Is Smaller Better? Cu2+/Cu+ Coordination Chemistry and Copper-64 Radiochemical Investigation of a 1,4,7-Triazacyclononane-Based Sulfur-Rich Chelator

The biologically triggered reduction of Cu2+ to Cu+ has been postulated as a possible in vivo decomplexation pathway in 64/67Cu-based radiopharmaceuticals. In an attempt to hinder this phenomenon, we have previously developed a family of S-containing polyazamacrocycles based on 12-, 13-, or 14-membered tetraaza rings able to stabilize both oxidation states. However, despite the high thermodynamic stability of the resulting Cu2+/+ complexes, a marked [64Cu]Cu2+ release was detected in human serum, likely as a result of the partially saturated coordination sphere around the copper center. In the present work, a new hexadentate macrocyclic ligand, 1,4,7-tris[2-(methylsulfanyl)ethyl)]-1,4,7-triazacyclononane (NO3S), was synthesized by hypothesizing that a smaller macrocyclic backbone could thwart the observed demetalation by fully encapsulating the copper ion. To unveil the role of the S donors in the metal binding, the corresponding alkyl analogue 1,4,7-tris-n-butyl-1,4,7-triazacyclononane (TACN-n-Bu) was considered as comparison. The acid–base properties of the free ligands and the kinetic, thermodynamic, and structural properties of their Cu2+ and Cu+ complexes were investigated in solution and solid (crystal) states through a combination of spectroscopic and electrochemical techniques. The formation of two stable mononuclear species was detected in aqueous solution for both ligands. The pCu2+ value for NO3S at physiological pH was 6 orders of magnitude higher than that computed for TACN-n-Bu, pointing out the significant stabilizing contribution arising from the Cu2+–S interactions. In both the solid state and solution, Cu2+ was fully embedded in the ligand cleft in a hexacoordinated N3S3 environment. Furthermore, NO3S exhibited a remarkable ability to form a stable complex with Cu+ through the involvement of all of the donors in the coordination sphere. Radiolabeling studies evidenced an excellent affinity of NO3S toward [64Cu]Cu2+, as quantitative incorporation was achieved at high apparent molar activity (∼10 MBq/nmol) and under mild conditions (ambient temperature, neutral pH, 10 min reaction time). Human serum stability assays revealed an increased stability of [64Cu][Cu(NO3S)]2+ when compared to the corresponding complexes formed by 12-, 13-, or 14-membered tetraaza rings.

In the present work, the synthesis, acid−base behavior, thermodynamics, kinetics, and structural properties of NO3S toward Cu 2+ and Cu + are reported.To assess the effect of the sulfanyl pendants on the Cu 2+/+ coordination, 1,4,7-tris-nbutyl-1,4,7-triazacyclononane (TACN-n-Bu) was studied for comparison as well (Figure 1C).Collectively, the study was executed through a combination of NMR and UV−vis spectroscopies, X-ray crystallography, and electrochemical techniques.To fully evaluate our hypothesis and appraise the potential of NO3S as a chelator for 64/67 Cu-based radiopharmaceuticals, its labeling performance and the stability of its [ 64 Cu]Cu 2+ complex in biological media were also investigated.

■ RESULTS AND DISCUSSION
NO3S and TACN-n-Bu: Synthesis, Acid−Base, and Structural Properties.NO3S was synthesized by complete alkylation of the unsubstituted precursor (i.e., TACN) with 2chloroethyl methyl sulfide as reported in Figure S2A.−25 NO3S was fully characterized in nonaqueous solvent by 1 H and 13 C{ 1 H} nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) as detailed in Figures S3−S5.TACN-n-Bu was synthesized by the complete alkylation of TACN with 2-bromobutane, as reported in Figure S2B.The full characterization of TACN-n-Bu by 1 H and 13 C{ 1 H} NMR spectroscopy in nonaqueous solvent and HR-ESI-MS is detailed in Figures S6−S8.
