Radiosynthesis of New [90Y]-DOTA-Based Maleimide Reagents

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Bioconjugate Chem. 2009, 20, 1340–1348

Radiosynthesis of New [90Y]-DOTA-Based Maleimide Reagents Suitable for the Prelabeling of Thiol-Bearing L-Oligonucleotides and Peptides Joern Schlesinger,† Cindy Fischer,† Inge Koezle,† Stefan Vonhoff,‡ Sven Klussmann,‡ Ralf Bergmann,† Hans-Jurgen Pietzsch,*,† and Joerg Steinbach† Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, Germany, and NOXXON Pharma AG, Berlin, Germany. Received March 4, 2009; Revised Manuscript Received June 8, 2009

We describe the radiosynthesis of two new [90Y]-DOTA-based maleimide reagents, suitable for the mild radiolabeling of L-RNAs and peptides modified with thiol-bearing linkers. The synthesis procedure of both maleimide-bearing 90Y complexes, [{(2S)-2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl}tetraacetato][90Y]yttrate(1-)([90Y]3) and [{(2S)-2-(4-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetrayl]tetraacetato}[90Y]yttrate(1-)([90Y]4), was optimized in terms of an easy purification method via solid-phase extraction (SPE). Application as well as reactivity of both maleimide reagents were initially evaluated by the prelabeling of glutathione (GSH) and a thiol-modified 12mer L-RNA as model substances. In comparison to the N-aryl maleimide-bearing complex [90Y]3, N-alkyl maleimide-bearing complex [90Y]4 showed an increased hydrolytic stability at pH g 7. A slightly higher reactivity was found for [90Y]3 by prelabeling of 0.1 and 1 µg glutathione, respectively, in phosphate buffer (pH 7.2) at room temperature. In terms of very high radiochemical yields, the direct radiolabeling of DOTA-L-RNA conjugate with [90Y]YCl3 proved to be more suitable than the prelabeling of the thiol-modified 12mer L-RNA derivative with [90Y]4.

INTRODUCTION Radioisotopes of yttrium and the lanthanides are increasingly becoming an important component of several medical applications, e.g., for molecular imaging and targeted radionuclide therapy of cancer diseases (1, 2). The trivalent radiometals are routinely conjugated to targeting molecules via the bifunctional chelator (BFC)1approach. A frequently utilized chelator for these radiometals is (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA). DOTA is known to form thermodynamically stable complexes with lanthanides and yttrium isotopes, and the corresponding radiometal complexes exhibit in many cases high kinetic inertness in ViVo (3). However, the slow complexation rates of trivalent radiometals with DOTA necessitate elevated temperatures (up to 80 °C) to achieve sufficient radiochemical yields (RCYs) (4). This may be a drawback, especially for the radiolabeling of thermally sensitive molecules, such as biopharmaceuticals (e.g., DOTA-modified antibodies or antibody fragments). On this account, previous works already focused on optimization of radiolabeling conditions to achieve high RCY without compromising the biological activity (5, 6). A promising alternative to the “direct” radiolabeling of DOTA(bio)conjugates (“postlabeling”) is the use of so-called “prelabeling” * Author to whom correspondence should be addressed. H.-J. Pietzsch, Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, PF 51 01 19, 01314 Dresden, Germany. ([email protected]). † Forschungszentrum Dresden-Rossendorf. ‡ NOXXON Pharma AG. 1 Abbreviations: BFC, bifunctional chelator; DIBD, (2S)-2-(4-{[4(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-DOTA; DID, (2S)-2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl)DOTA; DOTA, (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid; DTPA, diethylene triamine pentaacetic acid; EDTA, ethylene diamine tetraacetic acid; ESA, effective specific activity; GMBS, N-(γmaleimidobutyryloxy)succinimide ester; GSH, glutathione; RCP, radiochemical purity; RCY, radiochemical yield; SAP, square antiprism; TBE, Tris/Borate/EDTA; TEAA, triethylammonium acetate; TSAP, twisted square antiprism.

agents. Such a reagent is constituted of the radiometal complex and a reactive coupling moiety, e.g., a bromoacetyl or isothiocyanato group. In such a way, primary amine groups of thermally sensitive molecules can be radiolabeled under mild conditions (e37 °C) by reaction with the prelabeling agent. However, until now only a few radiometal-DOTA-based prelabeling agents were described (see Figure 1) (7-11). Another attractive coupling moiety of such a prelabeling agent could be the maleimide group. Maleimide reagents readily react with thiols (e.g., from cysteine residues of proteins or peptides) under physiological conditions (37 °C, pH 7.4) to form thioether bonds (12). In this work, we present the synthesis of two novel [90Y]-DOTAbased maleimide reagents (see Figure 2), suitable for a mild radiolabeling of a peptide or a thiol-modified L-RNA. An L-RNA is a mirror-image oligonucleotide constructed of L-configured riboses and characterized by an extraordinarily high stability against enzymatic degradation (13). By using 90Y-labeled L-RNAs as molecular probes, we pursue a strategy for an improved radioimmunotherapy of cancer using pretargeting methods (14, 15). However, the in ViVo application of the 90Y-labeled L-RNA requires a high effective specific activity (ESA), to prevent unlabeled L-RNA molecules from saturating the complementary L-oligonucleotide strands, which are present in the target tissue. Thus, both prelabeling and postlabeling of an L-RNA have been performed, to compare the two approaches with respect to the achievable RCYs and ESAs. The purpose of this work was to enhance RCY and ESA for 90Ylabeled L-RNAs, by using prelabeling methods. Therefore, application as well as reactivity of both maleimide reagents were initially evaluated by the labeling of glutathione (GSH) as a model substance.

