Effect of the second coordination sphere on new contrast agents based on cyclodextrin scaffolds for MRI signals

Article abstract of DOI:10.1039/c3ra40314a

Two new imaging tools using polydentate cyclodextrins were obtained using an innovative synthetic strategy. For the first time the influence of hydrogen bonding interactions of the cyclodextrin free rim was studied by MRI. The positive second coordination sphere effect was then quantified.

Full text of DOI:10.1039/c3ra40314a

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Effect of the second coordination sphere on new
contrast agents based on cyclodextrin scaffolds for
MRI signals3
Cite this: RSC Advances, 2013, 3, 4531
Received 21st January 2013,
Accepted 1st February 2013
a
a
a
a
a
´
Hussein Idriss, François Estour, Ibrahim Zgani, Cecile Barbot, Anais Biscotti,
Samuel Petit,b Chantal Galaup,c Marie Hubert-Roux,a Lionel Nicol,d Paul Mulderd
DOI: 10.1039/c3ra40314a
a
´
and Geraldine Gouhier*
www.rsc.org/advances
Two new imaging tools using polydentate cyclodextrins were
obtained using an innovative synthetic strategy. For the first time
the influence of hydrogen bonding interactions of the cyclodex-
trin free rim was studied by MRI. The positive second coordination
sphere effect was then quantified.
this cyclic oligosaccharide, Gd(III) complexes of b-CD decrease the
rotation rate in water, increase the water proton relaxation rates
(r1) and then enhance the contrast of images.
Nevertheless, efficient results have only been obtained with CAs
bearing more than one metal atom per CD, increasing the risk of
releasing free toxic gadolinium in the blood circulation.2
To reach our objectives we have synthesised heptacoordinated
Gd(III) CAs based on 2,3-dimethylated b-CD 1a and native b-CD 2a
thus containing only one metal ion (Fig. 1). The metal
complexation occurs thanks to seven acetate ligands grafted on
the b-CD primary face via stable ether functions under physiolo-
gical conditions. A molecular modeling study showed that all the
carboxylate functions could be localized at a distance of 2.4–2.5 Å
from the Gd3+ ion.5 From this design, two water molecules fill the
inner spheres of the metal ion center via the oxygen atoms.
The aim of this study was also to compare MRI results
obtained from these two new similar structures 1a and 2a and to
evaluate for the first time the influence of the hydroxyl groups
fixed to the crown of the CD on the MRI signal (Fig. 1).
One of the current aims of research in the field of magnetic
resonance imaging (MRI) is the design of new contrast agents
(CAs) in order to significantly improve the sensitivity of this
technique. The most important class of CAs for MRI is currently
represented by Gadolinium(III) (Gd) chelates including cyclic
tetraazacyclododecanetetraacetic acid (DOTA) or linear diethylene-
triaminepentacetic acid (DTPA) ligands.1 These clinically used
complexes are nine coordinated with eight polyaminopolycarbox-
ylate donor ligands and a water molecule occupying the ninth
coordination site. Despite their wide use, DOTA or DTPA could
lead to serious allergies after several injections.2 By increasing the
sensitivity of this medical diagnostic tool, it would then be
possible to inject a weaker amount of CA and to prevent
toxicological risks for the patients.
Functionalized cyclodextrins (CDs) have already been described
as interesting scaffolds to access to efficient CAs.3 Our challenge
was to design new CAs based on b-CD, with high relaxivities, in
order to increase the detection threshold. b-CD is a natural
macrocyclic molecule composed of seven glucose units b-(1–4) in
the shape of a conical cylinder delimiting an internal hydrophobic
cavity.4 The secondary face is more crowded than the primary side
due to the presence of a double number of hydroxyl groups and
constitutes the larger rim. Due to the higher molecular mass of
Four other metal ion complexes based on Europium Eu(III) 1b
and 2b and Lanthane La(III) 1c and 2c, respectively, have also been
isolated to complete the structural and analytical studies by
luminescence and NMR techniques (Fig. 1).