Subsequently, the acid−base properties of NO3S and TACN-n-Bu were appraised.This study is a prior fundamental step before the investigation of complexation equilibria, since the proton represents a competitor of the metal ion for the interaction with the donor sites having Brønsted and Lewis acid−base properties, such as the amines of the TACN scaffold.The acidity constants (pK a ) of NO3S in aqueous solution were determined at T = 25 °C by collecting 1 H NMR spectra at pH ranging from 0 to 14.The ionic strength (I) was adjusted using 0.15 M NaNO 3 .The formation of a precipitate was observed at basic pH because the completely deprotonated neutral form (L, Figure 1C) is sparingly soluble in water.Therefore, additional pK a measurements were performed in a 1:1 water/methanol mixture, where no precipitation was detected.Representative 1 H NMR spectra are reported in Figure S9A, and the signal attributions are summarized in Table S1.The acidity constants of TACN-n-Bu were derived upon analysis of the pH-potentiometric titrations data and pHdependent 1 H NMR spectra in water (Figure S9B).A detailed description of the proton resonances of TACN-n-Bu is reported in Table S2.The acidity constant values of both ligands are gathered in Table 1 along with the values of the unsubstituted macrocycle (i.e., TACN) and the methylfunctionalized one (i.e., 1,4,7-trimethyl-1,4,7-triazacyclonane, TACN-Me) derived from the literature. 30n the NMR spectra of NO3S, the SCH 3 resonance does not undergo significant changes in chemical shift at different pH values, likely because these protons are rather far from the ring, where the sites of protonation/deprotonation are located.However, the chemical shift of all of the other signals manifests a noticeable pH dependence.The most pronounced variation is experienced by the NCH 2 protons of the ring, possibly because the deprotonation processes led to a marked relaxation of the ring constraint caused by the H + −H + repulsions.An analogous pH dependency of the chemical shifts is observed for TACN-n-Bu, even if in this case a slowed exchange between the differently protonated forms can be recognized from the enlargement of the signals around pH = pK a,2 .Representative variations of the chemical shift of the different signals as a function of pH are reported in Figures S10 and  S11.The data were fitted, and the corresponding protonation constants (Table 1) were obtained.The derived distribution diagrams are shown in Figure S12.
If the pK a related to the second deprotonation process occurring in NO3S (pK a,2 = 2.70) is compared to that of TACN-n-Bu (pK a,2 = 5.0), where each S atom has been  replaced by one methylene group, then a noteworthy decrease can be observed (Table 1).Therefore, this behavior must be caused by the S atoms.An analogous effect was also formerly observed with the other S-containing macrocycles displayed in Figure 1A,B, and it was ascribed to the high polarizability and dimension of the S atoms. 22,25Actually, it was postulated that sulfurs caused an increased distortion of the ring by hindering the motion of the alkyl chains.This results in an enhanced electrostatic repulsion between the two charged nitrogens of H 2 L 2+ , thus favoring the release of one proton. 22,25It is noteworthy that the pK a,2 of TACN-n-Bu is nearly identical to that of TACN-Me, indicating that the length of the alkyl chain has no effect.Moreover, the pK a,2 values of TACN-n-Bu and TACN-Me are lower than that of the unsubstituted macrocycle, (i.e., TACN�pK a,2 = 6.86,Table 1): this effect is related to the alkylation of the secondary nitrogen atoms of the macrocycle, which destabilizes the generated alkylammonium ion and decreases its water solvation.
The solid-state structure of NO3S was determined via X-ray crystallography for crystals of the monoprotonated ligand obtained in CHCl 3 by the addition of NaPF 6 .Selected bond distances and angles are gathered in Table 2, while crystal data and refinement details as well as further structural information are provided in Tables S3−S5.The solid-state structure of [H(NO3S)](PF 6 ) is shown in Figure 2. As can be noticed, all of the S-containing chains are syn oriented, but the thioether arm on the opposite side with respect to the counterion slightly expands the cleft formed by the N 3 S 3 set of donors with an asymmetrical position.This is likely due to the small TACN ring size that hinders a closer contact among the arms.In fact, this distorted arrangement was not detected in the 12membered-ring analogue DO4S and can be rationalized from a steric point of view since the neighboring N atoms are 2.918 and 2.782 Å apart in DO4S and NO3S, respectively. 31As a result, in NO3S, the free-to-move side chains expand to circumvent this steric constraint.Intriguingly, two slightly different conformations have been refined for one of the Scontaining side chains of NO3S (Figure 2).
Cu 2+ -NO3S and Cu 2+ -TACN-n-Bu: Complexation Kinetics.The formation kinetics of the Cu 2+ complexes with NO3S and TACN-n-Bu was qualitatively appraised by UV−vis spectroscopy at ambient temperature and different pH values to explore the time necessary to reach equilibrium.Representative variations of the absorbance over time and the time courses of the complexation reactions are shown in Figures S13 and S14.The spectrum of NO3S and TACN-n-Bu as free ligands is also reported for comparison purposes in Figure S15.
The acquired data revealed that, at equimolar metal-toligand concentrations (10 −4 M), NO3S can quickly bind Cu 2+ at pH ≥ 4 as the equilibrium occurred in a few minutes (e.g., ∼10 min at pH = 4.0 and <1 min at pH = 7.1) while, at more acidic pH, the complexation kinetics were slower (e.g., ∼14 h at pH = 1.0).This slowed-down reactivity can be justified by considering the progressively more intense electrostatic repulsions generated between the N-bound H + of the differently protonated ligand forms and the incoming Cu 2+ cation.The charged nitrogens hinder the entry of the metal into the binding cleft, as also previously observed with the other S-rich polyazamacrocycles in Figure 1A,B. 24,25If the complexation kinetics of all of the S-rich chelators reported so far are directly compared, then NO3S exhibits the best performance at all pH values.Furthermore, the kinetic investigation revealed that the time necessary to reach equilibrium was appreciably longer when the S-side chains of NO3S were replaced by the n-butyl substituents in TACN-n-Bu: for example, the complexation reaction with TACN-n-Bu at neutral pH occurred in 8 h (vs <1 min with NO3S), as shown in Figure S14.This result points out the existence of a bonding interaction between the S donors and the metal cation.It can be hypothesized that the preorganization of NO3S, as detected in the solid state (vide supra), allows some or all S donors to efficiently interact with Cu 2+ forming an out-of-sphere complex.Afterward, this complex is evolved in the final geometry, thereby increasing the local Cu 2+ concentration around the N donor and accelerating the complexation event compared to TACN-n-Bu.
