Patterned surface derivatization using diels-alder photoclick reaction

Article abstract of DOI:10.1021/ja205652m

The utility of photochemically induced hetero-Diels-Alder reaction for the light-directed surface derivatization and patterning was demonstrated. Glass slides functionalized with vinyl ether moieties are covered with aqueous solution of substrates conjuga

Full text of DOI:10.1021/ja205652m

ARTICLE
pubs.acs.org/JACS
Patterned Surface Derivatization Using DielsꢀAlder
Photoclick Reaction
Selvanathan Arumugam and Vladimir V. Popik*
Department of Chemistry, University of Georgia, Athens, Georgia, 30602
S Supporting Information
b
ABSTRACT: The utility of photochemically induced hetero-
DielsꢀAlder reaction for the light-directed surface derivatiza-
tion and patterning was demonstrated. Glass slides functional-
ized with vinyl ether moieties are covered with aqueous solution
of substrates conjugated to 3-(hydroxymethyl)-2-naphthol
(NQMP). Subsequent irradiation via shadow mask results in
an efficient conversion of the latter functionality into reactive
2-napthoquinone-3-methide (oNQM) in the exposed areas.
oNQM undergoes very facile hetero DielsꢀAlder addition (kD-A ≈ 4 ꢁ 104 Mꢀ1sꢀ1) to immobilized vinyl ether molecules
resulting in a photochemically stable covalent link between a substrate and a surface. Unreacted oNQM groups are rapidly hydrated
to regenerate NQMP. The click chemistry based on the addition of photochemically generated oNQM to vinyl ether works well in
aqueous solution, proceeds at high rate under ambient conditions, and does not require catalyst or additional reagents. This
photoclick strategy represents an unusual paradigm in photopatterning: the surface itself is photochemically inert, while
photoreactive component is present in the solution. The short lifetime (τ ≈ 7 ms in H2O) of the active form of a photoclick
reagent in aqueous solution prevents its migration from the site of irradiation, thus allowing for the spatial control of surface
derivatization. Both o-napthoquinone methide precursors and vinyl ethers are stable in dark and the reaction is orthogonal to other
derivatization techniques, such as acetyleneꢀazide click reaction.
’ INTRODUCTION
and thiol-yne19 reactions, as well as photochemical generation of
reactive acetylenes10b and uncaging of hydroquinone moieties.20
We have recently reported a novel photoligation strategy
based on very facile hetero-DielsꢀAlder addition of 2-naptho-
quinone-3-methides (oNQMs) to vinyl ethers.21 Thus, irradia-
tion of substituted 3-(hydroxymethyl)-2-naphthols (oNQM
precursor, NQMP, 2) results in efficient (Φ = 0.20) dehydration
of the substrate and the formation of oNQM 1 (Scheme 1).22 In
the presence of vinyl ethers (3), oNQMs undergo very facile
(k ≈ 4 ꢁ 104 Mꢀ1sꢀ1) and quantitative DielsꢀAlder cycloaddi-
tion to yield photostable benzo[g]chromans (4, Scheme 1).21
The unreacted oNQM is rapidly hydrated (kH2O ≈ 145 sꢀ1) to
regenerate NQMP (2).22
In this report, we demonstrate a novel paradigm in light-
directed surface derivatization. Conventional photopatterning
relies on selective (using shadow mask or laser) activation of
light-sensitive compounds immobilized on the surface, which
then reacts with the second component in the dark step. In our
approach, surface is photochemically inert, thus simplifying
preparation and handling of the substrates, but photoreactive
NQMP (2) is present in solution. A very short lifetime of oNQM
(1) species prevents their migration from the site of irradiation,
allowing for high spatial resolution of derivatization. Additional
advantage of this approach is in quantitative regeneration of
“Click” reactions1 represent a set of very useful tools in
modern chemistry and biochemistry.2,3 Copper(I)-catalyzed
1,3-dipolar cycloaddition of azides to terminal acetylenes, also
known as azide click reaction, became the gold standard of click
chemistry and have found applications in many areas ranging
from material science3,4 to chemical biology5,6 and drug develop-
ment.7 While acetyleneꢀazide click chemistry has become
commonplace in surface derivatization, as well as polymer and
materials synthesis,3,4 the use of metal catalyst often limits the
utility of the method. Recently developed catalyst-free strategies
of azide click ligations8,9 were successfully applied to surface
derivatization.10 “Click” methods based on a DielsꢀAlder
cycloaddition are also gaining popularity since this reaction does
