Thomas W.
Price
ab,
Juan
Gallo
c,
Vojtěch
Kubíček
d,
Zuzana
Böhmová
d,
Timothy J.
Prior
e,
John
Greenman
a,
Petr
Hermann
d and
Graeme J.
Stasiuk
*ab
aSchool of Life Sciences, Department of Biomedical Sciences, University of Hull, Cottingham Road, Hull, HU6 7RX, UK. E-mail: g.stasiuk@hull.ac.uk
bPositron Emission Tomography Research Centre, University of Hull, Cottingham Road, Hull, HU6 7RX, UK
cAdvanced (magnetic) Theranostic Nanostructures Lab, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal
dDepartment of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030, 12840, Prague 2, Czech Republic
eChemistry, School of Mathematical and Physical Sciences, University of Hull, Cottingham Road, Hull, HU6 7RX, UK
First published on 17th November 2017
Gallium-68 (68Ga) has been the subject of increasing interest for its potential in the production of radiotracers for diagnosis of diseases. In this work we report the complexation of 68Ga by the amino acid based tripodal chelate H3Dpaa, and two bifunctional derivatives, H3Dpaa.dab and H4Dpaa.ga, under a range of conditions with particular emphasis on the rapid complexation of 68Ga at pH 7.4. 100 μM H3Dpaa achieved a radiochemical yield of 95% at pH 7.4 in 5 minutes at 37 °C. The bifunctional derivatives H4Dpaa.ga and H3Dpaa.dab achieved 94% and 84% radiochemical yields, respectively, under the same conditions. The resulting Ga(III) complexes show thermodynamic stabilities of logKGaDpaa = 18.53, logKGaDpaa.dab = 22.08, logKGaDpaa.ga = 18.36. Unfortunately, the resulting radiolabelled species do not present sufficient serum stability for in vivo application. Herein we show a flexible synthesis for bifunctional chelators based on amino acids that rapidly complex 68Ga under physiological conditions.
Of these positron emitting metal isotopes, 68Ga is of particular interest.6–9 The generator source of 68Ga allows for local production at the site of use, opening the possibility of individual hospitals producing their own radiotracers instead of relying upon centralised production facilities. Comparisons can be drawn to the successful 99mTc single photon emission computed tomography (SPECT) isotope.10 The 68 minute half-life of 68Ga is amenable to imaging with peptides and other molecules with relatively short blood circulation times in vivo.11
Traditional macrocyclic chelators, including 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA), have been successfully applied to 68Ga complexation.15–17 Conjugated DOTA derivatives have been applied to imaging of neuroendocrine tumours – with [68Ga]-DOTATATE being recently approved for use by the FDA. However, radiolabeling of DOTA with 68Ga requires relatively aggressive conditions, a pH of 4 and heating to over 80 °C for efficient radiolabeling.15,18 These conditions limit the range of targeting motifs that can be used with [68Ga]-DOTA to acid and temperature stable compounds.
A range of chelators have been tested for their 68Ga complexation abilities.4,12–14 Recent trends in chelate design for 68Ga have been to improve the radiolabeling procedure by reducing the temperature required for efficient complexation of 68Ga and raising the pH at which this occurs.4,12,19,20 This is a challenge due to the formation of kinetically inert gallium hydroxide species above pH 4.5.6,10 Radiolabelling at pHs and temperatures close to physiological conditions is necessary to maintain the structure of peptides and aptamers. Furthermore, radiolabelling at neutral pH would reduce the formulation required after synthesis of the radiotracer, simplifying the tracer production procedure.
