High throughput 'catch-and-release' synthesis within spatially discrete gel arrays

Article abstract of DOI:10.1016/j.tetlet.2010.09.024

A tetrafluorophenol acrylamide monomer unit was synthesised, co-polymerised and grafted onto a glass slide to form individual gel spots. As a proof of principle study, a small library of amides was rapidly synthesised within these gel spots using 'catch-and-release' chemistry, including the biologically interesting quorum sensing acyl-homoserine lactones. The tetrafluorophenol acrylamide gel provides an efficient platform to synthesise and screen small molecules for biological activity.

Full text of DOI:10.1016/j.tetlet.2010.09.024

Tetrahedron Letters 51 (2010) 5930–5932
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
High throughput ‘catch-and-release’ synthesis within spatially discrete
gel arrays
Christine M. Boehner a, David M. Marsden a, Hannah F. Sore a, David Norton b, David R. Spring a,
⇑
a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
b GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2010
Revised 4 August 2010
Accepted 6 September 2010
Available online 21 September 2010
A tetrafluorophenol acrylamide monomer unit was synthesised, co-polymerised and grafted onto a glass
slide to form individual gel spots. As a proof of principle study, a small library of amides was rapidly syn-
thesised within these gel spots using ‘catch-and-release’ chemistry, including the biologically interesting
quorum sensing acyl-homoserine lactones. The tetrafluorophenol acrylamide gel provides an efficient
platform to synthesise and screen small molecules for biological activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Combinatorial chemistry has developed over the past 20 years
into a tool for the high throughput synthesis of small molecules,
driven in part by the demands of the pharmaceutical industry to
generate rapidly large arrays of compounds for biological screen-
ing.1 A number of solid phase chemistry techniques have been
developed, for example, tetrafluorophenol (TFP) catch-and-release
beads for the synthesis of amide libraries.2 The TFP beads are made
from polystyrene, coupled to tetrafluoro-hydroxybenzoic acid. In
the catch-and-release process the TFP beads are firstly treated with
carboxylic acids to form the activated esters using either standard
coupling reagents or a direct reaction with pre-formed acid chlo-
rides (‘catch’). Secondly, these activated esters are reacted with
nucleophiles such as amines to generate the free amides in solu-
tion (‘release’).
Several on-chip cell-screening assays have been developed
including an array of transfected cells expressing defined locations
of DNA.3 In a similar approach, slides coated with a biodegradable
polymer have been used to screen small molecule activity in mam-
malian cells.4 Additionally, a library of acrylate polymers has been
attached to a chip and screened for biomaterial suitability in terms
of cell attachment, growth and differentiation.5 The development
of a methodology that would enable the incorporation of high
throughput compound synthesis followed by high throughput
biological screening on the same chip would be a significant
advancement. This sequential synthesis-screening technique has
the potential to be a more cost effective and rapid process for the
identification of early hits to feed into drug discovery programs.
A powerful synergy can be realised as the synthesis and screening
processes become more streamlined, faster and cost-effective. In
this context we have developed a polyethylene glycol (PEG) TFP
acrylamide 3D gel, which was grafted onto a microscope slide to
form spatially discrete spots. This array of gel spots acts as a series
of wall-less test tubes for the TFP synthesis of the amide library.
Each amide is held non-covalently within the gel spots but can dif-
fuse out easily for analytical detection or testing in cell-based
assays.
Acrylamide gels are typically formed by a vinyl-radical poly-
merisation between an individual monomer and cross-linking
units. They provide a robust and easily derivatisable platform for
a wide variety of applications from the electrophoresis of proteins
and DNA, to the solid-phase synthesis of small molecules.6,7
Our research began with the synthesis of the TFP monomer unit
needed to prepare the TFP acrylamide gel (Scheme 1). Boc-anhy-
dride was reacted with excess ethanediamine (1) to generate mono
Boc-protected amine 2. Subsequently, amine 2 was coupled with
acryloyl chloride under Schotten–Baumann conditions to form
acrylamide 3 in an excellent yield. The Boc-group was removed
using trifluoroacetic acid (TFA) and the isolated amine salt was
coupled directly with tetrafluoro-4-hydroxy-benzoic acid (4) to
produce the TFP acrylamide monomer unit (5) in a 43% overall
yield for the four-step sequence.
The TFP monomer unit (5) was initially co-polymerised to form
the free TFP gel. The polymerisation mixture consisted of PEG1900-
Da cross-linker (6), TFP acrylamide monomer unit (5) and 2-hydro-
xy-2-methylpropiophenone (7) as the UV initiator (Scheme 2). The
PEG1900Da cross-linker (6) is made up of repeating ethylene glycol
units (= 1900 Da molecular weight) and has been used in a variety
of applications including 3D hydrogel small-molecule microarrays
and solid-supported peptide synthesis.6,8 The free gel was formed
upon irradiation of the polymerisation mixture with UV-light.
The gel was then characterised in terms of its swelling and loading
capacity. Despite the presence of the hydrophilic PEG groups, the
gel did not swell considerably in water (1 mL/g). The hydrophobic
nature of the TFP group could account for the lack of swelling in
water. This hypothesis was confirmed as a control gel containing
⇑
Corresponding author. Tel.: +44 1223 336498; fax: +44 1223 336362.
E-mail address: spring@ch.cam.ac.uk (D.R. Spring).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.024

