X-ray photolysis to release ligands from caged reagents by an intramolecular antenna sensitive to magnetic resonance imaging

Article abstract of DOI:10.1002/anie.201102948

Sensitive to light: A metal-complex-sensitized organic probe was developed to release ligands on excitation by X-ray or γ irradiation (see picture). This overcomes a current limitation in permitting use of photolysis as an experimental tool in otherwise inaccessible materials that are not penetrated by light (ET=electron transfer). Copyright

Full text of DOI:10.1002/anie.201102948

Communications
DOI: 10.1002/anie.201102948
Molecular Probes
X-ray Photolysis To Release Ligands from Caged Reagents by an
Intramolecular Antenna Sensitive to Magnetic Resonance Imaging**
Morgane Petit, Guillaume Bort, Bich-Thuy Doan, Cꢀcile Sicard, David Ogden,
Daniel Scherman, Clotilde Ferroud, and Peter I. Dalko*
Light is important tool for the external control of biological
processes in life sciences. It is used directly for the optical
stimulation of neurons[1,2] for photolysis of caged com-
pounds,[3] actuators,[4] and switches.[5] The principal problem
that remains is the poor penetration of light in tissues, limited
to depths of around 100 mm even with near-IR wavelengths.[6]
Although the use of fiberoptics,[7] implantable photoelec-
trodes, laser-coupled microelectrode arrays,[7,8] and micro-
mechanical systems[9] improves the situation, release of
biologically active ligands with high spatiotemporal control
in otherwise inaccessible materials remains a challenge.[10]
The use of X-rays for the activation of molecular devices
has the potential to perform radiolysis in vivo.[11] Hard X-rays
used in radiography are known to penetrate soft tissues to
depths of centimeters and this has been exploited previ-
ously.[12] X-ray excitation can activate photosensitizers teth-
ered to lanthanum fluoride nanoparticles and generate toxic
singlet oxygen.[13a] Also the photothermal effect of X-ray
irradiation of noble-metal nanostructures may be used for
cancer treatment.[13b,c] More recently radiation-sensitive dis-
elenide-derived block copolymer aggregates were prepared
that load and release drugs in aqueous solution.[14] However,
the photoactivation of organic probes by X-rays with
subsequent release of covalently linked molecules has not
been reported because organic reagents are poorly absorbing
at these wavelengths.
We speculated, that photoactivated organic probes may
undergo one-electron reduction in the presence of Auger
electrons, and the radical anion intermediate may follow
reaction paths similar to those of photoexcited intermedi-
ates.[15] Auger electrons are generated by the interaction of X-
ray or g irradiation with heavy metals. The one-electron
reduction of hydroxymethyl quinolines would afford a radical
anion intermediate that may undergo fragmentation as
depicted in Scheme 1.[16] If the reaction proceeds in protic
solvent such as water the fragmentation products are
quenched after electron transfer rendering the process
irreversible.
Scheme 1. Auger electron-mediated electron transfer (ET) leading to
fragmentation of hydroxymethyl quinolines.
Here, we report the proof-of-principle of this concept and
show that quinolene-derived probes are activated through
interactions of X-ray or g irradiation with gadolinium (III)
complexes, which act as intramolecular antennae and convert
part of the energy to fragmentation processes.[17] For this the
GdIII complex is coupled to well-characterized aminoquino-
line-based photoprotecting groups,[18] normally cleaved by
UV light and only weakly by shorter wavelengths than
300 nm. Photolabile groups which are photolytically activated
by wavelengths in the 300–400 nm range are routinely used in
cell biology and neuroscience to release ligands with high
spatio-temporal resolution.[3a–d] In this case the activity of the
biomolecule is masked by the photosensitive group and the
light activation restores the activity by a process called
“uncaging” or “photorelease”.
