning ·
.
peptide · photodeprotection
Figure 3. a) Schematic representation of the photodeprotection of Si
wafers (blue) functionalized with NOTP (orange) using a shadow
mask (gray), and subsequent patterning with O-[(perfluorophenyl)-
methyl)] hydroxylamine hydrochloride. b) ToF-SIMS images after photo-
deprotection (left) and oxime ligation with the fluoro marker (center
and right). c) ToF-SIMS images after photodeprotection and oxime
ligation with aminooxy-GRGSGR (structure included in the Supporting
Information).
[1] C. G. Bochet, J. Chem. Soc. Perkin Trans. 1 2002, 125 – 142.
[2] M. Kessler, R. Glatthar, B. Giese, C. G. Bochet, Org. Lett. 2003,
5, 1179 – 1181.
[3] C. P. Holmes, J. Org. Chem. 1997, 62, 2370 – 2380.
[4] L. Kammari, T. Solomek, B. P. Ngoy, D. Heger, P. Klꢀn, J. Am.
ˇ
Chem. Soc. 2010, 132, 11431 – 11433.
[5] N. Kotzur, B. Briand, M. Beyermann, V. Hagen, Chem.
Commun. 2009, 3255 – 3257.
[6] a) D. D. Young, A. Deiters, Bioorg. Med. Chem. Lett. 2006, 16,
2658 – 2661; b) S. A. Sundberg, R. W. Barrett, M. Pirrung, A. L.
Lu, B. Kiangsoontra, C. P. Holmes, J. Am. Chem. Soc. 1995, 117,
12050 – 12057.
[7] A. G. Russell, M. J. Sadler, H. J. Laidlaw, A. Gutierrez-Loriente,
C. W. Wharton, D. Carteau, D. M. Bassani, J. S. Snaith, Photo-
chem. Photobiol. Sci. 2012, 11, 556 – 563.
[8] a) T. Pauloehrl, G. Delaittre, M. Bastmeyer, C. Barner-Kowollik,
Polym. Chem. 2012, 3, 1740 – 1749; b) G. Delaittre, T. Pauloehrl,
M. Bastmeyer, C. Barner-Kowollik, Macromolecules 2012, 45,
1792 – 1802.
secondary ion mass spectrometry (ToF-SIMS), which is
a powerful and highly sensitive technique for the spatially
resolved analysis of molecular patterns on solid substrates.[29]
In contrast to traditional fluorescence imaging, ToF-SIMS
data also provides detailed information on the chemical
composition, essential for analysis of non-fluorescent (bio)-
molecules.
A ToF-SIMS composition analysis of the nitro and
fluorine contents on the surface after irradiation and func-
tionalization readily reproduced the shadow mask structures
with a good spatial resolution between irradiated and non-
irradiated areas. Indeed, only the non-irradiated zone showed
[9] G. Mayer, A. Heckel, Angew. Chem. 2006, 118, 5020 – 5042;
Angew. Chem. Int. Ed. 2006, 45, 4900 – 4921.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
[10] S. Yamaguchi, S. Yamahira, K. Kikuchi, K. Sumaru, T. Kana-
mori, T. Nagamune, Angew. Chem. 2012, 124, 132 – 135; Angew.
Chem. Int. Ed. 2012, 51, 128 – 131.
[11] Y. Luo, M. S. Shoichet, Nat. Mater. 2004, 3, 249 – 253.
[12] a) A. P. Pelliccioli, J. Wirz, Photochem. Photobiol. Sci. 2002, 1,
441 – 458; b) D. D. Young, A. Deiters, Org. Biomol. Chem. 2007,
5, 999 – 1005.
[13] C. A. DeForest, K. S. Anseth, Nat. Chem. 2011, 3, 925 – 931.
[14] C. A. DeForest, K. S. Anseth, Angew. Chem. 2012, 124, 1852 –
1855; Angew. Chem. Int. Ed. 2012, 51, 1816 – 1819.
[15] V. Yesilyurt, R. Ramireddy, S. Thayumanavan, Angew. Chem.
2011, 123, 3094 – 3098; Angew. Chem. Int. Ed. 2011, 50, 3038 –
3042.
[22] M. Dietrich, G. Delaittre, J. P. Blinco, A. J. Inglis, M. Bruns, C.