EXPERIMENTAL PROCEDURES Materials. L-RNA [sequence: 5′-(1-hydroxy-7,8-dithia-tetradecyl) UGACUGACUGAC-3′, MW 4124] was synthesized at NOXXON Pharma AG (Germany). Synthesis of the DOTAmodified 12mer L-RNA 10 was described elsewhere (16). Glutathione (reduced) was from Fluka (Germany). (S)-2-(4-

10.1021/bc900095k CCC: $40.75  2009 American Chemical Society Published on Web 06/25/2009

[90Y]-DOTA-Based Maleimide Reagents

Figure 1. Chemical structures of previously described prelabeling agents based on radiometal-DOTA complexes: [88Y]-DOTA-(4-bromoacetamidobenzyl) ([88Y]-BAD) (7), [177Lu]-1,4,7,10-tetraaza-N-(1-carboxy3-(4-isothiocyanotophenyl)propyl)-N′,N′′,N′′-tris(acetic acid)cyclododecane ([177Lu]-DOTA-PA-NCS) (8), [225Ac]-DOTA-(2-(4-isothiocyanatobenzyl)) ([225Ac]-DOTA-Bn-NCS) (9), [90Y]-DOTA-Gly3-L-(4isothiocyanato)-phenylalanine amide ([90Y]-DOTA-peptide-NCS) (10, 11).

Figure 2. Chemical structures of the new [90Y]-DOTA-based maleimide reagents [90Y]3 and [90Y]4.

Aminobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (1) was purchased from Macrocyclics (Dallas, TX,

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USA). The radionuclide 90Y was purchased as [90Y]YCl3 in 0.04 M HCl (500 µL) from QSA Global GmbH (Germany) or Polatom (Poland). Measurements of 90Y were done in the 90Y channel of a dose calibrator ISOMED 2000 (Nuklear-MedizintechnikDresdenGmbH,Germany)bymeasuringthebremsstrahlung. Reactions and incubations were performed in 1.5 mL DNA low-binding tubes (Eppendorf, Germany) using a Thermomixer Comfort (Eppendorf, Germany). For evaporation of small sample volumes, a Concentrator 5301 (Eppendorf, Germany) was used. Lyophilization was carried out using an Alpha 2-4 LSC (Christ, Germany). L-RNA concentrations were determined via measurement of the UV absorbance at 260 nm using an Eppendorf (Germany) BIOPhotometer. Measurements of the pH value in small volumes were performed using the pH-sensor Biotrode (Metrohm, Germany). Chromatographic Purification and Analysis. HPLC analyses were performed with a WellChrom 2700 system (Knauer, Germany), coupled with the radioactivity detector VA-S-968 (Robotron, Germany). HPLC purifications were also performed with a WellChrom K-2201 system. The HPLC columns were as follows: (A) Aqua C18 column (Phenomenex, USA) 250 × 4.6 mm i.d., 5 µm, (B) Aqua C18 column (Phenomenex, USA) 250 × 10 mm i.d., 5 µm, and (C) XTerra MS C18 column (Waters, USA) 3.0 × 50 mm i.d., 2.5 µm. System A(I): flow 1 mL/min, UV 235 nm, 50 mM triethylammonium acetate buffer (TEAA) containing 7% CH3CN (a) and 50% CH3CN (b), gradient (a): 0 min 100%, 10 min 80%, 15 min 60%, 20 min 0%. System A(II): flow 1 mL/min, UV 240 or 260 nm, 50 mM NH4OAc buffer (pH 7.0) containing 5% CH3CN (a) and 50% CH3CN (b), gradient (a): 0 min 100%, 30 min 80%, 35 min 60%, 38 min 0%. System A(III): flow 1 mL/min, UV 240 nm, 0-25 min 50 mM TEAA (pH 7.0) containing 7% CH3CN. System B(I): flow 4 mL/min, UV 240 nm, 50 mM TEAA containing 7% CH3CN (a) and 50% CH3CN (b), gradient (a): 0 min 100%, 10 min 90%, 15 min 60%, 20-25 min 0%. System B(II): flow 4 mL/min, UV 240 nm, 50 mM TEAA containing 7% CH3CN (a) and 50% CH3CN (b), gradient (a): 0-20 min 100%, 40-55 min 0%. System C(I): flow 0.35 mL/min, UV 260 nm, 50 mM TEAA containing 7% CH3CN (a) and 50% CH3CN (b), gradient (a): 0 min 100%, 10 min 90%, 15 min 60%, 22 min 0%. TLC was performed on precoated RP18 plates (Merck, Germany) with System I (50 mM TEAA containing 20% CH3CN) and System II (MeOH/10% NH4OAc (9:1 v/v)). The dried TLC plates were exposed to phosphor imaging plates for 10 min and scanned with a Fujix BAS 5000 (Fujifilm Europe, Germany). Radiochemical yields (RCYs) of the reactions and radiochemical purity (RCP) of the compounds were calculated from the activity area of the product peak related to the total activity area using AIDA software (Raytest, Germany). Nonradioactive compounds were visualized under UV-light at 254 nm. Solid-phase extractions (SPE) were performed on SepPak Plus C18 and SepPak QMA Acell Plus cartridges (Waters, USA). Elution profiles of 1, Y2, Y3, and Y4 and the corresponding hydrolysis products Y12 and Y13 were initially monitored at ¨ KTAprime plus system 254 nm on the cartridges using an A (GE Healthcare, Germany). UV-chromatograms were evaluated with PrimeView software (GE Healthcare, Germany). Gel Electrophoresis. PAGE was performed with vertical electrophoresis unit TV200YK (Biostep, Germany) running native 20% polyacrylamide gels at 10 mA and 10 °C in 1× TBE running buffer. A 10 bp DNA ladder (Fermentas, USA) was applied as a molecular weight standard. After electrophoresis, gels were exposed to phosphor imaging plates for 2-4 min. The plates were scanned and the RCYs of the conjugations and RCP of the radiolabeled L-RNA derivatives were calculated as

