A generic small-molecule microarray immobilization strategy

Article abstract of DOI:10.1016/j.tetlet.2011.10.055

Small-molecule microarrays are often limited by the requirement for each compound undergoing immobilization to contain a common functional group or by the need to prepare glass slides containing photo-reactive groups. Herein, we present a generic strategy

Full text of DOI:10.1016/j.tetlet.2011.10.055

Tetrahedron Letters 52 (2011) 6819–6822
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A generic small-molecule microarray immobilization strategy
⇑
Mariona Vallès-Miret, Mark Bradley
EaStChem, School of Chemistry, University of Edinburgh, Joseph Black Building, West Mains Road, EH9 3JJ Edinburgh, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Small-molecule microarrays are often limited by the requirement for each compound undergoing immobi-
lization to contain a common functional group or by the need to prepare glass slides containing photo-reac-
tive groups. Herein, we present a generic strategy that allows any compound library to be immobilized. This
was achieved by printing a fluorous-tagged photo-reactive 3-aryl-3-trifluoromethyldiazirine, which
undergoes non-selective insertion into compounds following UV-activation, onto fluorous-functionalized
glass slides. The arrays could be reused following aqueous stripping and re-assessment of the compounds
with the same protein or another target of interest.
Received 27 June 2011
Revised 27 September 2011
Accepted 10 October 2011
Available online 17 October 2011
Keywords:
Small-molecule microarrays
Photoreactive functional groups
Microwave-assisted synthesis
Fluorous affinity
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Small-molecule microarrays are potentially a powerful tool for
the high-throughput screening of compound libraries with a vari-
ety of protein or cellular targets.1 While small-molecule micro-
arrays have allowed the identification of small molecule-protein
binding partners,2 this approach typically requires the inclusion
of a specific common handle in the compounds of interest to facil-
Scheme 1. Generic small-molecule immobilization strategy. Fluorous slides were prepared by treating glass slides with tridecafluoro-1,1,2,2-tetrahydrooctyl-dimethoxy-
chlorosilane (TFCS). Fluorous tagged diazirines were printed onto the fluorous slide with the small-molecules subsequently printed at the same location. Irradiation (350 nm)
generates a carbene which inserts non-selectively into the over-laid compound.
⇑
Corresponding author. Tel.: +44 131 650 4820; fax: +44 131 650 6453.
E-mail address: mark.bradley@ed.ac.uk (M. Bradley).
0040-4039/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.055

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M. Vallès-Miret, M. Bradley / Tetrahedron Letters 52 (2011) 6819–6822
itate immobilization onto a functionalized glass slide.3 One such
approach has used fluorous tagging of the compounds with micro-
array fabrication on fluoroalkyl modified glass slides.4 However,
more common approaches are based on the generation of combi-
natorial libraries which are then covalently attached onto function-
alized surfaces through a variety of selective chemical reactions
such as 1,4-additions, Diels–Alder, Staudinger or 1,3-dipolar addi-
tion chemistry or biotin/avidin interactions.5 A generic approach
for the stable capture of compounds has been the application of
photoreactive surfaces prepared from diazirines and aryl azides
which after irradiation generate highly reactive groups which
undergoes non-selective insertion reactions into a broad range of
molecules.6
Here, we describe the development of a generic method for
small-molecule immobilization which combines two strategies,
the covalent capture by photo-crosslinking through a diazirine
functional group tied in with the advantages of non-covalent fluor-
ous affinity interactions (Scheme 1). The photochemistry immobi-
lization approach allows the attachment of any type of compound
library without the need of library synthesis to include a common
handle, while widening the scope of the technique to include nat-
ural products; while fluorous chemistry prevents non-specific
interactions with proteins or cells, lowering the background and
increasing the signal-to-noise ratios. In addition, this hybrid meth-
od offers the possibility of recovering individual spots for further
analysis and protein stripping allowing recycling/reuse of the
array.
The strategy consisted of the immobilization of a fluorous
tagged diazirine on a fluorous surface. Irradiation of the diazirine
generates a carbene which immobilizes any proximal small mole-
cule in a non-selective manner. Experiments for the validation and
optimization of the approach were carried out using fluorescein
and biotin as model small molecules.
Scheme 2. Synthesis of {4-[3-(trifluoromethyl)-3H-diaziren-3-yl]phenoxy}acetic
acid (TFMAD) (2).
As the length of the spacer (see Scheme 1) would be expected to
have an effect on immobilization efficiency7 three different photo-
spacers were synthesized by solid phase synthesis using the Fmoc-
protected spacer unit (Fmoc-8-amino-3,6-dioxaoctanoic acid
spacer 1) and the photoreactive diazirine derivative {4-[3-(trifluo-
romethyl)-3H-diaziren-3-yl]phenoxy}acetic acid (2).
1H,1H,2H,2H-perfluorodecane, using PyBop and DIEA, and purified
via a fluorous silica column to give 18, 19, and 20.
The synthesis of the diazirine derivative 2 started with the pro-
tection of 4-bromophenol (3) with trimethylsilyl chloride followed
by a reaction with nBuLi and trifluoromethyl ethyl acetate to give 5
in 68% yield (Scheme 2). The phenol was then alkylated with t-bu-
tyl bromoacetate under microwave irradiation to give 6. Oxime 7
was generated by treatment with hydroxylamine hydrochloride
in pyridine and reacted with p-toluensulfonyl chloride to give 8.
Both reactions were performed under microwave irradiation. For-
mation of diazirine 9 was achieved by treatment of 8 with liquid
ammonia overnight. Iodine was used for the oxidation of 9 to give,
following removal of the t-butyl protecting group with trifluoro-
acetic acid, the trifluoromethyl aryldiazirine 2 in an overall yield
of 35%. Reaction times for some of the steps were shortened by
using microwave-assisted reactions.8b
Fluorous tagged biotin 21 and carboxyfluorescein 22 were syn-
thesized as positive controls in the same manner (Fig. 1).
In order to establish the best approach to immobilize com-
pounds different strategies for printing and washing the slides
were assessed using fluorous tagged carboxyfluorescein 22. Fol-
lowing printing, it was observed that the slides could be washed
with water without fear of compound diffusion, but any organic
solvent completely removed the immobilized compounds from
the slides.8
To determine the optimal photoreactive spacer length, com-
pounds 18, 19 and 20 were printed on the fluorous slides followed
by a solution of fluorescein. The spots were dried and irradiated for
40 min at 365 nm to generate the carbene and enable fluorescein
immobilization (Fig. 2).
The fluorous tagged photoreactive spacers were synthesized
from resin 11 (Scheme 3) which was esterified with 1 to give 12.
This was treated with 20% piperidine and the free amine was cou-
pled to {4-[3-(trifluoromethyl)-3H-diaziren-3-yl]phenoxy}acetic
acid (2) to give 14. The desired product 15 was obtained by cleav-
age from the resin with a standard acidic cocktail (48% TFA, 48%
CH2Cl2, 2% water, and 2% TIS).8 Two related constructs 16 and 17
were synthesized by the repeated addition of the Fmoc-8-amino-
3,6-dioxaoctanoic acid spacer 1 before capping with TFMAD and
cleavage (Scheme 3). The fluorous tagged photoreactive spacers
were synthesized by coupling 15, 16 and 17 to 1-amino-
This demonstrated that the shortest fluorous tagged photoreac-
tive spacer 18 gave the best results with the most intensive signals
with all concentrations of the dye. It also demonstrated that the
diazirine photoreactive spacer could capture small molecules and
display good sensitivity and signal-to-noise ratios with the printing
of 10 mM solutions, easily achievable with compound library
collections.
Photoreactive spacer 18 (5 Â 5 spots) was used to immobilize bio-
tin with optimization of the photoreactive spacer/biotin ratio. The
presence of biotin covalently attached to the surface was confirmed
by binding with Cy3-labeled Streptavidin (Fig. 3). This showed that

