DOTAGA-anhydride: Avaluable building block for the preparation of DOTA-like chelating agents

Article abstract of DOI:10.1002/chem.201200132

A DOTA derivative that contains an anhydride group was readily synthesized by reacting DOTAGA with acetic anhydride and its reactivity was investigated. Opening the anhydride with propylamine led to the selective formation of one of two possible regioisomers. The structure of the obtained isomer was unambiguously determined by 1D and 2D NMR experiments, including COSY, HMBC, and NOESY techniques. This bifunctional chelating agent offers a convenient and attractive approach for labeling biomolecules and, more generally, for the synthesis of a large range of DOTA derivatives. The scope of the reaction was extended to prepare DOTA-like compounds that contained various functional groups, such as isothiocyanate, thiol, ester, and amino acid moieties. This versatile building block was also used for the synthesis of a bimodal tag for SPECT or PET/optical imaging.

Full text of DOI:10.1002/chem.201200132

DOI: 10.1002/chem.201200132
DOTAGA–Anhydride: A Valuable Building Block for the Preparation of
DOTA-Like Chelating Agents
Claire Bernhard,[a] Mathieu Moreau,[a] Damien Lhenry,[a] Christine Goze,[a]
Frꢀdꢀric Boschetti,[b] Yoann Rousselin,[a] FranÅois Brunotte,[c] and Franck Denat*[a]
Abstract:
A
DOTA derivative that
periments, including COSY, HMBC,
and NOESY techniques. This bifunc-
tional chelating agent offers a conven-
ient and attractive approach for label-
ing biomolecules and, more generally,
for the synthesis of a large range of
DOTA derivatives. The scope of the
reaction was extended to prepare
DOTA-like compounds that contained
various functional groups, such as iso-
thiocyanate, thiol, ester, and amino
acid moieties. This versatile building
block was also used for the synthesis of
a bimodal tag for SPECT or PET/opti-
cal imaging.
contains an anhydride group was readi-
ly synthesized by reacting DOTAGA
with acetic anhydride and its reactivity
was investigated. Opening the anhy-
dride with propylamine led to the se-
lective formation of one of two possi-
ble regioisomers. The structure of the
obtained isomer was unambiguously
determined by 1D and 2D NMR ex-
Keywords: anhydrides · chelates ·
DOTA · imaging agents · protecting
groups
Introduction
tate pendant arms, thereby leading to DOTA–monoamide
units (DOTA-MA).[3] However, the transformation of one
of the carboxylic acid group into a carboxamide can de-
crease the stability of the complex by two or three orders of
magnitude.[3d] To overcome this problem, DOTA-like
BFCA, which contained four acetate side-arms, was synthe-
sized through the preparation of C-functionalized DOTA
macrocycles[4] or N-functionalized derivatives that contained
an additional pendant arm.[1c] The DOTAGA derivative
(GA=glutaric acid) was first synthesized by Maecke and
co-workers[5] by the reaction of tris-tBu-DOTA with a bro-
moglutaric diester derivative. Various DOTAGA bioconju-
gates were prepared and labeled with different radiometals
The use of so-called bifunctional chelating agents (BFCA)
has been the most-reliable and most-commonly used
method for attaching a metal ion (radiometal or paramag-
netic metal) onto a biomolecule for molecular-imaging ap-
plications (PET, SPECT, MRI). Such an approach has been
used for the development of metal-based imaging agents,
which are essential for diagnostic and therapeutic applica-
tions.[1] DOTA (1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tet-
raazacyclododecane) and its derivatives represent a major
class of chelating agents for biomedical applications, owing
to their excellent complexation properties towards many tri-
valent metal ions, and the high kinetic stability of the metal
complexes thus formed.[1c,2]
(
111In, 68Ga, 90Y).[6] In most cases, the conjugation of
DOTAGA onto a biomolecule was performed by using
DOTAGA derivatives in which the four coordinating ace-
tate arms were protected by tert-butyl ester groups.[5,7] The
remaining carboxylic acid group on the glutaric acid moiety
reacted with lysines in the presence of coupling reagents
(i.e., HBTU, EDCI, etc.). However, the deprotection condi-
tions that were used to cleave the ester groups were not
suitable for fragile biomolecules, such as antibodies. To
avoid this deprotection step, DOTAGA derivatives that con-
tained four free acidic groups and an activated group, which
was able to react specifically with an amino group, was pres-
ent in the biomolecule function (i.e., a pentafluorophenyl
ester or succinimidyl ester group) were developed.[8] The
synthesis of such compounds was not straightforward and
required protection/deprotection sequences and tedious
chromatographic work-up. Gd-DOTAGA-conjugates were
prepared by coupling the monohydrated Gd complex of
DOTAGA onto a macromolecule with no need for a protec-
tion step because the four pendant arms of DOTA were in-
volved in the coordination of the metal ion.[9] However, this
In most cases, the attachment of a DOTA-fragment onto
the biomolecule has involved one of the coordinating ace-
[a] Dr. C. Bernhard, M. Moreau, D. Lhenry, Dr. C. Goze,
Dr. Y. Rousselin, Prof. F. Denat
Institut de Chimie Molꢀculaire de
l’Universitꢀ de Bourgogne, UMR CNRS 6302
9 avenue Alain Savary, 21000 DIJON (France)
E-mail: franck.denat@u-bourgogne.fr
[b] Dr. F. Boschetti
CheMatech, 9 avenue Alain Savary
21000 DIJON (France)
[c] Prof. F. Brunotte
Service de mꢀdecine nuclꢀaire
centre Georges-FranÅois Leclerc
1 rue du Pr. Marion, 21000 DIJON (France)
Supporting information for this article, including characterization
data for compounds 2a and 3 (route B), NMR, MS, UV/Vis spectra,
and chromatograms, is available on the WWW under http://
dx.doi.org/10.1002/chem.201200132.
