Metal ion size profoundly affects H3glyox chelate chemistry

Article abstract of DOI:10.1039/d1ra01793d

The bisoxine hexadentate chelating ligand, H3glyox was investigated for its affinity for Mn2+, Cu2+ and Lu3+ ions; all three metal ions are relevant with applications in nuclear medicine and medicinal inorganic chemistry. The aqueous coordination chemistry and thermodynamic stability of all three metal complexes were thoroughly investigated by detailed DFT structure calculations and stability constant determination, by employing UV in-batch spectrophotometric titrations, giving pM values (pM = -log[Mn+]free when [Mn+] = 1 μM, [L] = 10 μM at pH 7.4 and 25 °C)-pCu (25.2) > pLu (18.1) > pMn (12.0). DFT calculated structures revealed different geometries and coordination preferences of the three metal ions; notable was an inner sphere water molecule in the Mn2+ complex. H3glyox labels [52gMn]Mn2+, [64Cu]Cu2+ and [177Lu]Lu3+ at ambient conditions with apparent molar activities of 40 MBq μmol-1, 500 MBq μmol-1 and 25 GBq μmol-1, respectively. Collectively, these initial investigations provide insight into the effects of metal ion size and charge on the chelation with the hexadentate H3glyox and indicate that further investigations of the Mn2+-H3glyox complex in 52g/55Mn-based bimodal imaging might be worthwhile.

Full text of DOI:10.1039/d1ra01793d

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PAPER
Metal ion size profoundly affects H3glyox chelate
chemistry†
Cite this: RSC Adv., 2021, 11, 15663
ab
c
d
Neha Choudhary,
Valery Radchenko,
Guadalupe Jaraquemada-Pelaez
Kendall E. Barrett,
Manja Kubeil,
be
c
d
Jonathan W. Engle,
Holger Stephan,
Marıa de
´
a
a
*
*
and Chris Orvig
´
The bisoxine hexadentate chelating ligand, H3glyox was investigated for its affinity for Mn2+, Cu2+ and Lu3+
ions; all three metal ions are relevant with applications in nuclear medicine and medicinal inorganic
chemistry. The aqueous coordination chemistry and thermodynamic stability of all three metal complexes
were thoroughly investigated by detailed DFT structure calculations and stability constant determination, by
employing UV in-batch spectrophotometric titrations, giving pM values (pM ¼ ꢀlog[Mn+
]
free when [Mn+] ¼ 1
mM, [L] ¼ 10 mM at pH 7.4 and 25 ꢁC) – pCu (25.2) > pLu (18.1) > pMn (12.0). DFT calculated structures
revealed different geometries and coordination preferences of the three metal ions; notable was an inner
sphere water molecule in the Mn2+ complex. H3glyox labels [52gMn]Mn2+, [64Cu]Cu2+ and [177Lu]Lu3+ at
Received 7th March 2021
Accepted 16th April 2021
ambient conditions with apparent molar activities of 40 MBq mmolꢀ1, 500 MBq mmolꢀ1 and 25 GBq mmolꢀ1
,
respectively. Collectively, these initial investigations provide insight into the effects of metal ion size and
DOI: 10.1039/d1ra01793d
charge on the chelation with the hexadentate H3glyox and indicate that further investigations of the Mn2+
H3glyox complex in 52g/55Mn-based bimodal imaging might be worthwhile.
–
rsc.li/rsc-advances
and preparation of new radioisotopes, there is access to a large
number of radiometals with varying physical (emission type and
energies, half-lives) and chemical properties (oxidation state,
metal hardness, size, coordination preferences) which can be
harnessed for their applications in nuclear medicine.2–4
Introduction
The radiotracer principle was rst described by George de Hev-
esy, who stated that radiopharmaceuticals are administered at
such low molar masses that they can participate in biological
processes and facilitate imaging, but they do not perturb native
biochemistry.1 Positron emission tomography (PET) relies on b+
emission of radionuclides; the emitted b+ collides with an elec-
tron leading to simultaneous emission of two g-rays of 511 keV
energy in opposite directions. This co-emission of two g-rays
leads to higher sensitivity and resolution of PET compared to
single photon emission computed tomography (SPECT) which
relies on the detection of unique emitted g-rays in all directions.
With the recent advent of advanced technologies in production
ImmunoPET has emerged as an attractive imaging modality
in the last decade because of the high affinity and specicity of
the antibodies and their longer plasma half-life.5 Therefore,
there has been burgeoning interest in longer-lived PET radio-
metals such as 89Zr (t1/2 ¼ 78.4 h), 64Cu (t1/2 ¼ 12.7 h) and 52gMn
(t1/2 ¼ 5.6 d) which match better with the biological half-lives of
antibodies.6–10 Another advantage offered by manganese-based
agents is their application for bimodal imaging using 55Mn
(high spin quantum number, S ¼ 5/2) which exhibits great
potential in Mn-based magnetic resonance imaging (MRI)
contrast agents.8,11,12 64Cu (t1/2 ¼ 12.7 h, Eb+ ¼ 278 keV (19%),
Ebꢀ ¼ 190 keV (39%), EC (61%)) has been a radionuclide of
great interest for ve decades because of its unique decay
scheme, which combines three types of decay (b+, bꢀ and EC),
and Auger electron emission with therapeutic potential.13 While
52gMn and 64Cu are two PET radiometals, 177Lu (t1/2 ¼ 6.7 d) is
a long lived radionuclide with its low energy bꢀ emission
aMedicinal Inorganic Chemistry Group, Department of Chemistry, University of British
Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T 1Z1, Canada. E-mail:
orvig@chem.ubc.ca; mdgjara@chem.ubc.ca
bLife Sciences Division, TRIUMF, 4004 Wesbrook Mall, Vancouver, British Columbia,
V6T 2A3, Canada
cDepartment of Medical Physics, University of Wisconsin, 1111 Highland Avenue,
Madison, WI 53711, USA
dInstitute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-
(Ebꢀ ¼ 134 keV, 100%) which makes it ideal for the treatment
avg
Rossendorf, Bautzner Landstraße 400, D-01328 Dresden, Germany
of small tumors using radiotherapy and the two useful g-
emissions (Eg1 ¼ 208 keV (10.41%) and Eg2 ¼ 113 keV (6.23%))
for SPECT imaging.14 Lutathera ([177Lu][Lu-DOTA-TATE]) is
a recently FDA-approved 177Lu-based-radiopharmaceutical for
eDepartment of Chemistry, University of British Columbia, 2036 Main Mall,
Vancouver, British Columbia, V6T 1Z1, Canada
† Electronic supplementary information (ESI) available: NMR spectra of
compounds 1–4, high resolution mass spectra, solution thermodynamic results,
radiolabeling studies, density functional theory (DFT) calculations. See DOI:
10.1039/d1ra01793d
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the treatment of somatostatin receptor positive gastroentero- (Scheme 1). The compound 2-(bromomethyl)quinolin-8-ol (2)
pancreatic neuroendocrine tumors.15
was prepared in two steps which involved a reduction of the 8-
Previously, we reported a versatile bisoxine hexadentate hydroxyquinoline-2-aldehyde using NaBH4 to give compound 1,
chelator, H3glyox (Chart 1); its metal complexes (Sc3+, Ga3+ and which was then brominated using PBr3. This synthetic scheme
In3+) displayed excellent thermodynamic stability in solution and provides a convenient method for the preparation of the alky-
against in vitro human serum challenge.16 Rapid radiolabeling of lating compound 2 and overcomes the limitations of free
[44Sc]Sc3+, [68Ga]Ga3+ and [111In]In3+ at ambient conditions (RT, radical bromination.18,19 Another advantage offered by this
15 min, pH 7) was observed with high specic activity and quan- synthetic route is that it makes possible functionalised deriva-
titative yields. Herein, we investigate the complexation ability of tives of the ligand by using other amino acids such as p-NO2–
H3glyox with the divalent metal ions (Mn2+ and Cu2+) and trivalent phenylalanine and N-alkylation using 2 in presence of base
metal ion (Lu3+) to gauge the effect of metal ion size, oxidation (K2CO3) and catalytic amount of KI. Compound 2 is not very
state and varying coordination preferences on the sequestering stable at room temperature and is prone to decomposition;
capabilities of H3glyox. All three metal ions (Mn2+, Cu2+ and Lu3+) therefore, it is recommended to use it immediately. No addi-
are medicinally relevant but have very distinct chemical properties. tional column purication was required for the synthesis of
Manganese and copper are rst row transition metal ions with compound 2 and the reactions provided high yields. H3glyox
2+
2+
˚
˚
ionic radii of 0.83 A (Mn , CN 6, high spin) and 0.73 A (Cu , CN 6) was prepared in overall 90% yield by SN2 reaction of glycine
respectively; while Lu3+ is the largest metal ion studied with ethyl ester and compound 2 in basic conditions, and subse-
H3glyox with an ionic radius 0.86 A (CN 6). These results are quent ethyl group deprotection under acidic conditions.
