Fluorescent probe: Complexation of Fe3+with the myo-inositol 1,2,3-trisphosphate motif

Article abstract of DOI:10.1039/b809238a

Natural myo-inositol phosphate antioxidants containing the 1,2,3-trisphosphate motif bind Fe³⁺ in the unstable penta-axial conformation. The Royal Society of Chemistry.

Full text of DOI:10.1039/b809238a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Fluorescent probe: complexation of Fe3+with the myo-inositol
1,2,3-trisphosphate motifw
David Mansell,a Nicholas Rattray,a Laura L. Etchells,a Carl H. Schwalbe,b
Alexander J. Blake,c Elena V. Bichenkova,a Richard A. Bryce,a
Christopher J. Barker,d Alvaro Dıaz,e Carlos Kremerf and Sally Freeman*a
´
Received (in Cambridge, UK) 30th May 2008, Accepted 3rd September 2008
First published as an Advance Article on the web 29th September 2008
DOI: 10.1039/b809238a
Natural myo-inositol phosphate antioxidants containing the
1,2,3-trisphosphate motif bind Fe3+ in the unstable penta-axial
conformation.
8 kcal molꢁ1 more favourable in energy than the penta-axial
conformation 1B (Scheme 1). This energetic preference increases
to 30 kcal molꢁ1 for the hexaanionic forms of 1A and 1B.
It has been proposed by Phillippy and Graf that the complex
of Fe3+ with Ins(1,2,3)P3 adopts the penta-axial conformation
(1B):10 the phosphate groups are orientated closer together,
allowing Ins(1,2,3)P3 to complex to Fe3+ with hexa-coordination
using two terminal oxygens from each phosphate. This structure
was based on the observation that azide has no effect on the
visible absorption spectrum of Ins(1,2,3)P3–Fe3+, in contrast to
the marked shift observed for Ins(1,2,6)P3–Fe3+ due to water
The natural product myo-inositol 1,2,3-trisphosphate (1)1
(Ins(1,2,3)P3, cellular concentration r 10 mM1) is a potent iron-
chelator (apparent Ki = 9.0 ꢀ 10ꢁ19 M) and antioxidant,
completely inhibiting iron-catalysed hydroxyl radical (HOꢂ)
formation.2,3 Our recent potentiometric studies demonstrate that,
unlike the more abundant myo-inositol hexakisphosphate (InsP6
E 20 mM4), Ins(1,2,3)P3 fulfils the requirements of a safe, low
molecular weight biological Fe3+ shuttle.5,6 Under conditions of
excess Mg2+ (e.g. cytosolic and nuclear regions of mammalian
1
displacement.10 To explore this further, we employed H NMR
studies to confirm the penta-equatorial conformation of
Ins(1,2,3)P3 in the absence of Fe3+. However, no structural
information on its Fe3+ complex could be obtained due to
peak broadening. An alternative experimental approach to
conformational studies of 1 was therefore required.
cells), a negligible proportion of InsP6 is bound to Fe3+ 7
. In
contrast, Fe3+ remains fully complexed with Ins(1,2,3)P3, both
under Mg2+-rich conditions and in acidic, Ca2+-rich media such
as in lysosomes.5 It was also predicted that iron bound to
Ins(1,2,3)P3 must be Fe3+, since its affinity for Fe2+ is not high
enough to allow interaction in the presence of excess cellular
Mg2+. Although the biological relevance of Ins(1,2,3)P3 binding
to iron is clear, the structure of its complex is uncertain and forms
the subject of this paper. We have previously shown that the
crystal structure of cyclohexylammonium Ins(1,2,3)P3 adopted the
expected stable penta-equatorial chair conformation (1A).8 Here
using density functional calculations at the UB3LYP/6-31+G*
level,9 we have modelled 1 as the hexamethyl phosphoester, to
represent protonation and counterion/solvent screening effects.
From the optimal geometries, we have estimated that 1A is
We have recently developed a fluorescent probe based on the
acid-triggered conformational flip of an orthoformate con-
strained myo-inositol ring using pyrene excimer fluorescence.11,12
This methodology inspired the design of a fluorescent probe to
study the Ins(1,2,3)P3 motif, using Fe3+ complexation as the
potential trigger for conformational change. 4,6-Bis-O-
pyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate (2) has been
synthesised as a fluorescent probe to monitor whether the ring
flip of myo-inositol occurs upon association with Fe3+
(Scheme 2). If Fe3+ binds to the more stable penta-equatorial
chair conformation (2A), a monomer emission signal of the
pyrene groups would be expected. If Fe3+ binds to the less stable
penta-axial conformation (2B) this will favour p–p stacking of
the pyrene groups promoting excimer fluorescence (Scheme 2).