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FULL PAPER
smart “metal-protection” approach could not be applied to
radiolabeling because the complexation of the radiometal
must be performed in the last step.
An alternative strategy that has not been explored so far
is to combine the high reactivity and facile preparation of li-
gands that contain an anhydride group with the good chela-
tion properties of DOTA and thus to prepare a new DOTA-
like derivative, namely DOTAGA–anhydride (Scheme 1).
Results and Discussion
Formation of DOTAGA–anhydride 1: DOTAGA–anhydride
1 was synthesized in a one-step reaction starting from the
DOTAGA macrocycle according to a literature procedure
(Scheme 2).[9] The strategy was similar to that reported for
the synthesis of DTPA–bisanhydride: the ammonium chlo-
Scheme 2. Synthesis of DOTAGA–anhydride (1): a) acetic anhydride,
pyridine, 658C, 18 h, 96% yield.
Scheme 1. DOTAGA and DOTAGA–anhydride.
ride salt of DOTAGA·2HCl was reacted with acetic anhy-
dride in the presence of pyridine at 658C to generate
a cyclic anhydride. The desired product was isolated by fil-
tration in 96% yield. The resulting anhydride was quite
stable and could be stored at 48C for several months with-
out degradation. The formation of the anhydride was con-
firmed by MS (ESI; see the Supporting Information, Fig-
ure S3).
Indeed, this approach was very efficient in the case of
a DTPA chelator. DTPA–bisanhydride has been successfully
used for labeling antibodies (e.g., trastuzumab),[10] antibiot-
ics,[11] enzymes,[12] and nanoparticles for MRI and therapy.[13]
We recently reported the preparation of ultrasmall nano-
particles that were decorated with DOTAGA units; the
DOTAGA–anhydride precursor enabled facile decoration
and their application for multi-imaging experiments.[14] 111In-
labeled DOTAGA–antibodies conjugates were also pre-
pared in our group, which showed the usefulness of
DOTAGA–anhydride for the efficient attachment of radio-
metals onto humanized mAb. Highly specific tumor target-
ing was observed in model human of breast tumors with
overexpressed HER2/neu antigen.[15]
Herein, we report a comprehensive study on the reactivity
of this new ligand, in particular concerning the selectivity of
opening the anhydride with nucleophiles. Opening the anhy-
dride group with propylamine was performed, which high-
lighted the formation of a single isomer. With the aim of ex-
tending the method and demonstrating its potential use, var-
ious nucleophiles were reacted with DOTAGA-anhydride.
Finally, new SPECT or PET/optical imaging bimodal precur-
sors were prepared by reacting DOTAGA–anhydride with
a bodipy unit.
Owing to its poor solubility in deuterated organic sol-
1
vents, the H NMR spectrum of compound 1 was recorded
in deuterium oxide (Figure 1a). The signal at d=4.45 ppm
was unambiguously assigned to the CH group of the anhy-
dride group by 2D COSY analysis. Because of the fairly
rapid hydrolysis of the anhydride moiety in D2O, a mixture
of DOTAGA and DOTAGA–anhydride was systematically
obtained (65:45 ratio after 2 min at 293 K), thus confirming
the high reactivity of the anhydride compound (see the Sup-
porting Information, Figures S1 and S2). Hydrolysis of the
anhydride could be followed by the disappearance of the
CH signal at d=4.45 ppm (Figure 1b) and by the increase
of a new peak at d=3.75 ppm, which corresponded to the
CH group in the hydrolyzed fragment.
Study of the regioselectivity of the anhydride-opening reac-
tion: Even though we had previously demonstrated the high
reactivity of the anhydride group towards nucleophilic
Figure 1. 1H NMR spectra of compound 1 (293 K, 500 MHz, D2O): a) after 2 min; b) change in the CH signal as a function of time.