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described above using AIDA software (Raytest, Germany). Subsequently, the gel was stained using ethidium bromide, and the DNA ladder was visualized under UV-light and photographed (Gene Flash, Syngene, UK). Mass Spectrometry. Mass spectra were recorded on a Quattro Ultima ESI-MS (Micromass, UK) and on a Daltonics Autoflex II TOF/TOF50 (Bruker). The MALDI-TOF matrix, 3-hydroxypicolinic acid, was purchased from Aldrich. The L-RNA probes were desalted for MALDI-TOF measurements using C18 purification tips (ZipTip, Millipore, USA) as described elsewhere (17). Chemistry. Synthesis, purification, and characterization of the ∆(δδδδ) (twisted square antiprism, TSAP) and Λ(δδδδ) (square antiprism, SAP) isomers of [(2S)-2-(4-aminobenzyl)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl]tetraacetato}yttrate(1-) (Y2) were described in-depth elsewhere (13). Following experimental procedures for synthesis of the TSAP and SAP isomers of Y3, Y4, Y6, Y7, and Y9, respectively, based on the corresponding TSAP and SAP isomers of Y2. Preparation of [{(2S)-2-[4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol1-yl)benzyl]-1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetrayl}tetraacetato]yttrate(1-) (Y3). The synthesis of Y3 was adapted and optimized from ref 18. Briefly, N-methoxycarbonylmaleimide (15 mg, 96 µmol) was dissolved in 150 µL 1,4-dioxane and added to a solution of Y2 (3.1 mg, 5.2 µmol) in 300 µL H2O and 150 µL saturated NaHCO3. The reaction mixture (pH 9.3) was incubated at 25 °C for a maximum reaction time of 15 min. The mixture was diluted with (250 µL) 1 M HCl to adjust pH 2. Subsequently, the mixture was fractionized by HPLC. HPLC fractions containing Y3 were concentrated by evaporation and lyophilized to obtain 2.9 mg (4.3 µmol) of Y3 as a white powder. ESI-MS: Y3 (TSAP and SAP isomer) m/z: 674 [M]-. HPLC: A(I): RT(Y3 TSAP) 17.1 min, RT(Y3 SAP) 17.3 min. B(I): RT(Y3 TSAP) 17.4 min, RT(Y3 SAP) 17.6 min. TLC: (I): Rf(Y3) 0.3, (II) Rf(Y3) 0.6. Preparation of [{(2S)-2-(4-{[4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-1,4,7,10-tetraazacyclododecane1,4,7,10-tetrayl]tetraacetato}yttrate(1-) (Y4). N-(γ-Maleimidobutyryloxy)succinimide ester (GMBS, 7.8 mg, 27.8 µmol) was dissolved in 250 µL DMSO and added to a solution of Y2 (2.6 mg, 4.4 µmol) in (750 µL) 67 mM sodium phosphate buffer (pH 7.2). The reaction mixture (pH 7.9) was incubated at 25 °C for 30 min. Subsequently, the mixture was fractionized by HPLC. HPLC fractions containing Y4 were concentrated by evaporation and lyophilized to obtain 3.0 mg (4.0 µmol) of Y4 as a white powder. ESI-MS: Y4 (TSAP and SAP isomer) m/z: 761 [M]-. HPLC: A(II): RT(Y4 TSAP) 33.5 min, RT(Y4 SAP) 35.8 min. B(II): RT(Y4 TSAP) 31.4 min, RT(Y4 SAP) 31.6 min. TLC: (I): Rf(Y4) 0.3, (II) Rf(Y4) 0.7. Preparation of Y-DID-GSH (Y6). The lyophilized compound Y3 (1.1 mg, 1.6 µmol) and GSH 5 (2.7 mg, 8.8 µmol) were dissolved in (600 µL) 67 mM sodium phosphate buffer (pH 6.8). The reaction mixture (pH 5.1) was incubated at 25 °C for 25 min. Subsequently, the product Y6 was fractionized by HPLC and lyophilized to obtain 1.4 mg (1.5 µmol) of Y6. ESI-MS: Y6 (TSAP and SAP isomer) m/z: 981 [M]-. HPLC: A(I): RT(Y6 TSAP) 7.1 min, RT(Y6 SAP) 9.0 min. TLC: (II) Rf(Y6) 0.1. Preparation of Y-DIBD-GSH (Y7). The lyophilized compound Y4 (1.0 mg, 1.3 µmol) and GSH 5 (3.0 mg, 9.8 µmol) were dissolved in (600 µL) 67 mM sodium phosphate buffer (pH 7.2). The reaction mixture (pH 6.6) was incubated at 25 °C for 25 min. Subsequently, the product Y7 was fractionized by HPLC and lyophilized to obtain 1.4 mg (1.3 µmol) of Y7. ESI-MS: Y7 (TSAP and SAP isomer) m/z: 1066 [M]-. HPLC: A(II): RT(Y7 TSAP) 27.9 min, RT(Y7 SAP) 28.3 min. TLC: (II) Rf(Y7) 0.1.

Schlesinger et al.

