Synthesis of Building Blocks for Solid-Phase Introducti...

Synthesis of Building Blocks for Solid-Phase Introduction of Diethylenetriaminepentaacetic Acid (DTPA) to Oligonucleotides and Oligopeptides

Synthesis of Building Blocks for Solid-Phase Introduction of Diethylenetriaminepentaacetic Acid (DTPA) to Oligonucleotides and Oligopeptides
Received December 29, 2005
Revised February 13, 2006
Web Release Date: April 15, 2006
Jari Peuralahti, Lassi Jaakkola, Veli-Matti Mukkala, and Jari Hovinen*
Bioconjugate Chem
ACS Publications
Copyright ? 2006 American Chemical Society
PerkinElmer Life and Analytical Sciences, Turku Site, POB 10, FIN-20101 Turku, Finland
Abstract:
Synthesis of building blocks that allow site-specific incorporation of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides using phosphoramidite and Fmoc chemistries, respectively, is described.
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Introduction
Because of its excellent metal-chelating properties diethylenetriaminepentaacetic acid (DTPA) is one of the most widely used organic ligands in magnetic resonance imaging (MRI) and positron emission tomography (PET) (1-3). Indeed, the first FDA approved contrast agent in clinical use is the Gd3+ DTPA chelate (4). The corresponding 111In and 68Ga chelates are suitable for PET applications (5-8), while Eu3+, Tb3+, Sm3+, and Dy3+ chelates can be used in dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) based applications (9). 99mTc DTPA, in turn, has been applied for single positron emission computed tomography (SPECT) (10-12). Bioactive molecules labeled with 111In or 117mSn DTPA may be used even as target-specific radiopharmaceuticals (13).
In several applications, covalent conjugation of DTPA to bioactive molecules is required. Accordingly, isothiocyanato, N-hydroxysuccinimide, or maleimide derivatives of the chelates are used in the labeling of the target molecules (14). Several bifunctional DTPA derivatives are currently commercially available. Because in all of these cases the labeling reaction is performed in the presence of an excess of an activated label, laborious purification procedures cannot be prevented. Especially when attachment of several label molecules is needed, purification and characterization of the desired biomolecule conjugate may be extremely difficult. The purification problems can be avoided by performing the labeling reaction on the solid phase. Hence, most of the impurities can be removed by washings when the biomolecule conjugate is still anchored to the solid support, and after release to the solution only one chromatographic purification is needed.
We have already developed a strategy for labeling of bioactive molecules with luminescent and nonluminescent lanthanide chelates on solid phase (15-20). The method involves synthesis of oligonucleotide and oligopeptide building blocks, which can be introduced to the biomolecule structure using commercial oligonucleotide and oligopeptide synthesizers by phosphoramidite and Fmoc chemistry, respectively. Upon completion of the chain assembly, the oligomer is deprotected and finally treated with the appropriate lanthanide(III) citrate to give rise to the desired biomolecule conjugate. Here our approach is used for the site-specific incorporation of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides.
Experimental Procedures
General Methods. Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck). Reagents for oligonucleotide and oligopeptide synthesis were purchased from Proligo and Nova Biochem, respectively. Sodium sulfinate resin (200-400 mesh, 1% DVB, 1.3 mmol g-1) was purchased from Tianjin Nankai Hecheng Science & Technology Company Limited (China). The oligopeptides and oligonucleotides were assembled on Applied Biosystems 433A and 3400 instruments, respectively, using recommended protocols. HPLC purifications were performed using a Shimadzu LC 10 AT instrument equipped with a diode array detector, a fraction collector, and a reversed phase column (LiChrocart 125-3 Purospher RP-18e 5 m). Mobile phase: (buffer A): 0.02 M triethylammonium acetate (pH 7.0); (buffer B): A in 50% (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to 21 min from 95% A to 100% B. Flow rate was 0.6 mL min.-1 All dry solvents were from Merck, and they were used as received. NMR spectra were recorded on a Brucker 250 spectrometer operating at 250.13 MHz for 1H and on a JEOL LA 400 spectrometer operating at 161.9 MHz for 31P. The signal of TMS was used as an internal (1H) and H3PO4 as an external (31P) reference. ESI-TOF mass spectra and IR spectra were recorded on Applied Biosystems Mariner and PerkinElmer Spectrum One instruments, respectively.