compared with the previous study16 to draw conclusions across the
17
˚
periodic table for the applicability of this unique ligand.
Metal complexes
Equimolar 1 : 1 concentration solutions of the ligand H3glyox
and the corresponding metal salts (MnCl2, CuSO4 and
Lu(NO3)3) dissolved in water/D2O were mixed together and the
pH was adjusted to 7 using 0.1 M NaOH to give the metal
Results and discussion
Synthesis and characterisation
An easier modular higher yielding synthetic route is reported complexes. LR-MS and HR-MS were used to conrm the
here for H3glyox from commercially available starting materials formation of the complexes, along with the change in
Chart 1 Structures of discussed chelators.
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Scheme 1 Improved high yield modular synthetic scheme for H3glyox, 4.
1
uorescence of the ligand observed under UV light. The addi- complexation with Lu3+ ion can be noted in the H NMR spec-
tion of CuSO4 to the ligand solution led to the quenching of the trum of the Lu3+–H3glyox complex but the complete coordina-
uorescence which was expected on Cu2+ complexation,20 tion of Lu3+ ion remains inconclusive; there was no success in
meanwhile no changes in uorescence intensity were observed obtaining 13C NMR or 2D NMR spectra due to the poor solu-
on addition of MnCl2,21 but there was a drastic increase in bility of the complex, leading to precipitation over time.
uorescence intensity on complexation of Lu3+ ion. In Fig. 1, the Comparing the 1H NMR spectra of the Ga3+–H3glyox and Lu3+
–
changes in chemical shi of the protons of the ligand on H3glyox systems, it can be hypothesized that due to the larger
Fig. 1 1H NMR spectra (300 MHz, room temperature, D2O, pD ¼ 7) of the ligand H3glyox (top in red) and the Lu3+–H3glyox complex (bottom in
blue).
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ionic size of Lu3+ (0.86 A) as compared to smaller Ga (0.62 A)
Paper
3+
˚
˚
The calculated structure in Fig. 2B reveals that the geometry
ion, the Lu3+ ion does not t well in the cavity of the ligand and around Mn2+ is distorted octahedral with ve donor atoms of
forms an asymmetric complex.16
the ligand and an additional O-donor atom of a water molecule.
On comparing bond distances in the calculation of
[Mn(glyox)(H2O)]ꢀ with those in the reported crystal structure of
[Sc(glyox)(H2O)],16 it is noted that the average Mn–O(ox) and
Mn–N(ox) bond distances are slightly shorter in
Density functional theory calculations
In the absence of crystal structures for the Ga3+, Mn2+, Cu2+ and
Lu3+ complexes, and inconclusive 1H NMR of the Lu3+–H3glyox
complex, DFT calculations were undertaken in order to study
the coordination environment differences of the metal
complexes in solution. The calculated structures are presented
in Fig. 2 along with their coordination spheres. Fig. 2A
demonstrates the distorted octahedral geometry (N2O4) around
Ga3+ in the calculated structure of [Ga(glyox)(H2O)], the two
oxine arms bind Ga3+ in a slightly distorted plane with axial
coordination from the carboxylic group and trans water mole-
cule, analogous to the crystal structure of [Ga(dpaa)(H2O)]
[Mn(glyox)(H2O)]ꢀ with an average bond distance of 2.067 A and
˚
2+
˚
˚
2.180 A, respectively, despite the bigger size of Mn (0.83 A, CN
6)17 vs. Sc3+ (0.74 A, CN 6). The bond distance of the amine
17
˚
˚
Mn–N3 is somewhat longer (2.764 A) than the analogous amine
Sc–N (2.515 A); the former is not considered as a coordinated
donor atom.16 Additionally, comparison of the calculated
structure of [Mn(glyox)(H2O)]ꢀ with the reported crystal struc-
ture of analogous [Mn(dpaa)(H2O)]ꢀ complex24 reveals that the
average Mn–O(ox) and Mn–N(ox) bond distances are shorter
˚
than the Mn–O(pa) and Mn–N(pa) bond distances with
24
22
˚
˚
˚
a difference of 0.199 A and 0.079 A, respectively. It is note-
worthy that the DFT calculated structures for both
[Mn(glyox)(H2O)]ꢀ and [Mn(dpaa)(H2O)]ꢀ consist of an addi-
tional water molecule coordinated to the metal centre.32
Cu2+ prefers ve-fold coordination with H3glyox, leading to
a distorted square pyramidal geometry (Fig. 2C), in which the
donor atoms N1, O1, O2 and O3 dene the basal plane and the
without coordination of the tertiary N atom (Ga–N3 ¼ 2.644 A).
The average Ga–O(ox) bond distances are similar for the re-
ported crystal structure of [Ga(hox)]+ and the calculated struc-
˚
ture of [Ga(glyox)(H2O)] with average bond distances of 1.959 A
23
˚
and 2.000 A, respectively. Meanwhile, the calculated average
Ga–N(ox) bond distances in [Ga(glyox)(H2O)], are slightly longer
+
23
˚
than in [Ga(hox)] with a difference of 0.257 A.