M. Vallès-Miret, M. Bradley / Tetrahedron Letters 52 (2011) 6819–6822
6821
Scheme 3. Synthesis of the photoreactive fluorous spacers by solid phase synthesis.
biotin was immobilized successfully and that a large excess of photo-
reactive spacer did not improve signal-to-noise ratios, with the best
ratios being 1:1 and 1:2 (photoreactive spacer/biotin).
The Streptavidin-Cy3 incubated slides could be reused. Thus the
slides could be stripped by treating with water at 70 °C for 2 h and
re-incubated with Cy3-Streptavidin, demonstrating efficient
re-binding (Fig. 4).
This approach, making use of carbene insertion chemistry, sim-
plifies small-molecule immobilization making it suitable for array-
ing and screening libraries of drug-like small molecules arrayed on
fluorous coated glass slides. The fluorous surface also has advanta-
ges of avoiding non-specific interactions with proteins and cells.
Furthermore the use of fluorous slides allows the recycling of the
slides either by total stripping (organic solvent) or removal of the
interrogation protein (water treatment at 70 °C) and reuse for re-
peated screening. We have also shown that a large excess of the
photoreactive spacer is not translated to better immobilization
and that the smaller spacer length is important for high ligand
binding and sensitivity of detection.
Figure 1. Positive controls prepared for the fabrication and optimization of the
fluorous-tagged microarrays.

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M. Vallès-Miret, M. Bradley / Tetrahedron Letters 52 (2011) 6819–6822
Figure 3. An inkjet printer was used to fabricate the array. The photospacer 18 was
used for the immobilization of a solution of 10 mM biotin. Columns (from left to
right) represent various photospacer/biotin ratios (1:1, 2:1, 3:1, 4:1, and 5:1) with
each row a series of replicates. The green rectangle represents spots of 10 mM
biotin solution printed without the photospacer (negative control). The red
rectangle represents printing of fluorous tagged biotin (21) (2 mM) (positive
control). The slides were left to dry and then irradiated for 40 min at 365 nm and
washed extensively before incubation with Streptavidin-Cy3 for 1 h at 37 °C and
scanned with a Cy3 filter.
Figure 2. Photoreactive fluorous spacer optimization. Photoreactive spacers 18, 19
and 20 were contact printed (10 mM solution in DMF) and allowed to dry.
Subsequently, different concentrations of carboxyfluorescein in DMF were printed
onto the photoreactive spacers and the slides were left to dry overnight. Irradiation
of the slide was carried out for 40 min at 365 nm before washing and scanning.
Negative controls (highlighted with green and red boxes) correspond to the printing
of the spacer 18 or unmodified carboxyfluorescein, respectively.
Figure 4. Slide recycling: (a) Initial incubation with Streptavidin-Cy3; (b) washing for 1 h at 70 °C; (c) washing for 2 h at 70 °C; and, (d) re-incubation with Streptavidin-Cy3.
4. (a) Ko, K. S.; Jaipuri, F. A.; Pohl, N. L. J. Am. Chem. Soc. 2005, 127, 13162–13163;
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