Cu 2+ -NO3S and Cu 2+ -TACN-n-Bu: Complexation Thermodynamics.The stability constants (logβ) of the Cu 2+ complexes formed by NO3S and TACN-n-Bu were determined via UV−vis spectrophotometric titrations.Due to both the slow complexation kinetics observed at acidic pH (vide supra) and the high complex stability, batch titrations were conducted and heating was applied to speed up reaching the equilibrium condition.
As shown in Figure 3A, after the addition of Cu 2+ to NO3S, from acidic to neutral pH, a distinct change in the electronic spectra with respect to the free ligand (reported in Figure S15A) was recognizable.
The change was evidenced by the appearance of three electronic transitions with a maximum absorption at λ max = 372 nm, indicative of the complexation event.These absorbances experienced a shift toward higher energies at more basic pH

Inorganic Chemistry
(Figure 3A).Furthermore, an isosbestic point is recognizable at λ = 340 nm (except at very acidic pH), highlighting the existence of two different cupric complexes, one predominant at acidic-neutral pH and the other under basic pH conditions.The variation of the absorbance at 372 nm as a function of pH is shown in Figure S16A.The stoichiometry of these two complexes was determined via UV−vis spectrophotometric titrations at different Cu 2+ -to-NO3S molar ratios and pH and via HR-ESI-MS analysis, as reported in Figure S17−S19.The metal-to-ligand ratio resulted 1:1 and the subsequent data treatment indicated their speciation as [CuL] 2+ and [CuL(OH)] + , respectively.The overall formation constants are reported in Table 3 while an example of speciation diagram is shown in Figure 4A.
The stability of the Cu 2+ complexes with TACN-n-Bu was assessed as well to investigate the role of the sulfanyl pendants in metal coordination.The UV−vis spectra recorded at different pH values are shown in Figure 3B, and the variation of the absorbance at λ = 295 nm is reported in Figure S16B.The results obtained are shown in Table 3 and Figure 4B.−25 As shown in Figure 4B and Figure 5, the presence of S in NO3S afforded not only a significant speciation change but also an extraordinary increase in the complex stability (6 orders of magnitude) with respect to TACN-n-Bu, thus demonstrating the key role of the sulfur donors in Cu 2+ stabilization.
The pCu 2+ calculation also allowed us to assess the effect of decreasing the ring size and number of N donors on the complex stability.When the performances of all of the pure Scontaining analogues are compared, the stability ranks in the following order: DO4S ≥ TRI4S > TE4S ≅ NO3S ≫ TACD3S (Figure 5).Although NO3S is not the chelator with the highest stability of the series, its pCu 2+ of 14.3 is still high enough to warrant further radiochemical investigations.The Cu 2+ -NO3S complexes are less stable also if compared with the Cu 2+ -NOTA ones (NOTA has the same ring scaffold as NO3S, but sulfanyl arms are replaced by carboxylates; see Figure S1).As for NOTA, a pCu 2+ value of 18.2 can be computed at pH = 7.4.This indicates a preference of Cu 2+ toward carboxylates rather than toward sulfur.However, the decreased stability of the Cu 2+ complexes formed by NO3S, when compared to that of NOTA, should be balanced by an increased stability of its Cu + complexes.Moreover, even if high thermodynamic stability is one of the key properties of a metal complex for in vivo applications, kinetic inertness might become the leading factor that guides its integrity in biological environments (vide infra).
Cu 2+ -NO3S: Solid-State and Solution Structures.The structure of Cu 2+ -NO3S was explored in the solid state by single-crystal X-ray diffraction.A view of the crystal structure of [Cu(NO3S)][Cu(NO 3 ) 4 ] is shown in Figure 6, and selected representative bond distances and angles are gathered in Table 2. Crystal data and refinement details as well as further structural information are provided in Tables S6 and  S8.