Fig. 2 DFT calculated structures of (A) [Ga(glyox)(H2O)], (B) [Mn(glyox)(H2O)]ꢀ, (C) [Cu(glyox)]ꢀ, (D) [Lu(glyox)(H2O)2]A and (E) [Lu(glyox)(H2O)2]B;
bond distances are found in Tables S5 and S6.†
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apical position is occupied by the N2 atom of the oxine arm. The the thermodynamic formation constant (log KML) for the metal
inclusion of either one or three explicit water molecules in the complex and the protonation constants of H3glyox (Table 1).16
calculations did not converge to a six-coordinate environment Herein, the aim was to determine the thermodynamic stability
in either case (Fig. S17 and S18†). It has been previously re- of the Mn+–H3glyox complexes (Mn+ ¼ Mn2+, Cu2+ and Lu3+). UV
ported that solvated Cu2+ ion prefers penta-coordination vs. the in-batch spectrophotometric titrations were applied to deter-
general assumption of octahedral solvation.25 Similarly, Cu2+ mine the stability constants and the results from the equilib-
complexes of monopicolinate derivatives of cyclen and cyclam, rium studies are summarised in Table 2. Aqueous solutions of
and [Cu(nompa)]+ (Chart 1) all exhibit ve-coordinate Cu2+ equimolar concentrations of H3glyox and each of three different
ion.26,27 Table S5† presents the calculated bond distances from metal ions were prepared at varying pH using standardised HCl
the DFT structures. Compared to the Cu–O(ox) bond distance of and NaOH solutions, and were allowed to reach equilibrium
2.142 A in the reported [Cu(hox)] structure,28 the Cu–O(ox) bond before measuring the UV-spectra.
˚
is slightly shorter in [Cu(glyox)]ꢀ complex with an average Cu–
On comparing the extensive spectral changes in the elec-
tronic spectra of the metal complexes with the spectra of the
2+
˚
O(ox) bond distance of 2.006 A. Due to the strong affinity of Cu
for the 8-hydroxyquinoline scaffold, the coordination uses the ligand,16 the complexation is observed directly (Fig. S9–S11†).
N2O2 donor set of the two oxine arms of the ligand and the O The stability constants of the metal complexes were determined
atom of the carboxylic group. The preorganization and rigidity using HypSpec2014 (ref. 30) by analysing the experimental data
of H3glyox leads to the longer distances for the coordination of along with the molar absorptivity of the different protonated
the amine N-atom and this is reected in the long Cu–N3 bond species of Hxglyox (Table 1).16 The speciation plots for the Mn2+
–
distance of 2.781 A which is not considered as coordinated.
H3glyox, Cu2+–H3glyox, and Lu3+–H3glyox systems are presented
˚
Fig. 2D and E illustrates the two geometrical isomers for in Fig. 3. In the aqueous environment, there is always compe-
[Lu(glyox)(H2O)2] (cis- and trans-diaqua) obtained through DFT tition between protons (H+) and the metal ions (Mn+) for the
calculations and the two structures exhibit a small energy basic donor atoms of the ligands. Therefore, the pM value31 (pM
difference (DG ¼ 3.85 kJ molꢀ1); bond distances are tabulated in ¼ ꢀlog[Mn+
]
free where [Mn+] ¼ 1 mM, [L] ¼ 10 mM at pH ¼ 7.4) is
Table S6.† As expected, Lu3+ forms an eight-coordinate complex a reliable thermodynamic parameter to evaluate the seques-
with the hexadentate glyox3ꢀ and two water molecules, in trating ability of the ligand for a specic metal ion at physio-
agreement with the reported crystal structure of the analogous logical pH and allows for a better comparison of the affinity of
[Lu(dpaa)(H2O)2] complex.29 There are no signicant differences different ligands with different denticities, basicity and metal
in the bond lengths for the calculated cis [Lu(glyox)(H2O)2] complex stoichiometries for a specic metal ion.
complex (Fig. 2D) and crystal structure of [Lu(dpaa)(H2O)2]. The
For the Mn2+–H3glyox system, the complex formation equi-
calculated mean bond distances for Lu–Oox and Lu–Nox in cis libria studies showed a trio of species Mn(Hglyox), [Mn(glyox)]ꢀ
[Lu(glyox)(H2O)2] are 2.256
A
and 2.431 A, respectively; and [Mn(glyox)(OH)]2ꢀ. The complexation begins around pH 3.5
˚
˚
compared to the mean bond distances of 2.576 A and 2.398 A from the free [Mn(H2O)6]2+ to form the monoprotonated
reported for Lu–Opa and Lu–Npa in [Lu(dpaa)(H2O)2] crystal Mn(Hglyox) complex. Around pH 5, the formation of the
structure.29 Due to the rigid and distorted planar structure of [Mn(glyox)]ꢀ species can be observed as there is an increase in
the ligand,16 the largest of the three metal ions studied here, the absorbance bands at lmax ¼ 255 and 370 nm characteristic
Lu3+, is exposed to coordination from solvent molecules. Hence, of quinolinate metal complexes.16,23,32 The formation of the
a lesser kinetic inertness of the Lu3+–H3glyox complex can be metal-hydroxo species [Mn(glyox)(OH)]2ꢀ starts aer pH 8 and
predicted from the incorporation of the two cis or trans water predominates at basic pH without the presence of free metal in
˚
˚
molecules completing the Lu3+–H3glyox coordination sphere.
solution (Fig. S9†). The absorbance band with lmax ¼ 255 nm
(Fig. S9C†) demonstrates the complexation of Mn2+ by the
ligand in a manner similar to the UV-Vis spectrum for
Mn(oxinate)2 complex formation.8
Solution thermodynamics
The evaluation of a chelator for its use in radiometal-based
radiopharmaceuticals heavily relies on the determination of
Consistent with the lack of ligand eld stabilisation in the
3d5 conguration of Mn2+, [Mn(glyox)]ꢀ is the least
Table 1 Stepwise protonation constants (log b) and protonation
constants (log K) of H3glyox at 25 ꢁC (ref. 16)
Table 2 Stability constants (log Kpqr) and corresponding stepwise
protonation constants log K1q1 (MHqL)a of H3glyox complexes with
Mn2+, Cu2+ and Lu3+ (T ¼ 25 ꢁC, I ¼ 0.16 M NaCl)
Equilibrium reaction
log ba
log Ka
L3ꢀ + H+ $ HL2ꢀ
HL2ꢀ + H+ $ H2Lꢀ
H2Lꢀ + H+ $ H3L
H3L + H+ $ H4L+
H4L+ + H+ $ H5L2+
H5L2+ + H+ $ H6L3+
10.66(1)b
20.36(1)b
27.87(2)b
33.28(1)b
36.61(1)b
39.27(1)b
10.66(1)
9.70(1)
7.51(2)
5.41(1)
3.33(1)
2.66(1)
Mn2+
Cu2+
Lu3+
log K101 (ML)
log K111 (MHL)
log K1ꢀ11 (M(OH)L)
pMb
16.75(1)
7.17(1)
10.45(2)
12.0
30.20 (1)
3.90 (1)
8.98 (1)
25.2
23.04(1)
4.30(1)
8.74(1)
18.1
a
a
From ref. 16. b In-batch UV spectrophotometric titrations at I ¼ 0.16 M
K1q1 ¼ [MHqL]/[MHqꢀ1L][H]q; (q ꢀ 1) ¼ ꢀ1 denotes OH. b pM31 dened
NaCl.