The 1,2,3,5-tetrakisphosphate myo-inositol derivative was
chosen over the Ins(1,2,3)P3 derivative due to the significantly
shorter synthesis of the fluorescent probe. We are confident that
myo-inositol 1,2,3,5-tetrakisphosphate is a good model of the
1,2,3-trisphosphate motif as it possesses similar anti-oxidant
properties and Fe3+ affinity to Ins(1,2,3)P3.2 The synthesis of
a School of Pharmacy & Pharmaceutical Sciences, University of
Manchester, Oxford Road, Manchester, UK M13 9PT.
E-mail: sally.freeman@manchester.ac.uk; Fax: 44 161 275 2396;
Tel: 44 161 275 2366
b School of Life and Health Sciences, Aston University, Birmingham,
UK B4 7ET
c School of Chemistry, The University of Nottingham, University
Park, Nottingham, UK NG7 2RD
d Rolf Luft Research Center for Diabetes and Endocrinology,
Karolinska Institutet, SE-171 76 Stockholm, Sweden
e
´
´
´
Catedra de Inmunologıa, Facultad de Quımica/Ciencias, Universidad
de la Repu´blica, Avenida Alfredo Navarro 3051, CP 11600
Montevideo, Uruguay
f Ca´tedra de Quı´mica Inorga´nica, Departamento Estrella Campos,
´
Facultad de Quımica, Universidad de la Republica, CC 1157
Montevideo, Uruguay
´
w Electronic supplementary information (ESI) available: Coordinates
and absolute energies for 7A and 7B. Crystal data for 3ꢃCDCl3. CCDC
690016. For ESI and crystallographic data in CIF or other electronic
format, see DOI: 10.1039/b809238a
Scheme 1 Penta-equatorial (1A) and penta-axial (1B) conformations
of Ins(1,2,3,)P3.
ꢄc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 5161–5163 | 5161

View Article Online
Scheme 2 Penta-equatorial (2A) and penta-axial (2B) conformations
of 4,6-bispyrenoyl Ins(1,2,3,5)P4. P = PO3
2ꢁ
.
ꢁ
(2) (Scheme 3) commences with 4,6-bispyrenoyl-myo-inositol
1,3,5-orthoformate (3), the excimer fluorescent properties of
which have been reported by our group.11,12
Fortuitously, crystals of (3ꢃCDCl3) were obtained and its X-ray
structure is described (Fig. 1).z The crystal structure elegantly
demonstrates the p–p stacking of the pyrene rings, and hence
rationalises its fluorescent properties. Bond angles at ortho-
formate oxygen atoms O1, O3 and O5 are approximately tetra-
hedral in 3, but the angle at O2, which bears the bulky silyl group,
is opened to 122.35(10)1, even wider than the value of 121.01(2)1
for the corresponding angle in the related feruloyl derivative.13 In
contrast to the feruloyl derivative, the two large pyrene rings of 3
are efficiently stacked (Fig. 1A), the dihedral angle between ring
planes being only 3.29(3)1. The pyrene rings of 3 resemble a
clamshell hinged along the C18ꢃ ꢃ ꢃC20 and C35ꢃ ꢃ ꢃC37 side
(Fig. 1B). C18 and C20 are only 3.307(2) and 3.276(2) A out of
the least-squares plane through all ring carbon atoms from C32 to
C47, but on the far side the distance from this plane increases to
3.578(2) and 3.592(2) A for C25 and C27. Since the stacking
separation exceeds the theoretical 2.52 A between adjacent
identical axial substituents on an undistorted cyclohexane ring,
and the average 2.54 A distance between axial H atoms observed
Fig. 1 Complementary ORTEP16 diagrams of (3ꢃCDCl3) showing the
atom numbering scheme. Ellipsoids are drawn at the 50% probability
level. In B the H atoms and selected atom labels are omitted for clarity.
in cyclohexane,14 adjustments are required. These start with the
splaying effect of expansion above the tetrahedral angle in
C4–C5–C6 to 113.75(13)1, C5–C4–O4 to 110.65(12)1 and
C5–C6–O6 to 114.51(14)1. Twisting about the three rotatable
bonds between C4 and C15 and those between C6 and C32 results
is a steady increase in separation distance from 2.565(2) A for
C4ꢃ ꢃ ꢃC6 to 2.804(2) A for O4ꢃ ꢃ ꢃO6 and 3.616(2) A for C14 and
C31 that still allows the pyrene rings to be nearly parallel.
