Formation and characterization of gallium(III) complexes with monoamide derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid: A study of the dependency of structure on reaction pH

Article abstract of DOI:10.1002/ejic.201000748

Two monoamide derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) conjugated with methylamine (4) or benzylamine (5) were synthesized by treating di-tert-butyl 1,4,7-triazacyclononane-1,4-diacetate (1) with 2-chloro-N-benzyl- or -N-methylacetamide, followed by an acid cleavage reaction. Complexes of 4 and 5 chelated to Ga³⁺ to give Ga-4 and Ga-5, respectively, in reaction solutions at different pH values (3 and 5). Complexes Ga-4 and Ga-5 were characterized by single-crystal X-ray diffraction and multinuclear NMR spectroscopy. In the solid state, these complexes were isostructural, and the coordination spheres of the metal ions exhibited distorted octahedral geometries. In the case of the Ga-4 complex, which was formed at both pH values, the metal ion is coordinated to the amide nitrogen atom of the modified pendent arm of 4. However, in the case of Ga-5, the metal ion is coordinated to a nitrogen or an oxygen atom of the amide linkage when the pH of the reaction soultion was 5 or 3, respectively. No significant difference was found between the ¹H NMR spectra of the complexes formed at pH = 3 and 5. However, ⁷¹Ga NMR spectra showed a broad resonance signal and a narrow singlet for the complex synthesized at the lower pH, but only a single narrow singlet for the complex prepared at neutral pH. Variable-temperature ¹H NMR spectra showed that complexes Ga-4 and Ga-5 are rigid in solution. The stability of these complexes in the physiological pH range and at high temperature suggests that NOTA can be used as a bifunctional chelating agent to label biomolecules with radioactive gallium by direct conjugation of the complex to target molecules. This finding could open up a wide range of applications for NOTA-type bifunctional chelating agents by eliminating the multi-step synthesis routes required to introduce extra linker groups to the labelling agent. Copyright

Full text of DOI:10.1002/ejic.201000748

FULL PAPER
DOI: 10.1002/ejic.201000748
Formation and Characterization of Gallium(III) Complexes with Monoamide
Derivatives of 1,4,7-Triazacyclononane-1,4,7-triacetic Acid: A Study of the
Dependency of Structure on Reaction pH
Dinesh Shetty,[a] Soo Young Choi,[b] Jae Min Jeong,*[a,c] Lathika Hoigebazar,[a]
Yun-Sang Lee,[a] Dong Soo Lee,[a] June-Key Chung,[a,c] Myung Chul Lee,[a] and
Young Keun Chung[b]
Keywords: Gallium / Chelates / X-ray diffraction / NMR spectroscopy / Amides
Two monoamide derivatives of 1,4,7-triazacyclononane-
1,4,7-triacetic acid (NOTA) conjugated with methylamine (4)
or benzylamine (5) were synthesized by treating di-tert-butyl
1,4,7-triazacyclononane-1,4-diacetate (1) with 2-chloro-N-
benzyl- or -N-methylacetamide, followed by an acid cleav-
age reaction. Complexes of 4 and 5 chelated to Ga3+ to give
Ga-4 and Ga-5, respectively, in reaction solutions at different
pH values (3 and 5). Complexes Ga-4 and Ga-5 were charac-
terized by single-crystal X-ray diffraction and multinuclear
NMR spectroscopy. In the solid state, these complexes were
isostructural, and the coordination spheres of the metal ions
exhibited distorted octahedral geometries. In the case of the
Ga-4 complex, which was formed at both pH values, the
metal ion is coordinated to the amide nitrogen atom of the
modified pendent arm of 4. However, in the case of Ga-5, the
metal ion is coordinated to a nitrogen or an oxygen atom of
the amide linkage when the pH of the reaction soultion was
5 or 3, respectively. No significant difference was found be-
tween the H NMR spectra of the complexes formed at pH =
1
3 and 5. However, 71Ga NMR spectra showed a broad reso-
nance signal and a narrow singlet for the complex synthe-
sized at the lower pH, but only a single narrow singlet for
the complex prepared at neutral pH. Variable-temperature
1H NMR spectra showed that complexes Ga-4 and Ga-5 are
rigid in solution. The stability of these complexes in the phys-
iological pH range and at high temperature suggests that
NOTA can be used as a bifunctional chelating agent to label
biomolecules with radioactive gallium by direct conjugation
of the complex to target molecules. This finding could open
up a wide range of applications for NOTA-type bifunctional
chelating agents by eliminating the multi-step synthesis
routes required to introduce extra linker groups to the label-
ling agent.
