Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

[TcII(NO)(trifluoroacetate)4F]2- - synthesis and reactions.

Fluoridotetrakis(trifluoroacetato)nitrosyltechnetate(ii) was prepared by the dissolution of Cs2[Tc(NO)F5] in trifluoroacetic acid and addition of (NBu4)F·3H2O. The compound crystallizes as a mixed Cs+/NBu4+ salt in the form of green crystals. Unlike the [Tc(NO)F5]2- salts, the product is soluble in organic solvents and can be used as a precursor for ongoing ligand exchange procedures with organic ligands. The corresponding reactions with triphenylphosphine (PPh3), 1,2-bis(diphenylphosphino)ethane (DPPE) or pyridyldiphenylphosphine (pyPPh2) give technetium(i) complexes of the compositions Cs[Tc(NO)(PPh3)2(CF3COO)2F], [Tc(NO)(DPPE)2(OOCCF3)](PF6) and [Tc(NO)(κN,P-pyPPh2)(κP-pyPPh2)(CF3COO)2]. The products were studied spectroscopically and by X-ray diffraction. The 99Tc NMR resonances of the novel Tc(i) nitrosyls appear between -627 and +952 ppm, which is at a remarkably high field and in the range where normally the signals of Tc(iii) compounds are observed.

Technetium complexes with arylselenolato and aryltellurolato ligands.

Reactions of (NBu4)[TcOCl4] or [TcCl3(PPh3)2(CH3CN)] with in situ-prepared lithium arylselenolates and -tellurolates give (NBu4)[TcVO(ArE)4] (E = Se, Te; Ar = phenyl) and [TcIII(ArE)3(PPh3)(CH3CN)] (E = Se, Te; Ar = phenyl, 2,6-Me2phenyl, mesityl) complexes, respectively. The products contain square-pyramidal (TcV compounds) and trigonal bipyramidal (TcIII complexes) coordinated technetium atoms. Density functional theory calculations indicate that the Tc-chalcogen bonds in the TcIII compounds have a greater bond order than those in the TcV compounds.

Thiourea derivatives as chelating agents for bioconjugation of rhenium and technetium.

Potential tetradentate thiocarbamoylbenzamidine derivatives H4L have been synthesized from the corresponding benzimidoyl chlorides and triglycine. They are suitable chelating agents for the oxidotechnetium(v) and oxidorhenium(v) cores and form stable, neutral [MO(HL)] complexes with an equatorial SN3 coordination sphere and an additional, uncoordinated carboxylic group, which can be used for bioconjugation. Representatives of the rhenium and 99Tc products have been isolated and analyzed with spectroscopic methods and X-ray diffraction. Bioconjugates of these complexes with angiotensin-II have been synthesized and structurally characterized. Analogous 99mTc complexes have been produced and tested in vitro and in vivo. The experiments confirm a considerable stability for the [99mTc(HL)] product as well as for its bioconjugate and recommend this class of compounds for further bioconjugation studies towards clinical applications.

Organometallic gold(iii) complexes with hybrid SNS-donating thiosemicarbazone ligands: cytotoxicity and anti-Trypanosoma cruzi activity.

Stable organogold(iii) compounds of the composition [AuIII(Hdamp)(L1)]Cl are formed from reactions of [AuCl2(damp)] with H2L1 (damp- = dimethylaminomethylphenyl; H2L1 = N'-(diethylcarbamothioyl)benzimidothiosemicarbazides). The cationic complexes can be neutralized by reactions with weak bases under the formation of [AuIII(damp)(L1)] compounds. The structures of the products show interesting features like relatively short AuH contacts between the methylene protons of the Hdamp ligand and the gold(iii) ions. Preliminary biological studies on the uncoordinated compounds H2L1 and their gold complexes indicate considerable cytotoxicity for the [AuIII(Hdamp)(L1)]Cl complexes against MCF-7 cells. The in vitro trypanocidal activity was evaluated against the intracellular form of Trypanosoma cruzi. The organometallic complexes display a remarkable activity, which is dependent on the alkyl substituents of the thiosemicarbazone building blocks of the ligands. One representative of the cationic [AuIII(Hdamp)(L1)]Cl complexes, where H2L1 contains a dimethylthiosemicarbazide building block, shows a trypanocidal activity against the intracellular amastigote form in the same order of magnitude as that of the standard drug benznidazole. Furthermore, no appreciable toxicity to mice spleen cells is observed for this compound resulting in a therapeutic index of about 30, which strongly recommends it as a promising candidate for the development of a future antiparasitic drug.