4-Iodophenylalanine Methyl Ester, 1. Thionyl chloride (10 mL) was added dropwise to anhydrous methanol (40 mL) on an ice-water bath. The mixture was allowed to warm to RT, and it was stirred for 30 min. 4-Iodophenylalanine (4.90 g, 16.8 mmol) was added, and the mixture was heated overnight at reflux. All volatiles were removed in vacuo. The residue was triturated with diethyl ether, filtered, and dried in vacuo. Yield was 5.10 g (99%). 1H NMR (DMSO-d6): 8.15 (2H, br s); 7.70 (2H, d, J 8.2); 7.06 (2H, d, J 8.2); 4.30 (1H, t, J 6.6); 3.70 (3H, s); 3.07 (2H, d, J 6.6). IR (KBr) 1788 cm-1 (C=O). ESI-TOF-MS for C10H13INO2 (M+H)+: calcd, 306.04; found, 306.01.
2-Amino-N-(2-aminoethyl)-3-(4-iodophenyl)propanamide, 2. Compound 1 (5.10 g, 16.7 mmol) was added portionwise to ethylenediamine (35 mL) during 2 h. The mixture was stirred overnight at RT and concentrated. Aqueous ammonia (7 M; 30 mL) was added, and the product was extracted to dichloromethane (4 ? 45 mL). The combined organic phases were dried over Na2SO4 and concentrated to give 5.57 g (93%) of compound 2. 1H NMR (CDCl3): 7.64 (2H, d, J 8.2); 7.49 (1H, br t); 6.98 (2H, d, J 8.2); 3.59 (1H, dd, J 4.3 and 8.9); 3.30 (2H, q, J 6.1); 3.18 (1H, dd, J 4.3 and 13.8); 2.80 (2H, t, J 6.4); 2.75 (1H, dd, J 8.9 and 13.1); 1.35 (4H, br s). IR (KBr) 1650 cm-1 (C=O). ESI-TOF-MS for C11H17IN3O (M+H)+: calcd, 334.04; found, 334.04.
N1-(2-Aminoethyl)-3-(4-iodophenyl)propane-1,2-diamine, 3. Compound 2 (4.79 g, 14.4 mmol) was dissolved in dry THF (120 mL) and deaerated with argon. Borane-THF (1 M, 120 mL) was added dropwise during 30 min, and the mixture was heated overnight at reflux. The excess of borane was destroyed by cautious addition of water. All volatiles were removed in vacuo. The residue was dissolved in 6 M HCl and heated at reflux for 3 h and concentrated. The residue was suspended in concentrated ammonia (100 mL) and extracted with chloroform (5 ?75 mL). The combined organic layers were dried over Na2SO4, concentrated, and dried in vacuo. Yield was 2.9 g (63%). 1H NMR (CDCl3): 7.61 (2H, d, J 8.3); 6.95 (2H, d, J 8.3); 3.06 (1H, m); 2.79 (2H, m); 2.79 (4H, m); 2.51 (2H, m); 1.39 (5H, br s). ESI-TOF-MS for C11H19IN3 (M+H)+: calcd, 320.06; found, 320.06.