Introduction
applications.[3] DOTA contains four amine groups and four
carboxylic acid groups that can act as coordination sites,
but only six of these (four amine and two carboxylic acid
groups) are involved in complex formation to produce a
Heterocyclic compounds such as NOTA and 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)
have been used as bifunctional chelating agents to label pep-
tides and proteins with radioactive metals.[1] In the present
study, we concentrated on NOTA derivatives because they
produce extremely stable complexes with 68Ga,[1b,2] which
was recently reported to have a huge potential in medical
hexacoordinated chelate complex with GaIII [4]
which allows
,
for the conjugation of a biomolecule to DOTA by forma-
tion of an amide bond between the biomolecule and one of
the free DOTA carboxylic acid groups (Figure 1).
Ligand NOTA also forms a hexacoordinated chelate
with GaIII [5]
However, metal–NOTA chelates contain no
.
free carboxylic acid groups for conjugation with a biomole-
cule. Thus, all reported chelating agents based on NOTA
include a branched linker such as isothiocyanate or an extra
acetic acid group. For example, isothiocyanatobenzyl-1,4,7-
triazacyclononane-1,4,7-triacetic acid (SCN-NOTA) has an
isothiocyanate residue, and 1,4,7-triazacyclononane-1,4-
diacetic-7-succinic acid (NODASA) has a branched acetic
acid group (Figure 1).[1c,6] However, it should be noted that
these NOTA derivatives require cumbersome, low-yielding,
multi-step syntheses, and thus they are intrinsically costly
to produce.
[a] Department of Nuclear Medicine, Radiation-Applied Life
Science, Institute of Radiation Medicine, Seoul National
University College of Medicine,
Seoul 110-744, South Korea
Fax: +82-2-745-7690
E-mail: jmjng@snu.ac.kr
[b] Intelligent Textile System Research Center and Department of
Chemistry, Seoul National University,
Silimdong Gwanak-gu, Seoul 151-747, South Korea
[c] Cancer Research Institute, Seoul National University, College
of Medicine,
Seoul 110-744, South Korea
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201000748.
5432
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GaIII Complexes with 1,4,7-Triazacyclononane-1,4,7-triacetic Acid Derivatives
was studied by crystallography and multinuclear NMR
spectroscopy conducted with sample solutions at different
pH values.
Results and Discussion
Synthesis and Characterization
We synthesized monoconjugated NOTA derivatives by
treating 1 equiv. of 1 with 1.1 equiv. of 2-chloro-N-methyl-
or -N-benzylacetamide in the presence of K2CO3
(Scheme 1). The GaIII complexes were obtained from aque-
ous solutions of the ligands 4 or 5 and Ga(NO3)3 by heating
the reaction solutions, which were at different pH values
(solutions were adjusted to pH = 3 or 5 by using 0.5 m
Na2CO3), in a boiling water bath for 15 min. The complexes
were purified by preparative HPLC and were then dissolved
in an ethanol/water mixture. Crystals were obtained by slow
evaporation of the solvents.
X-ray crystallography data showed no pH-dependant
structural features for Ga-4 (Figure 2), whereas the struc-
tures of Ga-5 differed when prepared at pH = 3 and 5 (Fig-
ure 3). Crystal data collection and refinement details for all
compounds are listed in Tables 1 and 2. A gallium ion fits
perfectly inside the nitrogen and oxygen coordination cavity
of NOTA. When NOTA derivatives with methylamine or
benzylamine bound to one of the pendent acetic acid
groups were introduced into the structure, one metal coor-
Figure 1. NOTA and DOTA derivatives as bifunctional chelating
agents.
The potential benefit to medicine of positron emission
tomography (PET) and 68Ga-based radiopharmaceuticals is
driving the developments of chelate complexes that can un-
dergo a wide range of chemical modifications.[7] Recent
studies have shown that some 68Ga-labelled peptides exhibit
distinctly better imaging properties than their 111In-labelled
analogues[4a,8] and 18F-based radiotracers.[9] One important
characteristic of 68Ga is its cyclotron-independent avail-
ability by the 68Ge/68Ga generator system. Isotope 68Ga is
an excellent positron emitter, with 89% positron branching
accompanied by low photon emission (1077 keV, 3.22%).[10]
Unlike other PET radioisotopes such as 18F or 11C, 68Ga
cannot bind covalently to target vectors. Instead it must be
complexed with a bifunctional chelating agent (BCA) prior
to being conjugated to a vector, which has the advantage
that labeling can be done straightforwardly, and imme-
diately before diagnostic examinations, thus the loss of
radioactivity is minimized.