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7835

F. Denat et al.
agents, the selectivity of the reaction had not been elucidat-
ed. Indeed, because the anhydride group in compound
1 was asymmetrical, ring opening by a nucleophile could
lead to two different isomers. To study this reaction in
detail, we treated DOTAGA–anhydride with propylamine.
As expected, HPLC analysis confirmed the formation of the
amide in 98% yield (see the Supporting Information, Fig-
ure S4), but this result did not support the conclusion about
the presence of isomers 2a, 2b, or both (Scheme 3).
peak was observed whatever the pD value. This result con-
firmed that only one product had formed during the reac-
tion. Indeed, even if the isochrony of the CH protons of two
isomers could accidentally occur for one pD value, it could
not be the case at four different pD values.
At this stage, the regioselectivity of the reaction had been
demonstrated but it remained unclear which isomer (2a or
2b) had formed. Thus, RM1 semi-empirical molecular-orbi-
tal modeling of DOTAGA–anhydride 1 was performed
(Figure 3). The model showed a higher accessibility of
carbon atom C1 than C2, which suggested that the sole
product of the reaction was isomer 2a, which arose from ex-
clusive nucleophilic attack on the carbonyl C1 atom. DFT
energies of final products 2a and 2b were also calculated
(see the Supporting Information). No significant difference
was noted and the steric hindrance around the C2 atom was
probably the main reason for the selective formation of
isomer 2a. To gain further insight into the regioselectivity of
the reaction, we unequivocally prepared isomer 2a from
DOTAGAACHTUNTGRNE(UNG tBu)4 macrocycle according to a modified litera-
ture procedure.[5] A coupling reaction with propylamine in
the presence of HBTU and HOBt, followed by treatment
with concentrated hydrochloric acid, afforded the ammoni-
um chloride salt of the desired deprotected product in 63%
yield (Scheme 4). The 1H NMR spectra of this compound
and isomer 2a were superimposable, which confirmed the
assignment of the isomer that had formed during the reac-
tion (see the Supporting Information, Figure S11).
Scheme 3. Reaction of DOTAGA–anhydride (1) with 2 equiv of propyla-
mine: a) propylamine (2 equiv), DMF, 508C, 2 h, 98% yield.
NMR experiments were carried out to determine the
number of isomers that were formed during the reaction.
1
1
1
ꢀ
H NMR and H H COSY spectra were recorded in D2O
at different pD values (Figure 2; also see the Supporting In-
formation, Figure S5).
Whatever the pD value, we were able to recognize two
sets of signals that were attributed to the hydrogen atoms of
two propyl fragments: the first set was assigned to the pro-
pylamide formed and the second set corresponded to the
propyl ammonium counterion. All of the NMR spectra
showed a complex signal in the region above d=2.8 ppm,
which corresponded to the diastereotopic hydrogen atoms
of the methylene groups on the macrocyclic backbone, as
well as the CH2 protons on the three carboxymethyl groups
and the CH proton (CHCH2CH2CO). The chemical shifts
were strongly dependent on the pD value, owing to the pres-
ence of many protonation sites on the macrocycle (tertiary
Scheme 4. Synthesis of isomer 2a from DOTAGAACTHNUTRGENUG(N tBu)4: a) propylamine
(1.1 equiv), HOBt, HBTU, EDCI, CH2Cl2, room temperature, 12 h, 64%
yield; b) HCl, room temperature, 30 min, 98% yield. HOBt=1-hydroxy-
benzotriazole, HBTU=O-(benzotriazol-1-yl)-tetramethyluronium hexa-
fluorophosphate, EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
monochloride.
To confirm these results, we carried out NOESY and
HMBC experiments on compound 2a, which was obtained
by opening compound 1 with propylamine. These NMR
spectra were recorded after elution on an ion-exchange
resin to remove the propylammonium cation (see the Sup-
porting Information, Figures S7 and S8). [D6]DMSO could
be used as a solvent for NMR analysis in this case. The
13C NMR high-frequency region of the HMBC spectrum is
shown in Figure 4a. The signal that corresponded to carbon-
yl atom C1 was identified from the cross-peak with the NH
1
1
ꢀ
amine functions and carboxylic acid groups). From H
H
COSY experiment that were performed at pD=8.5, the sig-
nals at d=1.75–1.85 ppm and d=2.15–2.20 ppm were attrib-
uted to protons Hb/Hb’ and Ha/Ha’, respectively. The cross-
peak on the COSY spectrum, which corresponded to the
correlation between the multiplet that was assigned to the
Hb/Hb’ protons and the CH proton, was particularly depen-
dent on the pD value (Figure 2). However, only one cross-
proton. Atom C1 also showed correlations with Ha/Ha’ and
2
Hc/Hc’, thereby corresponding to coupling constants J
N
3
a’,C1) and JACTHNUGRTNEUNG(Hc/c’,C1). Importantly, no cross-peak was ob-
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Synthesis of DOTA-Like Chelating Agents
FULL PAPER
shows the region of cross-peaks
that corresponded to correla-
tions that involved proton NH.