The orthoformate and tert-butyldimethylsilyl protecting
groups in 3 were removed simultaneously in the presence of
p-toluenesulfonic acid15 to give tetrol 4, which was treated
with dibenzyl N,N-diisopropylphosphoramidite, followed by
oxidation with m-chloroperoxybenzoic acid to give 5. Benzyl
deprotection of 5 was achieved by catalytic hydrogenation to
generate the free acid 6, which was converted to the sodium
salt (2) (Scheme 3).y
The fluorescence emission spectra of 2 were recorded in the
absence and presence of Fe3+ (Fig. 2). The emission spectrum
of 2 alone gave rise to blue fluorescence observed at 386 nm
and attributed to the locally excited state of the pyrene
monomer. The presence of Fe3+ caused a marked change in
fluorescence, with the observation of substantial quenching
of the fluorescence of the locally excited state at 386 nm
accompanied by the appearance of a new broad emission
band at 510 nm (green fluorescence). This was attributed to
the excimer emission of the two closely located pyrene groups
achieved in the penta-axial conformation (2B).
Due to the quenching properties of Fe3+, the fluorescence
emission spectrum of 2 was also recorded in the presence of Ga3+
(d10), a non-quenching analogue of Fe3+ (high-spin d5).17–19 Both
cations have the same charge, similar ionic radii and are known to
form isomorphous hexa-coordinate complexes.20 Based on this,
Ga3+ was predicted to behave similarly to Fe3+ with respect to
its binding to the fluorescent probe. This was indeed the case and
a similar emission spectrum was recorded in the presence of Ga3+
Scheme
3
Synthesis of the fluorescent probe 4,6-bis-pyrenoyl-
myo-inositol 1,2,3,5-tetrakisphosphate (2): (a) p-toluenesulfonic acid,
THF : MeOH (2 : 1); (b) i, dibenzyl N,N-diisopropylphosphoramidite,
0.45 M 1H-tetrazole, MeCN; ii, m-CPBA, DCM; (c) H2/Pd-C, ethanol;
(d) Dowex 50-X8 resin, mesh 20–50, Na+ form, elution with water.
ꢄc
This journal is The Royal Society of Chemistry 2008
5162 | Chem. Commun., 2008, 5161–5163

View Article Online
motif is achieved by adopting the penta-axial conformation.
Interest surrounds the precise biological function of
Ins(1,2,3)P3 (1), and in future research the fluorescent probe
may be of use to study the behaviour of 1 in cells.
N.R. thanks Morvus Technologies for a CASE studentship.
We thank ESPRC for award of a diffractometer.
Notes and references
z Crystal data for 3ꢃCDCl3: C47H38O8SiꢃCDCl3, M = 880.25, triclinic,
ꢀ
space group P1, a = 11.1679(9), b = 11.6838(9), c = 16.9249(13) A,
a = 72.420(2), b = 81.387(2), g = 87.634(2)1, V = 2081.5(3) A3, Z =
2, T = 150 K, Z = 2, D = 1.404 (calc.), m = 0.306 mmꢁ1. 18 993
reflections measured, 9307 unique (Rint = 0.0075); after final refine-
ment R = 0.041 for 6716 reflections with Fo 4 4s(Fo), wR(F2) =
0.114 for all 9307 reflections.
y For 2, dH (500 MHz, D2O, water supp.): 9.12 (2H, d, J 9.4 Hz, Pyr),
8.90 (2H, d, J 7.6 Hz, Pyr), 8.76–8.25 (14H, m, Pyr), 6.04 (2H, t, JAA
9.9 Hz, H-4/6), 5.34 (1H, br d, JPH 10.1 Hz, H-2), 4.91 (1H, dt E br q,
JAA E JPH 8.7 Hz, H-5), 4.79–4.68 (2H, br t, JAA E JPH 7.2 Hz,
H-1/3). dP (121 MHz, D2O): 1.79 (1P), ꢁ0.09 (2P), ꢁ1.08 (1P). IR
data: nmax/cmꢁ1 1703 (CQO); 1257 (m), 1226 (m), 1195 (m), (PQO);
1082 (s), 1048 (s), 1018 (s) (P–O–Bn).
Fig. 2 Emission spectra of 4,6-bispyrenoyl Ins(1,2,3,5)P4 (2) in the
absence (- - -) and presence (ꢃ ꢃ ꢃ) of Fe3+ (1 equiv.) and the presence
(—) of Ga3+ (1 equiv.). Spectra were recorded at a concentration of
1 mM in methanol at 20 1C. Excitation and emission slit widths were
3 nm (absence of metal) and 5 nm (presence of metal).
(Fig. 2). The excimer peak was observed at 515 nm with an
intensity more than double that observed with Fe3+. This
evidence supports the general principle that Fe3+ binds to the
penta-axial conformation.10
1 (a) C. J. Barker, P. J. French, A. J. Moore, T. Nilsson, P. O.
Berggren, C. M. Bunce, C. J. Kirk and R. H. Michell, Biochem. J.,
1995, 306, 557–564; (b) F. M. McConnell, S. B. Shears, P. J. L.
Lane and M. S. Scheibel, Biochem. J., 1992, 284, 447–455.