The purpose of the present study was to determine
whether NOTA derivatives, in which one of the carboxy
residues is conjugated with an amine, can make stable com-
plexes with gallium. To investigate this possibility, we syn-
thesized gallium complexes of NOTA derivatives that have
methylamine or benzylamine conjugated with one of their
Figure 2. Molecular structure of Ga-4 chelated at pH = 3 and 5
(complex structures were identical). Hydrogen atoms and counter-
three carboxylic acid groups. The coordination chemistry ions have been omitted for clarity.
Scheme 1. Synthesis of ligands 4 and 5.
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J. M. Jeong et al.
FULL PAPER
Figure 3. Molecular structures of Ga-5 chelated at pH = 3 (a) and pH = 5 (b). The gallium atom is coordinated to the oxygen atom (O5)
of the amide group when the complex is formed at pH = 3 and to the nitrogen atom (N4) of the amide group when formed at pH = 5.
Hydrogen atoms and counterions have been omitted for clarity.
dinating bond should be established between the metal ion Table 1. Selected bond lengths [Å] and bond angles [°] for com-
plexes Ga-4 and Ga-5.[a]
and either an amide nitrogen (N4) or an amide oxygen (O5)
atom.
Ga-4·H2O (pH = 5) Ga-5·CF3COO·H2O (pH = 3)
When chelation was performed at pH = 3, 4 showed gal-
Length [Å]
lium coordination with N4 (Figure 2), whereas 5 showed
coordination with O5 (Figure 3a). On the other hand, when
prepared at pH = 5, both complexes showed metal binding
to N4 along with five other donor atoms to form a hexa-
dentate complex (Figure 3b). The coordination of the
amide oxygen atom at low pH might be due to protonation
of O5, leading to the formation of an imide bond due to
isomerism that also causes the double bond character of
the carbon–nitrogen bond. It is noteworthy that the oxygen
atom of the amide group always coordinates with gallium
in the case of NOTA and [(nitroimidazolyl)ethyl]amine con-
jugates.[11] The coordination geometries for the metal cen-
ters in both Ga-4 and Ga-5 formed at both chelating pH
values were distorted octahedra with three amine-N atoms
occupying one face and the two carboxylate O-atoms and
either N4 or O5 occupying the opposite face. Distortion
of the coordination spheres from a regular octahedron was
evident from the compression of the N–Ga–N angles
(average angles are 83.22° for Ga-4 and 84.35° for Ga-5)
and expansion of the O1–Ga–O3, N4–Ga–O and O5–Ga–
O angles [O1–Ga–O3 94.22(8)°, N4–Ga–O 96.99° (av.) for
Ga-4; O1–Ga–O3 95.39(8)° and O5–Ga–O 94.65° (av.) for
Ga-5] from the ideal value of 90°. As a result, in Ga-4 the
trans angles of N1–Ga–O3, N2–Ga–N2 and N3–Ga–O1 are
164.23(8)°, 163.76(8)°, and 165.30(9)°, respectively. In Ga-5
formed at pH = 3 the three trans N–Ga–O angles are
165.01(9)°, 168.15(10)°, and 166.71(8)° for N1–Ga–O5,
N2–Ga–O1, and N3–Ga–O3, respectively.