not require catalysts, can proceed in high yield under physiolo-
gical conditions, and does not produce any byproduct.11 The
DielsꢀAlder click reaction has found applications in material
chemistry,11,12 derivatization of nanoparticles and surfaces with
various bioactive molecules,13 as well as the labeling of oligonu-
cleotides, proteins, and oligosaccharides.14 However, the gen-
eration of a reactive dieneꢀdienophile pair often requires either
thermal activation15 or the use of chemical promoters for the
in situ generation of highly reactive dienes.16
Light-induced “click” reactions permit spatial control of the
immobilization processes. Several photoclick strategies are under
development, including cycloaddition of alkenes to photo-
chemically generated nitrile imine,17 photoinitiated thiolꢀene,18
Received: June 29, 2011
Published: August 23, 2011
r
2011 American Chemical Society
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Scheme 1
Scheme 2. Synthesis of NQMP-PEG3-Biotin (2b)a
a Reagents and conditions: (a) TBDMS(OCH2CH2)3I, K2CO3, acetone, reflux, 65%; (b) i) LiAlH4, THF; HF/CH3CN aq., 85% over 2 steps; (c)
TsOH, 2,2-dimethoxypropane, acetone,90%; (d) TsCl, pyridine, DCM, 82% ; (e) NaN3, DMF,60 °C, 80%; (f) LiAlH4, Et2O, 85%; (g) d-biotin, EDC,
DMAP, DMF, 83% ; (h) Amberlyst-15, methanol, RT, 95%.
NQMP by hydration of unreacted oNQM molecules. Since only
a small fraction of NQMP is consumed in the derivatization
procedure, the labeling solution can be reused many times. The
addition of oNQMs to vinyl ethers is much faster than conven-
tional click reactions providing an opportunity for the develop-
ment of time-resolved methods. Finally, competition between
hydration and cycloaddition makes oNQMs very selective. Only
vinyl ethers are reactive enough to outcompete water. This is a
definite advantage over methods involving generation of other
reactive species, e.g., radicals, nitrenes, and carbenes.
of NQMP with various “payload” substrates. Thus, molecules
inert to hydride reduction can be coupled directly to 5. NQMP-
TEG (2c) or its protected analog 7 possess primary alcohol
moiety, which is suitable for Mitsunobu coupling or esterifica-
tion; acetylene click reaction can be employed to modify azide
derivative 9; molecules containing carboxylic acid or NHS-ester
groups can be readily coupled with protected NQMP-TEG-
NH2 (10).
Light-Directed Surface Derivatization. Vinyl ether derivati-
zation of commercial epoxide-functionalized glass slides was
achieved by treating the slides with 2-hydroxyethyl vinyl ether
or 6-methoxyhex-5-en-1-ol in the presence of catalytic amount of
anhydrous p-toluenesulfonic acid. Alternatively, incubation of
epoxy slides in 2-(2-(vinyloxy)ethoxy)ethanamine dichloro-
methane solution yields vinyl ether functionalized glass slides
without the need for a catalyst.24
’ RESULTS AND DISCUSSION
To illustrate the utility of photochemically induced hetero-
DielsꢀAlder cycloaddition for light-directed surface derivatiza-
tion, we have patterned fluorescently labeled Avidin onto vinyl
ether-derivatized glass slides.
The resulting vinyl ether-derivatized slides were covered with
aqueous solution of NQMP-TEG-biotin 2b (1 ꢁ 10ꢀ4 M), and
irradiated using 300 or 350 nm fluorescent lamps. Photochemical
dehydration of 3-(hydroxymethyl)-2-naphthol moiety in 2b
produces derivative of oNQM, which undergo very facile Dielsꢀ
Alder cycloaddition to vinyl ether groups on the surface result-
ing in covalent immobilization of biotin molecules. Unreacted
oNQM species rapidly react with water to regenerate 2b. Bio-
tinylated glass slides were then developed with FITC-avidin in
PBS solution for 15 min at 2 °C and thoroughly washed (Scheme 3).24
Surface derivatization was followed by FTIR and contact angle
measurements.24
Preparation of Materials. Synthesis of 6-(hydroxymethyl)-
naphthalene-1,7-diolꢀtri(ethylene glycol)-biotin conjugate (NQMP-
TEG-biotin, 2b) is outlined on the Scheme 2. Methyl-3,5-dihy-
droxy-2-naphthoate (5) was prepared according to previously
reported literature23 and was etherified to iodo-TEG-TBDMS,
followed by lithium aluminum hydride reduction and deprotec-
tion to give NQMP-TEG (2c). The glycol moiety was then
protected as acetone ketal and TEG terminal hydroxy group was
tosylated. Nucleophilic substitution of tosyl ester with azide ion
produced 9, which was further reduced to an amino derivative 10.
