Photoactivated bioconjugation between ortho -azidophenols and anilines: A facile approach to biomolecular photopatterning

Article abstract of DOI:10.1021/ja503056x

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease

Full text of DOI:10.1021/ja503056x

Article
pubs.acs.org/JACS
Photoactivated Bioconjugation Between ortho-Azidophenols and
Anilines: A Facile Approach to Biomolecular Photopatterning
Kareem M. El Muslemany,†,§ Amy A. Twite,†,§ Adel M. ElSohly,† Allie C. Obermeyer,†
Richard A. Mathies,† and Matthew B. Francis*,†,‡
†Department of Chemistry, University of California, Berkeley, California 94720-1460, United States
‡Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460, United States
S
* Supporting Information
ABSTRACT: Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug
screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed
to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily
accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective
manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to
aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins,
polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the
photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the
ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes.
Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a
photoresist or other blocking material.
through the use of multiple DNA sequences.22,23 In addition,
the DNA-based adhesion event has been shown to exhibit
INTRODUCTION
■
The ability to attach living cells to surfaces has enabled the
minimal changes in cellular behavior because it does not involve
study of many key behaviors in biology, including immune
native cell receptors.24 In previous studies, this strategy was
synapse formation,1 stem cell differentiation,2,3 cancer cell
used to measure the metabolism of single cells,21 conduct
motility,4 and drug response.5 In most of these studies, cells of
single-cell RT-PCR analysis,26 study the diffusion of paracrine
interest are exposed to surfaces, such as slides or supported
signaling molecules,22 and connect cells directly to AFM tips.20
lipid bilayers,6−8 that have been patterned with biomolecules
The technique has also been applied to the formation of three-
that engage cellular receptors in a well-defined way. Outside
these experiments, immobilized biomolecule arrays have also
dimensional cell clusters in suspension.27−29
This cell capture method benefits greatly from the availability
of streamlined techniques that can generate small and elaborate
shown promise in fundamental studies of biofuel produc-
tion,9−12 the investigation of antibody−antigen interactions,13
and the construction of biofuel cells based on enzymes.14
patterns of DNA with high precision and high throughput. This
is typically done through the use of photolithography,22,30−32
Although most of these studies have capitalized on interactions
soft lithography,33−35 Dip-Pen nanolithography,36,37 inkjet
between the surface integrins of adherent mammalian cells and
printing,38−40 and electron beam lithography.41−43 Several of
these methods have yielded impressive advances in the types of
arrays that can be generated.44−47 However, few if any can
successfully combine the ability to generate submicron feature
sizes with biomolecular compatibility, the use of ordinary
proteins bearing “RGD” peptide motifs,15−18 our lab has
developed an alternative strategy in which synthetic DNA
strands introduced on the cell surfaces bind to sequence
complements displayed on the binding surface.19−26 While it
does require the additional step of covalently modifying the
cells, demonstrated advantages of this approach include its
generality for all biological cell types, exceptionally high capture
efficiency, and ability to generate complex multicellular patterns
Received: March 26, 2014
© XXXX American Chemical Society
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laboratory equipment, and high patterning speed. This latter
feature is particularly important for the future commercializa-
tion of these platforms for diagnostic use, for example.
The use of photoinitiated coupling reactions offers significant
promise for surface patterning, as they can harness the inherent
spatial precision and parallelization of photolithography
without the need for photoresists. This feature serves to
increase biocompatibility and obviate the need for multiple
etching and washing steps. In this approach, the species of
interest is first placed on a photoreactive surface, after which
irradiation initiates the reaction to immobilize the molecule
through a covalent linkage. This process has already been
demonstrated with reactions that display low chemoselectivity,
relying on highly reactive radical or nitrene intermediates
generated from reagents like aryl azides48,49 and benzophe-
nones.50 These intermediates insert into C−H, O−H, or N−H
bonds found in the biomolecular targets. While these reactions
have enabled the coupling of photoreactive biomolecules,48−51
surfaces,52 and caged species,53 the low reaction specificity
limits their applicability. Photoinitiated orthogonal bioconjuga-
tion reactions have also found use in this space, primarily with
thiol−ene,54,55 cyclopropenone-azide,56 quinone methide,57,58
and thioaldehyde59,60 reactions. However, few of these
reactions have been evaluated using thin films that are
compatible with photomasking techniques, and many require
additional steps for biomolecule attachment or precursors that
are synthetically difficult to access.