Ga–N1
Ga–N2
Ga–N3
Ga–O1
Ga–O3
Ga–N4
Ga–O5
N4–C12
O5–C12
N4–C13
2.103(2)
2.123(2)
2.122(2)
1.9601(16)
1.9313(19)
1.948(2)
–
1.325(3)
1.243(3)
1.464(3)
2.084(2)
2.081(2)
2.105(2)
1.9135(19)
1.9218(17)
–
1.963(2)
1.294(4)
1.274(3)
1.465(4)
Angle [°]
N1–Ga–N2 83.49(9)
N2–Ga–N3 82.69(8)
N3–Ga–N1 83.49(9)
84.71(9)
84.40(8)
83.95(9)
83.92(9)
168.15(10)
97.72(8)
–
95.39(8)
93.95(9)
99.63(9)
83.21(8)
166.71(8)
–
O1–Ga–N1
O1–Ga–N2
O1–Ga–N3
O1–Ga–N4
O1–Ga–O3
O1–Ga–O5
O3–Ga–N1
O3–Ga–N2
O3–Ga–N3
O3–Ga–N4
N4–Ga–N1 101.20(9)
N4–Ga–N2 163.76(8)
N4–Ga–N3 82.42(8)
O5–Ga–N1
O5–Ga–N2
O5–Ga–N3
O5–Ga–O3
81.84(8)
96.58(7)
165.30(9)
99.47(8)
94.22(8)
–
164.23(8)
81.79(8)
100.19(8)
94.50(9)
–
–
–
–
–
–
–
165.01(9)
97.89(9)
81.63(9)
95.34(8)
[a] Data for Ga-5·2H2O (pH = 5) are not included because of the
The average Ga–N bond length is 2.116 Å in Ga-4 pre- disorder at the benzyl and solvent positions.
pared at pH = 5, which is slightly longer than that in Ga(N-
OTA) (av. Ga–N 2.090 Å),[5c,12] and in the case of Ga-5 as for Ga(NOTA), indicating the structural similarity be-
prepared at pH = 3, the average length was exactly the same tween Ga-5 and Ga(NOTA). The average Ga–O bond
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GaIII Complexes with 1,4,7-Triazacyclononane-1,4,7-triacetic Acid Derivatives
Table 2. Summary of the crystallographic data obtained for complexes Ga-4 and Ga-5.
Ga-4·H2O
(pH = 3)
Ga-4·H2O
(pH = 5)
Ga-5·CF3COOH·H2O Ga-5·H2O
(pH = 3)
(pH = 5)
Emprical formula
Formula mass
Space group
Crystal size [mm]
a [Å]
b [Å]
c [Å]
C13H23GaN4O6
401.07
P21/n
0.17ϫ 0.10ϫ0.06
7.5577(4)
15.0490(12)
14.0340(12)
90
C13H23GaN4O6
401.07
P21/n
0.27ϫ0.21ϫ0.18
7.5581(4)
15.0604(7)
14.0363(7)
90
C21H28F3GaN4O8
591.19
P21/n
0.27ϫ0.24ϫ0.19
8.9067(3)
23.1929(11)
11.8265(4)
90
C19H29GaN4O7
495.18
Pbca
0.34ϫ0.25ϫ0.17
10.3789(3)
14.2489(4)
32.3078(6)
90
α [°]
β [°]
94.992(5)
90
1590.1(2)
4
1.675
1.770
94.984(3)
90
1591.68(14)
4
1.674
1.769
93.237(2)
90
2439.13(16)
4
1.610
1.206
90
90
4777.9(2)
8
1.377
1.196
2064
γ [°]
V [Å3]
Z
ρcalcd. [g/cm3]
µ [mm–1]
F(000)
832
832
1216
T [K]
θ range [°]
Index range
293(2)
293(2)
293(2)
293(2)
3.03 Յ θ Յ 27.41
–9 Յ h Յ 9
–19 Յ k Յ 19
–18 Յ l Յ 17
9314
3.25 Յ θ Յ 27.47
–9 Յ h Յ 9
–17 Յ k Յ 19
–18 Յ l Յ 18
6548
2.45 Յ θ Յ 27.47
–11 Յ h Յ 11
–27 Յ k Յ 30
–15 Յ l Յ 15
9861
1.26 Յ θ Յ 27.58
–13 Յ h Յ 13
–18 Յ k Յ 18
–41 Յ l Յ 41
10163
Total reflections
Unique reflections
Rint
Transmission factors
Data/restraints/parameters
Goodness of fit on F2
R indices [IϾ2σ(I)]
3548
0.4353
0.901–0.753
3548/0/226
0.986
R = 0.0568, Rw
0.0903
3628
0.0392
0.741–0.647
3628/0/226
1.019
R = 0.0382, Rw
0.0739
R = 0.0658, Rw
0.0817
0.308/–0.356
5559
0.0608
0.803–0.737
5559/0/419
0.971
R = 0.0469, Rw
0.0726
R = 0.1041, Rw
0.0848
0.327/–0.322
5486
0.0469
0823–0.687
5486/0/275
1.963
R = 0.1495, Rw
0.4682
R = 0.1843, Rw
0.4838
1.918/–2.540
=
=
=
=
=
=
=
=
R indices (all data)
R = 0.1235, Rw
0.1073
Largest difference peak/hole [e/Å3] 0.520/–0.362
lengths in Ga-4 formed at pH = 5 and in Ga-5 formed at of Ga-5, the methylene hydrogen atoms of the benzyl group
pH = 3 are 1.946 Å and 1.933 Å, respectively. These values showed 2 distinct peaks centered at δ = 3.6 ppm due to the
are comparable to that of Ga(NOTA) (av. Ga–O 1.930 Å). flexibility of the benzyl group when the complex is prepared
The C12–N4 (bond between the amide carbon and nitrogen at pH = 3, which changed to a single peak at δ = 3.63 ppm
atoms) bond length is 1.294(4) Å for Ga-5 synthesized at in the spectrum for Ga-5 that is formed at pH = 5 (Fig-
pH = 3, which was shorter than the length of the same ure S4). The 13C NMR spectra of Ga-5 showed a shift of
bond in Ga-4 prepared at pH = 5 [1.325(3) Å]. Also, the the benzylic methylene carbon signal from δ = 46.45 ppm