Through-space
interactions
were identified between the
NH proton and Ha/Ha’, Hc/
Hc’, and Hd/Hd’.
Taken together, these studies
unambiguously demonstrated
that compound 2a was obtained
as the sole product in the reac-
tion of compound 1 with pro-
pylamine.
Synthesis of the DOTAGA
building blocks: We investigat-
ed the use of DOTAGA–anhy-
dride for the preparation of
new building blocks. The regio-
selectivity of the anhydride-
opening reaction allowed the
selective introduction of various
functional groups that were
suitable for bioconjugation.
First, we focused on the
opening reaction of the anhy-
dride with commercially avail-
able amines. Owing to the poor
solubility of DOTAGA–anhy-
dride in organic solvent, the re-
action was performed at high
temperature. For instance, com-
pound 1 was reacted with 4-ni-
trobenzylamine hydrochloride
in DMF at 658C in the presence
of three equivalents of triethyl-
amine (Scheme 5a). The crude
product, which was about 85%
1
1
1
ꢀ
Figure 2. a) H NMR spectrum of compound 2 (340 K, 500 MHz, D2O, pD =8.5); b) H H g-COSY 2D corre- pure by NMR spectroscopy (in-
lations of compound 2 at pD=1, 4.2, 8.5, and 12 (340 K, 500 MHz, D2O). The cross-peaks that corresponded
to the correlation between the CH and CH2 peaks are indicated by black dashed lines. The solvent peak was
omitted for clarity (for a color version, see the Supporting Information, Figure S6).
tegration of the aromatic pro-
tons), was purified by column
chromatography on a C18 re-
verse-phase column to remove
the starting material, thereby
affording compound 4 in 72% yield (96% purity, as mea-
sured by HPLC analysis).
To design DOTA precursors for attachment to a cysteine
group, which is an attractive target for labeling peptides or
antibodies,[16] and to gold nanoparticles,[17] we introduced
a thiol group onto the DOTAGA backbone (Scheme 5b).
We used the same conditions as those described for the syn-
thesis of compound 4 to prepare compound 5 in 45% yield.
This low conversion was presumably due to the formation of
dimers (through a disulfide bridge).
We also considered the opening of the anhydride with al-
cohols, such as EtOH, to afford their corresponding esters
(Scheme 5c). Owing to the lower reactivity of alcohols than
Figure 3. RM1 semi-empirical molecular-orbital model of DOTAGA–an-
hydride 1 (CPK).
served between the carbonyl atom of the amide moiety and
the CH proton, unlike what might have been expected for
isomer 2b. To further confirm this result, a NOESY spec-
trum was recorded under the same conditions. Figure 4b
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F. Denat et al.
Figure 4. a) 2D-HMBC spectrum of compound 2 (300 K, 500 MHz, [D6]DMSO). Only the region of cross-peaks that corresponded to the correlation be-
tween the C1 and H atoms (labeled) is represented for clarity. Correlations are indicated by dashed lines. b) 2D-NOESY spectrum (300 K, 500 MHz,
[D6]DMSO).
This reaction was also ex-
tended to the coupling of an
amino acid onto the DOTAGA
unit (Scheme 5d). For this pur-
pose, a protected DOTAGA–
nitrophenylalanine
derivative
was prepared by reacting
1 equiv of the appropriate nu-
cleophile with the DOTAGA–
anhydride. After purification by
column chromatography on
a C18 reverse-phase column, the
desired compound (7) was ob-
tained in 68% yield and charac-
terized by LCMS (see the Sup-
porting
Information,
Fig-
ure S19). The presence of two
chiral centers led to the forma-
tion of a mixture of diastereo-
isomers. This valuable building
block could then be inserted at
any position on a peptide or
PNA sequence.
Scheme 5. Reaction of compound 1 with various nucleophiles: a) 4-nitrobenzylamine hydrochloride (1 equiv),
NEt3 (3 equiv), DMF, 708C, 2 h, 72% yield; b) 2-aminoethanethiol hydrochloride (1 equiv), NEt3 (3 equiv),
DMF, 708C, 4 h, 45% yield; c) EtOH, NEt3 (3 equiv), DMF, 458C, 12 h, 35% yield; d) nitrophenylalanine
monoester hydrochloride (1 equiv), NEt3 (3 equiv), DMF, 708C, 12 h, 68% yield.