Conjugation of L-RNA 8 with Y4. The thiol-modified L-RNA was protected as a disulfide (5′-(1-hydroxy-7,8-dithia-tetradecyl) UGACUGACUGAC-3′). The protecting group was initially removed by treatment with dithiotreitol (DTT). Briefly, L-RNA (5 mg, 1.2 µmol) and DTT (9.3 mg, 60 µmol) were dissolved in (1.76 mL) 67 mM sodium phosphate buffer (pH 8.0) and incubated for 60 min at 25 °C. Subsequently, 5′-thiolhexyl L-RNA 7 was purified via size exclusion chromatography (SEC) ¨ KTAprime plus system (GE Healthcare, Germany) using an A with a Superdex 75 HR 10/30 column (GE Healthcare, Germany) and 67 mM sodium phosphate buffer (pH 7.0, saturated with nitrogen) as mobile phase at a flow rate of 30 mL/h. SEC-fractions containing 8 were lyophilized and stored at -24 °C. The identity of the 5′-thiolhexyl L-RNA 7 was verified by mass spectrometry. The lyophilized compound Y4 (0.12 mg, 160 nmol) and L-RNA 8 (0.24 mg, 60 nmol) were dissolved in (250 µL) 67 mM sodium phosphate buffer (pH 7.0). The reaction mixture (pH 6.6) was incubated for 25 min at 25 °C under argon atmosphere. Subsequently, the product Y9 was fractionized by HPLC and lyophilized to obtain 0.27 mg (57 nmol) of Y9. Maldi-TOF: 8 m/z 3991 [M + H]+, Y9 (TSAP and SAP isomer) m/z 4751 [M + H]+. SEC: VR(8) 12.4 mL. HPLC: A(II): RT(8) 25.4 min, RT(Y9 TSAP) 28.5 min, RT(Y9 SAP) 29.1 min. Determination of Hydrolytic Stability of Y3 and Y4. First, the corresponding hydrolysis products [(2Z)-4-oxo-4-{[4-({(2S)1,4,7,10-tetrakis[(carboxymethyl]-1,4,7,10-tetraazacyclododecan2-yl}methyl)phenyl]amino}but-2-enoato(4-)]yttrate(1-) (Y12) and [(2Z)-4-oxo-4-[(4-oxo-4-{[4-({(2S)-1,4,7,10-tetrakis[(carboxymethyl]-1,4,7,10-tetraazacyclododecan-2-yl}methyl)phenyl]amino}butyl)amino]but-2-enoato(4-)]yttrate(1-) (Y13) of the maleimides Y3 and Y4 were synthesized. Briefly, Y3 and Y4 were hydrolyzed after their synthesis by the addition of NaOH to the reaction mixtures (pH 9.0). The corresponding maleamic acid derivatives Y12 and Y13 were separated via HPLC and analyzed by ESI-MS. To determine the hydrolytic stability of Y3 and Y4 depending on time, Y3 (1 mg, 1.5 µmol) and Y4 (1 mg, 1.3 µmol) were separately dissolved in (500 µL) 67 mM sodium phosphate buffer (pH 7.0) and incubated at 25 °C for 96 h. Aliquots were sampled after 0, 1, 2, 3, 4, 5, 23, 48, and 96 h, and the samples were analyzed via HPLC. The experiments were repeated three times. HPLC fractions, containing separately Y3 and Y4 in a mixture of 50 mM TEAA/CH3CN (4:1 v/v, pH 6.25), were adjusted with acetic acid and triethylamine to pH 6.0, 6.6, 7.1, 7.5, 7.8, 8.0, and 8.4, respectively. All mixtures were incubated at 25 °C for 30 min and analyzed via HPLC. ESI-MS: Y12 (TSAP and SAP isomer) m/z 694 [M + 2H]+, Y13 (TSAP and SAP isomer) m/z 779 [M + 2H]+. HPLC: A(I): RT(Y12 TSAP) 10.5 min, RT(Y12 SAP) 11.6 min. A(II): RT(Y13 TSAP) 12.1 min, RT(Y13 SAP) 14.4 min. Radiochemistry. Preparation of [(2S)-2-(4-Aminobenzyl)-1,4, 7,10-tetraazacyclododecane-1,4,7,10-tetrayl]tetra-acetato}[90Y]yttrate(1-) ([90Y]2). Chelator 1 (100 µg, 0.15 µmol) in (500 µL) 0.6 M NaOAc buffer (pH 6.8) was added to 80 µL [90Y]YCl3 (13 MBq) in 0.04 M HCl. The mixture (pH 6.5) was incubated at 95 °C for 20 min. A solution of DTPA (15 µL, 0.45 µmol) was added, and the reaction mixture was incubated at 40 °C for 10 min to complex unreacted [90Y]Y3+. The radiometal complex [90Y]2 was separated from [90Y]Y-DTPA and 1 using a SepPak Plus C18 cartridge (Waters, USA). After elution of 1 and [90Y]Y-DTPA with (10 mL) 67 mM sodium phosphate buffer (pH 7.0), [90Y]2 was selectively eluted: (a) with 2 mL CH3CN/H2O (4:1 v/v) and evaporated to dryness under a stream of nitrogen at 95 °C for 40 min, or (b) fractionized with (2 mL) 67 mM sodium phosphate buffer/CH3CN (1:1 v/v, pH 8.1). The product [90Y]2 was found to be >99% radiochemically pure.

[90Y]-DOTA-Based Maleimide Reagents

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Scheme 1. Complexation of [90Y]Y3+ with Chelator 1 and Subsequent Synthesis of the Maleimide Reagent [90Y]3 by Reaction of the SPE-Purified Complex [90Y]2 with N-Methoxycarbonylmaleimide

Scheme 2. Synthesis of the Maleimide Reagent [90Y]4 by Reaction of [90Y]2 with GMBS

HPLC: A(III): RT([90Y]2 TSAP) 9.1 min, RT([90Y]2 SAP) 13.3 min. TLC: (I): Rf([90Y]2) 0.5, (I) Rf([90Y]2) 0.7. Preparation of [{(2S)-2-[4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol1-yl)benzyl]-1,4,7,10-tetraazacyclo-dodecane-1,4,7,10tetrayl}tetraacetato][90Y]yttrate(1-) ([90Y]3). The residue of the SPE-purified (scheme a) radiometal complex [90Y]2 (9.5 MBq) was redissolved in 100 µL H2O and 50 µL saturated NaHCO3. N-Methoxycarbonylmaleimide (5 mg, 32 µmol) was solved in 50 µL 1,4-dioxane and added to the solution of [90Y]2. The reaction mixture (pH 9.1) was incubated at 25 °C for 15 min. The mixture was carefully diluted with 1 M HCl (70 µL) to adjust pH < 3, and the tube was left open at room temperature for 20 min. Subsequently, the mixture was diluted with (20 mL) 50 mM NH4OAc solution (pH 6.0) and applied onto a SepPak Plus C18 cartridge. The cartridge was washed with (10 mL) 50 mM NH4OAc solution (pH 6.0) and (2 mL) 50 mM NH4OAc/ CH3CN (9:1 v/v, pH 6.5). The final product [90Y]3 was selectively eluted with (2 mL) 0.1 M NH4OAc/CH3CN (1:1 v/v, pH 6.5) and [90Y]3 was found to be >95% radiochemically pure. HPLC: A(I): RT([90Y]3) 17.4 min. TLC: (I): Rf([90Y]3) 0.3, (II) Rf([90Y]3) 0.6. Preparation of {[(2S)-2-(4-{[4-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-1,4,7,10-tetraazacyclododecane1,4,7,10-tetrayl]tetraacetato}[90Y]yttrate(1-) ([90Y]4). The SPE eluate, which contained the radiometal complex [90Y]2 (33.4 MBq) in (0.6 mL) 67 mM sodium phosphate buffer/CH3CN (1:1 v/v, pH 8.1), was added to GMBS (3 mg, 10.7 µmol). The reaction mixture (pH 7.4) was incubated at 25 °C for 90 min. Subsequently, the mixture was diluted with 20 mL H2O and applied onto a SepPak QMA cartridge. The cartridge was washed with 10 mL H2O, and the final product [90Y]4 was directly eluted on a SepPak Plus C18 cartridge with (6 mL) 0.5 M NaCl/CH3CN (95:5 v/v, pH 3.5). The SepPak Plus C18 cartridge was washed with (4 mL) 50 mM NH4OAc/CH3CN (9:1 v/v, pH 6.8), and compound [90Y]4 was selectively eluted with 2 mL H2O/CH3CN (4:1 v/v). The product [90Y]4 was found to be >98% radiochemically pure. HPLC: A(II): RT([90Y]4 TSAP) 34.5 min, RT([90Y]4 SAP) 36.3 min. TLC: (I): Rf([90Y]4) 0.3, (II) Rf([90Y]4) 0.7.