C12–O5 bond length in Ga-5 formed at pH = 3 is for Ga-5 prepared at pH = 5 to δ = 54.48 ppm for Ga-5
1.274(3) Å, which is slightly longer than the same bond in formed at pH = 3 due to differences in the double-bond
Ga-4 formed at pH = 5 [1.243(3) Å]. These findings provide character of the amide group (Figure S5). NOESY studies
evidence of –N=C–O–M bond formation when the gallium showed clearly a coupling interaction between the axial and
atom coordinates to the oxygen atom of the amide group.
equatorial hydrogen atoms of the ring methylene groups,
along with a coupling interaction between the axial hydro-
gen atoms of the ring methylene groups and the methylene
hydrogen atoms of pendent arms, which was also observed
NMR Characterization
1
for Ga–NOTA.[13] The H NMR spectrum of Ga-5 showed
The structures of Ga-4 and Ga-5 in aqueous solution
were elucidated by NMR spectroscopy (1H, 13C, NOESY,
HSQC, and 71Ga). Assignments of resonance signals were
based on NOESY and HSQC experiments conducted at
25 °C (Figures S6–S9). The proton resonance signals from
the macrocyclic backbones were tentatively assigned, due to
a doublet at δ = 3.49 ppm, which was assigned to two meth-
ylene protons on the modified pendent arm. The different
orientations of these protons suggests that the substituted
benzyl group is flexible.
71Ga NMR spectroscopic analysis of these complexes
superimposed axial and equatorial proton signals, in the 1H was performed in D2O over the pH range of 3–9 (Figure 4).
NMR spectra of Ga-4 and Ga-5 in D2O recorded at 25 °C. In the case of Ga-4, a small broad signal centered at δ =
1
No significant differences were observed in the H and 13C 175.2 ppm (ω1/2 ≈ 9000 Hz) and a relatively narrow singlet
NMR signals of isolated Ga-4 compounds synthesized at at δ = 171.1 ppm (ω1/2 = 283 Hz) were observed in the spec-
pH = 3 and at 5 (Figure S2, S3) and redissolved in D2O for tra recorded at low pH. These broad signals disappeared
analysis (NMR sample: pH ≈ 7). In the 1H NMR spectrum with increasing pH, and only a narrow singlet remained in
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J. M. Jeong et al.
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the spectrum collected at pH = 7. In the spectrum collected polar nuclei, especially when the nucleus is interacting with
at pH = 9, a singlet corresponding to [Ga(OH)4]– (δ = asymmetric electric field gradients. The widths of the 71Ga
223 ppm)[14] was observed. Similar findings were obtained NMR signals were determined by quadrapolar relaxation
for Ga-5. At low pH, a broad signal at δ = 173.8 ppm induced by the electric field gradients in the distorted Ga3+
(ω1/2 ≈ 4000 Hz) was observed, along with a narrow singlet coordination polyhedra, and these provide a sensitive mea-
at δ = 170.1 ppm (ω1/2 ≈ 279.1 Hz). This broad signal de- sure of the degree of distortion of the complexes in solution
creased in intensity as the pH increased, but did not disap- when corrected for changes in the rotational correlation
pear at pH = 7. At high pH values, this broad signal disap- time. Based on our findings, the widths of the peaks for
peared, and a signal corresponding to [Ga(OH)4]– was ob- Ga-4 and Ga-5 are between those for Ga(NOTA) (ω1/2
=
served in addition to a narrow signal that was assigned to 210 Hz) and Ga(NOTP) [NOTP = 1,4,7-triazacyclononane-
Ga-5. Furthermore, the δ values of these complexes were N,NЈ,NЈЈ-tris(methylenephosphonic) acid] (ω1/2 = 434 Hz)
found to be close to that of the Ga(NOTA) complex (δ = indicating the presence of stable Ga3+ complexes in aqueous
171 ppm).[13] The 71Ga NMR data indicated the presence solution, possibly with an octahedral or pseudo-octahedral
of two species in solution, possibly one of which includes geometry.
