Spatially controlled surface immobilization of nonmodified peptides

Article abstract of DOI:10.1002/anie.201302040

A phencyclone derivative is used to achieve light-controlled immobilization of peptides possessing only natural amino acids. The photoactive precursor (blue in picture) is formed in a Diels-Alder reaction and can undergo light-triggered ring-opening reactions with amines. Successful surface patterning with a genuine c(RGDfK) peptide (green) is evidenced by imaging time-of-flight secondary-ion mass spectrometry (ToF-SIMS). Copyright

Full text of DOI:10.1002/anie.201302040

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Angewandte
Communications
DOI: 10.1002/anie.201302040
Photopatterning
Spatially Controlled Surface Immobilization of Nonmodified
Peptides**
Thomas Pauloehrl, Alexander Welle, Michael Bruns, Katharina Linkert, Hans G. Bçrner,
Martin Bastmeyer, Guillaume Delaittre, and Christopher Barner-Kowollik*
Controlled surface modification through the precise position-
ing of chemical functionalities or relevant biomolecules holds
enormous promise for advances in ever-growing research
areas such as biointerface science, point-of-care applications,
biosensors, and nanotechnology.[1] Among the myriad of
modification techniques that have been developed, photo-
chemical approaches play a key role. They contribute not only
in a significant way to the existing repertoire of carbon–
carbon bond-forming reactions,[2] but also inherently confer
spatiotemporal control to chemical reactions and are as such
highly appealing for the production of patterns of carbohy-
drates, proteins, DNA fragments, or multiple cell lines.[3] The
use of light has a tremendous influence on the way bioma-
terials scientists can perform experiments, e.g., to control the
photodegradation of hydrogels[4] or the activity of cells.[5]
Today chemists have a number of methods available for
spatially controlled surface grafting, which mostly rely on the
release of appropriate functional groups upon a light stimulus,
such as azide-reactive cyclooctynes, ene-reactive nitrile
imines, or aminooxy-reactive aldehydes.[6] A novel approach
that photogenerates highly reactive intermediates with short
lifetimes, such as o-naphtoquinone methides, was also
reported.[7] Regeneration of the initial photoactive moiety
occurs through hydration resulting often in improved selec-
tivity and orthogonality as well as a better handling in an
environment that is not protected from light. Popik, Locklin,
and co-workers have followed such a path when they used 3-
(hydroxymethyl)-naphthalene-2-ol derivatives with vinyl
ether moieties for covalent surface derivatization.[8] In the
same context, we have recently employed the photoisomeri-
zation of o-methylbenzaldehyde to o-quinodimethanes and
demonstrated its ease and efficiency in the light-induced
modular ligation of polymers prepared by reversible addition-
fragmentation chain transfer polymerization (RAFT) and in
spatially controlled surface grafting.[9] Altogether, these
different photoreactions can be applied to photopatterning
in different contexts with high efficiency. A limiting factor of
their applicability in novel technologies is, however, that all of
the aforementioned transformations involve complex multi-
step synthesis of surface anchors functionalized with photo-
chemically active moieties and, often even more challenging,
individual modifications of (biologically) relevant substrates
with appropriate functionalities for coupling. These com-
pounds have to be precisely tailored to the concrete needs,
thus limiting their versatility. The idea of the current study is
to use potent dienes, namely phencyclone derivatives, as
a novel platform for light-triggered modifications in solution
and on surfaces. The outcomes of these chemical trans-
formations were assessed separately; specifically, the
approach initially investigated in solution proceeds in three
steps: A) a versatile and highly efficient Diels–Alder-based
formation of a photoactive precursor, B) a fast and mild
photoactivation to induce spatiotemporal control, and C) the
nucleophilic attachment of amines (Figure 1a). The construc-
tion of patterns of nonmodified biomolecules such as peptides
and proteins by means of their inherent amine functionality
would be highly desirable,[10] especially if the procedure does
not involve extremely reactive intermediates such as radicals,
which would clearly result in limitations of selectivity and
orthogonality. Upon performing a screening study of poten-
[*] T. Pauloehrl, Dr. G. Delaittre, Prof. Dr. C. Barner-Kowollik
Preparative Macromolecular Chemistry
Prof. Dr. M. Bastmeyer, Dr. G. Delaittre
Zoologisches Institut, Zell- und Neurobiologie, KIT
Karlsruhe (Germany)
Institut fꢀr Technische Chemie und Polymerchemie
Karlsruhe Institute of Technology (KIT)
Prof. Dr. M. Bastmeyer
Engesserstraße 18, 76128 Karlsruhe (Germany)
E-mail: christopher.barner-kowollik@kit.edu
Homepage: http://www.macroarc.de
Institut fꢀr Funktionelle Grenzflꢁchen, KIT
Eggenstein-Leopoldshafen (Germany)
Dr. G. Delaittre
Dr. A. Welle, Prof. Dr. C. Barner-Kowollik
Institut fꢀr Biologische Grenzflꢁchen (IBG), KIT
Hermann-von-Helmholtzplatz 1
Institute of Toxicology and Genetics (ITG), KIT
Eggenstein-Leopoldshafen (Germany)
[**] C.B.-K. acknowledges funding from the Karlsruhe Institute of
Technology (KIT) in the context of the BioInterfaces program of the
Helmholtz association.