The smaller macrocycle, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) has been applied to 68Ga complexation, and radiolabelling proceeds efficiently at room temperature, although acidic conditions are still required.16 Novel, non-macrocyclic chelates THP21 and DATA22 have been shown to rapidly complex 68Ga at higher pH values. While the conjugated DATA probe, DATA-TOC, has only been radiolabelled at pH 4–5,23 THP conjugates, THP-RGD and THP-TATE, were radiolabelled at pH 5–6.5.24,25 Despite this recent progress, rapid radiolabeling with 68Ga at pH 7 has not yet been widely realised, with few bifunctional chelators capable of achieving rapid complexation at neutral pH reported (structures of these bifunctional chelators are shown in Fig. S1†).26–28
Development of alternative chelates that can achieve this may allow for improved design of the imaging probe through different pharmacokinetic profiles/biodistributions.29 While THP-TATE can be radiolabelled at pH 6.5, its lipophilic nature results in a significantly different biodistribution when compared to DOTA-TATE, with longer renal and liver uptake. Balancing improved radiolabeling properties with ideal imaging properties requires the development of new chelates to optimize both properties.29
We report here the application of the chelate N,N-bis[(6-carboxypyridin-2-yl)methyl]glycine (H3Dpaa) and two bifunctional derivatives, H3Dpaa.dab and H4Dpaa.ga (Fig. 1) to 68Ga complexation. H3Dpaa is composed of two picolinic acid arms attached to a central glycine unit to produce a tripodal, hexadentate ligand. Picolinic acids have been demonstrated to be highly capable 68Ga coordinating arms.30 The aminebis(picolinic acid) motif has been applied to the complexation of a variety of metals with a number of different amines being used to form chelates with varying properties.31–42 The incorporation of a glycine residue into the chelate backbone provides both a carboxylic acid group that can bind strongly to Ga(III) due to a good hard acid/base match, and also a site which can be readily functionalised through application of other amino acids.31,33 H3Dpaa has previously been applied to complexation of lanthanide(III) ions such as gadolinium(III) (logKGdDpaa = 10.6)32,33 and terbium(III) (logKTbDpaa = 10.4).32 H3Dpaa has recently been applied to manganese(II) (logKMnDpaa = 13.2),34 lanthanum(III) (logKLaDpaa = 13.6)35 and gallium-67 (logKGaDpaa = 18.7)35 complexation showing the versatility of this ligand for metal coordination. Herein we show a flexible synthesis for bifunctional chelators that rapidly complex 68Ga under physiological conditions with a radiochemical yield of up to 95%. Unfortunately, the radiolabelled species [68Ga][Ga(Dpaa)], [68Ga][Ga(Dpaa.dab)] and [68Ga][Ga(Dpaa.ga)] show poor stability in serum competition studies and are unsuitable for advancing to in vivo studies.
Scheme 1 Synthetic scheme for the synthesis of ligands reported in this paper. (i) K2CO3, KI, MeCN, 60 °C, 12 h. (ii) 6 M HCl, reflux, 16 h, (iii) GaCl3, H2O, pH 4. |
Ga(III) complexes were synthesized by addition of GaCl3 to an aqueous solution of the ligand. The resulting complexes precipitated out of solution and were collected.
Evidence for complexation can be seen through the distinct NMR resonances of the two protons in the CH2 environment between the picolinate arms and the central amine. While these protons are equivalent (δH3Dpaa = 3.92, δH3Dpaa.dab = 4.41, δH4Dpaa.ga = 4.40) on the NMR time scale in the free ligands, in the Ga(III) complexes they are inequivalent (δ[Ga(Dpaa)] = 4.62 and 4.48, δ[Ga(Dpaa.dab)] = 4.56–4.33 and 4.07, δ[Ga(Dpaa.ga)] = 4.66 and 4.33) and show a strong geminal coupling to one another (2JHH = 16–17 Hz).