As a new photolithographic strategy for the generation of
complex patterns of DNA molecules on surfaces, we detail
herein a new photochemical bioconjugation reaction and
demonstrate its utility for immobilizing DNA-modified cells
in desired patterns. The process is based on the photoinitiated
reaction of aniline-modified glass surfaces with azidophenols,
enabling the preparation of a monolayer of covalently attached
single stranded DNA (ssDNA) on a surface upon UV
irradiation. Feature sizes as small as 20 μm are demonstrated,
and further improvements in resolution are likely possible. The
subsequent introduction of living cells modified with
complementary strands results in hybridization and immobili-
zation, allowing the straightforward generation of complex
cellular arrangements.
Figure 1. Rapid and efficient oxidative coupling reactions involving
anilines and iminoquinone intermediates (2). (a) The previously
reported reaction with aminophenol precursors requires ferricyanide as
an oxidant, ultimately yielding product 5. (b) The reaction of
azidophenol 6 is initiated by UV light and requires no additional
oxidant for the aniline coupling step to occur. This reaction
presumably involves nitrene 7, which rapidly rearranges to
iminoquinone 2. (c) In the absence of added oxidant, the oxidation
of initial adduct 4 can occur with O2 or through the redox transfer
pathway shown.
the amine to include a leaving group that would be lost upon
irradiation. Literature precedent suggested that, in fact,
azidophenols had the potential to undergo such a trans-
formation. In particular, early studies by Sander,69 Koseki,70
and Oleinik71 demonstrated that irradiation of o-azidophenol
(6, R1 = H) in an argon matrix liberates nitrogen gas to give the
corresponding nitrene (7). This intermediate rapidly rearranges
to form iminoquinone 2, providing a precedent that
azidophenols could indeed serve as photolabile iminoquinone
precursors. In other early work, Bayley has used a related
pathway for the photogeneration of inhibitors of photo-
synthetic electron transport.72 Our extensive studies involving
4-alkyl-substituted versions of the o-iminoquinone intermedi-
ates suggested that chemoselective bioconjugation to anilines
would be possible once these groups had been generated,
Figure 1b.
With this concept in mind, initial reaction studies involved
the irradiation of 4-methyl-2-azidophenol (6a, R1 = CH3) in the
presence of toluidine (3, Ar = 4-CH3C6H4) as an aniline
coupling partner at the absorbance maximum of 6a (Supporting
Information Figure S1). These reactants were exposed to 302
nm light for 5 min using a 6 W hand-held lamp in bis-tris buffer
at pH 6. The sole toluidine-containing product was identified as
5a (R1 = CH3), as characterized using reversed phase HPLC
and NMR (Figure S2). The success of the reaction was found
to be dependent on the ratio of the reacting species, with up to
95% HPLC yield being obtained with 1.5 mM 6a and 100 μM
3 (Figure S2).
RESULTS AND DISCUSSION
■
The development of an effective, light triggered surface
patterning strategy was predicated on our previous studies of
a series of aniline-based oxidative coupling methods for site-
specific bioconjugation.61−65 These reactions proceed via the
coupling of aminophenols and anilines (Figure 1a), presumably
involving iminoquinone intermediate 2. Although periodate ion
can also be used as a terminal oxidant,63 ferricyanide ion
mediates particularly clean coupling to afford p-iminoquinone 5
as a single, stable product.64 The reactions proceed very rapidly
(typically <2 min using periodate and <15 min using
ferricyanide) and can be used to modify biomolecules at low-
micromolar concentrations. In previous work, these coupling
strategies have been used to couple oligonucleotides,66
polymers,67 and chromophores64 to aniline groups introduced
on the surface of genome-free viral capsids and at the N-termini
of antibody Fc domains.68
The success of these reactions is based on the generation of
the key iminoquinone intermediate (2). While this is easily
done through the oxidation of o-aminophenol precursors, a
photochemical alternative could be envisioned by modifying
Although the reaction did not require an additional oxidant,
the p-iminoquinone species was nonetheless obtained. This
could occur from the aerobic oxidation of initial adduct 4 or
B
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from an intermolecular redox exchange with an additional
iminoquinone molecule (2), Figure 1c. While the latter
pathway yields a stable adduct involving the aniline group, it
also produces an aminophenol byproduct that can couple
competitively with an additional equivalent of 2 (albeit
apparently more slowly). In support of this pathway, small
amounts of dimeric species were isolated that were consistent
in mass with compound 10. The exact structure of this species
is still under investigation, as only minimal amounts are
available, and it has limited solution stability. In any case, this
secondary reaction potentially limits the efficiency of this
coupling strategy relative to our previous methods that involve
an excess of additional oxidants. Additional experiments to date
involving the addition of oxidants to the photochemical version
of the coupling reaction have produced complex product
mixtures, likely arising from the photochemical degradation of
the oxidants themselves.
irradiated capsids that lacked the pAF residues (see Figure S3).