Conjugation of Glutathione (GSH) 5 with [90Y]3 and [90Y]4. Dilution series with 1000, 100, 10, 1, and 0.1 µg GSH 5 (corresponding to 3300, 330, 33, 3.3, and 0.33 nmol 5) in (1 mL) 67 mM sodium phosphate buffer (pH 7.2) were prepared. Both maleimide reagents, [90Y]3 (0.2 MBq in (150 µL) 0.1 M NH4OAc/CH3CN (1:1 v/v, pH 6.5)) and [90Y]4 (0.2 MBq in 150 µL H2O/CH3CN (4:1 v/v, pH 7.0)), were separately added to each solution of 5. The reaction mixtures were incubated under argon atmosphere at 25 °C for 2 h. For determination of the RCYs, aliquots of the reaction mixtures with the corresponding products [90Y]Y-DID-GSH [90Y]6 and [90Y]Y-DIBDGSH [90Y]7, respectively, were analyzed after 5, 15, 30, 60, and 120 min by TLC. TLC: (II): Rf([90Y]6) 0.1, Rf([90Y]7) 0.1. Conjugation of L-RNA 8 with [90Y]4 via Prelabeling. Dilution series with 400, 200, 100, 40, 20, 8, 2, and 0.4 µg L-RNA 8 (corresponding to 100, 50, 25, 10, 5, 2, 0.5, and 0.1 nmol 8) in (100 µL) 67 mM sodium phosphate buffer (pH 7.0) were prepared. The maleimide reagent [90Y]4 (0.5 MBq in 65 µL H2O/CH3CN (4:1 v/v, pH 7.0)) was added to each solution of 8. The reaction mixtures were incubated under argon atmosphere at 25 °C for 2 h. For determination of the RCYs, aliquots of the reaction mixtures with the corresponding product [90Y]YDIBD-L-RNA [90Y]9 were sampled (after 60 and 120 min), evaporated (Concentrator 5314, Eppendorf, Germany), and analyzed via PAGE and HPLC. HPLC: A(II): RT([90Y]8 TSAP) 28.6 min, RT([90Y]8 SAP) 29.2 min. PAGE: see Supporting Information Figures S3 and S4. Preparation of the 90Y-Labeled L-RNA [90Y]11 via Postlabeling. Dilution series with 440, 220, 110, 44, 22, 4, 2, and 0.4 µg DOTA-L-RNA 10 (corresponding to 100, 50, 25, 10, 5, 2, 0.5, and 0.1 nmol 10) in 250 µL NH4OAc buffer (0.5 M, pH 7.0) were prepared. [90Y]YCl3 (8 µL, 2.4 MBq) in 0.04 M HCl was added to each solution of 10. After incubation of the reaction mixtures at 90 °C for 20 min, DTPA (10 µL, 30 mM in 0.5 M NH4OAc) was added, and the reaction mixtures were kept at room temperature for 10 min to complex unreacted [90Y]Y3+ as [90Y]Y-DTPA complex. Aliquots of the reaction mixtures were analyzed via PAGE and HPLC to determine the

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Figure 3. Isomers of the differently substituted [90Y]-DOTA complexes [90Y]2, [90Y]3, and [90Y]4 are represented schematically. Only the geometry around the N-CH2-COO group next to the (S)-configured carbon atom is given.

RCYs in dependence of the amount of 10. HPLC: C(I): RT([90Y]11) 15.3 min. PAGE: see Supporting Information Figure S5.

RESULTS AND DISCUSSION Synthesis of [90Y]3 and [90Y]4. A straightforward method to synthesize both [90Y]-DOTA-based maleimide reagents [90Y]3 and [90Y]4 is to initially complex [90Y]Y3+ with chelator 1 and subsequently transform the purified complex [90Y]2 into the corresponding maleimides, by using activating agents (see Schemes 1 and 2). As previously shown, [90Y]2 exists as two conformers, adopting the ∆(δδδδ) (twisted square antiprism, TSAP) and Λ(δδδδ) (square antiprism, SAP) configurations (see Figure 3) (13). On this account, two further conformers of [90Y]3 and [90Y]4, respectively, derived from both isomers of [90Y]1. Having shown that the differences in the helicity of the

Schlesinger et al.