Fig. 2 ORTEP representations of molecular structures of (A) [Ga(H2O)(Dpaa)]§ and (B) [Ga(H2O)(Dpaa.ga)]¶ obtained by single crystal X-ray crystallography (drawn with 30% certainty, solvent molecules omitted for clarity). |
Bond | Bond length/Å | |
---|---|---|
GaDpaa | GaDpaa.ga | |
O1–Ga1 | 2.0441(10) | 2.001(6) |
N1–Ga1 | 2.2354(11) | 2.191(7) |
N2⋯Ga1 | 2.4880(11) | 2.513(8) |
N3–Ga1 | 2.2017(12) | 2.180(7) |
O5–Ga1 | 2.0229(10) | 2.034(6) |
O3–Ga1 | 1.9173(10) | 1.942(6) |
O1W–Ga1 | 1.9109(10) | 1.952(5) |
Each structure features a distorted octahedral coordination of the Ga(III) as a consequence of the geometry of the ligand. The greatest distortion is obvious in the plane of the picolinic acids (Tables 1 and 2). In each case the N1–Ga–N3 angle is greater than 133° and consequently the N–Ga–O angles are much smaller than the ideal 90° expected for undistorted octahedral geometry.
Angle | Bond angle/° | |
---|---|---|
GaDpaa | GaDpaa.ga | |
O1–Ga1–N1 | 73.97(4) | 76.1(3) |
N1–Ga1–N3 | 135.16(4) | 133.7(3) |
N3–Ga1–O5 | 74.95(4) | 75.2(3) |
O5–Ga1–O1 | 75.92(4) | 75.5(3) |
O3–Ga1–O1 | 90.08(4) | 93.8(3) |
O3–Ga1–N1 | 88.09(4) | 86.0(2) |
O3–Ga1–N3 | 91.36(4) | 96.4(3) |
O3–Ga1–O5 | 92.23(4) | 90.7(2) |
O3–Ga1–O1W | 175.40(4) | 172.8(2) |
[Ga(Dpaa)] is relatively symmetric and there is a pseudo-mirror plane (through O3, N2, and O1W) present in the complex. The picolinate arms are close to planar; the angle subtended by the two mean planes of the picolinates is 9.53(3)°. In contrast the glutamic acid backbone introduces a twist in [Ga(Dpaa.ga)] removing the pseudo-mirror plane and pushing the picolinate rings further out of the same plane such that the angle between their mean planes is 15.85(3)°. It is important to note that the pendant carboxylate arm of the glutamic acid is not involved in Ga(III) coordination. Full crystallographic data can be found in the ESI.†
H3Dpaa | H3Dpaa.dab | H4Dpaa.ga | |
---|---|---|---|
a ([L] = 0.004 M, T = 25 °C, I = 0.1 M (NMe4)Cl). | |||
logK1 | 7.38 | 11.35 | 7.17 |
logK2 | 3.73 | 5.39 | 4.67 |
logK3 | 2.82 | 3.77 | 3.92 |
logK4 | — | 2.69 | 2.75 |
The additional carboxylate group in H4Dpaa.ga introduces an additional protonation constant in the weakly acidic region (pK2 = 4.67). However, it does not alter significantly the protonation constants of the ligand core (pK1 = 7.33) or the picolinate arms. The terminal amino group in H3Dpaa.dab is protonated above pH 11 (pK1 = 11.35). Presence of the additional protonated amino group significantly decreases basicity of the central amino group in the ligand core (pK2 = 5.39).
Complexation of Ga(III), Cu(II) and Zn(II) ions by the three ligands was studied by potentiometry. These metal–ligand systems were chosen due to their importance for the potential application of these ligands to nuclear medicine. The results are summarized in Table 4.
Metal ion | H3Dpaa | H3Dpaa.dab | H4Dpaa.ga |
---|---|---|---|
a Determined by UV-VIS titration [L] = [M] = 0.1 mM, T = 25 °C, pH = 2–7. b Determined by potentiometric titration ([L] = [M] = 0.004 M, T = 25 °C, I = 0.1 M (NMe4)Cl). c Constant (logKGaHL) describing equilibrium Ga(III) + (HL)2− [Ga(HL)]+ where the amine group deprotonation and hydroxido species formation are not considered. d Determined by UV-VIS titration ([L] = [M] = 0.01 mM, T = 25 °C, pH = 0–2). | |||
Ga(III) | 18.53a | 22.08b | 18.36b |
16.13b,c | |||
Cu(II) | 10.85b | 19.1b,d | 14.52b,d |
Zn(II) | 11.93b | 15.8b,d | 13.38b,d |
The study of the Ga(III)-Dpaa system was not straightforward due to low solubility of the uncharged [Ga(Dpaa)] species. Therefore, UV-VIS titration was performed at significantly lower concentration (Fig. S4†). Nitrogen atoms of the ligands are weakly basic and this leads to complexation of metal ions even in strongly acidic solutions. As a consequence, some of the complexes were fully formed in the beginning of potentiometric titrations. Thus, Cu(II) and Zn(II) systems with H4Dpaa.ga and H3Dpaa.dab were also studied by UV-VIS spectrophotometry at pH 0–2 (Fig. S6 and S8†). Spectrophotometry was not employed in Ga(III) systems for these ligands as stability constants could be determined from competition with hydroxide ions in the alkaline region (i.e. formation of [Ga(OH)4]−).