The reaction reached complete conversion after 5 min of
irradiation, with an optimal pH range of 5−7 (Figures S4−S6).
To access more complex substrates, o-azidophenol NHS
ester 12 was prepared and coupled to a cyclic RGD peptide
(yielding 6b), PEG polymer chains (6c,d), and the 5′-terminus
of a 20 nt DNA strand (6e, see Supporting Information for
experimental details). All of these reagents could be prepared
on the benchtop under normal conditions and were stable for
months when stored at −20 °C in the dark. A 5 min exposure
of pAF-containing MS2 capsids to 6b (1 mM) in the presence
of 302 nm light led to the formation of the expected product in
about 25% yield, Figure 2c. The coupling of pAF-MS2 to PEG
substrates 6c and 6d under similar conditions was monitored
using SDS-PAGE, followed by staining with Coomassie blue
(Figure 3a). Densitometry analysis indicated that 56% of the
With the basic reactivity pattern in place, the biocompatibility
of the reaction was next tested in the context of protein
bioconjugation. The first of these experiments involved
genome-free MS2 viral capsids (277 nM, 50 μM in capsid
monomer) that possessed a p-aminophenylalanine (pAF)
residue in position 19 of each monomer, Figure 2a.62 This
Figure 3. Modification of anilines with PEG polymers and DNA
strands bearing 2-azidophenols. (a) pAF19 MS2 (50 μM in aniline
groups) was buffered with pH 6 bis-tris (10 mM) and irradiated for 5
min with 302 nm light in the presence of 6c−6e. (b) Oligonucleotide
6e (sequence B, 500 μM) was irradiated at 302 nm for 5 min in the
presence or absence of 2.5 mM toluidine. (c) In parallel, unmodified
sequence B was subjected to the same irradiation conditions to
confirm sequence integrity. All of the reactions were analyzed by
MALDI-TOF MS. The peak assignments are (i) m/z = 6602: 5e, the
expected coupling product of 6e with toluidine; (ii) m/z = 6496: 6e
after azide photolysis (loss of N2); (iii) m/z = 6333−6336: amine-
modified sequence B; (iv) m/z = 6464; and (v) 6370: abasic analogs of
5e and photolyzed 6e that are typically seen upon MADLI-TOF MS
ionization.
Figure 2. Modification of aniline-containing MS2 viral capsids with
azidophenols under photochemical conditions. (a) Each of the 180
capsid proteins contained one aniline group in position 19. The intact
structure (50 μM in aniline concentration) was irradiated in the
presence of azidophenols. ESI-MS yielded the reconstructed mass
spectra for reaction with (b) 6a (R = CH3) and (c) 6b. A negative
control irradiating 6a with wild type T19 MS2 showed no reaction.
Expected mass for the coupling product with 6a: m/z = 13911;
expected mass for the coupling product with 6b: m/z = 14554.
monomers had been modified using the 2 kDa polymer and
that 39% were modified using the 5 kDa polymer. These yields
are lower than those achieved using aminophenols and external
oxidants (perhaps due to the competing coupling pathway
described above), but the results nonetheless confirm that
useful levels of site-selective modification can be achieved under
photochemical control. As a second substrate, an endocellulase
from Pyrococcus horikoshii (EGPh) bearing a p-aminophenyla-
lanine residue in position 2 was also modified with 6d in 60%
yield (Figure S7).