SPECT- and PET/optical-imag-
ing bimodal precursors: Finally,
this convenient method was
primary amines, a large excess of EtOH was used. The reac-
tion was performed in refluxing DMF, and purification by
column chromatography on a C18 reverse-phase column
gave the desired compound (6) in 35% yield.
used for the preparation of a new dual-mode probe that was
capable of simultaneous detection by optical imaging and
PET or SPECT scintigraphy.[18] There are no reports of bi-
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Synthesis of DOTA-Like Chelating Agents
FULL PAPER
Scheme 6. Synthesis of DOTAGA–bodipy, starting from bodipy system 8:[19] a) DOTAGA–anhydride (1), NEt3 (3 equiv), DMF, 708C, 12 h, 35% yield;
b) H2, Pd/C, H2O/EtOH (96:4), room temperature, 12 h, 61% yield; b) thiophosgene (3 equiv), CH2Cl2/H2O (1:1), room temperature, 1 h, 86% yield.
modal agents that contain DOTAGA units in the literature,
presumably owing to the difficulty in synthesizing this type
of compounds. Recently, we reported the synthesis of
second-generation water-soluble DOTA–bodipy deriva-
tives,[19] by coupling DOTA–NHS with a bodipy moiety, to
afford a fluorescent probe that exhibited strong fluorescent
properties and high stability.[20] The advantage of our ap-
proach was in the possibility of introducing any kind of mac-
rocyclic chelating agents in the last step. Similar systems
that contain DOTAGA units could be easily prepared by
opening the anhydride group of compound 1 (Scheme 6).
The free primary amine group in bodipy 8[19] was reacted
with anhydride 1 at 708C in the presence of triethylamine to
afford the desired bimodal system (9) in one step. The
“ready to be grafted” final product (11) was obtained by
Figure 5. Absorption spectra of compounds
9 and 10 in PBS 0.1m,
a classical procedure, that is, the reduction of the nitro
group into an aniline moiety, followed by transformation
into an isothiocyanate group.[21] As expected, the presence
of the four carboxylic pendant arms on the DOTAGA
moiety led to water-soluble systems, without the need to
add any hydrophilic groups onto the bodipy core.[22] Prelimi-
nary photophysical measurements were performed for
water-soluble bimodal systems 9 and 10. Absorption data of
compound 11 were not recorded owing to the low stability
of the activated NCS moiety in aqueous solution. Absorp-
tion spectra were recorded in MeOH, water, and under si-
mulated physiological conditions (phosphate buffer solution
(PBS), pH 7.4; see Figure 5 and the Supporting Information,
Figure S23). All of the systems considered exhibited the typ-
ical absorption features of a bodipy system,[23] that is,
a strong absorption transition in the range 520–530 nm,
which was attributed to the S0–S1 transition of the bodipy
unit, and a smaller transition in the range 340–420 nm,
which corresponded to the S0–S2 transition. The molar ab-
sorption coefficients in PBS were in the range 15000–
30000mꢀ1 cmꢀ1, which were lower than those in organic sol-
vent, but were in good agreement with those reported previ-
ously for water-soluble bodipy units.[19,22k]
pH 7.4, at 258C.
Conclusion
Bifunctional chelator DOTAGA–anhydride was readily pre-
pared thanks to a key cyclic-anhydride formation. The high
reactivity of the anhydride group and the regioselectivity of
the anhydride-opening reaction, as demonstrated by various
NMR experiments, made this compound a highly valuable
precursor for labeling biomolecules. Indeed, the reaction
with amines or other nucleophilic agents provided easy
access to bifunctional chelating agents by the introduction
of various functional groups that were suitable for bioconju-
gation, such as isothiocyanate or thiol groups. This approach
did not require the protection of the three acetate groups.
Moreover, when compared to other activated acid deriva-
tives, such as NHS or phenolic esters, the formation of the
amide bond between the anhydride and the amine groups
generated no side-product that needed to be separated from
the conjugate. The extension of the method for the prepara-
tion of chelators that contain other functional groups, such
as primary amines, through the reactions of DOTAGA–an-
hydride with ethylendiamine or maleimide derivatives that
are suitable for attachment to cysteine, is underway. The in-
corporation of an additional fluorescent moiety gave rise to
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F. Denat et al.
1H NMR (500 MHz, D2O, 300 K) d=1.94 (m, 1H), 2.05 (m, 1H), 2.48
a promising new class of SPECT/optical bimodal imaging
agents. The conjugation of such bimodal agent onto peptides
or antibodies through thiourea bond formation is also cur-
rently under investigation.