The stability constants of [Ga(L)] species are similar for both H3Dpaa and H4Dpaa.ga. This indicates a negligible role of the distant carboxylate in complexation reactions of H4Dpaa.ga in agreement with crystallographic data. For [Ga(Dpaa.ga)], the first protonation constant ([M(L)] + H [M(HL)], logK = 4.04, Table S2†) is comparable to that of free ligand, further supporting that the distant carboxylate group is not coordinated. In both systems, hydroxido species, [Ga(OH)(Dpaa)]− and [Ga(OH)(Dpaa.ga)]2−, are formed already in acidic region through a formal aqua ligand dissociation with corresponding pKa values of 4.41 and 5.27, respectively.
This points to an unsaturated coordination sphere of the metal ion in these complexes in which some of the ligand donor groups remain uncoordinated. The unsaturated coordination sphere is corroborated by the crystal structures obtained in which this site is occupied by a bound water molecule. Complexes in which the coordination sphere of Ga(III) is not fully satisfied by a chelate with six coordinating atoms have been reported previously, with modelling suggesting either water of chloride bound in the vacant site.43,44 Stability constants of the studied Ga(III) complexes are significantly higher than those reported for complexes of H3Dpaa with lanthanide(III) ions (logK = 10.6 and 10.4 for [Gd(Dpaa)] and [Tb(Dpaa)], respectively).32 This indicates that the ligands better suits “hard” and small metal ions such as Ga(III).
The stability constant for [Ga(Dpaa.dab)] complex (logKGaL = 22.08) is surprisingly much higher than those of the other two Ga(III) complexes. This is due to the different structure of the [GaL] species. The Ga(III) ion in [Ga(Dpaa)] and [Ga(Dpaa.ga)] complexes is coordinated by the fully deprotonated ligand. Ligand H3Dpaa.dab contains the highly basic terminal amino group. Consequently, the proton bound to the coordinated water molecule is more acidic than the proton bound to the amino group. The first protonation constant (logKa = 5.40, Table S2†) is similar to those describing formation of the monohydroxide species in both the Ga(III)-Dpaa and Ga(III)-Dpaa.ga systems and should be ascribed to the formation of the hydroxide species as well. Thus, the [Ga(Dpaa.dab)] complex is zwitterionic, with a hydroxide anion bound to the Ga(III) and with a protonated amine group. Dissociation constant of the amino group in the complex cannot be determined as it would dissociate at very high pH where the complex is fully decomposed to [Ga(OH)4]−. To compare the stability constants, it is more suitable to consider equilibrium between Ga(III) ion and monoprotonated ligand molecule (logKGaHL 16.13, Table 4) where influence of the above processes is not considered. The value is lower and in line with those for the other systems if the presence of a positive charge, due to a protonated amino group in the ligand molecule, is taken into account. This is reflected in the pM values which are 8.91, 6.34 and 8.21 (pH = 7.4, [Ga] = 10−6 M, [L] = 10−5 M) for [Ga(Dpaa)], [Ga(Dpaa.dab)] and [Ga(Dpaa.ga)], respectively. When comparing the formation constant of [Ga(Dpaa)] of 18.53 with that of [Ga(DOTA)] and [Ga(NOTA)] (logKGaL = 26.05 and 29.60, respectively)45,46 the thermodynamic stability is lower, but still it may be sufficient for the application due to the short half-life of 68Ga.