carboxylate side arms (Λ or ∆) does not affect the biodistribution of the corresponding radiolabeled L-RNA (13), both isomers of [90Y]2 were used for all radiosyntheses in this work without separation. Synthesis and SPE-Purification of [90Y]2. The synthesis of 90 [ Y]2 was performed in an analogous manner as previously described (13). By adding DTPA to the solution, unreacted [90Y]Y3+ was finally complexed as [90Y]Y-DTPA. Solid-phase extraction (SPE) was used to purify the radiometal complex [90Y]2 from the excess chelator 1 and [90Y]Y-DTPA complex. In this process, the negatively charged complex [90Y]1 was retarded on a SepPak Plus C18 cartridge applying an aqueous solution of the ion pair reagents TEAA, NH4OAc, and NaOAc, respectively, whereas 1 and [90Y]Y-DTPA were eluted with these eluents. This observation indicates that triethylammonium, ammonium, and Na+-cations provide a comparable interaction of [90Y]2 with the reversed-phase material by masking the negative charge of [90Y]2. Furthermore, no differences were observed for these cations in the performance of separation of [90Y]2 from the side products 1 and [90Y]Y-DTPA. For the synthesis of [90Y]3, the radiometal complex [90Y]2 was selectively eluted from the cartridge with a mixture of CH3CN and H2O (4:1 v/v) and evaporated to dryness. To minimize expenditure of time, [90Y]2 was alternatively eluted with 67 mM sodium phosphate buffer/CH3CN (1:1 v/v, pH 8.1) and used without any further processing for the radiosynthesis of [90Y]4. Synthesis of [90Y]3. To synthesize [90Y]3, the primary amine group of [90Y]2 reacted with N-methoxycarbonylmaleimide (see Scheme 1). This reaction is a comparably mild method for the transformation of amine groups into maleimides. As recently shown with N-methoxycarbonylmaleimide (18), radiochemical yields (RCYs) greater 90% were already achieved at room temperature after 10 min for a 18F-labeled maleimide reagent. On the basis of this work (18), we adapted and optimized these reaction conditions for synthesis of [90Y]3. The residue of the SPE-purified radiometal complex [90Y]2 was redissolved in NaHCO3 solution. After adding N-methoxycarbonylmaleimide, the mixture was incubated at room temperature and pH 9.1 for a maximum reaction time of 15 min. In a second step, the pH was lowered from 9.1 to ∼2 by addition of 1 M HCl. SPE Purification of [90Y]3. An efficient purification of [90Y]3 in regard to short purification time and high RCYs was performed with solid-phase extraction (SPE). For that purpose, elution profiles of 1, Y2, Y3, the corresponding hydrolysis product Y12, and N-methoxycarbonylmaleimide were initially analyzed on a SepPak Plus C18 cartridge. On the basis of the elution characteristics of these nonradiolabeled compounds, the following procedure was conducted to purify [90Y]3 with only

Figure 4. Radio-HPLC and radio-TLC chromatograms of the maleimide reagent [90Y]3. (A) Overlay of HPLC profiles of the TSAP and SAP isomer of Y3 as references. (B) SPE-purified [90Y]3. The TSAP and SAP isomer of [90Y]3 could not be separated under the used HPLC conditions. (C) Radio-TLC analysis of the reaction mixture containing [90Y]3 (1) and the SPE-purified [90Y]3 (2) with TLC system I.

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Table 1. Time-Dependent Radiochemical Yields (RCYs) of [90Y]4 (RCYs Were Determined via Radio-TLC Analysis) reaction time [min]

15

30

45

60

75

90

RCY [%]

30

38

46

58

62

66

one SepPak Plus C18 cartridge: (A) First, the concentration of dioxane was reduced from 25% in the reaction mixture to 0.5% by adding 20 mL NH4OAc solution. Higher dioxane concentrations would lead to an elution of [90Y]3. (B) Subsequently, the cartridge was washed with a solution of 10 mL 50 mM NH4OAc (pH 6.0) and a mixture of (2 mL) 50 mM NH4OAc and CH3CN (9:1 v/v, pH 6.5), to elute both unreacted N-methoxycarbonylmaleimide and the hydrolyzed species [90Y]12 from the cartridge. (C) Finally, compound [90Y]2 was selectively eluted with a mixture of (2 mL) 0.1 M NH4OAc and CH3CN (1:1 v/v, pH 6.5). A pH value of e6.5 in the final elution mixture proved to be important for preventing hydrolysis of the maleimide [90Y]3. The obtained maleimide reagent [90Y]3 was used for the prelabeling of GSH 5 without any further processing. Hence, in a typical experiment starting with the complexation of 11 MBq [90Y]YCl3 with 1, 3.7 MBq of [90Y]3 was obtained with a radiochemical purity of >95% within 145 min (Figure 4). Synthesis of [90Y]4. The transformation of the primary amine 90 [ Y]2 into a maleimide group can also be conducted with heterobifunctional reagents, e.g., N-(γ-maleimidobutyryloxy)succinimide ester (GMBS). At pH 7.0-7.5, GMBS readily reacts with both primary amines via the chemically reactive succinimidyl group and thiols via the maleimide group. The nucleophilic substitution of the primary amine [90Y]2, by reaction with the activated carbonyl carbon in GMBS, leads to the formation of [90Y]4 (see Scheme 2). To synthesize [90Y]4, SPE-purified [90Y]2 (33.4 MBq) in 0.6 mL sodium phosphate/CH3CN (1:1 v/v) was added directly to GMBS. Subsequently, the solution was vigorously mixed at room temperature. The pH value of the reaction mixture (pH 7.4) remained constant over the reaction time of 90 min. The kinetics of the reaction was monitored by radio-TLC analysis. The time-dependent RCYs of the maleimide formation are shown in Table 1. Compound [90Y]4 was obtained with the maximum RCY of 66% after 90 min. In a competing side reaction, GMBS was hydrolyzed to the corresponding byproduct N-hydroxysuccinimide and 4-maleimidobutyric acid, as confirmed by HPLC. Thus, reaction times longer than 90 min would not lead to higher RCYs. SPE Purification of [90Y]4. Separation of N-hydroxysuccinimide, 4-maleimidobutyric acid and the unreacted complex [90Y]2 from the maleimide reagent [90Y]4 was performed via SPE. For that purpose, elution profiles of these compounds on different cartridges were initially analyzed. On the basis of their