bound N3O3 (narrow signal), and the other must include a
Variable-temperature (VT) 1H NMR spectroscopy of
bound N4O2 species (broad signal) due to differences in the Ga-4 and Ga-5 in D2O (NMR sample solution: pH ≈ 7)
symmetry of the complexes. The 71Ga NMR study provides showed no significant change in the resonance signals in the
a clue to the geometry of the complex, which shows the spectra recorded within the temperature range of 5–85 °C
chemical shift and the symmetry of a complex, since the (Figure 5), indicating that the Ga-4 and Ga-5 complexes are
line width is extremely sensitive to molecular symmetry stable in solution, and that there is no dissociation of the
about the nucleus due to the rapid relaxation of the quadru- coordination bond between gallium and the amide nitrogen
or oxygen atoms. To investigate the stability of these com-
1
plexes, H NMR spectra were analyzed over the pH range
of 3–9 (Figure S10). There were no changes in the reso-
nance signals of these complexes up to pH = 7.6, whereas
Figure 4. 71Ga NMR spectra of Ga-4 (a) and Ga-5 (b) in D2O at
different pH values.
Figure 5. Variable-temperature 1H NMR spectra of Ga-4 (a) and
Ga-5 (b) in D2O at pH ≈ 7.
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GaIII Complexes with 1,4,7-Triazacyclononane-1,4,7-triacetic Acid Derivatives
lar ranges of 3° Ͻ θ Ͻ 27° for Ga-4·H2O, 2° Ͻ θ Ͻ 27° for Ga-
slight signal broadening and a noisy signal appeared at pH
= 8, possibly due to a fluxional effect. However, there was
no indication of complex decomposition in the range of
physiological pH. At pH = 9 both compounds showed de-
composition signals that were well supported by 71Ga
NMR spectroscopic data recorded at the same pH. In ad-
dition, no signal broadening and no coalescence point were
noticed in the VT NMR spectra, which suggest that coordi-
nated 4 and 5 remain firmly bound to gallium up to 85 °C.
5·CF3COOH·H2O, and 1° Ͻ θ Ͻ 27° for Ga-5·2H2O. A total of
ca. 10000 reflections were collected for each complex and about
half of these were unique. Data frames were integrated and cor-
rected for Lorentz and polarization effects with DENZO-SMN.[15]
Crystallographic drawings were created with the ORTEP[16] pro-
gram, and the structures were solved by direct methods with
SHELXS-97 and refined by full-matrix least squares on F2 with
SHELXL-97.[17] All non-hydrogen atoms were refined anisotropi-
cally, except for the benzyl group carbon atoms. All hydrogen
atoms were placed at calculated positions, except for those associ-
ated with water molecules.
Conclusions
General Synthesis and Characterization of Protected Ligands: To a
solution of 1 (0.3 g, 0.84 mmol) and K2CO3 (0.231 g, 1.68 mmol)
in acetonitrile (4 mL), 2-chloro-N-benzylacetamide (0.169 g,
0.92 mmol) or 2-chloro-N-methylacetamide (0.099 g, 0.92 mmol) in
acetonitrile (2 mL) was added dropwise over the course of 10 min.
The reaction mixture was then stirred at room temperature for 24 h.
The completion of the reaction was checked by TLC (CH2Cl2/
methanol, 9:1). The reaction mixture was then filtered, washed with
acetonitrile, concentrated in vacuo, and purified by silica gel col-
umn chromatography (CH2Cl2/MeOH). Products were eluted with
5% methanol to yield compounds 2 (0.187 g, 52%) and 3 (0.254 g,
60%).