In [Cu(NO3S)] 2+ , Cu 2+ is deeply embedded in the cleft formed by the three N donors of the macrocyclic ring and the three S atoms of the pendant chains in a symmetric N 3 S 3 hexacoordinate environment.The copper ion is 1.36 Å above the N3 plane and 1.31 Å below the S3 plane, and the S3 plane is rotated 46.9°clockwise with respect to the N3 plane.The average bond lengths are equal to 2.13 ± 0.03 Å for Cu−N and   1 and 3 or taken from the literature. 24,25.53 ± 0.06 Å for Cu−S.On the other hand, the autonomous [Cu(NO 3 ) 4 ] 2− complex is located 8.3 Å (Cu 1 −Cu 2 distance) from [Cu(NO3S)] 2+ , and Cu 2+ is coordinated by the four nitrate anions.However, in Cu 2+ -DO4S, each Cu 2+ was surrounded by four nitrogens of the macrocyclic ring (average N−Cu 2+ bond distances equal to 2.04 Å) and a nitrate anion in square-pyramidal geometry, while S did not form any bond with the metal center. 24n aqueous solution, the electronic spectrum of [Cu-(NO3S)] 2+ displayed a very intense band centered at λ max = 372 nm (ε 372 nm = 4.0 × 10 3 mol −1 •cm −1 •L calculated from Lambert−Beer's law, ε 372 nm = (3.5 ± 0.1) × 10 3 mol −1 • cm −1 •L computed from data treatment) accompanied by two lessintense UV and visible absorptions at λ = 285 nm (ε 285 nm = 1.4 × 10 3 mol −1 • cm −1 •L from Lambert−Beer's law) and λ = 635 nm (ε 635 nm = 1.6 × 10 2 mol −1 •cm −1 •L from Lambert− Beer's law).While the latter is characteristic of the d−d orbital transition of the metal center, the band at λ = 372 nm can be attributed to a S-to-Cu 2+ ligand-to-metal charge-transfer transition (based on the literature 24 ), thus further pointing out the existence of the S−Cu interaction in solution.Intriguingly, this absorption is shifted toward lower energy with respect to [Cu(DO4S)] 2+ or [Cu(TE4S)] 2+ which in solution possesses a mixed N 4 + N 4 S ax and a pure N 4 S ax coordination sphere, respectively (Figure S20). 24This confirms that NO3S has a structural arrangement around Cu 2+ that differs from that of the other two ligands.
Regarding the adsorption at λ = 285 nm, as shown in Figure S21, the same UV transition can be recognized if the spectra of [Cu(NO3S)] 2+ and its alkyl analogue [Cu(TACN-n-Bu)] 2+ are compared.Therefore, this band can likely be attributed to a N-to-Cu 2+ CT transition since TACN-n-Bu can provide only N donors.This finding further supports the attribution of the band at λ = 372 nm of [Cu(NO3S)] 2+ to a S-to-Cu 2+ transition; consequently, the [Cu(NO3S)] 2+ electronic spec-trum appears as the convolution of the S-to-Cu 2+ and N-to-Cu 2+ transitions.In conclusion, it can be postulated that the hexacoordinate structure detected in the solid state (vide supra), in which the metal ion is encapsulated in N 3 S 3 geometry, is maintained in aqueous solution.
Cu + -NO3S: Evaluation of the Short-and Long-Term Stability.Short-Term Stability.To assess the ability of NO3S to stably bind Cu + , cyclic voltammetry (CV) analysis was executed.The investigation was performed in aqueous solution at pH ranging from 3 to 12.The obtained cyclic voltammograms, reported in Figure 7, show a peak couple attributable to the quasi-reversible one-electron reduction−oxidation process of the Cu 2+ /Cu + redox pair (Table S9).No variation of the voltammetric behavior after multiple reduction−oxidation cycles was observed either with time or with the scan rate (except for the increase in the current intensity, i).This outcome is extremely significant because it proves the ability of NO3S to stabilize both copper oxidation states throughout the investigated pH range on the time scale of the CV experiments.
As illustrated in Figure 7 and Table S9, a pH-dependent variation of the cyclic voltammograms was found.At 4 ≤ pH ≤ 9, the cathodic (E p,c ) and anodic peak potentials (E p,a ) are independent of the proton content of the solution (Figure S22), thus implying that a single Cu 2+ /Cu + pair exists in this pH range.The Cu 2+ species is [Cu(NO3S)] 2+ (vide supra), and we assumed that the Cu + species is [Cu(NO3S)] + as previously observed with the other S-containing polyazamacrocycles. 24,25The standard potential of the [Cu(NO3S)] 2+ / [Cu(NO3S)] + couple was estimated from the peak potentials as E 1/2 = (E pa + E pc )/2.At pH > 9, changes in the voltammetric pattern begin to be observable as the peak potentials shift toward more negative values (Figure S22).This behavior can be explained by taking into consideration the Cu 2+ speciation, according to which the formation of [Cu(NO3S)(OH)] + starts to occur at basic pH (vide supra).
At pH < 4, some variations are also noticeable (Figure 7 and Figure S22).However, pH values lower than 3 were not examined, as free Cu 2+ starts to form.Remarkably, the pH variations do not affect the kinetics of the electron transfer (ET) which was always quite fast, with ΔE p = E pa − E pc values  slightly higher than the canonical 60 mV for Nernstian ET processes, as reported in Table S9.
