ISMSC 2007 - Università degli Studi di Pavia

PSA 63
A fluorescent-indicator displacement assay for detection of ATP and GTP
employing the cyclobisintercaland-type receptors
Anton Granzhan and Marie-Paule Teulade-Fichou
UMR 176 CNRS, Institute Curie, F-91405 Orsay, France
The detection of nucleotides and nucleosides represents an active research area, since these
analytes are involved in many biological processes. Along these lines, especially attractive are
the fluorescence-based detection strategies, particularly the ones based on the enhancement of
the fluorescence signal upon binding of the analyte [1,2]. The previous works from our group
have shown that the cyclobisintercaland-type hosts, such as BisA [3] and BisNP [4], bind
nucleotides with high binding constants. However, the quenching of the fluorescence of these
receptors, which accompanies the binding event, is not optimal for the fluorimetric applications,
since the quenching of fluorescence may be also caused by other stimuli, such as heavy ions,
functional groups that assist intersystem crossing, and others.
NH
NH
NH
N
N
H
N
HN
NH
BisA
O
NH
NH
BisNP
HN
HN
O
O 3S
OH
O3S SO3 3 Na
In the present work, we show that the cyclobisintercaland receptors bind the fluorescent
indicator HPTS (8-hydroxy-1,3,6-pyrenetrisulphonate, pyranine) with a 1:1 stoichiometry and
high binding constants in aqueous solutions (e.g. log K = 7.0 at pH 6 for BisA), which leads to
almost complete quenching of the fluorescence of the indicator. Remarkably, addition of
nucleotide triphosphates, such as ATP and GTP, to the BisA–HPTS complex leads to an
efficient displacement of the indicator, as these analytes bind to the receptor, and is
accompanied by a drastic recovery of the fluorescence. Thus, the fluorescence of the 1:1 BisA–
HPTS complex is enhanced about 2000-fold in the presence of only 0.2 mM ATP or GTP. In
contrast, much weaker enhancement of the fluorescence was observed in the case of other
nucleotide tri-, di-, and monophosphates. These data indicate that the BisA–HPTS complex
represents a highly sensitive fluorescent-indicator displacement assay [5] which may be used
for the detection of ATP and GTP.
[1] S. Atilgan, E. U. Akkaya, Tetrahedron Lett. 2004, 45, 9269–9271.
[2] P. P. Neelakandan, M. Hariharan, D. Ramaiah, J. Am. Chem. Soc. 2006, 128, 11334–11335.
[3] M.-P. Teulade-Fichou, J.-P. Vigneron, J.-M. Lehn, Supramol. Chem. 1995, 5, 139–147.
[4] M. Dhaenens, J.-M. Lehn, J.-P. Vigneron, J. Chem. Soc., Perkin Trans. 2 1993, 1379–1381.
[5] B. T. Nguyen, E. V. Anslyn, Coord. Chem. Rev. 2006, 250, 3118–3127.
HPTS
PSA 64
The supramolecular architecture of a guanosine derivative as a scaffold for
persistent radicals
Carla Graziano, Stefano Masiero, Silvia Pieraccini, Marco Lucarini, Gian Piero Spada
a Dipartimento di Chimica Organica "A. Mangini", Alma Mater Studiorum - Università di Bologna,
Via San Giacomo 11, 40126 Bologna, Italy (E-mail: carla.graziano@studio.unibo.it);
G-quartets formed by guanosines with an Hoogsteen like hydrogen bonding network are
observed in different environments such as telomeres of the human genome or G-quadruplex
formed by semisynthetic lipophilic guanosines (LipoG) in organic solvent in presence of alkali
ions.
Actually, LipoG’s are able to extract salts, like potassium picrate, by formation of G-quartets
stacked in columnar chiral aggregates whose stochiometry (figure 1) depends on the relative
amount of nucleobase and cation. 1
In particular, 2’,3’-O-isopropylideneguanosines differently substituted on either the nucleobase
or the ribose moiety, are able to self-assemble in solution by K + templation to give a
G-quadruplex with formula [LipoG]8K + Picr - . It consists in two G-quartets kept together by
coordination of the cation. 2 .
Here we report for the first time the synthesis and the supramolecular behaviour of a
2’,3’-O-isopropylideneguanosine derivative functionalized in 5’ position with a persistent
nitroxide radical 1.
Both EPR and circular dicroism studies confirm the formation in solution of the expected
octameric G-quadruplex after extraction of potassium picrate and sodium picrate. A more
detailed structural assignment was obtained by NMR on derivatives 2 and 3. The octamer
consists in two G-quartets templated by the central cation with D4-symmetry.
Figure 1
RO
H
H
O
O
H
N
N
H
O
O
N
NH
NH 2
O
1 R=
2 R= COC 9H 19
[1] J.T: Davis and G. P. Spada, Chem. Soc. Rev.; 2007, 36, 296-313.
[2] M.S. Kaucher, Y. Lam, S. Pieraccini and G.Gottarelli, J.T. Davis, Chem. Eur. J., 2004, 11,
164-173; X. Liu, I.C.M. Kwan, S. Wang and G. Wu, Org. Lett., 2006, 8, 3685-3688.
3 R=
O
N
O