as ꢀlog[Mn+
]
when [Mn+] ¼ 1 mM and [L] ¼ 10 mM at pH 7.4.
free
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Fig. 3 Speciation diagrams of the Mn+–H3glyox complexes where Mn+ ¼ Mn2+, Cu2+ and Lu3+; dashed line indicates pH 7.4.
log KML ¼ 16.83 for PCTA).34 On the other hand, the log KMnL
value for [Mn(glyox)]ꢀ is 3.56 units higher than for the struc-
turally analogous [Mn(dpaa)]ꢀ which indeed reects the effect
of the oxine vs. picolinic acid groups on complexation of Mn2+
Table
3 Stability constants (log KML), corresponding stepwise
protonation constants (log KMHL) and pMn values33 of relevant Mn2+
chelators
Chelator
H3glyoxa
H3dpaab
PyC3Ac
PC2A-EAd
PCTAd
ion.24 In Fig. 4B, pMn31 (pMn ¼ ꢀlog[Mnn+
]
free when [Mnn+] ¼ 1
log KML
log KMHL
log KM(OH)L
pMne
16.75(1)
7.17(1)
10.45(2)
8.03
13.19(5)
2.90(6)
11.97(6)
8.98
14.14
2.43
—
19.01
6.88
—
16.83
1.96
—
mM; [L] ¼ 10 mM at pH 7.4) vs. pH are plotted for the most
relevant Mn chelators to demonstrate the effect of the overall
basicity of the ligand on the metal scavenging ability at different
pH. The most signicant effect can be observed at physiological
pH where, even though the stability constant of [Mn(glyox)]ꢀ
(log KML ¼ 16.75) stands within the highest reported (Tables 3
and S7†), the high overall basicity of H3glyox with respect to the
other ligands (Table S7†), mainly imparted by the two basic
oxine–OH in the arms, makes the competition higher at phys-
iological pH and consequently, lowers the conditional stability.
Nevertheless, we see that still at higher pH the pMn continues to
increase as the metal is secured by the [Mn(glyox)(OH)]2ꢀ
species which reaches its maxima at pH ꢂ12. Additionally, since
the pH-dependent Mn2+ scavenging prole is very close (and
even superior at higher pH) to that of the also non-macrocyclic
chelator PyC3A,11 it is worth to further explore the potential of
this ligand for Mn2+ medicinal applications.
8.17
9.27
9.74
a
b
c
d
e
This work. From ref. 24. From ref. 11. From ref. 34. pMn33
dened as ꢀlog[Mn2+
]
when [Mn2+] ¼ [L] ¼ 10 mM at pH 7.4.
free
thermodynamically stable metal complex of H3glyox among all
six metal ions studied.16 But on the comparison (Table 3) with
other macrocyclic and non-macrocyclic aminopolycarboxylate
chelators (Chart 1) studied for Mn2+ complexation, and in
particular, with the highest pMn reported for the macrocycle
PCTA, H3glyox has a pMn value33 (pMn ¼ ꢀlog[Mnn+
]
when
free
[Mnn+] ¼ [L] ¼ 10 mM at pH 7.4, unique pMn conditions
different than for pM above with all other metal ions) just 1.71
units lower, notwithstanding having both nearly the same
thermodynamic stability (log KML ¼ 16.75(1) for H3glyox and
Fig. 4 (A) pM values31 vs. ionic radii17 for Mn+–H3glyox complexes (n ¼ 2 and 3) (CN ¼ 6), pM ¼ ꢀlog[Mn+
]
free when [Mn+] ¼ 1 mM and [L] ¼ 10 mM
at pH 7.4; (B) Mn2+ scavenging ability of different chelators as the pH is raised from 2 to 12, when [Mn2+] ¼ 1 mM and [L] ¼ 10 mM;31 dashed line
represents physiological pH 7.4.
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For each of the Cu2+–H3glyox and Lu3+–H3glyox systems, 0.1 M NaOAc within 15 minutes at pH 6 and [L] ¼ 10ꢀ5 M with
three metal–ligand species were observed (Fig. 3) – the proton- the apparent molar activity of 500 MBq mmolꢀ1. The second set
ated metal complex, Cu(Hglyox) and [Lu(Hglyox)]+, the metal of experiments varied temperature and reaction time vs. radi-
complex, [Cu(glyox)]ꢀ and Lu(glyox), and the hydroxo–metal olabeling efficiency. No signicant changes in radiolabeling
complex, [Cu(glyox)(OH)]2ꢀ and [Lu(glyox)(OH)]ꢀ. At neutral pH efficiency were observed when radiolabeling with [64Cu]CuCl2
7, the binary metal complexes [Cu(glyox)]ꢀ and Lu(glyox) are was done at room temperature, 37 ꢁC and 80 ꢁC, while the
completely formed.
radiochemical yields (RCY) observed for the time dependent
For the Cu2+–H3glyox system, different isosbestic points reactions are summarised in Table 4. Further, the ligand solu-
(Fig. S10†) can be seen in both the regions (225–300 nm and 300– tion concentration was varied to determine the ligand concen-
450 nm) of the spectra with the increase in pH from 0.37 to 11.07 tration dependant radiolabeling efficiency, [H3glyox] ¼ 10ꢀ4 to
which indicates presence of different metal complex species in 10ꢀ7 M and the results are summarised in Table 5. The radio-
equilibrium. On comparing pM values of the Cu2+ complexes of chemical yields were obtained by integrating the area under the
H3glyox, H2hox and H2CHXhox, all belonging to the same “ox” peaks observed for TLC (Fig. S13†) when eluted with 0.1 M EDTA
family with varying backbone, they follow the trend [Cu(glyox)]ꢀ (pH 5) ([64Cu][Cu(glyox)]ꢀ, Rf < 0.1; [64Cu]Cu-EDTA, Rf > 0.7),
(pCu ¼ 25.2, log KML ¼ 30.30) > Cu(CHXhox) (pCu ¼ 23.9, consistent with the well-resolved peaks in the HPLC trace
log KML ¼ 29.78) > Cu(hox) (pCu ¼ 22.8, log KML ¼ 22.80).28 This (Fig. S12†). Although H3glyox labels copper-64 at ambient
comparison demonstrates that the pre-organisation of the ligand conditions, it was found that the [64Cu(glyox)]ꢀ complex is
leads to stronger complexation of Cu2+.
unstable under acidic conditions (0.1% TFA in MQ water) as
For the Lu3+–H3glyox complexation equilibria, as pH seen in the radio-HPLC trace. This is consistent with the
increases from 3.74 to 6.54, there is an increase in absorption observations and inferences made from both DFT calculations
spectra at lmax ¼ 260 nm with new isosbestic points at l ¼ 252, and solution studies due the protonation of the carboxylate arm
338 nm (Fig. S11†). Although both H3glyox (hexadentate) and at pH below 5.