The stability constant of [Cu(NO3S)] + obtained from the cyclic voltammetric data is reported in Table 3.The computed pCu + is very large (14.0), and it is comparable to that obtained for Cu 2+ .If compared to the previously developed S-containing chelators, the pCu + also followed a trend similar to that already reported for Cu 2+ with DO4S > TRI4S > NO3S > TE4S (Figure 8).No stability is reported in the literature for the Cu + complexes formed by NOTA, but it is expected that they are much less stable than those formed by NO3S due to the wellknown preference of Cu + for soft donors such as sulfur rather than for hard donors such as carboxylates.
As a final consideration, by comparing the standard reduction potential (E 0 ) of [Cu(NO3S)] 2+ at physiological pH (assuming E 0 Cu/NO3S = E 1/2,Cu/NO3S = −0.121V vs SCE) with the threshold E 0 of the common biological reducing agents (E 0 = −0.64V vs SCE), it is readily apparent that it is prone to in vivo reduction. 2However, as demonstrated by CV and bulk electrolysis experiments (vide infra), the cleavage of the complex should be prevented by the capability of NO3S to stabilize both Cu 2+ and Cu + .
Long-Term Stability.To judge the long-term stability of [Cu(NO3S)] + and gain further insight into its solution speciation, bulk electrolysis of Cu 2+ -NO3S solutions was performed at several pH values, followed by the collection of 1 H NMR spectra.The marked spectral variation when compared with that of free NO3S (Figure S23) confirms the ability of the chelator to bind Cu + even during long time intervals.Additionally, the absence of pH-dependent spectral variations (Figure S24) points to the existence of a unique Cu + complex in the investigated pH range.The protons on the Sbound carbons (SCH 3 and SCH 2 ) are equivalent on the NMR time scale since they resonate as a singlet at 2.22 ppm and an asymmetric triplet at 2.87 ppm and experience a downfield shift upon metal binding (Table S10).The latter spectral feature proves the existence of Cu + −S bonds in solution, and their fine structure suggests that all of the S atoms are either statically bound to the metal center or are in rapid exchange during the time scale of the NMR experiment.The N-bound CH 2 of the pendant arms resonates as a single asymmetric triplet at 2.94 ppm, shielded with respect to the free ligand, implying either the concomitant static role of all of the N atoms in the metal binding or their rapid solution exchange.The NCH 2 protons in the macrocyclic backbone are shielded with respect to the free ligand and are split into two quasisymmetric multiplets centered at 2.65 and 2.80 ppm (Figure S23), indicating that the Cu + binding induces the magnetic nonequivalence of the ethylic fragments (which may be attributed to the axial and equatorial orientations of ring protons).The higher electron density experienced by the Nbound CH 2 protons of [Cu(NO3S)] + as compared to NO3S could be justified by considering that, while in the free ligand H + is located solely on the N atoms, in the complex the Cu + ion is concurrently interacting with both N and S donors, thus sharing the +1 charge among all of them.Combining all of these spectral features with the quasireversibility of the cyclic voltammograms, which indicates that the coordination environment around copper is rather unchanged upon the variation of the oxidation state, an average N 3 S 3 coordination environment can be assumed for [Cu(NO3S)] + .
[ 64 Cu]Cu 2+ -NO3S: Radiolabeling and Human Serum Integrity.To assess the ability of NO3S to chelate [ 64 Cu]Cu 2+ under extremely dilute radiochemical conditions, a radiolabeling investigation was conducted to explore the effects of temperature, pH, and chelator concentration on the radiochemical incorporation (RCI).Parallel experiments were performed as well using NODAGA, one of the current gold standards for [ 64 Cu]Cu 2+ chelation endowing a NOTA moiety, for comparison purposes.This ligand was also chosen as it contains the same TACN backbone of NO3S.The RCI was determined using both radio-UHPLC and radio-TLC analysis; a representative HPLC radiochromatogram is reported in Figure S25.
As displayed in Figure 9A, at room temperature and acidic pH (pH = 4.5) NO3S was able to quantitatively incorporate [ 64 Cu]Cu 2+ (RCI > 99%) at an apparent molar activity of up to 2.5 MBq/nmol in 10 min.The RCI progressively decreased when increasing the molar activity (i.e., lowering the chelator  3 or taken from the literature. 24,25organic Chemistry concentration) and dropped to <5% at molar activities >100 MBq/nmol.Although NODAGA achieved quantitative labeling at nearly the same maximum molar activity as did NO3S (i.e., 2.5 MBq/nmol), it demonstrated a lower labeling efficiency (Figure 9A).If compared with DO4S (i.e., our previously best-performing full S-substituted polyazamacrocycle), then NO3S showed superior features of being able to achieve quantitative incorporation at molar activity more than 2-fold higher. 26hen the temperature was increased to 95 °C (Figure 9B), a slight enhancement of the performances of NO3S was observed (e.g., the quantitative labeling shifted from 2.5 MBq/ nmol to around 5 MBq/nmol), and for NODAGA, the effect of the temperature variation on the maximal molar activity was less impacting.However, an improvement in its labeling trend was observed (e.g., the RCI shifted from 25% at RT to 50% at 95 °C at 10 MBq/nmol) (Figure 9A,B).As a result, NO3S still exceeded the performance of NODAGA at this temperature as well.