Barner-Kowollik, Adv. Funct. Mater. 2012, 22, 304 – 312.
[23] T. Pauloehrl, G. Delaittre, V. Winkler, A. Welle, M. Bruns, H. G.
Bçrner, A. M. Greiner, M. Bastmeyer, C. Barner-Kowollik,
Angew. Chem. 2012, 124, 1096 – 1099; Angew. Chem. Int. Ed.
2012, 51, 1071 – 1074.
[24] a) S. Arumugam, S. V. Orski, J. Locklin, V. V. Popik, J. Am.
Chem. Soc. 2012, 134, 179 – 182; b) S. Arumugam, V. V. Popik, J.
Am. Chem. Soc. 2011, 133, 15730 – 15736.
[25] L. Nebhani, C. Barner-Kowollik, Macromol. Rapid Commun.
2010, 31, 1298 – 1305.
[26] B. S. Bodnar, M. J. Miller, Angew. Chem. 2011, 123, 5746 – 5764;
Angew. Chem. Int. Ed. 2011, 50, 5630 – 5647.
[16] J.-M. Schumers, A. Vlad, I. Huynen, J.-F. Gohy, C.-A. Fustin,
Macromol. Rapid Commun. 2012, 33, 199 – 205.
[17] H. Zhao, E. S. Sterner, E. B. Coughlin, P. Theato, Macromole-
cules 2012, 45, 1723 – 1736.
[18] Y. V. Ilꢁichev, M. A. Schwçrer, J. Wirz, J. Am. Chem. Soc. 2004,
126, 4581 – 4595.
[19] M. C. Pirrung, Y. R. Lee, K. Park, J. B. Springer, J. Org. Chem.
1999, 64, 5042 – 5047.
[20] G. A. Lemieux, C. R. Bertozzi, Trends Biotechnol. 1998, 16, 506 –
513.
[21] a) K. L. Christman, R. M. Broyer, Z. P. Tolstyka, H. D. Maynard,
J. Mater. Chem. 2007, 17, 2021 – 2027; b) K. L. Christman, R. M.
Broyer, E. Schopf, C. M. Kolodziej, Y. Chen, H. D. Maynard,
Langmuir 2011, 27, 1415 – 1418; c) R. M. Broyer, E. Schopf,
C. M. Kolodziej, Y. Chen, H. D. Maynard, Soft Matter 2011, 7,
9972 – 9977.
[27] 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.
[28] a) P. Prompinit, A. S. Achalkumar, X. Han, R. J. Bushby, C.
Wꢂlti, S. D. Evans, J. Phys. Chem. C 2009, 113, 21642 – 21647;
b) S. A. Alang Ahmad, L. S. Wong, E. ul-Haq, J. K. Hobbs, G. J.
Leggett, J. Micklefield, J. Am. Chem. Soc. 2011, 133, 2749 – 2759.
[29] A. Benninghoven, Angew. Chem. 1994, 106, 1075 – 1096; Angew.
Chem. Int. Ed. Engl. 1994, 33, 1023 – 1043.
[30] a) D. S. Mantus, B. D. Ratner, B. A. Carlson, J. F. Moulder, Anal.
Chem. 1993, 65, 1431 – 1438; b) M. S. Wagner, D. G. Castner,
Langmuir 2001, 17, 4649 – 4660; c) H. E. Canavan, D. J. Graham,
X. Cheng, B. D. Ratner, D. G. Castner, Langmuir 2007, 23, 50 –
56.
[31] S. Kazanis, R. A. McClelland, J. Am. Chem. Soc. 1992, 114,
3052 – 3059.
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Angew. Chem. Int. Ed. 2012, 51, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Photoreactions on Surfaces
T. Pauloehrl, G. Delaittre, M. Bruns,
M. Meißler, H. G. Bçrner, M. Bastmeyer,
C. Barner-Kowollik*
&&&& — &&&&
(Bio)Molecular Surface Patterning by
Phototriggered Oxime Ligation
Making light work of ligation: A novel
method utilizes light for oxime ligation
chemistry. A quantitative, low-energy
photodeprotection generates aldehyde
which subsequently reacts with aminooxy
moieties. The spatial control allows pat-
terning on surfaces (see scheme) with
a fluoro marker and GRGSGR peptide
and can be imaged by time-of-flight
secondary-ion mass spectrometry.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!