H4octox (octadentate) differ in their denticity, they have quite
similar affinities for Lu3+; pM ¼ 18.1 for Lu(glyox) and
Radiolabeling with [52Mn]MnCl2
A radiochemically isolated [52gMn]Mn2+ sample35 was titrated
against varying concentrations of H3glyox (0–100 000 ng mLꢀ1
pM ¼ 18.2 for [Lu(octox)]ꢀ and the stability constants (log KML
)
are 23.04 and 24.66, respectively.32
)
Fig. 4A summarizes the effects of the metal ion size and
charge on chelation with H3glyox. As reported, the medium
sized trivalent metal ions (Sc3+, Ga3+ and In3+) form thermody-
namically stable and transchelation-resistant charge neutral
complexes.16 Meanwhile, the larger trivalent lanthanide, Lu3+ is
a poor match for the H3glox cavity size compared to the other
yielding apparent molar activities of 30 MBq mmolꢀ1 at pH 6.5
and 40 MBq mmolꢀ1 at pH 7. The labelling of [52gMn]Mn2+ by
H3glyox was compared with the “gold standard” chelator –
DOTA at similar conditions (30 min, 85 ꢁC, pH 7) and it was
found that DOTA yields apparent molar activity of 60 MBq
mmolꢀ1, slightly higher than that for [52gMn]Mn2+–H3glyox (40
MBq mmolꢀ1). It was observed that the molar activity of [52gMn]
Mn2+–H3glyox increased with the use of higher activity of
smaller metals. Both the divalent metal ions Mn2+ and Cu2+
however, form thermodynamically stable complexes
,
(log KMnL ¼ 16.75 and log KCuL ¼ 30.21) which suggested that
[
52gMn]Mn2+ per reaction. Radiolabeling was not attempted at
radiolabeling studies might be rewarding.
pH > 7 to avoid hydrolysis of the Mn2+ ion. The radiochemical
yields (RCY%) of chelated [52gMn]Mn2+ were determined via
TLC (silica gel plates) eluted with 0.1 M sodium citrate
Radiolabeling with [64Cu]CuCl2
Preliminary radiolabeling experiments were done in order to (Fig. S14†). The “free” [52gMn]Mn2+ travels up the plate as
determine the complexation kinetics and radiolabeling effi-
[
52gMn]Mn2+–citrate complex (Rf > 0.8) while the [52gMn]Mn2+–
ciency of H3glyox with [64Cu]CuCl2. The rst set of experiments DOTA and [52gMn]Mn2+–H3glyox complex stayed at baseline
involved radiolabeling in various buffer solutions (0.1 M MES, (Rf < 0.1). It has been noted previously that a rigid scaffold such
0.1 M NH4OAc and 0.1 M NaOAc) at pH 5.5 and 6; quantitative as bispidine around the Mn2+ ion endows the manganese
radiolabeling (RCY > 95%) was achieved in both 0.1 M MES and complex with increased kinetic inertness and similar to
Table 4 Radiochemical yields vs. time for the copper-64 complex of Table 5 Radiochemical yields vs. concentration for the copper-64
H3glyox (n ¼ 3)
complex of H3glyox (n ¼ 3)
Time (min)
[
64Cu]Cu2+ RCYa (%) [Ligand] (M)
[
64Cu]Cu2+ RCYa (%)
10
20
40
60
<60
100
100
100
10ꢀ4
10ꢀ5
10ꢀ6
10ꢀ7
100
100
>95
<10
a
a
Room temperature, 0.1 M NaOAc/AcOH, pH 6, [H3glyox] ¼ 10ꢀ5 M, 25
Room temperature, 0.1 M NaOAc/AcOH, pH 6, 15 min, 25 MBq [64Cu]
MBq [64Cu]CuCl2 per reaction.
CuCl2 per reaction.
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Table 6 Radiolabeling data for the [177Lu]Lu3+–H3glyox complex
the pM values, which are in the order pCu (25.2) > pLu (18.1) >
pMn (12.0). Although the thermodynamic stability of the Cu2+
–
Buffer
Time (min)
RCYa (%)
H3glyox complex is higher than that of the Mn2+ complex and in
agreement with the Irving–William series, the
[
64Cu]
0.1 M NaOAc pH 7.0
0.1 M NaOAc pH 8.5
0.1 M NaOAc pH 7.0
0.1 M NaOAc pH 8.5
15
15
30
30
75
70
80
60
[Cu(glyox)]ꢀ was found to decompose under mild acidic
conditions due to the formation of a protonated Cu(Hglyox)
species at pH < 5. However, radiolabeling of H3glyox with
[
52gMn]Mn2+, [64Cu]Cu2+ and [177Lu]Lu3+ yielded apparent
a
Room temperature, [L] ¼ 10ꢀ5 M, 2 MBq [177Lu]LuCl3 per reaction.
molar activities of 40 MBq mmolꢀ1, 500 MBq mmolꢀ1 and 25 GBq
mmolꢀ1, respectively. DFT calculations of the structures of the
three complexes revealed that the Mn2+ is hexacoordinated and
H3glyox, the radiolabeling of [52gMn]Mn2+ with a bispidine-
based ligand was done at pH 7, 70 ꢁC and 1 h.36 Meanwhile,
radiolabeling of [52gMn]Mn2+ with the open chain chelator,
is bound to
a water molecule similar to the reported
[Mn(dpaa)(H2O)]ꢀ structure,24 which makes the Mn2+–H3glyox
complex potentially relevant to Mn-based contrast agents.
Based on these interesting and encouraging preliminary
studies, the next step would be detailed studies to determine the
kinetic inertness, water exchange rate and relaxivity measure-
ments for application of H3glyox in Mn-based MRI contrast
agents. Meanwhile, it can be concluded that the highly preor-
ganized and rigid binding cavity of the ligand H3glyox is suit-
able for neither the smaller Cu2+ nor the larger Lu3+ ion.
CDTA
(trans-1,2-diamino-cyclohexane-N,N,N0,N0-tetraacetic
acid) is reported to be performed at pH 6, room temperature
and 30 minutes ([CDTA] ¼ 64 mM, ꢂ8 MBq [52gMn]MnCl2).10 No
data on apparent molar activities for 52gMn-labeled complexes
were found in the literature for other chelators discussed here
for comparison.10,36
Radiolabeling with [177Lu]LuCl3
Experimental section
Materials and methods
Encouraged by the relatively high pM value (pLu ¼ 18.1),
preliminary radiolabeling studies were done to investigate the
complexation kinetics of the hexadentate chelator H3glyox with
All solvents and reagents were purchased from commercial
suppliers (Sigma-Aldrich, TCI America, Fischer Scientic, Alfa
Aesar) and were used as received. Reactions were monitored by
TLC (MERCK Kieselgel 60 F254, aluminum sheet). Flash chro-
matography was performed using SilicaFlash F60 silica gel (40–
63 mM particle size), RediSep Rf HP silica columns, and a Com-
biFlash Rf column machine. Water used was ultrapure (18.2
[
177Lu]LuCl3. Although Lu3+ ion is a larger trivalent metal ion
with an ionic radius of 86–103 pm and coordination number 6–
9,17 the mismatch with the cavity size of H3glyox is evident from
the observed RCYs. Table 6 summarizes the RCYs (n ¼ 3) and as
expected, quantitative radiolabeling (RCY > 95%) was not ach-
ieved even by varying conditions – pH 7.0 and 8.5 at 15 and 30
minutes. The decrease in RCY was observed when the labelling
was performed at pH 8.5 aer 30 minutes compared to the RCY
at 15 minutes, this can be attributed to the labile nature of the
MU cmꢀ1 at 25 C, Milli-Q, Millipore, Billerica, MA).