{Tc(NO)(Cp)(PPh3)}(+) - a novel technetium(i) core.

Reactions between [Tc(I)(NO)X2(PPh3)2(CH3CN)] complexes (X = Cl, Br) and KCp form the pseudotetrahedral organotechnetium compounds [Tc(I)(NO)(Cp)(PPh3)X]. The halide ligands can readily be replaced by other halides or organometallic ligands giving access to a novel family of technetium(i) compounds with the robust {Tc(NO)(Cp)(PPh3)}(+) core.

2,6-Dipicolinoylbis(N,N-dialkylthioureas) as versatile building blocks for oligo- and polynuclear architectures.

Similar reactions of 2,6-dipicolinoylbis(N,N-diethylthiourea) (H2L(a)) with: (i) Ni(NO3)2·6H2O, (ii) a mixture of Ni(NO3)2·6H2O and AgNO3, (iii) a mixture of Ni(OAc)2·4H2O and PrCl3·7H2O and (iv) a mixture of Ni(OAc)2·4H2O and BaCl2·2H2O give the binuclear complex [Ni2(L(a))2(MeOH)(H2O)], the polymeric compound [NiAg2(L(a))2]∞, and the heterobimetallic complexes [Ni2Pr(L(a))2(OAc)3] and [Ni2Ba(L(a))3], respectively. The obtained assemblies can be used for the build up of supramolecular polymers by means of weak and medium intermolecular interactions. Two prototype examples of such compounds, which are derived from the trinuclear complexes of the types [MLn(III)(L)2(OAc)3] and [MBa(L)3], are described with the compounds {[CuDy(III)(L(a))2(p-O2C-C6H4-CO2)(MeOH)4]Cl}∞ and [MnBa(MeOH)(L(b))3]∞, H2L(b) = 2,6-dipicolinoylbis(N,N-morpholinoylthiourea).

Organometallic 99mTc-aquaion labels peptide to an unprecedented high specific activity.

UNLABELLED: A new peptide labeling method that uses the organometallic aquaion [99mTc(H2O)3(CO)3]+ has been developed. METHODS: A selection of amino acids was labeled at different concentrations with the organometallic aquaion, and the labeling yield was determined by high-performance liquid chromatography. This investigation has shown histidine to be a very potent ligand, with specific activities of up to 6 TBq/micromol (160 Ci/micromol) ligand. Histidine derivatives have been coupled to neurotensin(8-13) (NT[8-13]) and have been labeled with the aquaion, resulting in high specific activities with (N(alpha)-histidinyl)acetic acid-NT(8-13) similar to those with histidine. RESULTS: Histidine derivatives of NT(8-13) labeled using this approach fully retained their receptor affinity, showing KD values of all investigated NT analogs below 1 nmol/L on colon carcinoma HT29 cells. Biodistrbution experiments in BALB/c mice showed complete clearance of (N(alpha)-histidinyl)acetic acid-NT(8-13) from the blood after 24 h and no unwanted accumulation in any tissue. CONCLUSION: The novel labeling method using the organometallic 99mTc-aquaion combines the advantage of highest specific activities with minimal functionalization of proteins and peptides under retention of biologic affinity.

Technetium mixed-ligand complexes containing bidentate phosphines and monodentate thiol ligands. Preparation, in vitro data and detection of a certain heart affinity.

