Synthesis and evaluation of a scintillant-containing solid support for use in combinatorial chemistry

Article abstract of DOI:10.1016/S0040-4039(00)00141-6

A scintillant monomer, 2,5-diphenyl-4-vinyloxazole, has been synthesised and co-polymerised with 4-ethylstyrene, divinylbenzene and 4-vinylbenzyl chloride in the presence of a toluene porogen, to form a scintillant- containing macroporous resin. Despite prolonged exposure to organic solvent, this resin retains the ability to scintillate efficiently in the presence of ionising radiation. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00141-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 2253–2256
Synthesis and evaluation of a scintillant-containing solid support
for use in combinatorial chemistry
Bruce Clapham b,† and Andrew J. Sutherland a,∗
aChemical Engineering and Applied Chemistry, Aston University, Aston Triangle, Birmingham B4 7ET, UK
bDepartment of Chemistry and Physics, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
Received 22 December 1999; accepted 18 January 2000
Abstract
A scintillant monomer, 2,5-diphenyl-4-vinyloxazole, has been synthesised and co-polymerised with 4-
ethylstyrene, divinylbenzene and 4-vinylbenzyl chloride in the presence of a toluene porogen, to form a
scintillant-containing macroporous resin. Despite prolonged exposure to organic solvent, this resin retains the
ability to scintillate efficiently in the presence of ionising radiation. © 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: oxazoles; polymerisation; resins; combinatorial chemistry.
The scintillation proximity assay (SPA) developed and marketed by Amersham Pharmacia Biotech, is
a dynamic procedure for detecting and quantifying intermolecular interactions.1 In this elegant approach,
a receptor molecule is labelled with SPA beads. SPA beads are solid polymeric microspheres that contain
2,5-diphenyloxazole, a well-known scintillant molecule that is a major component of most commercially
available scintillation cocktails.2 In a typical SPA procedure, a tritiated ligand is incubated with a SPA-
labelled receptor. If the receptor binds the ligand (Fig. 1a), the tritium is brought into close proximity
to the encapsulated scintillant, resulting in the emission of light. This response may be monitored and
quantified in a scintillation counter. The short pathlength of tritium β-particles (average pathlength=1.5
µm in water3) ensures that a signal is detected only when the receptor actually binds the ligand. Unbound
material is too remote to elicit a significant signal (Fig. 1b).
SPA beads are prepared by incubating a DMSO solution of 2,5-diphenyloxazole with, for example,
Sepharose 4B beads. Addition of water to this system results in the DMSO being washed away from
the beads whilst the scintillant molecules, being insoluble in aqueous media, are precipitated within the
pores of the beads. Unfortunately, this manufacturing process ensures that SPA beads are completely
incompatible with the use of organic solvents. Exposure to organic solvent results in dissolution of
∗
†
Corresponding author. Tel: +44 121 359 3611; fax: +44 121 359 4094; e-mail: a.j.sutherland@aston.ac.uk (A. J. Sutherland)
Current address: Department of Chemistry, BCC582, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla,
CA 92037, USA. E-mail: bclapham@scripps.edu
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00141-6
tetl 16421