[*] M. Petit,[+] Dr. P. I. Dalko
Laboratoire de Chimie et Biochimie Pharmacologiques
et Toxicologiques, Universitꢀ Paris-Descartes (France)
E-mail: peter.dalko@parisdescartes.fr
G. Bort,[+] Prof. C. Ferroud
Service de Transformations Chimiques et Pharmaceutiques
Conservatoire national des arts et mꢀtiers (France)
Dr. B.-T. Doan, Prof. D. Scherman
Unitꢀ de Pharmacologie Chimique et Gꢀnꢀtique
et d’Imagerie, Universitꢀ Paris
Descartes, ENSCP-Chimie-Paristech (France)
Dr. C. Sicard
Laboratoire de Chimie Physique
Universitꢀ Paris-Sud, Orsay (France)
Dr. D. Ogden
Laboratoire de Physiologie Cꢀrꢀbrale
Universitꢀ Paris-Descartes (France)
As photoremoveable protecting groups 7-bromo-8-hy-
droxy-2-hydroxymethylquinoline (BHQ) and 7-(N,N-dime-
[+] These authors contributed equally to this work.
thylamino)-2-hydroxymethylquinoline
(DMAQ)
were
[**] D.O. and P.I.D. acknowledge the generous support of EU STREP
(PHOTOLYSIS-LSHM-CT-2007-037765) grant and M.P. acknowl-
edges FRM grant N8 FDT20091217593.
selected (Figure 1).[18a–c,e] These groups absorb efficiently in
the near-UV region at a wavelength of maximum absorbtion
(lmax) of 369 nm for BHQ with an absorbance, el (BHQ), of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201102948.
max
2600mÀ1 cmÀ1 [tris(hydroxymethyl)aminomethane hydrochlo-
9708
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Angew. Chem. Int. Ed. 2011, 50, 9708 –9711

Figure 1. Quinolin-based light-activated protecting groups.
Scheme 2. Synthesis of the Gd-DOTAGA-AQ dihydrocinnamate 5 and
the truncated control substrate 3b. a) Paraldehyde (3 equiv), HCl(cc),
RT 1 h then 808C, 3 h, (28%). b) SeO2 (1.3 equiv), dioxane, 808C, 3 h
(95%). c) NaBH4 (1.1 equiv), EtOH, RT, 1 h (78%). d) Tert-butyldime-
thylsilyl chloride (TBS-Cl, 1.2 equiv), imidazoline (1.2 equiv), N,N-
dimethylformamid (DMF), RT, 3 h (64%). e) Piperazine (5 equiv),
tris(dibenzylideneacetone)dipalladium (Pd2dba3, 10 mol%), PtBu3 (40
mol%) NaOtBu (1.2 equiv), toluene, 1108C, 16 h (68%). f) Boc2O
(1.1 equiv, boc is tert-butoxycarbonyl), DMAP (0.2 equiv), CH2Cl2, RT,
2 h (90%). g) TBAF (5 equiv), THF, RT, 5 h (89%). h) Ph(CH2)2COOH
(1.2 equiv), oxalyl chloride (1.5 equiv), DMF (cat), ether, 08C to RT,
then NEt3 (1.2 equiv), dimethylaminopyridine (DMAP, 0.2 equiv),
CH2Cl2, RT, 3 h (80%). i) HCl 6n, dioxane, RT, 10 min, then GdIII-
DOTAGA (1.5 equiv), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDCI·HCl, 3.0 equiv), hydroxybenzotriazole (HOBt, 0.6 equiv), DMF/
H2O of 2:1, RT, 3 h (44%).
ride (TRIS) buffer, pH 7.4, 298 K] and a lmax value of 368 nm
for DMAQ with an el (DMAQ) value of 4600mÀ1 cmÀ1
max
(KMOPS buffer, pH 7.2, 298 K). These groups are versatile
and have been used with molecules coupled to carboxylate,
phosphate, and diol units. For our purpose, the modified
aminoquinoline was tethered to GdIII-1,4,7,10-tetraazacyclo-
dodecane-1-glutaric
acid-4,7,10-triacetic
acid
[GdIII-
DOTAGA)][19] (Figure 1) prepared previously for magnetic
resonance imaging (MRI) applications.[20] The DOTAGA
analogue has a glutarate arm to bind a substrate without
lowering the stability of the complex or the exchange rate of
water on the Gd3+ ion. Macrocyclic GdIII complexes have high
thermodynamic and kinetic stabilities relative to linear GdIII
complexes that renders them compatible with in vivo appli-
cations.[21] Notably, gadolinium derivatives are also used as X-
ray contrast agents.[22]
For the fragmentation studies dihydrocinnamate was
selected as model leaving group because of its great hydro-
lytic stability relative to that of the acetate ester under
physiological conditions, and also because both photolysis
products can easily be monitored by HPLC.
propyl)-N’-ethylcarbodiimide hydrochloride (EDCI.HCl)
coupling agents (Scheme 2).