(m, 1H), 2.61 (m, 1H), 2.82–4.05 (m, 20H), 4.51 (d, 2J
ACHTUNGTRENNUNG
AB system, 1H; CH2ArNO2), 4.55 (d, 2J
1H; CH2ArNO2), 7.55 (d, 3J
ACHTUNGTRENNUNG
N
ACHTUNGTRENNUNG
8.6 Hz, 2H); IR (KBr): n˜ =1389 (N=O), 1517 (N=O), 1626 (C=O), 3097
(C H), 3394 cmꢀ1 (O H); MS (ESI): m/z: 611.26 [M+H]+, 633.24
[M+Na]+; HRMS (ESI): m/z: calcd for C26H38N6O11+H: 611.26713;
found: 611.26886; elemental analysis calcd (%) for C26H39N6O11·H2O:
C 49.68, H 6.41, N 13.37; found: C 49.50, H 6.14, N 13.59;
ꢀ
ꢀ
Experimental Section
Compound 5: M.p. 144(ꢁ1)8C; 1H NMR (600 MHz, D2O, 300 K): d=
3
1.81–2.07 (m, 2H), 2.30–2.56 (m, 2H), 2.69 (t, J
N
All of the chemicals that were used to prepare the title compounds, in-
cluding propylamine, 4-hydroxybenzotriazole, and N,N,N’,N’-tetramethy-
luronium-hexafluorophosphate, were purchased from Acros and Aldrich
and were used without further purification. 5-(tert-Butoxy)-5-oxo-4-
(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-
2.83–2.96 (m, 2H), 3.06–3.70 (m, 16H), 3.70–3.99 ppm (m, 9H); HRMS
(ESI): m/z: calcd for C21H37N5O9SNa: 588.22042; found: 588.22076;
HPLC (A: CH3CN, B: H2O+0.4% HCOOH; B: 98!0%): tR =5.05 min.
Compound 7: M.p. 155(ꢁ1)8C; 1H NMR (600 MHz, D2O, 300 K): d=
1.73–1.88 (m, 2H), 2.36–2.54 (m, 2H), 2.93–3.59 (m, 18H), 3.61–3.71 (m,
2H), 3.77 (s, 3H; OCH3), 3.77–3.87 (m, 5H), 4.79–4.81 (m, 1H;
yl) pentanoic acid (DOTAGAACTHNUTRGNE(NUG tBu)4), DOTAGA, and DOTA-NHS were
obtained from CheMatechꢂ and used without further purification.
Bodipy precursor 8 was prepared according to a literature procedure.[19]
Organic solvents were removed under reduced pressure on a rotary evap-
orator. Water was removed by lyophilization.
CHCOOMe), 7.51 (d, 3J(H,H)=8.8 Hz, 2H), 8.23 ppm (d, 3J
ACTHNUTRGENNUG ACHTUNGTRENNUNG(H,H)=
8.8 Hz, 2H); LCMS: (A: H2O+0.4% HCOOH, B: CH3CN+0.4%
HCOOH; B: 2!100%): tR =5.8 min, 683.8 [M+H]+.
Synthesis of ligand 1: Acetic anhydride (20 mL) was added to a suspen-
sion of DOTAGA·2HCl (10 g, 18.5 mmol) in pyridine (8.5 mL, 0.1 mol,
6 equiv) under a nitrogen atmosphere. The mixture was heated at 658C
for 18 h, then stirred at room temperature for 30 min. The suspension
was filtered, washed with acetic anhydride (30 mL), MeCN (50 mL), and
Et2O (80 mL). The precipitate was dried under vacuum to afford com-
pound 1 as a gray solid (9.5 g, 96%). Compound 1 was used without fur-
ther purification. M.p. 108(ꢁ2)8C; HRMS (ESI): m/z: calcd for
C19H30N4O9+Na+: 481.19050; found: 481.18922.
Compound 6: EtOH (4 mL) was added to a suspension of compound
1 (200 mg, 0.43 mmol) in DMF (20 mL) and the mixture was warmed at
458C for 12 h. After evaporation of the solvent, the crude solid was puri-
fied by column chromatography on a C18 reverse-phase column (H2O; A,
tR =4 min). The fraction was collected and the solution was lyophilized to
give compound 6 as a white solid (80 mg, 35%). 1H NMR (300 MHz,
3
D2O, 300 K): d=1.30 (t, J
N
1H), 2.02–2.15 (m, 1H), 2.51–2.76 (m, 2H), 2.94–3.28 (m, 6H), 3.31–4.00
(m, 14H), 4.01–4.10 (m, 1H, CH), 4.23 ppm (q, 3J
AHCTUNGTRENNUNG
OCH2CH3); MS (ESI): m/z: 525.15 [Mꢀ2H+Na]ꢀ, 541.17 [Mꢀ2H+K]ꢀ,
563.16 [Mꢀ3H+K+Na]ꢀ; elemental analysis calcd (%) for
C21H36N4O10·H2O·HCl: C 45.12, H 7.03, N 10.02; found: C 44.73, H 7.86,
N 10.08; HPLC (A: CH3CN, B: H2O+0.4% HCOOH; B: 98!0%): tR =
6.3 min.