ꢁ
1H and 13C NMR spectroscopy was performed using Bruker
Avance 300 and Bruker AV III HD 400 MHz spectrometers.
Chemical shis (d) are quoted in ppm relative to residual
solvent peaks as appropriate; coupling constants (J) are
provided in Hertz (Hz). 1H NMR signals were designated as
follows: s (singlet), d (doublet), t (triplet), q (quartet), quin
(quintet), sxt (sextet), spt (septet), m (multiplet), or a combina-
tion of these, with br representing a broad signal. Low resolu-
tion ESI-MS was performed on a Waters 2965 HPLC-MS with the
sample prepared in methanol or acetonitrile (ACN). Results are
labeled with m/z (abundance percentage) values ꢀ [M + X]ꢃ.
High resolution ESI-MS was performed on a Waters/Micromass
LCT TOF-MS with the sample prepared in methanol. Results are
[
177Lu]Lu3+–H3glyox complex and subsequent transchelation of
Lu3+ ion by EDTA (pH 7, mobile phase) leading to lower RCYs.
The radiochemical yields were determined by integrating the
area under the peaks observed in the iTLC plates (Fig. S15,†
[
177Lu]Lu(glyox), Rf < 0.1; [177Lu]Lu(EDTA), Rf > 0.8). A high
apparent molar activity of 25 GBq mmolꢀ1 was achieved by using
10ꢀ6 M ligand concentration at ambient conditions (15 min, RT,
and pH 7).
Conclusions
It has been an ongoing challenge for the medicinal inorganic labeled with m/z (abundance percentage) values. Semi-
chemistry community to stabilize the labile coordination preparative reverse phase high-performance liquid chromatog-
complexes of endogenous metal ions such Mn2+ and Cu2+ in raphy (HPLC) for H3glyox was performed on a Phenomenex
relevant biological environments. These results indicate that Synergi hydro-RP 80 A, 250 ꢄ 21.2 mm column connected to
further investigation of the preorganized H3glyox as a potential a Waters 600 controller, a Waters 2487 dual wavelength absor-
chelator for 52g/55Mn-based dual MRI-PET tracer application is bance detector, and a Waters delta 600 pump. The HPLC
worthwhile, supported by the DFT calculated structure of solvents were (A) H2O containing 0.1% triuoroacetic acid (TFA)
[Mn(glyox)(H2O)]ꢀ and efficient radiolabeling of [52gMn]Mn2+. and (B) CH3CN containing 0.1% TFA. Silica gel impregnated
In-batch UV spectrophotometric titrations were conducted to TLC plates (MERCK Kieselgel 60 F254, aluminum sheet) were
access the thermodynamic stability of the metal complexes by used to analyse [64Cu]Cu2+ and [177Lu]Lu3+ radiolabeling reac-
determining the formation constants, speciation diagrams and tion progress and the complex stability tests were counted on
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a BioScan System 200 imaging scanner equipped with a BioScan
Autochanger 1000. For the evaluation of [52gMn]Mn2+, TLC
plates were scanned on a Packard Cyclone™ phosphor-storage
plate equipped with Opti-quant™ soware. Linear extrapola-
tion was used to determine the amount of chelator needed to
Ethyl bis((8-hydroxyquinolin-2-yl)methyl)glycinate (3).
Compound 2 (0.54 g, 2.26 mmol) was dissolved in 30 mL dry
ACN and to this solution was added glycine ethyl ester (0.20 g,
1.43 mmol), excess K2CO3 (0.90 g, 6.51 mmol) and KI (0.37 g,
ꢁ
2.26 mmol). The mixture was reuxed overnight at 55 C; the
complex 50% and 100% of [52gMn]Mn2+
177Lu]LuCl3 was purchased from Isotope Technologies
.
reaction was then quenched with distilled water (10 mL) and
the product was extracted with DCM (3 ꢄ 25 mL). The solvent
was removed under a vacuum to give crude 3, which was then
puried by column chromatography (eluted with a gradient of
100% hexane to 100% ethyl acetate) to afford 3 as an off-white
solid (0.47 g, 1.14 mmol, 61%, Rf ¼ 0.50 in 20% EtOAc in
hexane). 1H NMR (300 MHz, CDCl3, RT): d 8.11 (d, J ¼ 8.5 Hz,
2H), 7.72 (d, J ¼ 8.5 Hz, 2H), 7.41 (m, 2H), 7.31 (d, J ¼ 8.2 Hz,
2H), 7.17 (d, J ¼ 7.5 Hz, 2H), 4.19 (m, 6H), 2.03 (s, 2H), 1.27 (t, J
¼ 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3, RT): d 171.3, 160.6,
157.1, 145.3, 140.8, 136.5, 136.4, 134.1, 128.9, 128.8, 127.0,
125.8, 122.9, 122.2, 60.7, 60.3, 55.4, 14.4. LR-ESI-MS calcd for
[
Garching (ITG). All the isotopes used were no-carrier added
(n.c.a.). The production of [64Cu]CuCl2 was performed at a TR-Flex
cyclotron from Advanced Cyclotron Systems Inc (ACSI, Canada)
according to a reported procedure via 64Ni(p,n)64Cu nuclear
reaction and module-assisted separation as described in detail
recently.37 No-carrier-added [52gMn]Mn2+ was produced by irradi-
ation of a metallic chromium target of natural isotopic abundance
(83.8% 52Cr; 9.5% 53Cr; 4.3% 50Cr; and 2.4% 54Cr) with 16 MeV
protons as described previously in a report.35 Deionized water was
ltered through the PURELAB Ultra Mk2 system.
C
24H23N3O4: 417.4. Found: 440.3 [M + Na]+.
Bis((8-hydroxyquinolin-2-yl)methyl)glycine, H3glyox (4).