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Fig. 1. Schematic representation of the scintillation proximity assay (SPA)
the precipitated scintillant molecules rendering the beads useless for subsequent assay. Indeed, even in
aqueous solvents leaching of this type has been reported as problematic.4
The fact that exposure to organic solvents renders conventional SPA beads inactive precludes their use
as supports for solid phase combinatorial chemistry and this has limited the application of SPA in the
field of combinatorial chemistry. Our interest in developing new assay techniques for application in solid
phase combinatorial chemistry prompted us to investigate whether chemically-functionalised polystyrene
based beads, that incorporate 2,5-diphenyloxazole molecules covalently, could be used for solid phase
synthesis and still retain the ability to scintillate in the presence of ionising radiation. We wish to report
here the construction and evaluation of a chemically-functionalised scintillant-containing support5 and
in the following communication6 we describe the use of this resin in solid phase synthetic chemistry and
a subsequent scintillation-based assay.
To construct a scintillant-containing macroporous resin we first required the ready availability of an
appropriate monomer that contained a 2,5-diphenyoxazole scintillant moiety. Recently,7 we reported
the synthesis of aldehyde 2 formed by oxidising alcohol 1 with pyridinium chlorochromate (PCC).
Consequently, 2,5-diphenyl-4-vinyloxazole 3 was selected as the monomer of choice since it was
anticipated that this monomer could be synthesised readily from aldehyde 2 via a standard Wittig
olefination reaction.8
Whilst the small-scale PCC oxidation of alcohol 1 proceeds in good yield (67%),7 on a multi-gram
scale the reaction was capricious and yields lower. Fortunately, when 2-iodoxybenzoic acid (IBX)9 was
used in place of PCC for this oxidation, multi-gram amounts of aldehyde 2 could be synthesised from
alcohol 1 reproducibly and in excellent yield. As we had hoped, the subsequent Wittig reaction of
aldehyde 2 proceeded smoothly, again on a multi-gram scale, giving the desired scintillant monomer
3 in good yield10 (Scheme 1).
Scheme 1. Synthesis of 2,5-diphenyl-4-vinyloxazole 3
Having established an efficient synthetic route to monomer 3, we proceeded with the construction
of a chemically-functionalised scintillant-containing polystyrene-based resin. In general, the resins
used in solid phase synthetic chemistry and combinatorial chemistry are constructed by a suspension
polymerisation procedure. Accordingly, monomer 3 was co-polymerised with 4-vinylbenzyl chloride
4, 4-ethylstyrene 5 and a cross-linker, divinylbenzene 6, using standard suspension polymerisation
techniques.11 This procedure was carried out in the presence of toluene, which served as a porogen
and water containing poly(vinyl alcohol) (90% hydrolysed, average Mw, 90 000–146 000) which acted as
a droplet stabiliser (Scheme 2).

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Scheme 2. Synthesis of scintillant-containing macroporous resin 7
The 20% cross-linked, chemically-functionalised scintillant-containing macroporous polymeric beads
7 that resulted from the suspension polymerisation were collected by filtration, washed exhaustively,
dried and then sieved to provide resin beads with sizes ranging between 300 and 500 µm in diameter. The
beads were then subjected to Soxhlet extraction using tetrahydrofuran. At various time intervals, a sample
of resin was removed from the Soxhlet thimble and these samples, together with a blank control resin
(prepared in an identical fashion to the scintillant containing resin but with monomer 3 being replaced
by styrene), were subjected to a scintillation counting assay. Three portions of each resin sample, of
known mass, were placed in scintillation vials. To each vial, an equal aliquot of a 14C hexadecane/toluene
stock solution was added and the vials monitored in a scintillation counter. The averaged counts per
minute (cpm) obtained per mg of resin are shown in Fig. 2. As the figure shows clearly, even after
prolonged Soxhlet extraction times, the scintillant-containing macroporous resin 7 still retains the ability
to scintillate efficiently in the presence of ionising radiation (column c). In addition, the counts obtained
for the scintillant-containing resin 7 (columns a, b and c) are far in excess of the background counts
obtained for the corresponding non-scintillant-containing control resin (column d).
Fig. 2. Scintillation counting efficiencies of scintillant-containing resin 7 (after varying periods of Soxhlet extraction) and a
non-scintillant-containing control resin. a=Resin 7, Soxhlet time=0 h; b=resin 7, Soxhlet time=24 h; c=resin 7, Soxhlet time=48
h; d=non-scintillant-containing control resin. The activity of the 14C hexadecane/toluene stock solution was 121 000 cpm per
aliquot (assessed in triplicate by counting in commercial scintillation cocktails). In all cases, the total mass of resin per vial was
measured to the nearest 0.1 mg with masses varying between 5.1 and 8.4 mg
In conclusion, we have synthesised 2,5-diphenyl-4-vinyloxazole 3 and used this monomer to construct
chemically-functionalised macroporous polystyrene beads that scintillate efficiently in the presence of
ionising radiation. In addition, we have demonstrated that this scintillant-containing resin retains the
ability to scintillate even after prolonged exposure to organic solvent. In the following communication,
we describe how this chemically-functionalised scintillant-containing resin may be used successfully for
both solid phase synthetic organic chemistry and subsequent scintillation-based assay.6

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Acknowledgements
We should like to thank Dr. M. C. Perry and Mrs K. C. Farrow (both of Aston University) for NMR
spectra and mass spectra, respectively. We should also like to thank Dr. A. V. Hine and Prof. N. K.
H. Slater (both of Aston University) for helpful discussions. We are grateful to Aston University and
The Nottingham Trent University for financial assistance and to the Research Enhancement Fund of the
Nottingham Trent University for the award of a Research Studentship to B.C.
References
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