The photolysis and radiolysis of compounds 3b and 5 were
performed in TRIS buffer under UV, X-ray, or g irradiation
conditions, as illustrated in Scheme 3. For near-UV irradia-
tion of 3b and 5 a 366 nm lamp (8 W; Carl Roth) was used, X-
rays of 17.5 keV were produced by an X-ray generator
(Diffractis 583 Enraf Nonius), and g radiolysis experiments at
1.17 MeV were performed on a panoramic 60Co source
(IL60PL Cis-Bio International). The dose rates of 21 and
28 GyminÀ1, respectively, were determined with a Fricke
dosimeter.
The photolysis of 3b and 5 by irradiation at 366 nm (TRIS
20 mm, NaCl 100 mm, pH 7.4) was monitored by disappear-
ance of the starting materials and the appearance of the
uncaged hydroxy compounds 6a and 6b (see Table 1), and by
dihydrocinnamate 7 through liquid-chromatography-coupled
mass spectrometry (LC-MS) analysis. The structure of the
products 6a and 6b were also proven by comparison of the
HPLC-MS data with samples prepared by chemical synthesis.
Compound 3b undergoes photolysis at 366 nm with a
quantum sensitivity (eQu) of 213, which is comparable to the
eQu value of 211 of the parent DMAQ-OAc compound.[18a]
The presence of the Gd tether reduces, however, the
sensitivity to photolysis of the quinoline group as GdIII-
The synthesis of the Gd-DOTAGA-AQ-caged dihydro-
cinnamate 5 was realized from 7-bromoquinaldine (2) which
was prepared in quantities of grams by the Doebner–Miller
synthesis from 3-bromoaniline (Scheme 2) under modified
Doreꢀs conditions.[18a] The bromoquinaldine 2 was trans-
formed to the corresponding hydroxymethylene compound
by an oxidation–reduction sequence, and the free alcohol was
secured as tert-butyldimethylsilyl (TBS) ether. The piperazine
group was introduced under conditions of Buchwald–Hartwig
amination (tris(dibenzylideneacetone)dipalladium (Pd2dba3),
P(tBu)3, NaOtBu, toluene, 1108C, 68%),[23] and the secondary
amine was converted to the tert-butoxycarbonyl (Boc)
derivative 3a using a standard protocol. The introduction of
the dihydrocinnamate model substrate was realized after
cleveage of the TBS protecting group in the presence of
excess tetrabutylammonium fluoride (TBAF, 1m in THF) and
the dihydrocinnamate was introduced after activation of
oxalyl chloride (80%). The assembly of 3a and GdIII-
DOTAGA acid 4 were realized in the presence of hydrox-
ybenzotriazole (HOBt) hydrate and N-(3-dimethylamino-
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Communications
Figure 2. Progressive radiolysis of 0.4 mm of 5. The remaining fraction
(in %) is plotted against the cumulative dose of X-rays of 17.5 keV
delivered at 21 GrayminÀ1 (full triangles) and the g photons of
1.17 MeV at 28 GrayminÀ1 (full squares). Experimental details are
given in the Supporting Information.
Scheme 3. Photo- and radiolysis of caged dihydrocinnamates 6a and
6b.
Table 1: Photolysis of compounds 3b and 5.
observation suggests that the GdIII complex plays the role of a
sensitizer.