GaIII complexes of monopendent-arm-modified NOTA
derivatives are stable and rigid at physiological pH and at
temperatures up to 85 °C. The described direct conjugation
of NOTA with amines of biomolecules provides a novel
tool for the design of small bioactive molecules, as well as
a more convenient means of binding NOTA to biomolec-
ules than using NOTA derivatives containing an extra
linker that require multi-step syntheses. This study could
lead to the development of a new range of cost-effective
and target-specific PET tracers.
N-Methylacetamide 2: 1H NMR (CDCl3, 300 MHz, 25 °C): δ =
1.39 (s, 18 H), 2.61 (d, J = 6 Hz, 3 H, NHCH3), 2.72–2.82 (m, 12
H), 3.21 [s, 2 H, CH2C(O)NH], 3.24 [s, 4 H, CH2C(O)O], 9.09 [br,
1 H, C(O)NH] ppm. 13C NMR (CDCl3, 300 MHz, 25 °C): δ = 25.8
(3 C), 28.1 (9 C), 54.1 (2 C), 55.1 (2 C), 55.5 (2 C), 58.3 (2 C), 60.7,
80.9 (2 C), 171.0 (2 C), 173.2 ppm. ESI-MS: calcd. for
[C21H41N4O5]+ ([M + H]+) 429; found 429.3. HRMS: calcd. for [M
+ H]+ 429.3077; found 429.3079.
N-Benzylacetamide 3: 1H NMR (CDCl3, 300 MHz, 25 °C): δ = 1.38
(s, 18 H), 2.59 (br. s, 8 H), 2.75 (br. s, 4 H), 2.95 [s, 4 H, CH2C-
(O)O], 3.30 [s, 2 H, CH2C(O)NH], 4.40 (d, J = 6 Hz, 2 H, benzyl),
7.11–7.28 (m, 5 H, aromatic), 9.31 [br., 1 H, C(O)NH] ppm. 13C
NMR (CDCl3, 300 MHz, 25 °C): δ = 28.0 (9 C), 43.2 (2 C), 54.3
(2 C), 55.4 (2 C), 56.5 (2 C), 58.3 (2 C), 61.0, 80.7 (2 C), 127.1,
127.9 (2 C), 128.3 (2 C), 138.8, 171.0 (2 C), 172.5 ppm. ESI-MS:
calcd. for [C27H45N4O5]+ ([M + H]+) 505; found 505.4. HRMS:
calcd. for [M + H]+ 505.3390; found 505.3394.
Experimental Section
Materials and Physical Methods: Compound di-tert-butyl 1,4,7-tri-
azacyclononane-1,4-diacetate (1) was purchased from ChemaTech
(Dijon, France). Starting material 2-chloro-N-methylacetamide was
purchased from Frinton Laboratories, Inc. (Vineland, New Jersey,
U.S.A.). All other chemicals and solvents were purchased from
Sigma–Aldrich (St. Louis, MO, U.S.A.). HPLC-grade solvents were
purchased from Fisher (Seoul, Korea). H and 13C NMR spectra
1
were recorded with a Jeol 300 JNM spectrometer (300 MHz for 1H,
75 MHz for 13C). In addition, NOESY, HSQC, and 71Ga NMR
spectroscopic data were obtained with a Bruker (Germany) Avance
600 NMR spectrometer (600 MHz for correlation NMR, and
183 MHz for 71Ga NMR). 1H resonance shifts were measured rela-
tive to tetramethylsilane (TMS), and 67Ga chemical shifts were
measured relative to the [Ga(H2O)6]3+ species present in a 0.1 m
Ga(NO3)3 D2O solution. In the case of the Ga-4 and Ga-5 NMR
analysis over a range of pH values, the isolated samples were redis-
solved in D2O after synthesis at pH = 3 and 5, and the NMR
sample solution pH was controlled by addition of either NaOD or
DCl to the solution. Electrospray ionization mass spectra (ESI-
MS) were acquired with a Waters ESI ion-trap spectrometer de-
signed to detect positive and negative ions. The samples were di-
luted 1:100 with water and injected directly into the source. High
resolution mass spectra were obtained with a Jeol JMS-AX505WA
HP 5890 series II spectrometer with fast atom bombardment
(FAB+) ionization detection.