The stability constants obtained for [Cu(Dpaa)] and [Zn(Dpaa)] complexes are significantly lower than that of the Ga(III) complex (Tables S2 and S3†). This may be rationalized due to the low flexibility of the ligand preventing the complex from fulfilling the ideal coordination geometry of these two ligands and due to the high charge density of Ga(III) compared to Cu(II) and Zn(II). The ligands are highly charged with hard oxygen donor atoms and interaction of the ligands with Ga(III) is highly electrostatic in its nature and, therefore, thermodynamic stability of the Ga(III) complexes is increased compared to Cu(II) and Zn(II) complexes. This preference for Ga(III) complexation is encouraging for biomedical imaging using [68Ga][Ga(Dpaa)] as Cu(II) and Zn(II) are two of the most abundant transition metal ions in vivo.47
Whilst the Cu(II) and Zn(II) complexes of H3Dpaa.dab and H4Dpaa.ga are less thermodynamically stable than the Ga(III) complexes, the resulting complexes are more stable than those seen with H3Dpaa. This suggests that the additional coordinating arms may be involved in the complexation of these two metals.
Fig. 3 Speciation diagrams for Ga(III) – ligand systems. (A) H3Dpaa, (B) H3Dpaa.dab(NH2), (C) H4Dpaa.ga (T = 25 °C, I = 0.1 M (NMe4)Cl, [L] = [Ga(III)] = 0.004 M). |
This difference between buffered and aqueous solutions may be explained by a weak gallium–phosphate complex being formed. This may act as a “pre-coordination” complex, preventing the rapid formation of gallium hydroxide species that would result in slower complexation due to the kinetically inert gallium–hydroxide bonds.19
The pH of the radiolabelling reaction also has a significant effect on the concentration of ligand required for efficient radiolabelling (Fig. 5). When radiolabelling at pH 4, efficient complexation is achieved at ligand concentrations as low as 500 nM, with radiochemical yields >90% achieved in 15 minutes at ambient temperature. However, the radiochemical yield sharply decreases below this concentration with no radiolabelling seen when [H3Dpaa] = 100 nM. In contrast, at pH 7.4 the radiochemical yield after 15 minutes at ambient temperature is maintained above 90% at 50 μM, however drops below 90% at ligand concentrations of 10 μM.
The ability to rapidly complex 68Ga at neutral pH has the potential to simplify the production of 68Ga labelled radiopharmaceuticals by reducing the post-reaction conditioning required. To develop this further, the complexation of 68Ga by 100 μM H3Dpaa in saline and phosphate buffered saline (PBS) was assessed. High radio-chemical yields of 99% and 95% respectively were achieved with mild heating (37.7 °C) after 5 minutes. Furthermore, the pH of the PBS solution remained at pH 7.4 after complexation, although the pH of the saline solution was lower (pH 5.5) after addition of the ligand.
The bifunctional chelates H3Dpaa.dab and H4Dpaa.ga achieved 99% RCYs after 5 minutes at pH 4 and ambient temperature, and the RCY remained as high as 84% and 94% respectively at pH 7.4 in PBS after 5 minutes at 37 °C (Table 5 and Fig. S9†). Specific activities of 20.0 GBq μmol−1 (541 mCi μmol−1) and 28.9 GBq μmol−1 (781 mCi μmol−1) were achieved with H3Dpaa.dab and H3Dpaa.ga respectively (Fig. S12 and S13†) after radiolabelling at pH 4 (T = 25 °C, t = 5 minutes, I = 0.1 M acetate buffer), however H3Dpaa achieved a specific activity of only 3.9 GBq μmol−1 (105 mCi μmol−1)(Fig. S11†).