EDC-promoted coupling of the latter with d-biotin, followed
by ketal hydrolysis produced target NQMP-TEG-biotin (2b,
Scheme 3).
Two procedures were employed to achieve patterned immo-
bilization of FITC-avidin on vinyl ether-derivatized slides. In the
first method, 2 μL droplets (∼1 mm in diameter) of 0.1 mM
It is important to note, that synthetic intermediates shown
in the Scheme 2 can serve as entry points for the conjugation
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Scheme 3. Schematic Representation of Preparation and Light-Directed Biotinylation of Vinyl Ether-Coated Slides Followed by
Immobilization of FITC-Avidina
a The insert shows fluorescent images of vinyl ether-coated slides spotted with 2 μL drops of 10ꢀ4 M NQMP-TEG-biotin aqueous solution and flood
irradiated (A); or covered with 10ꢀ4 M NQMP-TEG-biotin aqueous solution and irradiated via “A” mask (B) or 12.5 μm pitch copper grid (both
enlarged). Insert “D” shows fluorescent intensity profile along the line perpendicular to the pattern “C”.
NQMP-TEG-biotin (2b) aqueous solution were placed on the
slides, which were then flood irradiated for 10 min with fluor-
escent UV lamp, washed, and stained with FITC-avidin24
(Scheme 3, Insert A). The actual photopatterning has been
achieved by the irradiation of the slides covered with solution of
2b via a shadow mask. The “latent” image was also developed
using FITC-avidin (Scheme 3, inserts B and C). We used two
different masks for irradiation: first one had ∼2 mm high letter
“A” cut in a steel plate. For the second, we used 12.5 μm pitch
copper grid. As insert C in Scheme 3 illustrates this avidin
photopatterning method readily reproduce features as small as
5 μm (the width of the grid bars; Scheme 3, insert D). This is a
remarkable result, since the photoreactive compound is not
immobilized on the surface but rather present in low viscosity
solution. High resolution photopatterning in this case relies on a
short lifetime (τ ≈ 7 ms) of oNQM in aqueous solution, which
Figure 1. Relative fluorescent intensity of FITC-avidin-stained vinyl
prevents its migration from the site of irradiation.
ether-derivatized glass slides after various time of irradiation in 0.1 mM
NQMP-TEG-biotin (2b) solution. Numerical values of average fluor-
escent intensity of the slide are shown next to each image.
To evaluate the efficiency of photochemical surface derivatiza-
tion, vinyl ether-coated slides covered with 0.1 mM aqueous
solution of NQMP-TEG-biotin (2b) and irradiated for various
periods of time. Slides were then washed, treated with FITC-
avidin, and fluorescence intensity was recorded. The relative
fluorescence intensity of photoderivatized slides increased with
the exposure time reaching saturation at approximately 10 min.
Longer irradiation times have virtually no effect on the fluores-
cence of the slides (Figure 1). The vinyl ether-derivatized slide
incubated for 30 min in solution of 2b in the dark shows no
detectable Fluorescein emission (0 min, Figure 1).
solution and washed. One of the slides was further irradiated in
aqueous PBS solution for 30 min. Both slides were then devel-
oped with FITC-avidin solution. The fluorescent intensity of
the irradiated slide was 99% of the control one. This experi-
ment confirms that the formation of DielsꢀAlder adduct on the
surface is photochemically irreversible. Since concentration
of the NQMP-TEG-biotin (2b) in the solution remains virtually
unchanged after photolysis, saturation of fluorescence after
10 min of irradiation indicates that all accessible vinyl ether
groups on the surface have reacted with oNQMs. Growth of
To test the photostability of photochemically induced linkage,
two slides were biotinylated using 10 min irradiation in 0.1 M 2b
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Figure 2. Relative fluorescent intensity of FTIC-Avidin-stained glass
slides biotinilated under different conditions.
Figure 4. Vinyl ether-derivatized slides AꢀD were spotted with NQMP
2b solution and irradiated with 300 nm light. Slide “D” was further
irradiated in NQMP 2c solution. Slides were stained with FTIC-Avidin
and washed in PBS solution for h (A and D), 6 h (B), and 16 h (C).