The change in pH from a mildly acidic to a neutral environment strongly enhanced the [ 64 Cu]Cu 2+ incorporation of NODAGA.Under this condition and at ambient temperature (Figure 9C), NODAGA was able to quantitatively incorporate the radiometal at a 10-fold-higher apparent molar activity than at pH = 4.5 (i.e., 25 MBq/nmol), slightly surpassing the performance of NO3S (quantitative incorporation around 10 MBq/nmol).At 50 MBq/nmol, the RCI with NODAGA was still high (82%) and decreased to <5% at 100 MBq/nmol.Under the same conditions, NO3S exhibited RCIs equal to 39 and 28%.Heating to 95 °C considerably increased the performances of both chelators, obtaining a quantitative RCI at 50 MBq/nmol and nearly identical labeling behavior (Figure 9D).Also at neutral pH, NO3S outperformed the results previously obtained with DO4S since the latter achieved quantitative incorporation at the maximal molar activity of 10 MBq/nmol at both RT and 95 °C. 26he human serum stability of [ 64 Cu][Cu(NO3S)] 2+ was evaluated to assess its integrity in the presence of biologically relevant ligands and metal ions that can respectively transchelate and transmetalate the complex in vivo.The obtained results, compared with those obtained with NODAGA and DO4S (data for the latter were taken from our previous work 26 ), are shown in Figure 10.
[ 64 Cu][Cu(NO3S)] 2+ was demonstrated to be fairly labile in human serum since the percentage of [ 64 Cu]Cu 2+ bound to the chelator was around 60% after 24 h (Figure 10B), while DO4S formed a less-stable complex as its concentration dropped to 20% after the same time (Figure 10A).An even lower integrity was attained when the ring size was increased, passing from DO4S to TRI4S, as detailed in our previous work, attesting to NO3S as the most kinetically inert among all of the Scontaining fully substituted polyazamacrocycles studied so far. 26When the results obtained with NO3S and DO4S are compared, it should be noted that the copper complex formed by DO4S is thermodynamically more stable than that formed by NO3S (Figure 5).However, herein we found that the latter is more kinetically inert in human serum.This enhanced inertness can be attributed to the saturated N 3 S 3 coordination sphere around the metal center afforded by NO3S, which is likely beneficial to slowing down the complex disruption observed with DO4S.
Finally, the state-of-the-art chelator NODAGA afforded a more robust complex than NO3S, maintaining full integrity even at the later investigated time points (Figure 10C), still confirming the preference of Cu 2+ for harder donor atoms such as oxygen.The origin of the lower stability of the [ 64 Cu]Cu 2+ complexes displayed in human serum by our sulfur-containing compounds is difficult to rationalize without dedicated experiments.On the basis of our CV results, we can exclude the occurrence of Cu 2+ /Cu + reduction or other redox processes.Demetalation might be due to transchelation reactions occurring with proteins present in human serum with a high affinity for copper.
NMR. 1 H NMR spectra of free NO3S and TACN-n-Bu (C L = 1.0 × 10 −3 M) were collected at T = 25 °C and I = 0.15 M NaNO 3 at different pH in 90% H 2 O + 10% D 2 O. Small additions (∼μL) of HNO 3 and/or NaOH were used to adjust the pH.The latter was measured as for the potentiometric measurements.Under highly acidic conditions, the pH was computed from the HNO 3 concentration (pH = −logC H + ).An excitation sculpting pulse scheme was used to suppress the water signal. 32tability Constants of Cu 2+ Complexes.UV−vis pH-spectrophotometric titrations were carried out as previously reported by means of the out-of-cell method at T = 25 °C and I = 0.15 M NaNO 3 . 24,25riefly, stock solutions of NO3S or TACN-n-Bu were mixed with Cu(NO 3 ) 2 in independent vials at a 1:1 metal-to-ligand ratio (C Cu 2+ = C L = 1.0 × 10 −4 M).The pH was adjusted as described in the NMR experiments.The vials were sealed and heated to T = 60 °C in a thermostatic bath to ensure complete complexation and then cooled to ambient temperature.The absorption spectra were recorded, and equilibrium was considered to be reached when no variations in either the pH or the electronic spectra were detected over time.
Stoichiometry of Cu 2+ Complexes.The stoichiometry of the Cu 2+ complex at pH = 7.1 was determined as described in our previous works. 24,25Briefly, solutions were prepared in which the metal-toligand ratio was varied at constant pH.To accurately determine the stoichiometry of the Cu 2+ -NO3S complex formed at basic pH (pH = 12), the Job method was applied, since under these conditions the similar absorbances of the complex and the free (excess) Cu 2+ at λ = 280 nm make the previous method unfeasible.In this case, a series of independent solutions at different metal-to-ligand molar ratios were prepared, keeping the sum of their concentrations constant (C Cu 2+ + C NO3S = 2.0 × 10 −4 M).The stoichiometry was determined by plotting the absorbance at the characteristic wavelength as a function of the metal-to-ligand ratio.
