"[M(CO)3]+" (M = Re, Tc) Moiety for the Labeling of Biomolecules

Radiochimica Acta 79, 99-103 (1997) © R. Oldenbourg Verlag, München 1997 + Potential of the "[M(CO)3] " (M = Re, Tc) Moiety for the Labeling of Biomolecules* By R. Alberto1·4**, R. Schibli1, U. Abram3, A. Egli1, F. F. (Russ) Knapp2 and P. A. Schubiger1 1 Paul Scherrer Institute, Division of Radiopharmacy, CH-5232 Villigen/Switzerland 2 Oak Ridge National Laboratory, Health Sciences Research Division, Oak Ridge TN 37831 3 University of Tübingen, Institute of Inorganic Chemistry, D-72076 Tübingen/Germany 4 Universität Zürich, Institute of Inorganic Chemistry, CH-8057 Zürich/Switzerland (Received December 10, 1997; accepted in revised form May 22, 1997) Rhenium / Technetium / Organometallics / their general application. Since the most convenient Direct labeling / Proteins labeling procedures are with radioisotopes such as mIn or 90Y, basically present in form of their aqua- ions, we are interested in rhenium or technetium ana- Summary logues bearing, a defined number of substitution labile 2 ,88 water molecules in the first coordination sphere. It is The synthesis of [MX3(CO)3] - (M = Re, Re, "Tc) directly from the corresponding permetallates is described. Intermediates of further interest that ligand replacement on such a and byproducts have been isolated and characterized. Prelimi- precursor can be performed with simple chelators, re- nary direct labeling studies at r.t. revealed a slow but irreversible 188 + sulting in complexes with high kinetic rather than in incorporation of "[ Re(CO)3] " into intact antibodies. The best such of high thermodynamic stability. The latter point yield was 40% after 20 h with 0.8 mg/ml of intact antibody. To in particular makes clear that such a metal center must elucidate possible coordination sites in proteins, the synthesis 6 3 and formation of model complexes has been investigated by have a d (or d ) electronic configuration and, thus, means of Ή NMR spectroscopy and X-ray structure analysis. A its complexes belonging to the class or organometallic high tendency for imidazole (im) coordination has been found compounds of the group 7 transition elements. and is examplified in the model complexes [ReBr(histamine)- (CO)3] and [Re2^-OH)2(im)2(CO)6], which structures will be presented. Additionally, complexation with the macrocyclic thio- + ether ligand [20-aneS6] was studied in order to establish a post- The "/ac-[M(CO)3] " moiety, synthesis labeling protocol with this type of chelator. The structure of and properties [(20-aneS6-OH){Tc(CO) } ]2+ will be presented. 3 2 Little is known about the behaviour of organometallic Re- or Tc-complexes in water. Among the numerous compounds in the +1 valency of these two elements Introduction only a few seem to fulfill the above-mentioned prop- A wide variety of methods for the labeling of proteins erties. In the earlier literature, the complex [NEt4]2 and small biologically active molecules with metallic [ReBr3(CO)3] has been described, but almost no fur- radionuclides have been described [1—3]. The syn- ther investigations have been performed with this start- thesis of metal complexes in general or organometallic ing material [12—14]. The reaction conditions for the complexes in particular, which are able to act as spec- preparation of this important synthon were optimized ific probes in biological systems, is one of the chal- [15], but the method is not suitable for routine appli- lenging topics in coordination chemistry directed to- cation since the synthesis demands high CO pressure ward application [4, 5]. The key role of Afunctional and high temperature. We therefore developed a syn- ligands in that respect is well known and has been thesis starting directly from [NBu4][M04] under at- subject of several review articles discussing advan- mospheric CO pressure. Details of the procedure are tages and disadvantages of the different protocols [6- given in Scheme 1. 8]. A broad variety of Afunctional chelators mainly based on open chained tetradentate N,S systems such [NBU4]X, THF, CO 1atm as N2S2 (diamide dimercaptide) and N3S (triamide [Re04r mercaptide) providing high thermodynamic stability RT, 1-2h, BHä'THF ~ [ReX3(CO)3] " have been prepared and successfully applied with macroscopic > 90%; X = CI,Br !88/i86Re or 99mTc for ^g (p0St_ or pre_) labeling of antibodies [9—11]. Disadvantages concerning the synthesis, the handling (tendency to be oxidized) and the [188Re0 ]' THF,c01atm 188 4 [ ReX3(CO)3f availability of some of these ligands limit (sometimes) RT, 15min, BH3*THF no-carrler-added 30-90% * Presented at the Symposium on Radiochemistry and Radio- 2 immunotherapy, Orlando, Florida, USA, August 1996. Scheme 1. Synthesis of [MX3(CO)3] ~ under ambient con- ** Author for correspondence. ditions. 