Results and discussion
The 2,3-dimethylated b-CD was successfully synthesized in three
steps from native b-CD 3 as already described.6 Seven ligands were
then grafted on the lower face with 55% yield by reaction with
aNormandie Univ, COBRA, UMR 6014, FR 3038, INSA Rouen, CNRS, IRIB, IRCOF 1
`
rue Tesniere 76821 Mont-Saint-Aignan, France. E-mail: geraldine.gouhier@univ-
rouen.fr; Fax: 33-235522959; Tel: 33-235522909
b
´
EA 3233, SMS, 76821 Universite of Rouen, France
c
´
SPCMIB, UMR CNRS 5068 Universite Paul Sabatier, 118, route de Narbonne 31062
Toulouse Cedex, France
dINSERM U1096, Institute for Research and Innovation in Biomedicine, UFR de
´
´
Medecine et de Pharmacie, 76821 Universite de Rouen, France
Electronic supplementary information (ESI) available: Experimental section,
3
structure characterization, NMR, luminescence analysis, MRI analysis and
complexometric titration. See DOI: 10.1039/c3ra40314a
Fig. 1 Metal complexes based on CD scaffolds.
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ethyl diazoacetate in the presence of tetrafluoroboric acid in
anhydrous dichloromethane at room temperature according to
Zavada’s procedure.7
However the application of this methodology to native b-CD 3
brought about difficulties to obtain selective per-O-6-functiona-
lized compounds due to the insolubility of 3 in dichloromethane.
The use of other solvents such as DMF, DMSO or pyridine did not
improve the reaction. The functionalization of the primary face of
3 was then tested by addition of sodium iodoacetate in the
presence of pyridine in DMF but without success.8 In order to
enhance the solubility of the macrocycle in dichloromethane, the
secondary face was temporarily protected by silyl9 or acetyl
groups10. However the complete substitution by seven carboxylate
arms was not observed even after optimization tests such as the
variation of the number of reagent equivalents (1-5 per b-CD) and/
or of the reaction time (24–72h). In all cases mixtures of
substituted CDs (3 to 7 ethyl acetate groups) inseparable by
column chromatography were observed by mass spectrometry
analysis.
Looking for an alternative strategy we have selected the allylic
group as a convenient synthon. To our knowledge allylic
functionalization had never been used as a temporary protective
group for CDs synthesis. Only mono, perallylated, or selectively
alkylated b-CDs in various positions have already been reported in
successive reaction schemes such as oxidation, reduction and
addition leading to many multifunctionalized CDs.11 The suitable
compound 5 was obtained with 45% yield from 6-O-persilylated
b-CD 4 (Scheme 1).12 Quantitative deprotection of the primary face
in the presence of ammonium fluoride led to new compound 6
being soluble in dichloromethane. The introduction of seven
acetate functions on the primary face of 6 using Zavada’s
procedure led to derivative 7 with 36% yield.7 A mixture of
compounds bearing four to six functions was also isolated, but an
oversubstitution of these derivatives was tested without success.
In order to access to the desired compound 8, complete
deprotection of 7 had to be adjusted. Deallylation conditions have
been optimized using Pd(PPh3)4 in acetic acid. After purification
by chromatography, compound 8 was isolated in the presence of
catalyst. The use of supported Pd(PPh3)4 on insoluble polymer did
not facilitate the purification step.13 The elimination of residual
palladium required two successive filtrations on Dowex1 50WX4
and diethanolamine polystyrene (PS-DEAM) resins.14 Finally the
compound 8 was isolated with 70% yield.
Scheme 1 Synthesis of per-6-ethylacetate CD 8.
For access to metal complexes 1a–c and 2a–c, a saponification
reaction of the ester functions of 8 and 9 was quantitatively carried
out (Scheme 2). Gd(III), Eu(III) or La(III) chloride hexahydrate salts
were then complexed by 10 and 11 in water under controlled pH.
Mass spectrometry analyses and luminescence studies proved a
stoichiometry 1/1 M/CD for complexes 1 and 2 respectively (ESI ).