General Procedure for the Synthesis of Ligands 4 and 5: Compound
2 or 3 was dissolved in a mixture of TFA and CH2Cl2 (TFA/
CH2Cl2, 1:1; 12 mL), and the mixture was stirred at room tempera-
ture for 20 h. The solvent was then removed to give compounds 4
(0.18 g, 96%) and 5 (0.25 g, 98%) as TFA salts containing 1 equiv.
of TFA.
1
N-Methylacetamide 4: H NMR (D2O, 300 MHz, 25 °C): δ = 2.61
(s, 3 H, NHCH3), 3.13–3.22 (m, 12 H), 3.60 [s, 2 H, CH2C(O)NH],
3.75 [s, 4 H, CH2C(O)O] ppm. 13C NMR (D2O, 300 MHz, 25 °C):
δ = 26.3 (3 C), 51.3 (2 C), 51.5 (2 C), 51.6 (2 C), 57.2 (2 C), 58.5,
169.3, 171.2 (2 C) ppm. ESI-MS: calcd. for [C13H25N4O5]+ ([M +
H]+) 317; found 317.2. HRMS: calcd. for [M + H]+ 317.1825;
found 317.1828.
X-ray Crystallographic Analysis: Data collection and structure
analysis were conducted at the Organometallic Laboratory, Seoul
National University. Single-crystal diffraction data for all gallium
complexes were measured with an Enraf–Nonius CCD single-crys-
tal X-ray diffractometer at room temperature. Unit-cell parameters
and intensity-data collections were performed with graphite-mono-
chromated Mo-Kα radiation (λ = 0.71073 Å). Preliminary orienta-
tion matrices and unit-cell parameters were obtained from the
peaks on the first 10 data frames, and these were then refined
against the complete datasets, which were collected over the angu-
N-Benzylacetamide 5: 1H NMR (D2O, 300 MHz, 25 °C): δ = 2.78–
2.89 (m, 12 H), 3.42 [s, 2 H, CH2C(O)NH], 3.45 [s, 4 H, CH2C-
(O)O], 3.81 (s, 2 H, benzyl), 7.73–7.78 (m, 5 H, aromatic) ppm. 13
C
NMR (D2O, 300 MHz, 25 °C): δ = 43.6 (2 C), 49.2 (2 C), 51.3 (2
C), 51.4 (2 C), 56.9 (2 C), 58.6, 114.6 (q, CF3COO–), 127.8 (2 C),
128.0, 129.1 (2 C), 137.7, 162.7 (q, CF3COO–), 168.8, 170.6 (2 C)
ppm. ESI-MS: calcd. for [C19H29N4O5]+ ([M + H]+) 393; found
393.2. HRMS: calcd. for [M + H]+ 393.2138; found 393.2140.
Eur. J. Inorg. Chem. 2010, 5432–5438
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
5437

J. M. Jeong et al.
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Synthesis and Characterization of Ga-4 Complexes: A solution of
Ga(NO3)3·xH2O (0.030 g, 0.12 mmol) in water (0.5 mL) was added
to a solution of 4 (0.05 g, 0.12 mmol) in water (1 mL). The pH of
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and tested with microfine pH paper (Sigma–Aldrich, HydrionTM
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syringe filters (Whatman, 0.45 μm) and purified by RP-HPLC
(XTerra® prep RP18, 19ϫ250 mm; water/EtOH eluent: 100%
water for 5 min and 0 to 80% EtOH for 25 min). Collected frac-
tions were lyophilized to obtain the complexes as white solids
(0.03 g, 68%). Crystals for X-ray crystallography were obtained by
slow concentration of EtOH/H2O (80:10, v/v) solutions of the com-
plexes. 1H NMR (D2O, 300 MHz, 25 °C): δ = 2.86 (s, 3 H,
NHCH3), 3.01–3.18 (m, 6 H), 3.30–3.35 (m, 6 H), 3.66 [s, 4 H,
CH2C(O)O], 4.04 [s, 2 H, CH2C(O)NH] ppm. 13C NMR (D2O,
300 MHz, 25 °C): δ = 28.4 (3 C), 53.6 (2 C), 53.7 (2 C), 53.9 (2 C),
60.3, 62.5, 173.2, 175.2 (2 C) ppm. ESI-MS: calcd. for
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EtOH solutions of the complexes at room temperature. H NMR
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3.11–3.31 (m, 8 H), 3.58 [d, J = 18 Hz, 2 H, CH2C(O)NH], 4.0 (s,
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Published Online: October 27, 2010
5438
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Eur. J. Inorg. Chem. 2010, 5432–5438