Practical synthesis of a new analytical construct: Thiopyrimidine safety-catch linker for facile monitoring of solid-phase chemistry

Article abstract of DOI:10.1016/S0040-4039(00)01508-2

We report here a new analytical construct based on a thiopyrimidine safety-catch linker. This construct adds to the existing portfolio of linkers available for facile monitoring of reactions conducted on solid support. A practical solution phase synthesis of the precursor is described, together with the proof of concept and cleavage protocols. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01508-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8609–8613
Practical synthesis of a new analytical construct:
thiopyrimidine safety-catch linker for facile monitoring of
solid-phase chemistry
Olivier Lorthioir,* Stephen C. McKeown, Nigel J. Parr, Melanie Washington and
Stephen P. Watson
Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
Received 12 July 2000; accepted 30 August 2000
Abstract
We report here a new analytical construct based on a thiopyrimidine safety-catch linker. This construct
adds to the existing portfolio of linkers available for facile monitoring of reactions conducted on solid
support. A practical solution phase synthesis of the precursor is described, together with the proof of
concept and cleavage protocols. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: pyrimidine; solid-phase synthesis; mass spectrometry; safety-catch; analytical construct.
Combinatorial techniques are now widely accepted as a powerful strategy in drug discovery
programs.1–4 However, successful as it is proving to be, monitoring of reactions conducted on
solid-phase remains problematic.5 Recently ‘analytical constructs’ have been successfully intro-
duced to address such problems of analysis.6,7
A typical analytical construct (Fig. 1) contains a conventional linker, ‘linker 2’, allowing the
release of substrates in a classical manner, but also an orthogonal linker, ‘linker 1’, cleavable by
a specific reagent in an additional ‘analytical mode’. If the latter mode is used, an analytical
fragment incorporating the substrate is produced, which is highly sensitised to electrospray mass
spectrometry and easily identified by its isotopic label. We have recently reported the use of
nitroveratryl-,6 2-nitrophenyl sulfonamide-8 and Dde- based linkers,8 cleavable by photolysis,
thiolysis or hydrazinolysis, respectively. All of these possess a high degree of orthogonality with
conventional linkers 2. However, for its general application to solid-phase chemistry, this
analytical construct methodology requires a portfolio of linkers 1 compatible with the reaction
sequence envisaged.
* Corresponding author. Tel: +44 (0) 1438 76 3608; e-mail: ol65917@glaxowellcome.co.uk
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01508-2

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Figure 1. Analytical construct dual linker
Herein we describe a new analytical construct linker and its facile solution phase synthesis.
This thiopyrimidine linker (Fig. 2), which exploits a safety-catch mechanism for cleavage,9 has
now been added to our portfolio of linkers dedicated to tackle analysis problems on solid
support. The design of this new linker was based on previously reported chemistry developed for
the production of pyrimidine libraries.10–12 Analytical cleavage of the thiopyrimidine linker is
effected by nucleophilic displacement using 1-methylpiperazine after activation by OXONE®
oxidation of the alkylthio linkage. 1-Methylpiperazine provides, in this particular case, both the
cleavage reagent and the sensitiser (Fig. 2).
Figure 2. Thiopyrimidine analytical construct
1. Synthesis of thiopyrimidine construct precursor
While in principle the thiopyrimidine construct could be synthesised directly on resin, a
solution phase approach is preferred for its preparation for two reasons. Firstly, the linker can
be purified and characterised before its attachment to the resin, eliminating any impurity which
could arise from a stepwise construction on the solid support. Secondly, the versatile intermedi-
ate obtained can be loaded onto any resin of choice.
4-(Chloromethyl)benzoic acid (1) was protected using tert-butyl 2,2,2-trichloroacetimidate13 in
the presence of a catalytic amount of boron trifluoride diethyl etherate (Scheme 1). The
trichloroacetamide by-product was simply removed by passing the mixture through a plug of
silica. Thiourea alkylation12 provided the required thiouronium salt (2), which required no
further purification. Activated diketoderivative (3) was obtained by Knoevenagel condensation
of methyl acetoacetate and N,N-dimethylformamide dimethyl acetal. Cyclocondensation of

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thiouronium salt (2) with diketoderivative (3)14 provided, after saponification, the expected
alkylthiopyrimidine (4). Coupling of the labelled amine, removal of protecting groups by TFA
and protection of the amine with either Boc or Fmoc group finally afforded protected linker (5).
It is noteworthy that (5) can be obtained in good overall yield and excellent purity with
primarily aqueous/organic extraction techniques and no difficult chromatography. In addition
we have readily prepared 200 g of intermediate alkylthiopyrimidine (4) by scaling up the
previous procedure.
Scheme 1. Solution phase route to thiopyrimidine linker
2. Proof of concept: linker stability and ESI-MS analysis
To validate the use of thiopyrimidine based analytical construct we prepared a model
substrate on a resin incorporating our new construct (Fig. 3).15 The reaction sequence consisted
of coupling the acid labile aldehyde linker to the analytical construct resin, followed by
attachment of 4-methylbenzylamine to the resin by reductive amination, coupling of N-a-Fmoc-
N-a-methyl-L-alanine and finally capping with 4-nitrobenzoic acid to afford resin (6) (Fig. 3).
The resin was then treated with OXONE® to oxidise the sulfur atom (we found electron rich
linkers and substrates unstable to m-CPBA) and cleavage with 1-methylpiperazine affords the
analytical fragment (7) (Fig. 3). An easily identified doublet, exhibiting a peak split of 2, is by
far the main signal in the spectrum, the signal-to-noise ratio being very high, even at this single
bead level.

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Figure 3. Analytical fragment release and ESI-MS spectrum at the single bead level of a model substrate
In summary, we have developed a new analytical construct ‘linker 1’ and designed a practical
route for its synthesis. This linker represents a highly versatile building block now available to
us in two protected forms and bulk quantities to couple to any solid support and subsequently
to any conventional linker 2. Importantly, the preparation of the construct resin is rapid and
straightforward (cf. well-behaved coupling of standard linkers to resin by simple amide bond
formation). The new thiopyrimidine-based linker reported here provides further flexibility/
orthogonality to the application of analytical constructs and is now part of the portfolio of
linkers dedicated to such a modular approach. We have recently used the present linker for the
synthesis and successful single bead level analysis of a 64 member model library.15 Further
applications of this strategy are in progress and represent significant steps towards solving our
current solid phase analysis problems at the single bead level.
Acknowledgements
We would like to thank Nicole Hamblin for fruitful discussions about thiopyrimidine
chemistry and Yih-Sang Pang for his assistance with Electrospray MS.
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