[a]
[b]
[b]
[c]
[d]
lmax
[nm]
el
e366
[mÀ1 cmÀ1
Qu
eQu
max
[mÀ1 cmÀ1
]
]
[mÀ1 cmÀ1
]
Under conditions of X-ray or g irradition in the presence
of oxygen, radiolysis of water induces the formation of several
species and mostly of HOC or O2CÀ radicals. Hydroxyl radicals
are very strong oxidants. Therefore, to exclude their role in
the fragmentation process of compound 5, we performed the
same experiment in phosphate buffer because TRIS buffer is
known to scavenge most of these radicals. The identical
G values obtained (data not shown) suggest that radiolytic
fragmentation is not dependent on the presence of hydroxyl
radicals in the solution, and fragmentation is attributed to the
direct interaction of radiation with compound 5.
5
3b
340
350
3100
7320
2100
5750
0.020
0.037
42
213
General conditions: TRIS 20 mm, NaCl 100 mm pH 7.4. [a] Wavelength
at maximum absorption. [b] Absorbance at the given wavelength.
[c] Uncaging quantum efficiency. [d] Sensitivity upon irradiation at
366 nm.
DOTAGA-senzitized 5 undergoes photolysis with a eQu value
of 42. Yet, the high aqueous solubility of compound 5
represents a major advantage over other caged compounds
that exhibit often poor solubility which reduces their effi-
ciency in biological studies.
MRI characteristics of compound 5 were evaluated by
in vitro r1 and r2 relaxivity measurements, where r1 is the
longitudinal and r2 is the transversal relaxivity parameter. The
obtained r1 value of 7.4 mmÀ1 sÀ1 and r2 value of 9.6 mmÀ1 sÀ1 of
5 at 300 MHz agrees with a low molecular weight and a low
rotational correlation time tR relative to the values (r1 =
3.9 mmÀ1 sÀ1 and r2 = 4.5 mmÀ1 sÀ1 at 300 MHz, 300 K) of
Progressive radiolysis of the Gd antenna-sensitized com-
pound 5 at X-ray irradiation of 17.5 keV showed clean
fragmentation with the appearance of the radioproducts 6a
and dihydrocinnamate 7 (Scheme 3 and Figure 2). When 3b
was irradiated under identical conditions, 6b and 7 appeared
solely as the products of hydrolysis at a conversion rate of less
than 1% per hour, and this observation was identical to that
of a control experiment in the dark. Likewise, when
compound 5 was submitted to radiolysis at g irradiation of
1.17 MeV, fragmentation was observed and 6a and dihydro-
cinnamic acid 7 (Scheme 3 and Figure 2) were produced. No
radiolysis was observed, however, when compound 3b was
irradiated without the Gd-DOTAGA sensitizer. Figure 2
shows the fractional loss of 5 plotted against the dose received
by continuous irradiation at a dose rate of 21 GrayminÀ1 at
17.5 keV (full triangles) and at 28 GrayminÀ1 at 1.17 MeV
radiation (full squares). Uncaging profiles of compound 5
with X-ray and g irradiations are very similar with calculated
G values of À93 nmolJÀ1, where the G value denotes the
number of moles of species produced/consumed per Joule of
absorbed energy. In these experiments the release of dihy-
drocinnamic acid 7 by X-ray or g irradiation occurred only if
the caged compound was labeled by the GdIII complex. This
DOTAREMꢁ
(GdIII-1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid, Gd-DOTA). Likewise, the radio-
product 6a shows relatively low relaxivity values r1 of
5.7 mmÀ1 sÀ1 and r2 of 7.4 mmÀ1 sÀ1 that agree with a lower
rotational correlation time of 6a relative to 5.
In conclusion, a first energy dispersive X-ray-activated
probe (EDiXA) irradiated by X-rays or g photons has been
prepared to address the problem of controlled photorelease
of biologically active ligands deep within the tissues of the
body. We have demonstrated the release of dihydrocinnamate
in aqueous solution at physiological pH. Because caged
compounds are used principally as research tools in molecular
biology, cell physiology, and neuroscience, X-ray cages may
extend the range to in vivo experiments. This method has the
potential to improve photodynamic therapies by extending
the depth of penetration of the radiation and by expanding
the range of molecules that are released; particularly it has
the potential application in chemotherapy of delivering toxic
drugs to their sites of action as nontoxic caged precursors.
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Received: April 29, 2011
Published online: July 21, 2011
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Keywords: gadolinium · magnetic resonance imaging ·
photochemistry · photolysis · radiolysis
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