Synthesis of compound 2a (route A): Propylamine (46.5 mL, 0.56 mmol,
2 equiv) were added to a suspension of compound 1 (150 mg, 0.28 mmol)
in DMF (2 mL). The mixture was stirred at 508C for 2 h. The solvent was
evaporated to dryness to give compound 2a (+CH3CH2CH2NH3+) as
a yellow solid (160 mg, 98%). 1H NMR (500 MHz, D2O, 340 K, pD=
4.2): d=0.72 (t, 3J
1.35 (qt, 3J(H,H)=7.4 Hz, 3J
7.4 Hz, 3J
(H,H)=7.1 Hz, 2H), 1.68–1.78 (m, 1H), 1.79–1.90 (m, 1H),
2.15–2.23 (m, 1H), 2.24–2.31 (m, 1H), 2.82 (t, JACTHGUNTRNE(NUG H,H)=7.1 Hz, 2H), 2.97
A
ACHTUNGTRENNUNG
Compound 9: DOTAGA–anhydride (1; 383 mg, 0.967 mmol, 1.1 equiv)
and NEt3 (527 mL, 4.39 mmol, 5 equiv) were added to a solution of
bodipy–ethylenediamine (8; 500 mg, 0.879 mmol, 1 equiv) in DMF
(50 mL), then the mixture was stirred 12 h at 708C. After the evaporation
of DMF, the crude product was purified by column chromatography on
silica gel (Cl2Cl2/EtOH/NH4OH, 2:7:1) to give compound 9 as a mixture
of two diastereoisomers as a red solid (360 mg, 40%). M.p.>2008C;
A
R
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
3
(t, 3J
ACHTUNGTRENNUNG(H,H)=7.1 Hz, 2H), 2.76–2.37 (m, 17H), 3.40–3.67 ppm (m, 6H);
MS (ESI): m/z: 518.28 [M+H]+, 540.26 [M+Na]+; HPLC (A: CH3CN, B:
H2O+0.4% HCOOH; B: 98!0%): tR =6.3 min.
1H NMR (300 MHz, MeOD, 300 K): d=0.99 (t, 3J
1.30 (s, 6H), 1.97–2.11 (m, 2H), 2.35 (q, 3J
(H,H)=7.5 Hz, 4H), 2.48 (s,
6H), 2.61–2.82 (m, 2H), 2.82–3.02 (m, 2H), 3.03–3.30 (m, 14H), 3.34–
3.87 (m, 16H), 5.03 (m, 1H), 7.41 (d, 3J(H,H)=8.1 Hz, 2H), 7.59 (d, 3J-
(H,H)=8.5 Hz, 2H), 7.97 (d, 2H, 3J(H,H)=8.1 Hz), 8.12 ppm (d, 3J-
ACHTUNGTREN(NUGN H,H)=7.5 Hz, 6H),
Compound 2a was deprotonated on an amberlite ion-exchange resin
(eluent: CH3COOH/water, 5%). 1H NMR (500 MHz, DMSO, 300 K):
AHCTUNGTRENNUNG
d=0.81 (t, 3J(H,H)=7.4 Hz, 3H), 1.39 (qt, 3J(H,H)=7.4 Hz, 3J
ACHTUNGTRENNUNG ACHTUNRTGENNUNG ACHTNUGERTN(NUGN H,H)=
AHCTUNGTRENNUNG
7.1 Hz, 2H), 1.69–1.80 (m, 1H), 1.82–1.94 (m, 1H), 2.15–2.30 (m, 2H),
2.71–3.19 (m, 17H), 3.31–3.59 (m, 8H), 7.95 (brs, 1H; NH), 9.16–
13.40 ppm (m, 4H; COOH); 1H NMR (500 MHz, D2O, 300 K, pD=3):
d=0.88 (t, 3J=7.4 Hz, 3H), 1.51 (qt, 3J=7.4 Hz, 3J=7.1 Hz, 2H), 1.87–
2.08 (m, 2H), 2.39–2.59 (m, 2H), 2.93–4.02 ppm (m, 25H); 13C{1H} NMR
(125 MHz, DMSO, 300 K): d=11.4 (CH3), 22.4, 32.4, 40.4, 46.8, 49.4,
50.8, 51.1, 54.7, 51.1 (CH2), 62.5 (CH), 170.4, 171.1, 171.6, 172.8 ppm (C=
O); elemental analysis calcd (%) for C22H39N5O9·3.1H2O: C 46.08,
H 7.95, N 11.74; found: C 46.62, H 8.26, N 12.21.
A
ACHTUNGTRENNUNG
E
[M+Naꢀ2H]ꢀ, 1159.48 [M+2Naꢀ3H]ꢀ; UV/Vis (MeOH): lmax (e)=523
(53100), 377 (sh, 6300), 324 nm (sh, 7600mꢀ1 cmꢀ1); UV/Vis (0.1m PBS):
lmax (e)=526 (17000), 406 (sh, 4300), 275 nm (sh, 1520mꢀ1 cmꢀ1).