The stability of the radiolabelled complexes formed were assessed against biological competitors, apo-transferrin and foetal bovine serum (FBS). Some stability to the iron transport protein apo-transferrin was seen, 92% of 68Ga activity was associated with the [68Ga][Ga(Dpaa)] complex after 2 hours of incubation (Fig. S14†). In FBS complete decomplexation of the 68Ga was seen within 30 minutes for all chelate derivatives (Fig. S15†). This suggests that having the vacant coordination site filled by H2O (Fig. 2), allows for 68Ga to be more readily taken up by competitor proteins found in the serum and therefore H3Dpaa is not the ideal system for 68Ga application in vivo.
1H NMR (400 MHz, CDCl3, 298 K): 7.95 (dd, 2H, J = 6, 2.5 Hz), 7.76 (m, 4H, py), 4.46 (q, 4H, 3JHH = 7.1 Hz), 4.22 (qd, 2H, 3JHH = 7.1 Hz, J = 1.8 Hz), 4.16 (d, 2H, 2JHH = 15.5 Hz), 4.10 (d, 2H, 2JHH = 15.5 Hz), 4.01 (qt, 2H, 3JHH = 7.1 Hz, J = 3.7 Hz), 3.45 (dd, 1H, J = 9.2, 6.1 Hz), 2.50 (m, 2H), 2.13 (tq, 1H, 2JHH = 14.1 Hz, 3JHH = 7.5 Hz), 2.02 (dqt, 1H, 2JHH = 14 Hz, 3JHH = 7.5 Hz, J = 1.6 Hz), 1.44 (t, 6H, 3JHH = 7.1 Hz), 1.33 (t, 3H, 3JHH = 7.1 Hz), 1.18 (t, 3H, 3JHH = 7.1 Hz) 13C NMR (100 MHz, CDCl3, 298 K): 173.04, 172.36, 165.26, 160.23, 147.76, 137.21, 125.93, 123.35, 62.33, 61.73, 60.65, 60.25, 57.08, 30.66, 24.79, 14.38, 14.25, 14.08.
1H NMR (400 MHz, D2O (pD = 7.1), 298 K), δ: 7.72–7.55 (m, 4H), 7.42–7.32 (m, 2H), 4.47–4.33 (m, 4H), 3.77–3.63 (m, 1H), 2.43–2.27 (m, 2H), 2.27–2.06 (m, 2H). 13C NMR (100 MHz, D2O (pD = 7.1), 298 K), δ: 181.70, 176.22, 171.57, 153.47, 151.81, 138.88, 126.17, 123.11, 69.52, 57.14, 34.51, 25.67 MS (ESI), m/z: 418.04 [M + H]+. Elemental analysis (C/H/N), %: expected for H4Dpaa.ga·2HCl (C19H21Cl2N3O8): 46.55/4.32/8.57, found: 46.22/4.40/7.99.
1H NMR (400 MHz, D2O (pD = 8.8), 298 K), δ: 7.72–7.62 (m, 4H), 7.38 (br d, 2H, 3JHH = 7.8 Hz), 3.92(br s, 4H), 3.27 (br s, 2H). 13C NMR (100 MHz, D2O (pD = 8.8), 298 K), δ: 172.96, 152.71, 138.14, 125.87, 122.35, 60.07, 59.22 MS(ESI), m/z = 346.4 [M + H]+. Elemental analysis (C/H/N), %: expected for H3Dpaa·1.3(HCl)0.25(diethyl ether) (C17H19.05Cl1.3N3O6.25): 49.62/4.67/10.21, found: 49.63/4.40/9.94.
1H NMR (400 MHz, D2O (pD = 1.6), 298 K), δ: 8.09 (t, 2H, 3JHH = 7.8 Hz), 7.96 (d, 2H, 3JHH = 7.8 Hz), 7.71 (d, 2H, 3JHH = 7.8 Hz), 4.41 (s, 4H), 4.00 (t, 1H, 3JHH = 7.3 Hz), 3.37–3.18 (m, 2H), 2.40–2.23 (m, 2H). 13C NMR (100 MHz, D2O (pD = 1.6), 298 K), δ: 174.50, 164.62, 154.61, 145.26, 143.74, 128.24, 124.92, 64.99, 55.53, 37.66, 26.10. MS (ESI) m/z: 389.4 [M + H]+. Elemental analysis (C/H/N), %: expected for H3Dpaa.dab(HCl)3(Acetone)0.55 (C19.65H28.3Cl3N4O7.55): 43.09/5.21/10.23, found 42.85/5.15/9.93.