C. M. Boehner et al. / Tetrahedron Letters 51 (2010) 5930–5932
5931
(i) TFA
F
F
OH
(ii)
F
F
HOOC
F
4
acryloyl chloride,
Na2CO3,
F
F
OH
F
1,4-dioxane
Boc2O
EDC, HOBt, NEt3,
DMF, 50 ºC
O
O
H
N
CH2Cl2:H2O 1:1
NHBoc
NH2
NHBoc
H2N
H2N
N
H
N
H
70%
76%
81%
O
5
1
2
3
Scheme 1. Synthesis of the TFP (TetraFluoroPhenol) acrylate monomer unit (5). TFA = trifluoroacetic acid, Boc = tert-butoxycarbonyl, EDC = 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride, HOBt = 1-hydroxybenzotriazole.
O
H
N
O
O
O
N
H
F
F
F
F
PEG1900Dan 6
O
R2
F
OH
F
F
O
F
R1
R1
N
H
F
O
O
F
OH
F
O
O
O
O
H
N
14-21
OH
N
H
O
F
O
7
5
R2NH2
12-13
R1COCl
8-11
(i) Polymerisation mixture + initiator
75 mm
Hydrophobic cover slide
(ii)
pre-functionalised
TFP gel spots -
'wall-less test-tubes'
active ester - 'catch'
amide - 'release'
& UV light (λ = 254 nm)
acrylic ester silane slide
R1COCl =
R2NH2
=
O
O
O
O
NH2
NH2
N
Cl
Cl
Cl
Cl
Br
N
8
9
10
11
12
13
Scheme 2. Generation of TFP 3D gel spots for ‘catch’ and ‘release’ synthesis. A pre-functionalised glass slide was generated by treatment with methylacryloxypropyltri-
methoxysilane in acidic ethanol/water mixture. The pre-polymerisation mixture which consisted of the PEG cross-linker (6), TFP monomer (5) and 1% UV-initiator (7) in DMF
was applied in 4 lL volume using a Gilson pipette to create an array of up to 16 spots/slide. A hydrophobic slide was used to cover the spots before polymerisation using UV
light. The cover slide was removed and the spots were treated with one of four acid chlorides (R1COCl, 8–11), followed by one of two amines (R2NH2, 12–13). LCMS spectra of
washings were used to show evidence for synthesis of the amides (14–21).
two standard acrylamide monomer units but no TFP acrylamide
monomer unit (5) swelled considerably in water (7.9 mL/g). The
TFP acrylamide gel was found to swell effectively in either DMSO
or DMF solutions (DMF = 4.6 mL/g). The loading capacity of the
TFP acrylamide gel was calculated to be 0.7 mmol/g; this compared
favourably with reported loadings of 1.0 mmol/g for the commer-
cial TFP beads.9
After polymerisation the TFP acrylamide gel was incorporated
onto glass slides as gel spots. In order to co-polymerise the gel onto
the glass platform, the slides were first derivatised with 3-methyl-
acryloxypropyltrimethoxysilane as shown in Scheme 2. The func-
tionalised glass slides were then spotted with the polymerisation
mixture and covered with a hydrophobic cover slide to prevent
excessive exposure to the air. It is essential to prevent excess air
getting into the reaction as oxygen inhibits the free radical poly-
merisation. The glass slides were finally irradiated with UV light
to generate the gel spot array.
Following the generation of the gel spot array, the amides were
synthesised within each gel spot using the catch-and-release TFP
chemistry. The same amides were also synthesised in solution for
analytical comparison to those produced using the TFP acrylamide
gel. Each individual spot was treated with one of four different acid
chlorides (8–11) to generate the intermediate activated esters. It
should be noted that carboxylic acids were also successfully loaded
onto the TFP acrylamide gel using diisopropylcarbodiimide-medi-
ated coupling; however, acid chlorides were favoured as the elec-
trophile as coupling reagents were not required.
thesis of the amide library within the gel was confirmed by com-
parison to those amides made in solution; the recorded mass and
retention times of the amides were identical independent of the
method used to form them. The approximate yield was calculated
to be in the range 40–60%.
Quorum sensing is the chemical language used by bacteria to
communicate and thus co-ordinate behaviour.10 The gram negative
bacteria Serratia and Chromabacterium violaceum use the acyl-
homoserine lactone (AHL)-signalling molecules butanoyl-
L-homo-
serine-lactone (BHL) and hexanoyl- -homoserine lactone (HHL),
L
respectively, to communicate. The inhibition of AHL signalling
and consequently quorum sensing could provide the next genera-
tion of anti-bacterial agents against multi drug resistant bacteria.11
To expand upon our proof of principle study we next examined the
synthesis of biologically active quorum-sensing small molecules in
the TFP acrylamide gel. In a similar strategy, a recent report by Pra-
neenararat et al. describes the synthesis of quorum-sensing mole-
cules on planar cellulose supports.12
Pleasingly, the natural quorum-sensing signalling molecules
BHL and HHL were successfully synthesised within the TFP
acrylamide gel spots and this was confirmed by LCMS analysis
(Scheme 3, compounds 25 and 26, respectively). This approach of-
fers the potential to synthesise and screen13 sequentially the gel
spots containing the AHL analogues for phenotypic activity on
the same chip. Quorum sensing is an ideal pathway for cell-based
screening because its activation leads to the expression of certain
detectable phenotypes.14
Every gel spot containing a different active ester was reacted
with one of the two primary amines (12 or 13) to generate eight
distinct amides (14–21), one in each gel spot. The successful syn-
In summary, we have developed a new and effective method for
the synthesis of small molecules using TFP acrylamide 3D gels. A
library of amides were generated within TFP-based gel spots using