76344 Eggenstein-Leopoldshafen (Germany)
Dr. M. Bruns
Institut fꢀr Angewandte Materialien (IAM) and
Karlsruhe Nano Micro Facility (KNMF), KIT
Eggenstein-Leopoldshafen (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201302040.
K. Linkert, Prof. Dr. H. G. Bçrner
Laboratory for Organic Synthesis of Functional Systems
Department of Chemistry
Humboldt-Universitꢁt zu Berlin (Germany)
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Figure 1. a) Overall concept for light-triggered modifications of the GRGDS peptide 2. b) ESI-MS spectra of a maleimide end-capped GRGDS
peptide 2 and after reaction with phencyclone derivative 1 (1.05 equiv). Subsequent decarbonylation and dehydrogenation to 4 was performed in
water by an in situ photoreaction (2 h, 320 nm) in the presence of 1,4-benzoquinone (3 equiv). Amine attachment to 5 was achieved by ring
opening employing an excess of an amine derivative at 458C. c) Kinetic investigations of the Diels–Alder reaction of 1 with 2 by UV/Vis
spectroscopy (top) and of the in situ activation of 3 by irradiation in the presence of 1,4-benzoquinone (3 equiv) to give 4 (bottom). The structural
formulas of all compounds are included in Scheme S1 in the Supporting Information.
tial phencyclone candidates, we have identified the easily
synthesizable[11]
1,3-bis(4-methoxyphenyl)-2H-cyclopenta-
[l]phenanthren-2-one (MCPO) 1 compound to be the most
efficient precursor (see Figure S13 in the Supporting Infor-
mation).
The initial light-triggered modifications were performed
in solution utilizing the maleimide-functionalized GRGDS
peptide 2, which aided us to evaluate the possibility of
performing our proposed reaction sequence in the presence of
diverse amino acid residues in polar solvents. The synthetic
procedure for the attachment of the photoactive moiety is
completely dark green solution turned colorless within 40 h
reaction time, thereby indicating the efficient consumption of
1. As a further test for a successful attachment of the
photoactive precursor, an electrospray mass spectrometry
(ESI-MS) analysis was performed. Figure 1b depicts the mass
spectra of the starting GRGDS peptide 2 alongside the
quantitatively formed Diels–Alder cycloadduct 3. Upon
confirming the successful attachment of the photoactive
species, we turned our attention to systematically investigat-
ing the photochemical behavior of the latter by employing
a low-cost 36 W compact fluorescent lamp (lmax = 320 nm;
Figure S9 in the Supporting Information) as the UV source. In
general, bridged carbonyl compounds such as norbornen-7-
one derivatives can eliminate carbon monoxide upon heating
or mild irradiation.[12] In the case of 3 we observed light-
triggered decarbonylation, accompanied by a low amount of
dehydrogenation product 4 (see Figure S14 in the Supporting
Information). To produce the fully conjugated triphenylene
imide 4 in a quantitative manner, one can perform a one-pot
straightforward and was achieved by stirring
2 with
1 (1.05 equiv of 1, MeCN/H2O 7:1 v/v). UV/Vis spectroscopy
is a useful technique for the online measurement of the
concentration of 1, which is colored by virtue of its n!p*
transition. The progress of the cycloaddition reaction could
thus be monitored by measuring the loss of the long-wave-
length absorbance of the phencyclone chromophore in the
visible spectrum (644 nm, see Figure 1c) while the initially
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in situ decarbonylation/dehydrogenation photoreaction. In
detail, 3 was irradiated at ambient temperature in water for
2 h in the presence of 1,4-benzoquinone (1:3 mol/mol).