Synthesis and characterisation
Compound 3 (0.47 g, 1.14 mmol) was dissolved in HCl (10
mL, 6 M) and reuxed overnight. The solution was then
cooled and the solvent was evaporated under reduced pres-
sure to give a yellow coloured solid product 4 (0.42 g,
1.09 mmol, 96%). The purity of compound 4 was conrmed
by the presence of a single sharp peak in the HPLC trace
(Fig. S19†) with tR ¼ 12.1 min (eluents: (A) 0.1% TFA in H2O
and (B) 0.1% TFA in ACN with a linear gradient 5% to 100% B
for 25 min and ow rate set to 1 mL minꢀ1). 1H NMR (300
MHz, RT, D2O): d 8.58 (d, J ¼ 7.7 Hz, 2H), 7.79 (d, J ¼ 7.9 Hz,
2H), 7.51 (t, J ¼ 7.2 Hz, 2H), 7.36 (d, J ¼ 7.6 Hz, 2H), 7.21 (d, J
¼ 6.7, 2H), 4.57 (s, 4H), 4.09 (s, 2H). 13C NMR (75 MHz, RT,
D2O): d 176.1, 155.1, 150.4, 146.9, 146.6, 130.9, 128.8, 128.4,
122.3, 119.2, 117.0, 58.5, 57.3. HR-ESI-MS calcd for
2-(Hydroxymethyl)quinolin-8-ol (1). 8-Hydroxyquinoline-2-
aldehyde (0.35 g, 2.05 mmol) was dissolved in 20 mL CH3OH
in a 100 mL round bottom ask and NaBH4 (0.10 g, 2.66 mmol)
was added gradually to the reaction ask which was placed in
an ice bath and stirred constantly for 5 hours. The reaction
progress was monitored using TLC (Rf ¼ 0.5, stationary phase –
silica gel and mobile phase – 1 : 1 EtOAc/hexane). On
completion of reaction aer 5 hours, the solvent was removed
under vacuum and the crude solid was dissolved in 10 mL
distilled water and the product was extracted using DCM
(20 mL ꢄ 3). The combined organic layers were dried over
anhydrous MgSO4 and the solvent was removed under vacuum
to give white solid product 1 (0.35 g, 2.00 mmol, 98%). 1H NMR
(300 MHz, CD3OD, RT): d 8.21 (d, J ¼ 8.5 Hz, 1H), 7.54 (d, J ¼
8.5 Hz, 1H), 7.38 (m, 2H), 7.11 (d, J ¼ 7.1 Hz, 1H), 4.90 (s, 2H).
13C NMR (75 MHz, CD3OD, RT): d 158.8, 152.8, 137.6, 136.7,
128.3, 126.9, 118.9, 117.7, 110.9, 64.6. LR-ESI-MS calcd for
C
22H19N3O4: 389.1400. Found: 390.1451 [M + H]+. Anal. calcd
for [H3glyox$2.8 HCl$1.3 CH3OH]: C, 52.49; H, 5.1; N, 7.88.
Found: C, 52.48; H, 5.11; N, 7.93.
1
[
natLu(glyox)]. A H NMR spectrum of Lu3+–H3glyox was ob-
C
10H9NO2: 175.2. Found: 176.3 [M + H]+.
tained by mixing together 1 : 1 (L : M) solutions of ligand and
Lu(NO3)3 prepared in D2O (10 mM) and 0.1 M NaOD was added
2-(Bromomethyl)quinolin-8-ol (2). Compound 1 (0.32 g, 1.82
1
mmol) was dissolved in 25 mL dry ACN in a 100 mL round
bottom ask and placed on an ice bath. PBr3 (0.19 mL, 2.00
mmol) was added dropwise with constant stirring to the reac-
tion mixture which was le to stir. Aer 3 hours, TLC analysis
(Rf ¼ 0.8, stationary phase – silica gel and mobile phase – 1 : 1
EtOAc/hexane) indicated the completion of reaction. The reac-
tion was quenched by addition of saturated Na2CO3 (30 mL) and
the mixture was stirred for another 10 minutes. The reaction
mixture was transferred to a separating ask and DCM (3 ꢄ 20
mL) was used to extract out the product. The organic layers were
combined and dried over anhydrous MgSO4 and the solvent was
removed under vacuum to slightly yellow colored product 2
(0.40 g, 1.78 mmol, 98%). 1H NMR (300 MHz, CD3OD, RT):
d 8.17 (d, J ¼ 8.5 Hz, 1H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.49 (t, J ¼
7.9 Hz, 1H), 7.36 (d, J ¼ 9.4 Hz, 1H), 7.22 (d, J ¼ 7.6 Hz, 1H) 4.73
(s, 2H). 13C NMR (75 MHz, CD3OD, RT): d 157.8, 151.7, 137.4,
127.9, 127.6, 124.1, 119.4, 118.2, 111.1, 64.9. LR-ESI-MS calcd
for C10H879BrNO: 238.0. Found: 239.1 [M + H]+.
to increase the pD of the solution to 7. H NMR (300 MHz, RT,
D2O): d 8.38 (d, J ¼ 8.6 Hz, 0.5H), 8.28 (d, J ¼ 8.6 Hz, 1H), 7.61 (t,
J ¼ 8.5 Hz, 0.5H), 7.42 (t, J ¼ 8.4 Hz, 6H), 7.13 (m, 2H), 4.68 (s,
2H), 4.12 (s, 2H), 3.26 (s, 2H). HR-ESI-MS calcd for
C
22H16175LuN3O4: 561.0548. Found: 562.0546 [M + H]+.
H[natMn(Hglyox)]. H3glyox (10 mg, 0.02 mmol) was dissolved
in 1 mL of distilled water in a scintillation vial, and MnCl2-
$4H2O (3.95 mg, 0.02 mmol) was added, followed by pH
adjustment to ꢂ7 using 0.1 M NaOH. The solution was le to
stir for 1 h, leading to the precipitation of the metal complex
which was conrmed using LR-ESI MS. HR-ESI-MS calcd for
C
22H1755MnN3O4: 442.0599. Found: 443.0601 [M + H]+.
Na[natCu(Hglyox)]. H3glyox (10 mg, 0.02 mmol) was dissolved
in 1 mL of distilled water in a scintillation vial, and CuSO4‧5H2O
(5.00 mg, 0.02 mmol) was added, followed by pH adjustment to
ꢂ7 using 0.1 M NaOH. The solution was le to stir for 1 h,
leading to the dark green colored precipitate which was ltered
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and collected. Formation of the metal complex was conrmed the pH range ꢂ0.37–11.07. A Ross combination pH electrode
using LR-ESI MS as well as quenching of uorescence under was calibrated for hydrogen ion concentration using HCl as
a UV lamp. HR-ESI-MS calcd for C22H1763CuN3O4: 450.0515, described before38 and the results were analysed by the Gran39
Found: 473.0412 [M + Na]+.
procedure. For the most acidic samples, the equilibrium H+
concentration was calculated from solution stoichiometry and
was not measured with the glass electrode. For the samples
containing the metal complexes, the measurements were
performed only when equilibrium was achieved. The stability
constants for the metal complexes were calculated from the
experimental data using the HypSpec2014 program.30 Proton
Radiolabeling
For concentration-dependent labeling of copper-64, different
aliquots were added from the stock solution of H3glyox (2 mg
dissolved in 1 mL 0.1 M HCl and 2 mL MQ H2O, ꢂ10ꢀ3 M),
mixed with [64Cu]CuCl2 (25 MBq) and diluted to a nal
volume of 500 mL using 0.1 M NaOAc buffer solution (pH 6).