residue was introduced using the amber codon suppression
techniques developed by the Schultz lab,73 providing 180
aniline groups on the external surface of each 27 nm protein
shell. These capsids were combined with compound 6a (R1 =
CH3, 1 mM) in bis-tris buffer at pH 6. Following irradiation at
302 nm with a hand-held lamp for 5 min, excellent conversion
was observed to the expected product, Figure 2b. In contrast,
no coupling was observed in the absence of irradiation or for
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Figure 4. Cell immobilization via DNA hybridization on glass slides. (a) Aniline-coated glass surfaces were generated starting from aldehyde glass or
through treatment with silane 14 followed by a TCEP wash. UV irradiation of the aniline surfaces coated with a thin film of azidophenol DNA
yielded covalent modification of the glass surface. (b) After photopatterning, S. cerevisiae modified with a complementary DNA sequence was then
exposed to the surface strands to allow hybridization to occur. (c) DNA-labeled S. cerevisiae was exposed to slide surfaces treated with (from left to
right) (i) matched sequence 6e and irradiation (positive experiment), (ii) matched sequence 6e without irradiation, (iii) mismatched sequence 6e
and irradiation, and (iv) O-benzylhydroxylamine, followed by matched sequence 6e and irradiation. (d−g) UV irradiation of dehydrated films of 6e
on aniline glass through a photomask, followed by addition of S. cerevisiae and gentle rinsing, resulted in the pattern shown (yeast appear as dark
regions). Patterns were made of (d) the Earth with a dashed line indicating pattern edge, (e) a marker with 30 μm features, (f) a triangle with sharp
vertices, and (g) a series of lines 20 μm in width. Aniline glass was generated from (d,e) alkoxyamine 13 or (f,g) silane 14.
While the experiments above confirm that the reaction is
compatible with peptides and proteins containing most amino
acids, it was found to be incompatible with free cysteine
sulfhydryl groups, as multiple addition products were observed.
However, free cysteines can be capped with maleimides prior to
irradiation, preventing further reactivity (Figure S8).
bioconjugates were analyzed carefully as the exposure of
oligonucleotides to UV light is known to result in thymine
cyclobutane dimers and Dewar valence isomers.76,77 Using a
combination of dynamic light scattering measurements and
SDS-PAGE gel shift assays, it was demonstrated that the DNA-
MS2 construct was able to undergo successful hybridization
after the photochemical bioconjugation step (Figure S10). To
demonstrate that ability further, the melting temperature of two
different ssDNA sequences and their complements was found
to be unaltered by the irradiation step (Figures S11 and S12).
While particularly problematic sequences, such as poly(T)
motifs, may still be incompatible with this technique, these
results suggest that a very large number of capture sequences
can be used.
The adaptation of this reaction strategy for the covalent
attachment of DNA to surfaces required the development and
optimization of four independent steps: (1) the generation of
an aniline-functionalized surface, (2) the deposition of a thin
film of DNA conjugate 6e (Figure S13), (3) the exposure of
selected regions of the surface to light to initiate the reaction,
and (4) the development of a rinsing protocol to remove
unreacted DNA strands. The requisite aniline functionality was
introduced onto glass surfaces using one of two methods. In the
first, aldehyde-silanized glass was exposed to alkoxyamine
reagent 13 in the presence of additional aniline78,79 (Figure 4a).
In the second approach, freshly plasma-cleaned glass slides were
These protein-based experiments demonstrate that the
aniline coupling reaction can be photoinitiated and still retain
much of the high chemoselectivity, speed, and conversion levels
that are characteristic of the parent strategy. To test its
compatibility with the DNA strands required for surface
patterning, 20 nt ssDNA 6e was first coupled to toluidine. After
5 min of irradiation at 302 nm using a hand-held lamp,
MALDI-TOF MS analysis indicated clean conversion to the
expected product, Figure 3b (species i). In the absence of the
aniline coupling partner, no reaction was observed other than
the loss of nitrogen from the azide group (species ii). The
parent DNA strand showed no change in mass upon irradiation
under these conditions (Figure 3c). Strand 6e was also coupled
successfully to MS2 capsids containing pAF groups, as
confirmed using an SDS-PAGE gel shift assay (Figure 3a,
lane 4). Densitometry analysis indicated that about 14% of the
monomers had participated in the reaction, corresponding to
25 DNA strands on each capsid.
Since DNA has successfully been used with primarily long-
wave UV light in other applications,30,32,74,75 our resulting
D
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exposed to a solution of phenylazide silane 14 in acidic
methanol (Figure S13a). In the latter case, the azide groups
were subsequently reduced to anilines using TCEP before use.
The final contact angles of the modified surfaces were estimated
as 40° in both cases, suggesting the successful formation of the
organic monolayer.
the reagents make it a good candidate for widespread adoption
and application. The demonstrated use of this method in
combination with DNA-mediated cell patterning allows for
significantly faster and more precise immobilization of cells
with respect to the current state of the art. This UV-initiated
reaction provides a robust solution to patterning that is not
easily achieved using current techniques and has great potential
for simple and rapid fabrication of spatially resolved, multi-
functional surfaces.