1H NMR (400 MHz, D2O (pD = 6.0), 298 K) δ: 8.30–8.11 (m, 4H), 7.81–7.73 (m, 2H), 4.66 (br d, 2H, 2JHH = 17 Hz), 4.33 (br d, 2H, 2JHH = 17 Hz), 3.05–2.97 (m, 1H), 2.44–2.33 (m, 1H), 2.26–2.15 (m, 1H), 2.06–1.88 (m, 2H). 13C NMR (100 MHz, D2O (pD = 6.0), 298 K) δ: 180.94, 178.13, 168.10, 152.32, 151.03, 144.69, 144.45, 142.83, 142.76, 127.57, 126.38, 123.11, 123.06, 62.08, 58.28, 53.07, 34.80, 22.33. MS (ESI), m/z: 483.94 [69GaM + H]+, 485.97 [71GaM + H]+. Elemental analysis (C/H/N), %: expected for GaDpaa.ga(COOH)·3.5H2O·0.5MeOH (C19.5H25GaN3O12): 41.59/4.47/7.46, found: 41.56/4.68/7.42.
1H NMR (400 MHz, D2O (pD = 8.8), 298 K) δ: 8.21 (t, 2H, 3JHH = 7.3 Hz), 8.14 (d, 2H, 3JHH = 7.3 Hz), 7.75 (d, 2H, 3JHH = 7.3 Hz), 4.62 (dd, 2H, 2JHH = 16.0 Hz, 4JHH = 2.75 Hz), 4.48 (br d, 2H, 2JHH = 16.0 Hz), 3.39 (d, 2H, 4JHH = 2.3 Hz). 13C NMR (100 MHz, D2O (pD = 8.8), 298 K) δ: 176.86, 168.40, 151.26, 144.57, 142.35, 126.76, 122.99, 60.60, 59.89. MS (ESI), m/z: 411.95 [69GaM + H]+, 413.89 [71GaM + H]+. Elemental Analysis (C/H/N), %: expected for GaDpaa·H2O·3HCl (C16H17Cl3GaN3O7): 35.63/3.18/7.79, found: 35.85/3.09/7.59.
1H NMR (400 MHz, D2O (pD = 1.1), 298 K) δ: 8.32–8.24 (m, 2H), 8.24–8.12 (m, 2H), 7.88–7.75 (m, 2H), 4.56–4.33 (m, 3H), 4.07 (br d, 1H, 2JHH = 17.4 Hz), 3.18–3.12 (m, 1H), 3.11–3.02 (m, 2H), 2.28–2.07 (m, 3H). 13C NMR (100 MHz, D2O (pD = 1.1), 298 K) δ: 177.21, 167.18, 167.05, 153.37, 152.71, 145.06, 144.69, 143.97, 128.49, 127.67, 123.77, 123.63, 61.19, 65.00, 51.95, 38.11, 30.30, 24.07. MS (ESI), m/z: 454.43 [69GaM + H]+, 456.39 [71GaM + H]+. Elemental Analysis (C/H/N), %: expected for GaDpaa.dab(NH2)·H2O·0.9HCl (C18H19.9Cl0.9GaN4O7): 42.73/3.97/11.07, found: 43.02/3.63/10.87.
UV-VIS spectra were recorded on spectrophotometer Specord 50 Plus (Analytik Jena AG). Temperature was maintained by Peltier block. UV-VIS titration of the Ga(III)-H3Dpaa system ([L] = [M] = 0.0001 M) was performed at pH range 2.4–6.9 in 0.1 M (NMe4)Cl, pH was adjusted with ∼0.2 M (NMe4)OH using a glass electrode. UV-VIS titrations of the Cu(II) and Zn(II) systems with H4Dpaa.ga and H3Dpaa.dab ([L] = [M] = 0.00001 M) were performed at pH range 0.0–2.0 without ionic strength control, pH was calculated from added amount of HCl.