Aer incubation for 15 min at RT, the radiolabeling yields
were determined using iTLC (silica gel plates developed with
aqueous 0.1 M EDTA solution, pH 5; [64Cu][Cu(glyox)]ꢀ, Rf <
0.1; [64Cu]Cu–EDTA, Rf > 0.7). For labeling of lutetium-177,
a total volume of 500 mL of the reaction mixture was ob-
tained by addition of an aliquot from the ligand stock solu-
tion (10ꢀ5 M), [177Lu]LuCl3 (2 MBq) and 0.1 M NaOAc/HOAc
buffer (pH 7, 8.5). The RCYs were obtained by spotting an
aliquot from reaction mixture aer 15 min and 30 min on
a TLC plate (silica gel) and developing with aqueous EDTA
solution (0.1 M, pH 7). The [177Lu]Lu(glyox) complex stayed
on the baseline (Rf < 0.1) while “free” [177Lu]Lu3+ travelled up
the plate as [177Lu]Lu3+–EDTA complex (Rf > 0.7). For labeling
dissociation constants corresponding to hydrolysis of Mn2+
,
Cu2+, and Lu3+ aqueous ions included in the calculations were
taken from Baes and Mesmer.40 The species formed in the
studied systems are characterized by the general equilibrium
in eqn (1).
pM + qH + rL 4 MpHqLr (charges omitted)
(1)
By convention, a complex containing a metal ion M, proton H,
and ligand L has the general formula MpHqLr. The stoichio-
metric index p might also be 0 in the case of ligand protonation
equilibria, and negative values of q refer to proton removal or
hydroxide ion addition during formation of the complex. The
overall equilibrium constant for the formation of the complexes
MpHqLr from its components is designated as log b. Stepwise
equilibrium constants log K correspond to the difference in log
units between the overall constants of sequentially protonated (or
hydroxide) species. pM value is dened as ꢀlog[Mn+]free calcu-
lated at specic conditions ([Mn+] ¼ 1 mM, [Lxꢀ] ¼ 10 mM, pH
7.4)31 except where pMn values amongst ligands are compared
([Mn2+] ¼ 10 mM, [Lxꢀ] ¼ 10 mM, pH 7.4).33
with 52gMn,
a
reported procedure was followed.35 In
summary, 100 mL of chelator solution was added to 1 mL vials
in varying concentrations (0–100 000 ng mLꢀ1) and 100 mL of
[
52gMn]Mn2+ solution (ꢂ2.7 MBq) was added to each vial.
Finally, the pH of the solution was adjusted to pH 7 with
addition of 100–200 mL of 1 M HEPES/NaOH buffer (pH 7). It
was observed that the molar activity of [52gMn]Mn2+–H3glyox
complex increased with the use of higher activity of [52gMn]
Mn2+ per reaction. The apparent molar activity for [52gMn]
Mn2+–H3glyox complex determined from titration of 52gMn
solutions with H3glyox can be expressed as twice the ratio of
the 52gMn over moles of chelator required to chelate 50% of
the [52gMn]Mn2+. Radiolabeling was not attempted at pH > 7
in order to avoid hydrolysis of the Mn2+ ion. Vials were
agitated, assayed and heated for 30 minutes at 85 ꢁC. The
percentage of chelated [52gMn]Mn2+ was determined via TLC
(silica gel plates; Fig. S14†) eluted with 0.1 M aqueous sodium
citrate (pH 5.5). The “free” [52gMn]Mn2+ travels up the plate as
DFT calculations
All DFT simulations were performed as implemented in the
Gaussian 16 revision c01.41 Each structure was optimised and
free energies were calculated using DFT with the hybrid meta
generalized gradient approximation with the TPSSh exchange-
correlation functional42 and the def2-TZVP basis set43 for all
non-metal atoms. Effective core potentials (ECPs) were used to
account for scalar relativistic effects in metal core electrons (Mn
and Cu,44,45 Ga46,47 and Lu48,49). Metal basis sets were down-
loaded from the Basis Set Exchange website.50–52 The integral
equation formalism of the polarisable continuum model
(IEFPCM) was used as an implicit water model in all calcula-
tions to simulate the average dielectric effects of the solvent.
Default IEFPCM parameters were used, as implemented in
Gaussian (3 ¼ 78.36, van der Waals surface without “added
[
52gMn]Mn2+–citrate complex (Rf > 0.8) while the [52gMn]
Mn2+–DOTA and [52gMn]Mn2+–H3glyox complexes stayed at
baseline (Rf < 0.1).
Solution thermodynamics
Metal stability constants were calculated from UV spectro- spheres”). For Cu2+–H3glyox, calculations included 0, 1 and 3
photometric titration data obtained using a Cary 60 UV-Vis explicit water molecules while for Mn just one explicit water
spectrophotometer in the spectral range of 200–450 nm. The molecule was included. Lu3+–H3glyox calculations included 2
path length was 1 cm for all samples. Individual samples (5 explicit water molecules. Before optimisation, the randomly
mL) containing the ligand (H3glyox, [L] ¼ 2.5 ꢄ 10ꢀ5 M) and placed water molecules were placed closer to the metal with the
the corresponding metal complexes (M ¼ Mn2+, Cu2+ and Lu3+
;
aim to try to coordinate it to Cu2+, Mn2+ or Lu3+ or to hydrogen
[ML] ¼ 2.5 ꢄ 10ꢀ5 M) were prepared in MQ water by adjusting bond centres, such as oxinates and carboxyl groups. Normal
the pH using standardized HCl or NaOH solutions, and NaCl self-consistent eld (SCF) and geometry convergence criteria
was added to maintain a constant 0.16 M ionic strength over were conducted for all the calculations. The calculated
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structures were visualized using GaussView version 6.0 (ref. 53)
and Avogadro version 1.2.0.54
information-approved-drugs/fda-approves-lutetium-lu-177-
dotatate-treatment-gep-nets, accessed 13 July 2020.
´
16 N. Choudhary, M. d. G. Jaraquemada-Pelaez, K. Zarschler,
X. Wang, V. Radchenko, M. Kubeil, H. Stephan and
C. Orvig, Inorg. Chem., 2020, 59, 5728–5741.
17 R. D. Shannon, Acta Crystallogr., Sect. A: Cryst. Phys., Diffr.,
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Conflicts of interest
The authors declare no competing nancial interest.
Acknowledgements
We gratefully acknowledge both the Canadian Institutes for 19 N. Choudhary, A. Dimmling, X. Wang, L. Southcott,
Health Research (CIHR) and the Natural Sciences and Engi-
neering Research Council (NSERC) of Canada for grant support,
V. Radchenko, B. O. Patrick, P. Comba and C. Orvig, Inorg.
Chem., 2019, 58, 8685–8693.
¨
NSERC for an IsoSiM CREATE at TRIUMF studentship (NC), and 20 D. Brox, A. Kiel, S. J. Worner, M. Pernpointner, P. Comba,
MITACS for a Globalink Graduate fellowship (NC). We thank B. Martin and D. P. Herten, PLoS One, 2013, 8, e58049.
WestGrid and Compute Canada for access to their computa- 21 L. Prodi, F. Bolletta, M. Montalti and N. Zaccheroni, Coord.
tional resources. Karin Landrock is acknowledged for excellent Chem. Rev., 2000, 205, 59–83.
technical assistance, as is the HZDR for supporting the IsoSiM 22 D. M. Weekes, C. F. Ramogida, M. d. G. Jaraquemada-Pelaez,
International Research Experience programme; TRIUMF
receives federal funding via a contribution agreement with the
National Research Council of Canada.
B. O. Patrick, C. Apte, T. I. Kostelnik, J. F. Cawthray,
L. Murphy and C. Orvig, Inorg. Chem., 2016, 55, 12544–
12558.
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23 X. Wang, M. d. G. Jaraquemada-Pelaez, Y. Cao, J. Pan,
K. S. Lin, B. O. Patrick and C. Orvig, Inorg. Chem., 2019, 58,
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