Average signal-to-background fluorescent intensity ratios are shown
under each image.
Protein patterning procedures often require extensive washing
to remove substrate nonspecifically absorbed on the surface. In
our experiments, the FITC-Avidin-stained surfaces (e.g., A and B,
Scheme 3) were washed by sonicating the glass slides in PBS
solution for 30 min followed by overnight incubation in fresh
phosphate buffer. From practical point of view, a shorter washing
procedure could enhance the efficiency of photoclick protein
patterning. In order to reduce nonspecific protein binding, we
have developed a photochemical PEGylation procedure. After
the initial patterning of the substrate on the vinyl ether-deriva-
tized surfaces, slides are subjected to flood irradiation in the
aqueous solution of NQMP-TEG (2c, Figure 4). This procedure
makes previously unexposed areas highly hydrophilic and sig-
nificantly reduces protein binding. Longer PEG units attached to
NQMP 2 can further increase this effect.
To test the efficiency of the photo-PEGylation procedure,
four commercial epoxy-coated glass slides were treated with
2-(2-(vinyloxy)ethoxy)ethanamine under same conditions
(Scheme 3). The resulting vinyloxy-derivatized slides were
spotted with 2 μL droplets of 0.1 mM aqueous solution of
NQMP-TEG-biotin (2b) and irradiated for 3 min with 300 nm
fluorescent UV lamps. One of these slides was then immersed in
0.1 mM aqueous solution of NQMP-TEG (2c) and irradiated for
additional 3 min. After FITC-avidin labeling, the slide was rinsed
with water and incubated in PBS solution for 1 h. The resulting
fluorescent image of this slide shows high contrast between
biotinilated and biotin-free areas (Figure 4 D). Three other slides
were treated with FITC-Avidin, rinsed, and incubated in PBS
solution for 1, 6, and 16 h, respectively (Figure 4AꢀC). The
1- and 6-h long washing are clearly insufficient to remove
nonspecifically absorbed avidin from the slide. Only 16 h washing
procedure produces contrast approaching that of photo-PEGy-
lated slide. These experiments show that flood irradiation of
biotin-patterned vinyloxy-slide does not decrease the amount of
immobilized biotin molecules but drastically reduces nonspecific
binding of avidin to the slide.
Figure 3. UV-spectrum of 0.05 mM aqueous solution of NQMP-TEG-
biotin (2b).
fluorescence intensity follows zeroth-order kinetics (Figure 1),
indicating that the formation and diffusion of oNQM to the
surface, rather than cycloaddition to immobilized vinyl ether
groups, is a rate-limiting step in the derivatization process.
It is important to note that all experiments shown discussed
above (Scheme 3C and Figure 1) were conducted using the same
solution of 2b. Since the amount of the substrate required to
derivatize a monolayer is so minute, no detectable changes of
concentration of NQMP 2b were detected by UV measurements
in the course of photobiotinylation experiments. HPLC analysis
shows no significant accumulation of byproducts even after eight
photolyses, underscoring the cleanness of the photodehydrationꢀ
hydration reaction of 2b.
The irradiation time can be adjusted using different light
sources or concentrations of NQMP. Medium pressure mercury
lamp equipped with 295 nm cut off glass filter allows us to cut
exposure time to 3 min at 0.1 mM NQMP 2b (Figure 2).
However, if longer wavelength irradiation is required, 350 nm
light can be employed for patterning. This process, however, is
less efficient. Using 350 nm lamps with intensity similar to
300 nm fluorescent tubes discussed above, the complete fictio-
nalization is achieved at ∼100 min of irradiation. The reduced
rate of surface derivatization is, apparently, due to the lower
extinction coefficient of NQMP 2b at 350 nm (Figure 3). In fact,
at 10-fold higher concentration of 2b (1 mM), complete con-
version at this wavelength is achieved in 10 min (Figure 2).
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Scheme 4
Figure 5. Hydrolytic stability of photochemically biotinylated slides.