The titration and UV-VIS data were treated simultaneously with OPIUM program package.53,54 Calculated constants are concentration constants defined as βhl = [HhLl]/[H]h·[L]l or βhlm = [HhLlMm]/[H]h·[L]l·[M]m and standard deviations are given directly by the program. pM values were also calculated by OPIUM from determined protonation and stability constants.
The full version of the OPIUM program is available (free of charge) on http://www.natur.cuni.cz/_kyvala/opium.html.
TLC analysis was performed on Kieselgel 60 F254 plates (Merck) with an eluate of 0.1 M citric acid in water. HPLC analysis was carried out using an Agilent Zorbax Eclipse XDB-C18 column and a solvent system of water + 0.1% TFA and methanol.
Footnotes |
† Electronic supplementary information (ESI) available. CCDC 1549314 ([Ga(Dpaa)]), 1530703 ([Ga(Dpaa.ga)]) and 1530704 (H4Dpaa.ga). For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7dt03398b |
‡ H4Dpaa.ga crystal structure data: refined formula C19H19N3O8; Mr = 417.37; crystal dimensions 0.275 × 0.130 × 0.056 mm3; monoclinic; P21/n; a = 7.0852(6) Å, b = 32.290(4) Å, c = 7.6786(6) Å, β = 94.108(7)°, V = 1752.2(3) Å3; Z = 4; ρcalcd = 1.582 g cm−3; μ = 0.125 mm−1; Mo Kα radiation λ = 0.7173 Å; T = 150 K; 2θmax = 51.10°; no. of reflections measured (independent) = 9845 (3229); Rint = 0.0571; R = 0.0401; wR2 = 0.0612; ρmax/min = 0.172/−0.197 e Å−3; data collected using a Stoe IPDS2 diffractometer; structure solved by routine dual space methods and refined against all observed F2 values. |
§ GaDpaa crystal structure data: refined formula C16H20Ga1N3O10; Mr = 484.07; crystal dimensions 0.040 × 0.005 × 0.005 mm3; triclinic; P; a = 7.06810(10) Å, b = 8.21920(10) Å, c = 15.9952(2) Å, α = 93.6180(10)°, β = 93.7920(10)°, γ = 91.9130(10)°, V = 924.69(2) Å3; Z = 1; ρcalcd = 1.739 g cm−3; μ = 1.425 mm−1; synchrotron radiation λ = 0.6889 Å; T = 100 K; 2θmax = 72.358°; no. of reflections measured (independent) = 20701 (8614); Rint = 0.057; R = 0.0425; wR2 = 0.1062; ρmax/min = 1.606/−0.543 e Å−3; data collected at Diamond synchrotron UK, station I19; structure solved by routine dual space methods and refined against all observed F2 values. |
¶ GaDpaa.ga crystal structure data: refined formula C19H24Ga1N3O12.5; Mr = 564.12; crystal dimensions 0.060 × 0.005 × 0.005 mm3; orthorhombic; Pccn; a = 20.7850(12) Å, b = 30.2224(18) Å, c = 7.2085(6) Å, V = 4528.2(5) Å3; Z = 8; ρcalcd = 1.646 g cm−3; μ = 1.186 mm−1; synchrotron radiation λ = 0.6889 Å; T = 100 K; 2θmax = 49.67°; no. of reflections measured (independent) = 12211 (4251); Rint = 0.2109; R = 0.0999; wR = 0.2359; ρmax/min = 2.429/−1.265 e Å−3; disordered water was modelled using the SQUEEZE routine; data collected at Diamond synchrotron UK, station I19; the crystal suffers from radiation damage but structure solution and refinement were routine; structure solved by routine dual space methods and refined against all observed F2 values. |
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