TcO-4 is reduced by a mixture of phosphine and thiol ligands to yield cationic complexes in which both ligands are coordinated. Polar and lipophilic properties of the products can easily be controlled by variation of either of the ligands. The yields are always high. Potential heart affinity of the compounds was screened by means of the isolated perfused rat heart. Some of the complexes show significant heart uptake and good retention in the myocardial tissue.

Gold complexes with potentially tri- and tetradentate phosphinothiolate ligands.

Reactions of [Au(PPh3)Cl], (Bu4N)[AuCl4] and the organometallic gold complex [Au(damp-C1,N)Cl2] (damp- = 2-(N,N-dimethylaminomethyl)phenyl) with the potentially tri- and tetradentate proligands PhP(C6H3-SH-2-R-3)2 (H2L1a, R = SiMe3; H2L1b, R = H) and P(C6H4-SH-2)3 (H3L2) result in the formation of mono- or dinuclear gold complexes depending on the precursor used. Monomeric complexes of the type [AuL1Cl] are formed upon the reaction with [Au(damp-C1,N)Cl2], but small amounts of dinuclear [AuL1]2 complexes with gold in two different oxidation states, +1 and +3, have been isolated as side-products. The dinuclear compounds are obtained in better yields from [AuCl4]-. A dinuclear complex having two Au(III) centers can be isolated from the reaction of [Au(PPh3)Cl] with H3L2, whereas from the reaction with H2L1b the mononuclear [Au(Ph3P)HL1b] is obtained, which contains a three-coordinate gold atom. Comparatively short gold-gold distances have been found in the dinuclear complexes (2.978(2) and 3.434(1) A). They are indicative of weak gold-gold interactions, which is unusual for gold(III).

Influence of the denticity of ligand systems on the in vitro and in vivo behavior of (99m)Tc(I)-tricarbonyl complexes: a hint for the future functionalization of biomolecules.

Functionalization of biologically relevant molecules for the labeling with the novel fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) precursor has gained considerable attention recently. Therefore, we tested seven different tridentate (histidine L(1)(), iminodiacetic acid L(2)(), N-2-picolylamineacetic acid L(3)(), N, N-2-picolylaminediacetic acid L(4)()) and bidentate (histamine L(5)(), 2-picolinic acid L(6)(), 2,4-dipicolinic acid L(7)()) ligand systems, with the potential to be bifunctionalized and attached to a biomolecule. The ligands allowed mild radiolabeling conditions with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+) (30 min, 75 degrees C). The ligand concentrations necessary to obtain yields of >95% of the corresponding organometallic complexes 1-7 ranged from 10(-)(6) to 10(-)(4) M. Complexes of the general formula "fac-[(99m)TcL(CO)(3)]" (L = tridentate ligand) and "fac-[(99m)Tc(OH(2))L'(CO)(3)]" (L' = bidentate ligand), respectively, were produced. Challenge studies with cysteine and histidine revealed significant displacement of the ligands in complexes 5-7 but only little exchange with complexes 1-4 after 24 h at 37 degrees C in PBS buffer. However, no decomposition to (99m)TcO(4)(-) was observed under these conditions. All complexes showed a hydrophilic character (log P(o/w) values ranging from -2.12 to 0.32). Time-dependent FPLC analyses of compounds 1-7 incubated in human plasma at 37 degrees C showed again no decomposition to (99m)TcO(4)(-) after 24 h at 37 degrees C. However, the complexes with bidentate ligands (5-7) became almost completely protein bound after 60 min, whereas the complexes with tridentate coordinated ligands (1-4) showed no reaction with serum proteins. The compounds were tested for their in vivo stability and the biodistribution characteristics in BALB/c mice. The complexes with tridentate coordinated ligand systems (1-4) revealed generally a good and fast clearance from all organs and tissues. On the other hand, the complexes with only bidentate coordinated ligands (5-7) showed a significantly higher retention of activity in the liver, the kidneys, and the blood pool. Detailed radiometric analyses of murine plasma samples, 30 min p.i. of complex fac-[(99m)TcL(1)(CO)(3)], 1, revealed almost no reaction of the radioactive complex with the plasma proteins. By contrast, in plasma samples of mice, which were injected with complex fac-[(99m)Tc(OH(2))L(5)(CO)(3)](+), 5, the entire radioactivity coeluded with the proteins. On the basis of these in vitro and in vivo experiments, it appears that functionalization of biomolecules with tridentate-chelating ligand systems is preferable for the labeling with fac-[(99m)Tc(OH(2))(3)(CO)(3)](+), since this will presumably result in radioactive bioconjugates with better pharmacokinetic profiles.