2 I. D. Spiers, C. J. Barker, S.-K. Chung, Y.-T. Chang, S. Freeman,
J. M. Gardiner, P. H. Hirst, P. A. Lambert, R. H. Michell and D.
R. Poyner, Carbohydr. Res., 1996, 282, 81–99.
3 I. D. Spiers, S. Freeman, D. R. Poyner and C. H. Schwalbe,
Tetrahedron Lett., 1995, 36, 2125–2128.
4 A. J. Letcher, M. J. Schell and R. F. Irvine, Biochem. J., 2008,
DOI: 10.1042/BJ20081417.
To obtain detailed structural information, calculations on high-
spin Fe3+ complexes of penta-axial hexamethyl phosphoester at
the UB3LYP/6-31+G* level were performed. Conforming to the
1 : 1 Ins(1,2,3)P3–Fe3+ stoichiometry suggested by potentiometric
titrations5 and the absence of water within the coordination
sphere implied by azide competition experiments,10 we obtained
after conformational searching two essentially isoenergetic
Fe3+-coordinating penta-axial geometries, 7A and 7B (Fig. 3).
Distinct from the structure suggested by Phillippy and Graf,10 for
which we were unable to detect a stable minimum, both 7A and
7B involve inositol phosphoester oxygens as well as terminal
oxygens in the coordination of Fe3+. Similar Fe3+-coordination
is predicted for hexa-anionic Ins(1,2,3)P3. Structure 7A adopts a
distorted tetrahedral coordination involving one phosphoester and
three terminal oxygens in the coordination of Fe3+. Structure 7B,
which is 0.1 kcal molꢁ1 higher in energy than 7A, adopts a
distorted trigonal bipyramidal coordination around Fe3+ invol-
ving participation of two phosphoester and three terminal
oxygens. Further experimental studies are required to unambi-
guously establish the structure of the Ins(1,2,3)P3–Fe3+ complex.
This study provides evidence that Fe3+ binding to
myo-inositol phosphates possessing the 1,2,3-trisphosphate
5 N. Veiga, J. Torres, D. Mansell, S. Freeman, S. Domı
Barker, A. Dıaz and C. Kremer, J. Biol. Inorg. Chem., DOI:
10.1007/s00775-008-0423-2.
´
nguez, C. J.
´
6 C. J. Barker, J. Wright, P. J. Hughes, C. J. Kirk and R. H. Michell,
Biochem. J., 2004, 380, 465–473.
7 J. Torres, S. Domı
Irvine, A. Dıaz and C. Kremer, J. Inorg. Biochem., 2005, 99,
828–840.
nguez, M. F. Cerda, G. Obal, A. Mederos, R. F.
´ ´
´
8 I. D. Spiers, S. Freeman and C. H. Schwalbe, J. Chem. Soc., Chem.
Commun., 1995, 2219–2220.
9 M. J. Frisch and co-workers, Gaussian 03, 2004. See ESI for full
citationw.
10 B. Q. Phillippy and E. Graf, Free Radical Biol. Med., 1997, 22,
939–946.
11 M. Kadirvel, E. V. Bichenkova, A. D’Emanuele and S. Freeman,
Chem. Lett., 2006, 35, 868–869.
12 M. Kadirvel, B. Arsic, S. Freeman and E. V. Bichenkova, Org.
Biomol. Chem., 2008, 6, 1966–1972.
13 A. Hosoda, Y. Ozaki, A. Kashiwada, M. Mutoh, K. Wakabayashi,
K. Mizuno, E. Nomura and H. Taniguchi, Bioorg. Med. Chem.,
2002, 10, 1189–1196.
14 R. Kahn, R. Fourme, D. Andre and M. Renaud, Acta Crystallogr.,
Sect. B: Struct. Crystallogr. Cryst. Chem., 1973, 29, 131–138.
15 T.-H. Kim and A. B. Holmes, J. Korean Chem. Soc., 2006, 50,
129–136.
16 C. K. Johnson, ORTEP-II, Report ORNL-5138, Oak Ridge
National Laboratory, Oak Ridge, TN, USA, 1976.
17 B. Bodenant, F. Fages and M. H. Delville, J. Am. Chem. Soc.,
1998, 120, 7511–7519.
18 F. Fages, B. Bodenant and T. Weil, J. Org. Chem., 1996, 61,
3956–3961.
19 F. Fages, S. Leroy, T. Soujanya and J.-E. Sohna Sohna, Pure Appl.
Chem., 2001, 73, 411–414.
20 B. A. Borgias, S. J. Barclay and K. N. Raymond, J. Coord. Chem.,
1986, 15, 109–123.
Fig. 3 Optimal UB3LYP/6-31+G* geometries (A) of the hexa-
methyl phosphoester of Ins(1,2,3)P3–Fe3+(1) in the high-spin state.
ꢄc
This journal is The Royal Society of Chemistry 2008
Chem. Commun., 2008, 5161–5163 | 5163