DataSheet1.PDF

<p>Due to its 4 carbonic acid groups being available for bioconjugation, the cyclen tetraphosphinate chelator DOTPI, 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetrakis[methylene(2-carboxyethylphosphinic acid)], represents an ideal scaffold for synthesis of tetrameric bioconjugates for labeling with radiolanthanides, to be applied as endoradiotherapeuticals. We optimized a protocol for bio-orthogonal DOTPI conjugation via Cu(I)-catalyzed Huisgen-cycloaddition of terminal azides and alkynes (CuAAC), based on the building block DOTPI(azide)₄. A detailed investigation of kinetic properties of Cu(II)-DOTPI complexes aimed at optimization of removal of DOTPI-bound copper by transchelation. Protonation and equilibrium properties of Ca(II)-, Zn(II), and Cu(II)-complexes of DOTPI and its tetra-cyclohexylamide DOTPI(Chx)₄ (a model for DOTPI conjugates) as well as kinetic inertness (transchelation challenge in the presence of 20 to 40-fold excess of EDTA) were investigated by pH-potentiometry and spectrophotometry. Similar stability constants of Ca^II-, Zn^II, and Cu^II-complexes of DOTPI (logK_(CaL) = 8.65, logK_(ZnL = 15.40, logK_(CuL) = 20.30) and DOTPI(Chx)₄ (logK_(CaL) = 8.99, logK_(ZnL) = 15.13, logK_(CuL) = 20.42) were found. Transchelation of Cu(II)-complexes occurs via proton-assisted dissociation, whereafter released Cu(II) is scavenged by EDTA. The corresponding dissociation rates [k_d = 25 × 10⁻⁷ and 5 × 10⁻⁷ s⁻¹ for Cu(DOTPI) and Cu(DOTPI(Chx)₄), respectively, at pH 4 and 298 K] indicate that conjugation increases the kinetic inertness by a factor of 5. However, demetallation is completed within 4.5 and 7.2 h at pH 2 and 25°C, respectively, indicating that Cu(II) removal after formation of CuAAC can be achieved in an uncomplicated manner by addition of excess H₄EDTA. For proof-of-principle, tetrameric DOTPI conjugates of the prostate-specific membrane antigen (PSMA) targeting motif Lys-urea-Glu (KuE) were synthesized via CuAAC as well as dibenzo-azacyclooctine (DBCO) based, strain-promoted click chemistry (SPAAC), which were labeled with Lu-177 and subsequently evaluated in vitro and in SCID mice bearing subcutaneous LNCaP tumor (PSMA+ human prostate carcinoma) xenografts. High affinities (3.4 and 1.4 nM, respectively) and persistent tumor uptakes (approx. 3.5% 24 h after injection) confirm suitability of DOTPI-based tetramers for application in targeted radionuclide therapy.</p

Data_Sheet_1_Equilibrium Thermodynamics, Formation, and Dissociation Kinetics of Trivalent Iron and Gallium Complexes of Triazacyclononane-Triphosphinate (TRAP) Chelators: Unraveling the Foundations of Highly Selective Ga-68 Labeling.doc

<p>In order to rationalize the influence of Fe^III contamination on labeling with the ⁶⁸Ga eluted from ⁶⁸Ge/⁶⁸Ga-generator, a detailed investigation was carried out on the equilibrium properties, formation and dissociation kinetics of Ga^III- and Fe^III-complexes of 1,4,7-triazacyclononane-1,4,7-tris(methylene[2-carboxyethylphosphinic acid]) (H₆TRAP). The stability and protonation constants of the [Fe(TRAP)]³⁻ complex were determined by pH-potentiometry and spectrophotometry by following the competition reaction between the TRAP ligand and benzhydroxamic acid (0.15 M NaNO₃, 25°C). The formation rates of [Fe(TRAP)] and [Ga(TRAP)] complexes were determined by spectrophotometry and ³¹P-NMR spectroscopy in the pH range 4.5–6.5 in the presence of 5–40 fold H_xTRAP^(x−6) excess (x = 1 and 2, 0.15 M NaNO₃, 25°C). The kinetic inertness of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ was examined by the trans-chelation reactions with 10 to 20-fold excess of H_xHBED^(x−4) ligand by spectrophotometry at 25°C in 0.15 M NaCl (x = 0,1 and 2). The stability constant of [Fe(TRAP)]³⁻ (logK_FeL = 26.7) is very similar to that of [Ga(TRAP)]³⁻ (logK_GaL = 26.2). The rates of ligand exchange reaction of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ with H_xHBED^(x−4) are similar. The reactions take place quite slowly via spontaneous dissociation of [M(TRAP)]³⁻, [M(TRAP)OH]⁴⁻ and [M(TRAP)(OH)₂]⁵⁻ species. Dissociation half-lives (t_1/2) of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ complexes are 1.1 × 10⁵ and 1.4 × 10⁵ h at pH = 7.4 and 25°C. The formation reactions of [Fe(TRAP)]³⁻ and [Ga(TRAP)]³⁻ are also slow due to the formation of the unusually stable monoprotonated [^*M(HTRAP)]²⁻ intermediates [^*logK_Ga(HL) = 10.4 and ^*logK_Fe(HL) = 9.9], which are much more stable than the [^*Ga(HNOTA)]⁺ intermediate [^*logK_Ga(HL) = 4.2]. Deprotonation and transformation of the monoprotonated [^*M(HTRAP)]²⁻ intermediates into the final complex occur via OH⁻-assisted reactions. Rate constants (k_OH) characterizing the OH⁻-driven deprotonation and transformation of [^* Ga(HTRAP)]²⁻ and [^*Fe(HTRAP)]²⁻ intermediates are 1.4 × 10⁵ M⁻¹s⁻¹ and 3.4 × 10⁴ M⁻¹s⁻¹, respectively. In conclusion, the equilibrium and kinetic properties of [Fe(TRAP)] and [Ga(TRAP)] complexes are remarkably similar due to the close physico-chemical properties of Fe^III and Ga^III-ions. However, a slightly faster formation of [Ga(TRAP)] over [Fe(TRAP)] provides a rationale for a previously observed, selective complexation of ⁶⁸Ga^III in presence of excess Fe^III.</p