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C. M. Boehner et al. / Tetrahedron Letters 51 (2010) 5930–5932
petri dish
O
O
F
F
F
F
agar with
lawn of bacteria
N
H
activation of
quorum sensing
F
OH
F
F
O
F
n
O
n
O
O
O
O
BHL 25 n=2
HHL 26 n=4
Cl
O
n
gel slide
22 n=2
23 n=4
H2N
O
24
TFP gel spots -
'wall-less test-tubes'
phenotypic assay
active ester - 'catch'
-homoserine lactone) and HHL (hexanoyl-L-homoserine lactone) in TFP acrylamide gel spots. Gel spots were loaded with butyryl
amide - 'release'
Scheme 3. Synthesis of BHL (butanoyl-
L
chloride (22) or hexanyl chloride (23) before reaction with aminobutyrolactone (24) to produce BHL (25) and HHL (26), which were validated by LCMS. BHL and HHL could be
detected using quorum sensing phenotypic assays using Serratia sp. ATCC39006 (BHL) or Chromabacterium violaceum (HHL). For example, C. violaceum produces a purple
violaceum halo under HHL mediated quorum sensing.
(b) Salvino, J. M.; Kumar, N. V.; Orton, E.; Airey, J.; Kiesow, T.; Crawford, K.;
Mathew, R.; Krolikowski, P.; Drew, M.; Engers, D.; Krolikowski, D.; Herpin, T.;
Gardyan, M.; McGeehan, G.; Labaudiniere, R. J. Comb. Chem. 2000, 2, 691–697.
catch-and-release chemistry. Bioactive quorum-sensing molecules
were successfully synthesised in preparation for phenotypic assay
screening.
3. Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107–110.
4. Bailey, S. N.; Sabatini, D. M.; Stockwell, B. R. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 16144–16149.
5. Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863–866.
6. Meldal, M. Tetrahedron Lett. 1992, 33, 3077–3080.
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We are grateful for financial support from EPSRC, BBSRC, Glaxo-
SmithKline, the Cambridge Trust and the Augustus Newman
Foundation.
10. (a) Nealson, K. H.; Platt, T.; Hastings, J. W. J. Bacteriol. 1970, 104, 313–322; (b)
Smith, D.; Wang, J.-H.; Swatton, J. E.; Davenport, P.; Price, B.; Mikkelsen, H.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.09.024.
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