Scheme 3. Maleamic Acid Derivatives [90Y]12 and [90Y]13 Derived from the Basic Hydrolysis of the Maleimide Reagents [90Y]3 and [90Y]4

elution characteristics, the following procedure was conducted to purify [90Y]4 efficiently with an anion-exchanger (SepPak QMA Acell Plus) and a C18-cartridge (SepPak Plus C18): (A) Prior to loading the reaction mixture onto the anion-exchanger the concentration of phosphate ions was reduced from 67 to 6 mM by adding 20 mL H2O, thus preventing an elution of [90Y]4 during the loading step. (B) The anion-exchanger was washed with 10 mL H2O to elute unreacted GMBS. (C) The maleimide reagent [90Y]4 was eluted directly onto the C18-cartridge with a mixture of (6 mL) 0.5 M NaCl and CH3CN (95:5 v/v). (D) Subsequently, the C18-cartridge was washed with (4 mL) 50 mM NH4OAc/CH3CN (9:1 v/v), to elute all byproduct from the cartridge. Under these conditions, almost 70% of the product [90Y]4 remained on the C18-cartridge. (E) Finally, compound [90Y]4 was selectively eluted with 2 mL H2O/CH3CN (1:1 v/v, pH 7.0) from the cartridge. Hence, in a typical experiment starting with the complexation of 39 MBq [90Y]YCl3 with 1, 8 MBq of [90Y]4 was obtained with a radiochemical purity of >98% within 170 min (Figure 5). Hydrolytic Stability of [90Y]3 and [90Y]4. During the preparation process, the maleimide reagents [90Y]3 and [90Y]4 hydrolyzed at pH values greater than 7.0 into the corresponding nonreactive maleamic acids [90Y]12 and [90Y]13 (Scheme 3) (12, 19). Differences were found in the hydrolytic stability of the N-aryl maleimide [90Y]3 and the N-alkyl maleimide [90Y]4. By examining heterobifunctional linkers, Kitagawa et al. observed a higher hydrolytic stability of N-alkyl against N-aryl maleimides in aqueous solutions at pH 8.0 (20). Thus, only 8-12% hydrolysis products were found for N-alkyl

Figure 5. Radio-HPLC and radio-TLC chromatograms of the maleimide reagent [90Y]4. (A) Overlay of HPLC profiles of the TSAP and SAP isomer of Y4 as references. (B) SPE-purified [90Y]4. (C) Radio-TLC analysis of the reaction mixture containing [90Y]4 (1) and the SPE-purified [90Y]4 (2) with TLC system I.

1346 Bioconjugate Chem., Vol. 20, No. 7, 2009

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reaction time and pH seems to be important for the synthesis of [90Y]3, since the formation of [90Y]3 competes with the basecatalyzed ring-opening of the maleimide group in the first reaction step (see Scheme 1). HPLC analyses also confirmed that 10% of the corresponding maleamic acid derivative Y12 was already formed 20 min after addition of N-methoxycarbonylmaleimide to the nonradioactive precursor Y2 (data not shown). Therefore, in order to get higher RCYs a reaction time of 15 min and pH 9 should not be exceeded in the first reaction step. Prelabeling of GSH as Model Peptide with [90Y]3 and 90 [ Y]4. To evaluate the application and reactivity of both maleimide reagents [90Y]3 and [90Y]4, the prelabeling approach was initially tested with GSH 5 as a model substance. The conjugations of both maleimide reagents [90Y]3 and [90Y]4 to 5 were conducted with different amounts of 5 in 1 mL of phosphate buffer (pH 7.2) at room temperature (see Scheme 4). The kinetics of these reactions was monitored via radioTLC analysis. Time-dependent RCYs of both prelabeling reactions are summarized in Table 2. For [90Y]6 and [90Y]7, RCYs greater than 85% were reached already after 5 min reaction time in batches with 10 to 1000 µg 5. Slight differences in the obtained RCYs were only observed for both maleimide reagents at amounts of 0.1 and 1 µg 5. At these low amounts of 5, higher RCYs were achieved for maleimide reagent [90Y]3 than for [90Y]4. This points to a slightly higher reactivity of [90Y]3 compared to [90Y]4. In contrast, the higher hydrolytic stability of [90Y]4 showed no influence on the RCYs. In summary, higher RCYs were obtained for both conjugations with increasing amounts of 5 and longer reaction times. Similar results were found for the radiolabeling of GSH 5 with both [90Y]-DOTA-based maleimide reagents [90Y]3 and [90Y]4 as already known for a [18F]-based maleimide (21). 90 Y-Labeling of an L-RNA via the Pre- and Postlabeling Approach. For the evaluation of achievable RCYs and ESAs both pre- and postlabeling were performed with a 12mer L-RNA as model substance. Due to its higher hydrolytic stability, only [90Y]4 was used for the prelabeling of the 12mer L-RNA 8. On

Figure 6. Hydrolytic stability of the maleimides Y3 and Y4: (A) as a function of time at pH 7.0, (B) as a function of pH value for 30 min (n ) 3).

maleimides, whereas 41-69% of the N-aryl maleimides were hydrolyzed under the same conditions (after 30 min at 30 °C). The highest stability was observed for all maleimides in weak acid solutions at pH 6.0. To verify the hydrolytic stability of the maleimide reagents [90Y]3 and [90Y]4, hydrolysis rates of the nonradioactive maleimides Y3 and Y4 were analyzed as a function of time and pH (see Figure 6). In agreement with the previous observations of Kitagawa et al. (20), N-alkyl maleimide Y4 showed increased stability at pH 7.0 over a period of 96 h, in comparison to N-aryl maleimide Y3. Hydrolysis ratios of 73% for Y3, but only of 27% for Y4 were estimated after 23 h incubation time. As expected, Y4 was more stable than Y3 at higher pH values (7.8-8.4). Therefore, a careful control of

Scheme 4. Conjugation of Glutathione (GSH) 5 with [90Y]3 and [90Y]4, Respectively

Table 2. RCYs (%) of Conjugates [90Y]6 and [90Y]7 as a Function of the Time and Amount of 5 (Presented as Data ( SD, RCYs Were Determined via Radio-TLC Analysis (n ) 3 ([90Y]6), n ) 4 ([90Y]7)) 5 min

10 min

30 min

60 min

120 min

GSH [µg]

[90Y]6

[90Y]7

[90Y]6

[90Y]7

[90Y]6

[90Y]7

[90Y]6

[90Y]7

[90Y]6

[90Y]7

0.1 1 10 100 1000

20 (2.6) 74 (9.2) 86 (4.0) 87 (4.6) 89 (1.5)

14 (3.5) 44 (4.9) 87 (5.9) 88 (4.7) 90 (5.0)

29 (5.0) 85 (4.5) 88 (2.5) 88 (2.5) 91 (2.3)

20 (3.4) 65 (9.2) 88 (4.7) 89 (4.6) 91 (4.5)

35 (8.5) 88 (3.1) 89 (2.1) 89 (1.7) 91 (2.3)