Derivatization procedures discussed above produce glass slides
with vinyl ether moieties attached to the surface via the vinylic
oxygen atom (L1, Scheme 4). 2-Alkoxy substituent (O-L1) in
benzo[g]chroman 4, which is formed in the photoclick reaction,
is acid labile and can be hydrolyzed off at pH 1 (Scheme 4).21
This hydrolytic liability of the linker 4 permits complete
removal of the previously immobilized molecules from the glass
surface. We have employed acid cleavage procedure to evaluate
the efficiency of the photoclick immobilization. For this purpose
slides of known epoxide group density (VWR # 16001ꢀ030,
density = 2 ꢁ 1013 molecules per mm2, surface area =1875 mm2)
were functionalized with vinyl ether groups and subsequently
irradiated with 300 nm lamps for 10 min in 0.1 mM aqueous
solution of NQMP 2a. The resulting slides that contained
benzochroman 4a on the surface were incubated in 0.1N perchloric
acid solution overnight to induce the release of 2-hydroxyben-
zochroman 12a (Scheme 4). The concentration of 12a in the
supernatant solution was determined from its absorbance at
281 nm.24 The surface density was calculated from triplicate
measurements to be (1.40 ( 0.06) ꢁ 1013 of 4a molecules per
mm2. In other words, all three steps of the process, i.e., vinyl ether
functionalization of epoxy slides, photoclick derivatization, and
acid-catalyzed release of the substrate, are very efficient proceed-
ing with at least 70% overall yield.
imager (Figure 5). The fluorescent intensity of the acid-treated
slide was less than 2% of the second one, indicating almost
quantitative loss of biotin molecules from the surface.
Photo-DielsꢀAlder click reaction can also be employed in
applications that require hydrolytically stable linker between a
surface and immobilized substrate. In this case, vinyl ether groups
should be attached to the surface via β-carbon atom rather than
ether linkage (e.g., 30 Vs 3 in Scheme 4). We have prepared
30-derivatized surfaces by treating the epoxide glass slides with
6-methoxyhex-5-en-1-ol in methylene chloride in the presence of
catalytic amount of anhydrous p-toluenesulfonic acid. These
slides were then irradiated in an aqueous solution of NQMP
2b to yield biotinylated slides. One set of these slides was stained
with FITC-avidin after irradiation; the second set was incubated
in 0.1 M perchloric acid solution overnight before staining. As
clearly seen from the Figure 5, incubation in acidic solution did
not significantly affect the fluorescent intensity of photobiotiny-
lated slides, indicating that density of biotin groups on the surface
remained the same. This observation underscores the stability of
linker 4b0 (Scheme 4) to acid-catalyzed hydrolysis.
’ CONCLUSIONS
The photoclick immobilization strategy described in this work
is based on the fast and efficient hetero-DielsꢀAlder cycloaddi-
tion of photochemically generated o-naphthoquinone methides
(oNQMs) to immobilized vinyl ether moieties. Since vinyloxy-
derivatization of the surface is achieved via a simple and efficient
process and a wide variety of substrates can be attached to
naphthoquinone methide precursors, 3-(hydroxymethyl)-2-
naphthols (NQMP), this method offers a new platform for
To evaluate the yield of the acid-induced detachment proce-
dure, vinyl ether-derivatized slides were photobiotinilated by
irradiation in NQMP 2b solution. One set of the resulting slides
was then incubated overnight in 0.1 M perchloric acid. The acid-
treated and the control slides were stained with FITC-avidin,
washed in PBS solution overnight, and scanned using Typhoon
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light-directed surface functionalization. Photo-DielsꢀAlder pat-
terning approach is orthogonal to other derivatization techniques
and can be used in conjunction with well-developed acetyleneꢀ
azide click chemistry. The high rate of oNQMs cycloaddition to
vinyl ether moieties makes this photochemical derivatization
strategy very efficient. The unreacted oNQMs are quenched with
water to regenerate NQMP. The competition between hydration
and cycloaddition makes oNQMs very selective: only vinyl ethers
are reactive enough to outcompete addition of water to the
substrate. We also describe a new photopatterning paradigm, in
which photochemically inert surface is selectively irradiated in a
solution of a photoactive component. The short lifetime of
photogenerated reactive species (oNQM) limits their migration
from the site of irradiation and permits high spatial resolution of
the patterning process. The photoclick immobilization technique
comprising o-napthoquinone methide precursor and vinyl
ether can be tailored to produce permanent or a hydrolytically
labile linkage.
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Corresponding Author
vpopik@uga.edu
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’ ACKNOWLEDGMENT
Authors thank Professor Jason Locklin and Ms. Sara Orski for
the useful discsussions and assistance in surface characterization;
Professor Geert-Jan Boons and Dr. Margreet Wolfert for help
with slide imaging experiments; Professor Yan Geng and
Ms. Jennifer Haley for providing fluorescent microscope for
our experiments; Georgia Cancer Coalition for the support of
this project.
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