Angewandte
Chemie
DOI: 10.1002/anie.201200467
Small-Molecule Probes
Chemical Probes for the Recognition of Cannabinoid Receptors in
Native Systems**
Lidia Martꢀn-Couce, Mar Martꢀn-Fontecha, ꢁscar Palomares, Leyre Mestre, Arnau Cordomꢀ,
Miriam Hernangomez, Sara Palma, Leonardo Pardo, Carmen Guaza, Marꢀa L. Lꢂpez-
Rodrꢀguez,* and Silvia Ortega-Gutiꢃrrez*
Dedicated to Professor Miguel ꢄngel Miranda on the occasion of his 60th birthday
The endogenous cannabinoid system (ECS) regulates a broad
number of physiological processes, and perturbations in its
normal functioning are linked to many disorders.[1] In this
respect, the development of high-sensitivity and high-
throughput analytical tools that afforded a broader view of
the ECS would be highly valuable. As such, small-molecule
fluorescent probes could provide dynamic information con-
cerning the direct spatial and temporal expression levels of
cannabinoid receptors as has been recently reported for some
specific classes of enzymes.[2] Accordingly, the development of
small-molecule probes that are able to recognize cannabinoid
receptors is an area of current interest, because they could
complement and even overcome some of the drawbacks of
the available antibodies.[3] Recent attempts toward this goal
have been mainly focused on the CB2 receptor (CB2R = can-
nabinoid receptor type 2). However, these probes show
moderate affinities [Ki = (260–387) nm], and their application
in native systems is limited.[4] Similarly, endocannabinoid-
based probes display modest affinities for CB1 and CB2
receptors [(84.7–450) nm],[5] a fact that could limit their use
in complex systems. Therefore, we focused our efforts on
the synthetic high-affinity cannabinoid ligands HU210
[Ki(CB1R) = 0.061 nm, Ki(CB2R) = 0.52 nm],[6] and HU308
[Ki(CB1R) > 10000 nm, Ki (CB2R) = 22.7 nm][7] (Scheme 1).
Among the different tags, biotin was selected owing to its
versatility for detection by a variety of readily available
(strept)avidin conjugates. A closer look to the structure of
these ligands revealed that the most straightforward possibil-
ity was to attach the tag at the free hydroxy groups
(Scheme 1).
[*] Dr. L. Martꢀn-Couce, Prof. Dr. M. Martꢀn-Fontecha,
Prof. Dr. M. L. Lꢁpez-Rodrꢀguez, Dr. S. Ortega-Gutiꢂrrez
Department of Organic Chemistry
Universidad Complutense de Madrid
28040 Madrid (Spain)
E-mail: mluzlr@quim.ucm.es
Scheme 1. Structures of the synthetic cannabinoid ligands HU210 and
HU308 and of synthesized probes 1–3.
Dr. ꢃ. Palomares, S. Palma
Department of Biochemistry and Molecular Biology
Universidad Complutense de Madrid
28040 Madrid (Spain)
Ligand HU210 was prepared as previously described.[8]
For the introduction of the tag, we made some attempts to
selectively acylate the allylic hydroxy group using either
Mitsunobu conditions[9] or the HfCl4·2THF catalyst.[10] How-
ever, none of them gave good yields in our hands and we
carried out the reaction between ligand HU210 and N-
(+)-biotinyl-6-aminohexanoic acid in the presence of 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylami-
nopyridine. Under these conditions, formation of bisacylated
product was not observed, starting material was partly
recovered, and compounds 1 and 2 were obtained after
separation by column chromatography with 25% and 14%
yields, respectively. Ligand HU308 was obtained as previously
described[7] from commercially available a-pinene. Esterifi-
cation of ligand HU308 with N-(+)-biotinyl-6-aminohexanoic
acid gave derivative 3 in 55% yield. Compounds 1–3 were
Dr. L. Mestre, Dr. M. Hernangomez, Dr. C. Guaza
Cajal Institute, CSIC
Dr. Arce 37, 28002 Madrid (Spain)
Dr. A. Cordomꢀ, Prof. Dr. L. Pardo
Universitat Autꢄnoma de Barcelona
08193 Bellaterra, Barcelona (Spain)
[**] This work was supported by grants from the Spanish Ministerio de
Economꢀa y Competitividad (MINECO, SAF2010-22198, SAF2010-
17501, and SAF2008-04053, and predoctoral fellowship to L.M.-C.)
and Comunidad de Madrid (S2010/BMD-2353). The authors thank
the Spanish Society of Medicinal Chemistry (SEQT) and Glaxo-
SmithKline for the young researcher award to L.M.-C. S.O.-G. and
O.P. are Ramon y Cajal Scholars funded by MINECO and the
European Social Fund.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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