Following surface derivatization, a thin film of DNA was then
deposited on the surface using a solution of 6e in a 1:1 mixture
of water and methanol (see Figure S14). The solution was
distributed with a 1 cm-thick rectangular piece of polydime-
thylsiloxane as a spreader, and evaporated to leave a thin layer
of DNA. Covalent surface modification was then initiated by
ultraviolet irradiation with a mercury arc lamp through a mask
with an aligner for 1 min, resulting in DNA patterning only in
regions corresponding to transparent regions of the mask. The
DNA species that did not react with the surface were then
removed by rinsing with a 0.4% aqueous solution of sodium
docecyl sulfate, followed by deionized water. The DNA-
modified surface was then blocked for 30 min with a 1%
solution of bovine serum albumin in Dulbecco’s phosphate
buffered saline to minimize nonspecific interactions. Subse-
quent introduction of Saccharomyces cerevisiae modified to
display the complementary DNA strand23 allowed for efficient
and rapid patterning of cells only on the regions of the slide
that were exposed to the light source, as shown in Figure 4.
A number of control experiments were performed to confirm
the DNA-dependent nature of the patterning (Figure 4c). No
DNA was deposited in the nonilluminated areas, and the
unreacted aniline groups did not bind the DNA-coated cells
(especially after BSA blocking). In addition, no patterning was
observed after irradiation in the presence of noncomplementary
strand 6e (“mismatched”) or after the irradiation of glass
modified with nonreactive benzylalkoxyamine 15 in place of
aniline alkoxyamine 13. Analogous control experiments for
glass modified with silane 14 yielded similar results (Figure
S14b).
CONCLUSION
■
In summary, we have developed a new photoinitiated reaction
involving azidophenols and anilines, and we have demonstrated
its utility for the photopatterning of DNA strands on surfaces
with subsequent cell immobilization. We are currently
developing azidophenol analogs with longer wavelength
sensitivity to broaden substrate compatibility. The general
nature of this method should facilitate its use in a variety of
applications, and our lab is currently focusing on extending the
scope of this technique to include the rapid generation of
multistrand DNA patterns for applications in cell and enzyme
patterning. As we have previously demonstrated the use of
DNA as a templating architecture for a variety of cell types,
including adherent and nonadherent mammalian cells, we
envision using this technique to study cellular interactions
resulting from the patterning of multiple cell types in close
proximity. Finally, the technical simplicity and large number of
substrates already demonstrated with this method (i.e., small
molecules, polymers, proteins, and nucleic acids) suggest that it
could provide a broadly applicable solution to photopatterning.
ASSOCIATED CONTENT
* Supporting Information
Full experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
A significant advantage of this approach is the ability to
photopattern complex features quickly, as demonstrated by the
generatation of several patterns of living yeast cells using masks
during the irradiation steps. Figure 4d exemplifies the ability to
generate accurate arbitrary patterns with minimal levels of
background binding. In addition, Figures 4e−g show the ability
of the method to generate feature sizes as small as 20 μm while
maintaining a high degree of cell binding. Aniline glass prepared
using the oxime formation or through the use of silane 14 and
subsequent TCEP reduction functioned equivalently in these
experiments.
An additional attractive feature of this new patterning
method is its efficient use of the DNA-azidophenol reagent,
which becomes increasingly important for the coverage of large
slide areas. In our previous work, we have used a reductive
amination method to attach amine-labeled DNA to aldehyde-
coated glass.20−26 Those optimized conditions require 1 nmol
of DNA reagent to cover 1 cm2 of slide, and even this amount
leads to incomplete coverage when applied to large areas. Due
to the higher efficiency of this photochemical method, as little
as 128 pmol of DNA can be used to cover the same 1 cm2 area.
Importantly, significantly greater cell pattern density can be
achieved despite the lower quantity of reagent that is applied
(for example, see Figure 4f).
AUTHOR INFORMATION
Corresponding Author
mbfrancis@berkeley.edu
■
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Synthetic Biology Institute at
UC Berkeley. AAT and ACO were supported by Graduate
Research Fellowships from the NSF. Gary J. Tong provided P.
horikoshii endocellulase cultures. Prof. Michelle Chang and
Prof. Carolyn Bertozzi provided instrumentation access.
Zhongrui Zhou and Tony Iavarone at the QB3/Chemistry
Mass Spectrometry preformed sample analysis. The Biomo-
lecular Nanotechnology Center provided additional instrumen-
tation access.
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