25 (1.9) 76 (9.6) 88 (4.2) 90 (4.4) 91 (4.7)

37 (9.6) 89 (1.5) 90 (2.0) 90 (2.0) 91 (2.3)

30 (5.3) 82 (7.0) 88 (4.2) 90 (3.9) 92 (3.7)

39 (11.9) 89 (1.5) 90 (2.0) 91 (2.3) 91 (2.3)

34 (9.0) 84 (4.9) 89 (3.9) 90 (4.2) 92 (3.7)

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Bioconjugate Chem., Vol. 20, No. 7, 2009 1347

Scheme 5. 90Y-Labeling of L-RNA via the Pre- and Postlabeling Approacha

a Prelabeling approach: Compound [90Y]9 was formed by reaction of L-RNA 8 with [90Y]4 under mild conditions. Postlabeling approach: [90Y]Y3+ labeling of DOTA-L-RNA 10 results in formation of [90Y]11.

Figure 7. RCYs (%) of [90Y]9 and [90Y]11 as a function of the amount of L-RNA derivatives 8 and 10. The results were obtained by PAGE analysis (n ) 4 ([90Y]9), n ) 3 ([90Y]11)).

this account, the prelabeling of 8 with [90Y]4 was compared with the direct radiolabeling of DOTA-L-RNA 10 with [90Y]YCl3. Prelabeling of a Thiol-Modified L-RNA 8 with [90Y]4. The 12mer L-RNA was derivatized with a 5′-hexyl disulfid group, and after removal the protective group, the free thiol group of 8 was used to attach the maleimide reagent [90Y]4. The conjugation was performed at room temperature in 0.1 mL phosphate buffer at pH 7.2 (Scheme 5). The dependence of the RCY of the prelabeling reaction on the amount of 8 was estimated in range 0.1-100 nmol of 8. The progress of the reaction was monitored by gel electrophoresis after 2 h reaction time (Supporting Information Figure S3B). The difference in the retention factor values of [90Y]4 (RF 0.39; 0.70) and the product [90Y]9 (RF 0.61) allowed a quantitative analysis of the reaction (Supporting Information Figure S4). The obtained RCYs of the conjugation, as a function of the amount of 8, is shown in Figure 7. Postlabeling of DOTA-L-RNA 10 with [90Y]YCl3. The 90Ylabeling of DOTA-L-RNA 10 was performed in an analogous manner to that previously described (16). The dependence of the RCY of the postlabeling reaction on the amount of 10 was monitored in the range 0.1-100 nmol of 10 and estimated by gel electrophoresis (Supporting Information Figure S5B). Before analyzing the reaction aliquots, unreacted [90Y]Y3+ was first complexed as [90Y]Y-DTPA by adding DTPA to the solution. The RCYs of the complexation, obtained from PAGE analyses, are graphically shown in Figure 7. Comparison of Both Radiolabeling Methods. In the range 0.1-100 nmol of L-RNA, higher RCYs of 90Y-labeling were

Figure 8. Image fusion of the autoradiogram of the exposed 20% polyacrylamide gel and the photographed 10 bp DNA ladder (MW) after staining with ethidium bromide. Applied samples in the lanes: maleimide reagent [90Y]4, 90Y-labeled L-RNA [90Y]9 before (aliquot of the crude reaction mixture) and after HPLC separation.

obtained by the postlabeling approach. Using 2.4 MBq [90Y]Y3+, RCYs higher than 97% were already achieved with 0.5 nmol 10. A higher effective specific activity (ESA) of up to 4 GBq/ µmol [90Y]11 was obtained with the postlabeling approach. Furthermore, an additional HPLC separation of [90Y]11 (RT 15.3 min) from excess 10 (RT 16.8 min) would theoretically enhance the ESA to 29 GBq/µmol (Supporting Information Figure S2, HPLC system C(I)). In contrast, prelabeling experiments with 0.5 MBq [90Y]4 gave lower RCYs of 90Y-labeling in the range 0.1-100 nmol L-RNA. The maximum RCY of 84% was obtained at 50 nmol 8. In comparison to 35% and 97% RCY for postlabeling at 0.1 and 0.5 nmol of L-RNA 10, respectively, 23% and 62% RCY was calculated for prelabeling at the same amounts of L-RNA 8. HPLC separation of [90Y]9 resulted in an ESA of 0.7 GBq/ µmol [90Y]9 and a radiochemical purity of 99%, as determined by HPLC and PAGE analysis (Figure 8). The ESA calculated for the HPLC purified [90Y]9, was approximately six times lower than the 4 GBq/µmol found for [90Y]11. HPLC analysis showed

1348 Bioconjugate Chem., Vol. 20, No. 7, 2009

a ratio of both isomers of [90Y]9 of 1:5 (TSAP/SAP) after HPLC separation (Supporting Information Figure S1C).

CONCLUSIONS The purpose of the present work was to investigate the applicability of two new [90Y]-DOTA-based maleimide reagents, the N-aryl maleimide [90Y]3 and N-alkyl maleimide [90Y]4, for the prelabeling approach. On this account, we were able to radiolabel GSH and a 12mer L-RNA as model substances using these new prelabeling agents. Both maleimide reagents showed a high potential for that purpose. However, concerning high RCYs, the direct labeling of DOTA-L-RNA with [90Y]YCl3 proved to be more efficient than the prelabeling of the thiolmodified 12mer L-RNA derivative with [90Y]4 at low activity levels. With regard to 90Y-labelings of thermally sensitive molecules, such as biopharmaceuticals, prelabeling could have more advantages than the direct radiolabeling, due to the milder labeling conditions. To enhance both RCY of the prelabeling approach and ESA of the corresponding 90Y-labeled molecules, further optimizations of the SPE-separation process of both maleimide reagents [90Y]3 and [90Y]4 seem to be necessary.

ACKNOWLEDGMENT This work was supported by the EU-FP6 integrated project BioCare contract no. 505785. Supporting Information Available: HPLC chromatograms, gel electrophoresism, and autoradiography data of the 90Ylabeled L-RNAs [90Y]9 and [90Y]11. This material is available free of charge via the Internet at http://pubs.acs.org.

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