Derivatives of 1,3,5-triamino-1,3,5-trideoxy-cis-inositol as versatile pentadentate ligands for protein labeling with Re-186/188. prelabeling, biodistribution, and X-ray structural studies.

The pentadentate H3bhci [1,3,5-trideoxy-1, 3-bis((2-hydroxybenzyl)amino)-cis-inositol] and its bifunctionalized analogue H3bhci-glu-H [1,3,5-trideoxy-1, 3-bis((2-hydroxybenzyl)amino)-5-glutaramido-cis-inositol] were synthesized, and their coordination chemistry was investigated with inactive rhenium, with no carrier added Re-188 and with carrier added Re-186. The neutral Re(V) complexes [ReO(bhci)] and [ReO(bhci-glu-H)] are formed in good yields starting from [ReOCl3(P(C6H5)3)2] or in quantitative yield directly from [186/188ReO4]- in aqueous solution by reduction with Sn(II) or Sn(0). The X-ray structures of [ReO(bhci)] and [ReO(bhci-glu-H)] were elucidated revealing pentadentate "side on" coordination of the ligands to the "Re=O" core. The basic cyclohexane frame adopts a chair form in the case of [ReO(bhci)] and a twisted boat form in the case of [ReO(bhci-glu-H)]. [ReO(bhci)] crystallizes in the monoclinic space group C2/c with a = 27.425(3), b = 14.185(1), c = 19.047(2) A, and beta = 103.64(2) degrees and [ReO(bhci-glu-H)] in the monoclinic space group P21/c with a = 13.056(3), b = 10.180(1), c = 22.378(5) A, and beta = 98.205(9) degrees. Both 188Re complexes are stable in human serum for at least 3 days without decomposition. After injection into mice, [ReO(bhci-glu)]- is readily excreted through the intestines, while [ReO(bhci)] is excreted by intestines, liver, and the kidneys. TLC investigations of the urine showed exclusively the complexes [ReO(bhci-glu-H)] and [ReO(bhci)], respectively, and no decomposition products. For derivatization of antibodies, the carboxylic group of [ReO(bhci-glu-H)] was activated with N-hydroxysuccinimide, which required unusually vigorous reaction conditions (heating). The anti colon cancer antibody mAb-35 [IgG and F(ab')2 fragment] was labeled with [186/188ReO(bhci-glu)] to a specific activity of up to 1.5 mCi/mg (55 MBq/mg) with full retention of immunoreactivity. Labeling yields followed pseudo-first-order kinetics in antibody concentration with the ratio of rates between aminolysis and hydrolysis being about 2. Biodistributions of 186Re-labeled intact mAb-35 as well as of its F(ab')2 fragment in tumor-bearing nude mice revealed good uptake by the tumor with only low accumulation of radioactivity in normal tissue.

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules.

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.

Synthesis and biological studies of neutral technetium (V) complexes containing NNOS donor sets.

Complexation of ligands containing an N3S donor set has been affected with [99mTc]. These are part of a ligand series of analogous structures which exhibit similar chemistry and potentially interesting biology. The complexes which have been characterized with [99Tc] as [TcOL] are neutral and lipophilic and their biological behaviour has been assessed in rats. After HPLC purification of the no-carrier added preparation, brain uptake of the tracers was greater than 1% at 15 min p.i. Muscle activity was significant with slow blood clearance.