Inspection of the ESI-MS spectra in Figure 1b reveals a full
peak shift of 30 amu towards lower mass-to-charge ratios,
which corresponds to the loss of CO and H2. Complete
disappearance of the quasimolecular ion [3+H]+ leaving
[4+H]+ demonstrates that full conversion was achieved after
less than 120 min irradiation time at ambient temperature
(Figure 1c). A more in-depth analysis of the photoreaction in
various solvents, through a two-step proce-
functionality prompted us to translate it to the spatially
constrained immobilization of amines onto surfaces to
produce molecular patterns. Note that, in contrast to the
initial solution experiments where the peptide was bearing
the photoactive moiety, this time we employed a nonmodified
peptide able to react with photoactivated substrates through
its lysine residue. The synthetic route to functionalized silicon
surfaces is straightforward. A maleimide end-capped silane
was prepared from commercially available substances
(Scheme 1) and was converted to the MCPO end-capped
dure (adding the dehydrogenation agent after
the light-triggered decarbonylation) and the
utilization of different dehydrogenation
agents (e.g., alkylmaleimides) may be found
in the Supporting Information. We observed
in all of these cases an efficient transforma-
tion from the photoactivatable compound 3
to photoactivated 4 in a quantitative and
solvent-independent manner. Secondary pho-
tolysis, which can be a major issue in the
Scheme 1. Synthesis of the MCPO-functionalized silane.
widely employed o-nitrobenzyl photoprotect-
ing moiety,[13] is avoided even after prolonged irradiation by
means of the overall high photolytic and thermal stability of
all products, which are namely CO, H2, and the triphenylene
imide 4. The latter moiety is however capable of undergoing
ring-opening reactions with amines in analogy to the well-
known phtalimide system.[14] Inspection of Figure 1b (for the
attachment of other amines refer to Figure S19 in the
Supporting Information) reveals the successful formation of
the monoadduct of 2-(4-fluorophenyl)ethanamine 5 as the
main product, whereas double attachment resulted in cleav-
age and a minor fraction of amine end-capped peptide 6. The
solution-based experiments convincingly demonstrate the
highly efficient attachment of a photoactive precursor
(which does not require complex synthesis), its rapid and
quantitative photoactivation, and its catalyst-free coupling
with amines. A limiting factor of the approach in solution is,
however, the necessity to employ an excess of amine to
efficiently ring-open the triphenylene imide 4. The current
strategy is thus not the system of choice in cases where
product separation is difficult, e.g., when polymer–polymer
conjugations are targeted. However, the full strength of the
given method for light-directed functionalization is evidenced
when the reaction sequence is performed on a surface. Excess
of amine reagent is easily removed by simple washing, and
double nucleophilic ring-opening by amines is less pro-
nounced owing to steric hindrance. The clear dichotomy
between the photoactivation and the chemical attachment in
the current strategy would also allow for the construction of
patterns of light-sensitive (bio)molecules that cannot be
obtained by more conventional strategies. Importantly, and
being the basis for any spatially resolved grafting of molecules
onto surfaces, the nonirradiated photosensitive precursor 3
was found to be fully inert in the presence of excess 2-(4-
fluorophenyl)ethanamine at 458C (see Figure S20 in the
Supporting Information).
silane in a Diels–Alder cycloaddition with 1. Finally the silane
was dissolved in anhydrous toluene and employed to treat
activated silicon wafers. Upon successful silanization, the
photopatterning was achieved by irradiation of the silicon
wafers for 3 h under normal atmospheric conditions (no inert
gas, ambient temperature) and without solvent. Two shadow
masks were utilized for the locally constrained surface
activation: one featuring squares with 50 mm pitches in
x/y coordinates and one with a macroscopic pattern. After
irradiation, patterning was achieved by immersing the wafers
in a 1,4-benzoquinone solution (dehydrogenation) and finally
in a methanolic solution of the respective amine for 18 h at
458C. Analysis of the photopatterning was achieved by
imaging time-of-flight secondary-ion mass spectrometry
(ToF-SIMS), which is a highly surface-sensitive and label-
free technique for the spatially resolved analysis of solid
substrates.[15] AToF-SIMS composition analysis of the surface
reproduced the shadow mask structures with an excellent
spatial resolution (edge steepness: 4.5 mm) between irradiated
and nonirradiated areas (see Figure 2b, top). Indeed, only the
nonirradiated zone showed the presence of the maleimide–
ꢀ
phencyclone Diels–Alder cycloadduct (C35H24NO5 ), while
only the irradiated squares exhibited a fragment at 30 amu
lower mass-to-charge ratio, corresponding to triphenylene
ꢀ
imide (C34H22NO4 ), which is formed by loss of H2 and CO.