Due to the paramagnetic property of Gd(III), NMR analyses have
been carried out on 1c and 2c complexes. The 13C NMR spectrum
3
Through this original synthetic strategy, we developed an
alternative methodology that could be applied in order to
selectively prepare various CDs derivatives bearing groups only
on the primary rim. The new important key intermediate 6
extends the panel of known selective protective groups to be easily
removable such as acetyl, benzyl or silyl functions usually used in
the synthesis of modified CDs. Moreover the high solubility in
organic media of this allylic precursor is an alternative to the
permanent methylation functionalization commonly used to avoid
the CD solubility problems. Thus, this new methodology could be
useful in order to reach new CD structures with other flexibilities,
solubilities and new binding properties.4
Scheme 2 Synthesis of complexes 1 and 2.
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showed significant chemical shifts of the carbon atoms around
the carboxylate functions proving the complexation of the
lanthanide with the seven ligands. Finally hydration numbers
have been calculated for luminescent Eu3+ complexes 1b and 2b
by using the well-established isotope effect and the Horrocks
After complexometric titration using EDTA in the presence of
xylenol orange as an indicator, weak percentages of 0.6% of free
Gd(III) for 1a and of 0.4% of free Eu(III) for 2b were measured
validating the MRI results observed.
equation (ESI ). Indeed the comparison of the luminescence
3
Conclusions
lifetimes of these europium complexes obtained in H2O and D2O
allows an assessment of the hydration state. Hydration numbers
of 2.48 and 2.18 were obtained for 1b and 2b, respectively, and
confirm the presence of heptacoordinated active sites.
In summary, we developed a new synthetic method using allylic
functions to access to C6-functionalized CDs that enlarge the
panel of known selective protections and can be a solution to solve
the common problem of weak solubility in organic solvents of
these scaffolds.
The MRI results obtained with two new CAs confirmed the
potential of this strategy for relaxation enhancement using
polydentate hydrophilic CDs as new imaging tools. A comparative
study underlined the individual contributions of the rim of CD on
the MRI signal and allowed the first quantification of the positive
influence of the second coordination shell of the CD. The relaxivity
gain could be attributed to a high number of water molecules
being involved in a network of hydrogen bonding interactions.
A nuclear magnetic relaxation dispersion profile (NMRD)
measurement will pinpoint the respective roles of the three
hydration spheres contributions (inner, second and outer
coordination spheres).
MRI tests were carried out at 0.5 Tesla on minispec mq 20 in
TRIS buffer under physiological pH and at 37 uC. The relaxivities
(r1) of 1a and 2a CAs were 4.56 and 6.53 mM21 s21 respectively.
These results correspond to an increase of 30% and 87% of the
signal imaging compared to the reference value of DOTA (Chart 1).
This variation observed between DOTA and CAs 1a–2a could be
explained by a higher molecular size of the CD scaffold increasing
the reorientational correlation times (tr) and consequently the
relaxivities.3 Nevertheless the high signal variation of 43% between
CAs 1a and 2a is mainly due to the second coordination sphere
effect because the variation of mass between the adducts here is
negligible.15 Indeed 2a may yield stronger interactions with water
molecules on the upper face of the macrocycle, and lengthens
their lifetime rate in the proximity of the paramagnetic centre.
Such hydrogen-bonding networks involving the coordinated
water(s) are then reinforced and perturb the relaxivity.16 In order to
reduce the water molecules around the complexes and present in
the cavity of the macrocycle, dioxane was selected due to its high
miscibility with water and its slow self-diffusion.17 A similar drop
Acknowledgements
We are grateful to Haute-Normandie Region via the FEDER for
the post-doctoral fellowship (I. Z.), the French Ministry of
Research for the PhD grant (H. I.), Interreg AIChem for the
PhD grant (A.B.), OSEO, SEINARI, and the Institute for
Research and Innovation in Biomedicine of Rouen for their
financial supports.
of the relaxivities for 1a and 2a at 2.2 mM21s21 and 2.5 mM21s21
,
respectively, was observed proving the key role of the outersphere
for these two complexes.
Water molecules around 1a and 2a complexes may present
different rotational mobilities due to the distortion of the shape of
the CD slowing down the dynamics of rotation and enhancing the
contrast imaging.
Notes and references
As the presence of free Gd3+ in CAs may have no negligible
impact on the contrast in the MR image and could provide
erroneous information on the contrast effect induced, a titration
has been carried out following the protocol described by Barge.18
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Chart 1 Comparison of MRI results between DOTA, 1a and 2a.
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