Compound 10: Compound 9 (200 mg, 0.179 mmol, 1 equiv) was dissolved
in a solution of H2O/EtOH (96:4, 10 mL), activated palladium (30 mg,
0.028 mmol, 0.15 equiv) was added to the bodipy and the mixture was
stirred under a hydrogen atmosphere overnight. The solution was filtered
through Clarcelꢂ, and the solvents were evaporated. The crude product
was purified by column chromatography on silica gel (EtOH/CH2Cl2/
NH4OH, 7:2:1) to afford the two diastereoisomers as a red powder
(120 mg, 61%). M.p.>2008C; 1H NMR (300 MHz, MeOD, 300 K) d=
General procedure for the opening reaction of compound 1 with a pri-
mary amine: Ligands 4, 5, and 7 were prepared by mixing a solution of
DOTAGA–anhydride 1 in DMF at 708C with a solution of the amine salt
(1 equiv) in DMF at 708C in the presence of triethylamine (3 equiv). The
mixture was heated for a minimum of 2 h until the two starting com-
pounds had completely solubilized. After the evaporation of DMF, the
crude solid was purified by reverse phase column chromatography on
C18. After evaporation of MeCN, the aqueous solution was lyophilized to
afford a white solid in 35–72% yield.
1.02 (t, 3J
(m, 6H), 2.38 (q, 3J
21H), 3.36–3.70 (m, 7H), 4.78 (m, 1H), 6.71 (d, 3J
7.08 (d, 3J(H,H)=8.1 Hz, 2H), 7.46 (d, 3J
(H,H)=8.1 Hz, 2H), 7.99 ppm
(d, 3J
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
A
ACHTUNGTRENNUNG
Compound 4: M.p. 165(ꢁ2)8C; 1H NMR (300 MHz, DMSO, 300 K): d=
A
1123.49 [M+Kꢀ2H]ꢀ, 1145.47 [M+Na+Kꢀ3H]ꢀ; UV/Vis (MeOH): lmax
(e)=523 (43100), 377 (sh, 5500), 321 nm (sh, 4800mꢀ1 cmꢀ1); UV/Vis
(0.1m PBS): lmax (e)=526 (20500), 373 (sh, 4700), 322 (sh, 5200mꢀ1 cmꢀ).
1.78 (m, 1H), 1.91 (m, 1H), 2.27 (m, 1H), 2.51 (m, 1H), 2.56–3.16 (m,
15H), 3.24–3.62 (m, 8H), 4.32 (dd, 3J(H,H)=6.2 Hz, 2J
ACTHNUGTRENNUGN ACHTUNGTRENNNUG
AB system, 1H; CH2ArNO2), 4.41 (dd, 3J
G
ACHTUNGTRENNUNG
16.0 Hz, AB system, 1H; CH2ArNO2), 7.54 (d, 3J
R
Compound 11: Compound 10 (50 mg, 0.046 mmol) was dissolved in a mix-
ture of water and CH2Cl2 (5 mL). Then, a solution of thiophosgene
8.17 (d, 3J
C
A
7840
www.chemeurj.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 7834 – 7841

Synthesis of DOTA-Like Chelating Agents
FULL PAPER
(10.6 mL, 0.14 mmol, 3 equiv) in CH2Cl2 (2 mL) was added and the mix-
ture was stirred for 1 h at room temperature. The solvents were evaporat-
ed and the product was dried under vacuum to give compound 11 as
a pink powder (45 mg, 86%). MS (ESI): m/z: 1129.5 [M+H]+, 1151.5
[M+Na]+, 1173.5 [M+2NaꢀH]+.
Perriat, O. Tillement, Angew. Chem. 2011, 123, 12507; Angew.
Chem. Int. Ed. 2011, 50, 12299.
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Tizon, F. Boschetti, O. Duchamp, F. Brunotte, F. Denat, Bioconju-
gate Chem. DOI: 10.1021/bc200680X.
[16] Z. Miao, M. R. McCoy, D. D. Singh, B. Barrios, O. L. Hsu, S. M.
Cheal, C. F. Meares, Bioconjugate Chem. 2008, 19, 15; b) S. Lacerda,
M. P. Campello, I. C. Santos, R. Delgado, Polyhedron 2007, 26, 3763.
L. Li, S. W. Tsai, S. A. L. Anderson, D. A. Keire, A. A. Raubitschek,
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Acknowledgements
[17] a) P. J. Debouttiꢅre, S. Roux, F. Vocanson, C. Billotey, O. Beuf, A.
Favre-Reguillon, Y. Lin, S. Pellet-Rostaing, R. Lamartine, P. Perriat,
O. Tillement, Adv. Funct. Mater. 2006, 16, 2330; b) C. Alric, J. Taleb,
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Support was provided by the CNRS, the University of Burgundy, and the
Conseil Rꢀgional de Bourgogne through the 3MIM Project. Marie Josꢀ
Penouilh is acknowledged for technical support (NMR spectroscopy ex-
periments).
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Received: January 12, 2012
Published online: May 21, 2012
Chem. Eur. J. 2012, 18, 7834 – 7841
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
7841