100 R. Alberto, R. Schibli, U. Abram, A. Egli, F. F. (Russ) Knapp and P. A. Schubiger The synthesis can be performed on the macroscopic as well as on the microscopic level with gener- 188 186 ator produced Re or with Re from high flux reac- 3CC tors. However, under these conditions, two byproducts were detected by TLC analysis of the final organic reaction solution. To elucidate their nature, the reaction was performed with "Tc. "Tc has been chosen ST3 since it allows application of the same radioanalytical methods as for 188/186Re and, thus, a direct instant comparison. Although the chemistry of the two elements - 10-· is sometimes different, the carbonylation revealed the same byproducts in detectable amounts when per- forming the reaction in the presence of [X]", but in quantitative yield in the absence of any [X]". The two incubation time [h] compounds were identified as the mixed hydrido- Fig. 1. Direct labeling yield as a function of antibody concen- carbonyl complexes [TcH(CO)4]3 and [NBu4][Tc3H4- (CO) ], The structure of the former byproduct has tration: a) 800 μg b) 400 μg c) 300 μg and d) 200 μg/ml, 0.01 Μ 12 phosphate buffer at pH = 7.4 and 25 °C. been elucidated by X-ray structure analysis [16]. A complete reaction scheme for the quantitative syn- 2 thesis of [MX3(CO)3] ~ is given in Scheme 2. The two ing reagent such as iminothiolane or by reduction of intermediates were, after completion of the synthesis, 2 the disulfide bridges has recently been described. It transformed to [MX3(CO)3] ~ by addition of base/acid. could be shown that incorporation takes place but is Subsequently, the organic solvent was evaporated and slow [18—20]. This corresponds to the experience, that then replaced by a buffer medium of choice. rhenium reacts in general much slower than technetium. In comparison, qualitative labeling experi-' nbu4ci/Thf .99. THF/CO ments were performed to investigate the direct interac- T0O4]· 188 + co/BH,. ex. BH3.THF tion of [ Re(OH2)3(CO)3] with intact antibodies. The labeling process consisted in mixing the buffer solution with a underivatized protein of choice. Direct 188 + labeling of proteins with [ Re(OH2)3(CO)3] fol- [Tc2CI3(CO)6r PcHiCOWd + nWXW lowed the same trend as observed with the reduced ex. ones but with the decisive advantage that the protein i. NaOH H20 NEUCI did not have to be derivatized prior to labeling. As in ii. HCl the former case, incorporation was found to be slow. :2 - H20 After purification of the labeled protein by means of [TcCI3(CO)3] 1 [Tc(OH2)3(CO)3r I size exclusion chromatography, stability tests were performed in PBS and blood serum at 37 °C. TLC Scheme 2. Complete reaction scheme and characterized interme- analysis (to detect small decomposition products) and diates. FPLC (trans chelation to serum proteins) revealed no significant loss of [188Re(OH ) (CO) ]+ over the inves- As described elsewhere [17] the three [X]~ are 2 3 3 tigated time scale (24-48 h). The labeling yield de- readily substituted by water (or other solvent) mole- pended on the phosphate buffer concentration, which cules, thus, the "semi aqua-ion" [M(OH ) (CO) ]+ 2 3 3 is reasonable taking the weak interaction of the formed. This compound is water and air stable and [H P0 ]"/[HP0 ]2~ anions with the cationic carbonyl fulfills the stability and coordination requirements 2 4 4 complex into account. Furthermore, the labeling yield pointed out above (Scheme 3). was a function of protein concentration. The highest yield in the investigated range was about 40% incorpo- H,0 ration after 20 h at r.t. (Fig. 1). The good linearity ,co substitution (yield vs. protein concentration) supports a pseudo labile < first-order kinetic model. Also in agreement with this Re® h shielded; r substitution stable model is the fact that the labeling yield was found to H 0' CO 2 depend only slightly on the absolute amount of radio- CO activity (Fig. 2). Increasing the amount of 188Re left + Scheme 3. Coordinative properties of [M(OH2)3(CO)3] . the yield almost untouched but increased the specific activity of the radioimmunoconjugate by the corresponding factor. In the investigated range the highest specific activity achieved was about 130 MBq/mg of 188 + Direct labeling of proteins with [ Re(OH2)3(CO)3] protein. Biological in vitro activity was verified to be 188 retained in three different experiments with the method Direct labeling of proteins with Re in parallel to introduced by Lindmo et al. [21]. 99mTc by introduction of thiol groups with a derivatiz- + Potential of the "[M(CO)3] " (Μ = Re, Tc) Moiety for the Labeling of Biomolecules 101 • 500 μρ AK-47 + + [Re(sol)3(CO)3] [Re(sol)2(im)CO)3] II 200 μρ AK-47 Β ο | + im £ 30 £ [ReBr(im)2(CO)3] JÜ- [Re(sol)(im)2(CO)3f ο Scheme 4. Stepwise coordination of im to the [Re(HOCH ) - J> 20 3 3 φ (CO)3r.

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