Bromine compounds with their inherent isotopic pattern can
be unambiguously detected by ToF-SIMS, therefore, the
photopatterned wafer was immersed in a solution of 2-(4-
bromophenyl)ethanamine as a molecular marker to spatially
map the amine-reactive areas. Clearly, only the irradiated part
exhibited bromine functionalization after immersion of the
wafer in the solution of 2-(4-bromophenyl)ethanamine (Fig-
ure 2b, bottom). To demonstrate the feasibility of a covalent
and spatially resolved attachment of nonmodified peptides by
this phototriggered approach, we repeated the irradiation/
dehydrogenation procedure (irradiation: no inert gas, ambi-
ent temperature, no solvent; dehydrogenation in a 1,4-
These findings and the potential ability to pattern non-
modified biomolecules by means of their inherent amine
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benzoquinone solution) for a freshly prepared MCPO-
functionalized wafer. Finally, the surface was treated with
a solution of N,N-diisopropylethylamine (DIPEA) and the
extracellular matrix protein-mimicking c(RGDfK) peptide in
DMF. In a ToF-SIMS analysis we could confirm the successful
photodeprotection by using a negative contrast for the Diels–
Alder cycloadduct (C35H24NO5ꢀ, see Figure S24 in the
Supporting Information) as well as by its retro-Diels–Alder
follow-up product 1 (C31H22O3ꢀ, see Figure 2c, top left)
formed during analysis. ToF-SIMS also provided evidence
that the peptide was immobilized in a pattern corresponding
to the mask features. In that case, composition analysis was
based on the presence of CH3N2+ and C8H10N+ (Figure 2c, top
right), characteristic secondary ions for arginine- or phenyl-
alanine-containing peptides, respectively.[16] Sums of all
positive ion fragments that can be assigned to the peptide
are depicted in Figure 2c. The same surface modification
sequence can also be applied for the locally constrained
immobilization of a highly charged peptide comprising the
active amino acid sequence of statherin, which inhibits
secondary calcium phosphate precipitation. In this case
again, the substrate was efficiently immobilized in a patterned
way (see Figure 2d, for a full overview of all observed
fragments for both peptides in ToF-SIMS negative and
positive modes, refer to Figures S22, S25, and S26 in the
Supporting Information).
Herein we introduced a novel technique allowing the
photopatterning of peptides strictly composed of naturally
occurring residues, representing the first important feature of
our method, since it avoids the expensive preparation or
purchase of nonnatural amino acids or the modification of
peptide C or N termini. In addition, the synthetic route
towards the relevant photoreactive compounds is technically
simple and achieved at a rather low cost. Finally, the
photopatterning sequence itself involves catalyst-free and
rather mild conditions. The photoreaction is fully compatible
to all investigated solvents including water, avoids the
formation of harmful photolysis by-products, and can be
performed under ambient atmosphere and temperature. The
combination of these features renders the system extremely
attractive for industrially relevant peptide micro- or nano-
array fabrication.
Received: March 11, 2013
Revised: May 31, 2013
Published online: July 24, 2013
Keywords: patterning · peptides · phencyclone · photochemistry
.
Figure 2. a) Representation of the phototriggered surface grafting.
b) ToF-SIMS images of silicon wafers patterned utilizing two shadow
masks. Photopatterning was achieved as described in (a) by immers-
ing the wafers in a 1,4-benzoquinone solution (top). The patterned
surfaces were then immersed in 2-(4-bromophenyl)ethanamine to
visualize the amine-reactive areas (bottom). c) ToF-SIMS images of
silicon wafers patterned with c(RGDfK) and d) with a statherin
sequence utilizing a shadow mask.
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