Data Processing.−25 All equilibrium constants refer to the overall equilibrium pM m+ + qH + + rL l− ⇌ M p H q L r pm+q−rl , where M = Cu 2+ and L = NO3S or TACN-n-Bu, respectively, and are defined as cumulative formation constants ( X-ray Crystallography.Crystals of NO3S suitable for X-ray diffraction were obtained in CHCl 3 by the addition of NaPF 6 in methanol after 24 h at 0 °C.Crystals of Cu 2+ -NO3S were prepared by mixing Cu(NO 3 ) 2 •3H 2 O (27.8 mg, 0.115 mmol, 1 equiv) dissolved in water (2 mL) with NO3S (40.5 mg, 0.115 mmol, 1 equiv) dissolved in CHCl 3 (2 mL).The solution was reacted for 30 min, filtered, and kept in a freezer for 2 days (−20 °C).The obtained crystals were washed with iced methanol (4 mL) (yield 43.7%).X-ray measurements were conducted at room temperature on a Nicolet P3 instrument using a numerical absorption correction with graphite monochromate Mo Kα radiation as previously described. 24The SHELX 93 crystallographic software package was used. 33The graphical representation and the edition of CIF files were created with Mercury software. 34The structures were deposited with CCDC numbers of 2067485 for [H(NO3S)]PF 6 and 2067484 for [Cu-(NO3S)][Cu(NO 3 ) 4 ].
Cyclic Voltammetry and Electrolysis.Cyclic voltammetry (CV) experiments were carried out as reported in our former works. 24,25riefly, a six-necked cell equipped with three electrodes and connected to an Autolab PGSTAT 302N potentiostat interfaced with NOVA 2.1 software (Metrohm) was used.A glassy-carbon working electrode (WE) fabricated from a 3-mm-diameter rod (Tokai GC-20), a platinum wire as a counter electrode (CE), and a saturated calomel electrode (SCE) as the reference electrode (RE) were employed.All CVs were performed at ambient temperature in aqueous 0.15 M NaNO 3 at C Cu 2+ = C NO3S = 1.0 × 10 −3 M. The pH of the solutions was adjusted with small aliquots (μL) of NaOH and/or HNO 3 solutions.CV with scan rates ranging from 0.005 to 0.2 V/s was recorded in the region from −0.5 to 0.5 V.At this potential range, the solvent with the supporting electrolyte and the free ligand were found to be electroinactive.
Bulk electrolysis of the preformed Cu 2+ -NO3S complexes was carried out in a two-compartment cell.The WE was a large-area glassy carbon, and the CE was a Pt gauze in 0.15 M NaNO 3 , separated from the WE solution by two glass frits (G3).The RE was SCE.The electrolysis was performed at a fixed potential equal to E = −0.35V and monitored by linear scan voltammetry on a rotating disk electrode.The electrolysis was considered to be complete when the cathodic current reached 2% of the initial value.At the end of each electrolysis, the solution was transferred to the NMR tube under an inert atmosphere (Ar).After the transfer, the atmosphere was filled with inert gas. 1 H NMR spectra of the in situ-generated Cu + complexes at different pH values were collected at T = 25 °C as detailed in the previous section.No variations of the 1 H NMR spectra were detected after 2 days (tubes were stored under a normal atmosphere).The stability constant of the Cu + -NO3S complex was obtained as described in our previous work. 24 64 Cu]Cu 2+ Radiolabeling and Human Serum Integrity.Caution! [ 64 Cu]Cu 2+ is a radionuclide that emits ionizing radiation, and it was manipulated in a specif ically designed facility under appropriate safety controls.
Radiolabeling data were collected through the addition of [ 64 Cu]CuCl 2 diluted in 0.05 M HCl (1 MBq, 10 μL) to a solution containing the ligand (20 μL) diluted in sodium acetate buffer (90 μL, pH = 4.5) or sodium phosphate buffer (90 μL, pH = 7).Different apparent molar activities were tested from 0.1 to 1000 MBq/nmol, corresponding to a final ligand concentration ranging from 1.0 × 10 −4 to 1.0 × 10 −9 M. The reaction mixtures were allowed to react for 10 min at ambient temperature or 95 °C.All radiolabeling reactions were repeated at least in triplicate.Radiochemical incorporation (RCI) was determined via radio-UHPLC using the analytical method previously described. 26Alternatively, radio-thin layer chromatography (radio-TLC) on silica gel 60 RP-18 F254S plates with sodium citrate (1 M, pH = 4) as the eluent was employed.Under these conditions, free [ 64 Cu]Cu 2+ migrates with the solvent front (R f = 1) while [ 64 Cu]Cu 2+ complexes remain at the baseline (R f = 0).A Cyclone Plus Storage Phosphor System (PerkinElmer) was used to analyze the radio-TLC plates after their exposure to a super-resolution phosphor screen (type MS, PerkinElmer; Waltham, MA, USA).All of the data were processed with OptiQuant software (version 5.0, PerkinElmer Inc.; Waltham, MA, USA).
Human Serum Integrity.The integrity of [ 64 Cu][Cu(NO3S)] 2+ and [ 64 Cu][Cu(NODAGA)] − (as controls) was assessed over time by incubation of the preformed complexes (prepared using the radiolabeling protocol described above) in human serum at T = 37 °C (1:1 V/V dilution).The radiometal-complex stability was monitored at different time points over the course of 24 h via radio-TLC following the protocol reported for radiolabeling studies.

■ CONCLUSIONS
The biologically triggered reduction of Cu 2+ to Cu + has been postulated as a possible demetalation pathway in 64/67 Cu-based radiopharmaceuticals.To hinder this phenomenon, we have previously developed a family of N-and S-containing macrocycles capable of efficiently accommodating both Inorganic Chemistry oxidation states.Unfortunately, under highly dilute radiochemical conditions, a marked radiometal release was observed in human serum likely because of the partially saturated coordination sphere around the metal center.In this work, we have hypothesized that switching to a smaller macrocyclic backbone could avoid the hitherto observed demetalation by fully encapsulating and saturating the coordination sphere of the copper ion.For this purpose, the new hexadentate macrocyclic ligand NO3S was synthesized by introducing three sulfanyl pendants on a TACN backbone.
While conserving a high thermodynamic stability for both copper oxidation states, NO3S proved to be superior to the previously studied full S-substituted chelators in the radiolabeling performances with [ 64 Cu]Cu 2+ and in the human serum integrity.These findings suggest that a copper ion fully encapsulated in a N 3 S 3 hexacoordinate environment as in [Cu(NO3S)] 2+ could afford a more inert complex in vivo than Cu complexes possessing a N 4 + N 4 S ax or N 4 S ax coordination sphere such as [Cu(DO4S)] 2+ .The results obtained herein prove that the TACN ring is a more promising backbone (with respect to cyclen and to larger rings) to build up new copper Scontaining chelators.The overall lower performances evidenced by NO3S when compared to NODAGA and the lower stability of the Cu 2+ -NO3S complexes when compared to the Cu 2+ -NOTA complexes suggest that the presence of oxygen donors appended on the TACN backbone is beneficial to the formation of stable Cu 2+ complexes, but at least one sulfur atom might likely be essential to stabilizing them upon in vivo reduction to Cu + .Hence, the development and study of hybrid sulfur-carboxylic TACN derivatives might be a useful strategy to fulfill all of the requested demands.
01 d a L represents the completely deprotonated form of the ligand as shown in Figure 1C.The reported uncertainty was obtained by the fitting procedure and represents one standard deviation unit.b From Bianchi et al. (I = 0.1 M KNO 3 , T = 25 °C). 30c Obtained via 1 H NMR. d Obtained via pH potentiometry in 1:1 water/methanol.e Obtained via pH potentiometry.f Not determined due to precipitation of the sparingly soluble, completely deprotonated L.

Figure 5 .
Figure 5.Comparison of the pCu 2+ values for the S-containing ligands investigated in previous works (DO4S, TRI4S, TE4S, and TACD3S) with NO3S and TACN-n-Bu.The reported pCu 2+ values were calculated at C L = 1 × 10 −5 M and C Cu 2+ = 1 × 10 −6 M and pH 7.4 using the stability constants reported in Tables1 and 3or taken from the literature.24,25

Figure 8 .
Figure 8.Comparison of the pCu + values for the S-containing ligands investigated in the previous works (DO4S, TRI4S, and TE4S) with NO3S.The reported pCu + values were calculated at C L = 10 −5 M and C Cu + = 10 −6 M at pH = 7.4 using the stability constants reported in Table3or taken from the literature.24,25

Figure 9 .
Figure 9.Comparison of the [ 64 Cu]Cu 2+ incorporation yields at different molar activities for NO3S and NODAGA at pH = 4.5 and (A) RT and (B) 95 °C and at pH = 7.0 and (C) RT and (D) 95 °C (10 min reaction time).

Figure 10 .
Figure 10.Integrity of (A) [ 64 Cu][Cu(DO4S)] 2+ , (B) [ 64 Cu][Cu-(NO3S)] 2+ , and (C) [ 64 Cu][Cu(NODAGA)] − in human serum.Data reported in (A) were taken from our previous work.26 Figures of other chelators used in copper-based radiopharmaceuticals; schemes of the syntheses of NO3S and TACN-n-Bu; corresponding NMR and ESI-MS spectra; NMR and UV−vis spectra of the free ligands and of the metal−ligand complexes at various pH values (tables and figures) and corresponding fitting lines where applicable; voltammetric data; crystallographic data; and a representative HPLC radio chromatogram (PDF)