Pentamethyl 2-(4-Iodobenzyl)diethylenetriaminepenta(acetate), 4. Compound 3 (2.7 g, 8.5 mmol) was suspended in dry DMF (80 mL), and the mixture was deaerated with argon. DIPEA (16.2 mL, 93 mmol), potassium iodide (1.54 g, 9.3 mmol), and methyl bromoacetate (7.2 mL, 75.8 mmol) were added, and the mixture was stirred overnight at RT. All volatiles were removed in vacuo. The residue was suspended on dichloromethane (140 mL), washed with sat. NaHCO3 (3 ?55 mL), dried over Na2SO4, and concentrated. Purification on silica gel (eluent petroleum ether, bp 40-60 C/ethyl acetate/ triethylamine 5:1:1, v/v/v) yielded 3.1 g (55%) of compound 4 as an oil. 1H NMR (CDCl3): 7.58 (2H, d, J 8.2); 6.97 (2H, d, J 8.2); 3.69 (6H, s); 3.66 (6H, s); 3.63 (3H, s); 3.57 (2H, s); 3.56 (2H, s); 3.54 (4H, s); 3.53 (2H, s); 3.10 (1H, p, J 6.6); 2.80 (1H, m) 2.74 (4H, m); 2.60 (1H, m); 2.47 (2H, m). ESI-TOF-MS for C29H39IN3O10 (M + H)+: calcd, 680.17; found, 680.18. IR (film) 1747 cm-1 (C=O).
2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3-(5-hexyn-1-yl)uridine, 5. 2'-Deoxy-5'-O-(4,4'-dimethoxy-trityl)uridine (5.0 g, 9.4 mmol), 5-hexyn-1-ol (1.25 mL, 11.3 mmol), and triphenylphosphine (3.0 g, 11.3 mmol) were dissolved in dry THF (50 mL). Diethylazodicarboxylate (1.80 mL, 11.3 mmol) was added dropwise during 30 min, and the reaction was allowed to proceed for 2 h at RT. All volatiles were removed in vacuo. Purification on silica gel using diethyl ether as the eluent gave 4.15 g (72%) of the title compound. 1H NMR (DMSO-d6): 7.70 (1H, d, J 8.1); 7.39 (2H, m); 7.31 (2H, m); 7.25 (5H, m); 6.90 (4H, d, J 8.0); 6.18 (1H, t, J 6.3); 5.49 (1H, J 8.1); 5.38 (1H, d, J 4.6; exch. with MeOD); 4.31 (1H, m); 3.90 (1H, m); 3.78 (2H, m); 3.74 (6H, s); 3.26 (1H, dd, J 5.4 and 10.7); 3.19 (1H, dd, J 2.9 and 10.7); 2.23 (3H, 3 ? m); 1.63 (2H, p); 1.47 (1H, t); 1.43 (2H, m). ESI-TOF-MS for C36H39N2O7 (M+H)+: calcd, 611.27; found, 611.29.
2'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3-{pentamethyl-2-[4-(5-hexyn-1-yl)benzyl]diethylene-triaminepentaacetato}uridine, 6. Compounds 5 (1.15 g, 1.80 mmol) and 4 (1.00 g, 1.47 mmol) were dissolved in the mixture of dry THF (10 mL) and triethylamine (6 mL) under argon. Pd(Ph3P)2Cl2 (22 mg, 0.03 mmol) and CuI (14 mg, 0.07 mmol) were added, and the mixture was stirred overnight at RT. The solid material was removed by filtration, and the filtrate was concentrated in vacuo. The residue was dissolved in dichloromethane (30 mL), washed with water (2 ? 20 mL), and dried over Na2SO4. Purification on silica gel (eluent petroleum ether, bp 40-60 C/ethyl acetate/triethylamine 2:5:1, v/v/v) gave 1.30 g (74%) of compound 6. 1H NMR (CDCl3): 7.74 (1H, d, J 8.2); 7.40-7.23 (11H, m); 7.11 (2H, d, J 8.2); 6.84 (4H, d, J 8.0); 6.32 (1H, t, J 6.4); 5.47 (1H, J 8.2); 4.55 (1H, m); 4.01 (1H, m); 3.97 (2H, m); 3.79 (6H, s); 3.68 (6H, s); 3.66 (6H, s); 3.62 (3H, s); 3.57 (2H, s); 3.55 (2H, s); 3.53 (4H, s); 3.47 (2H, m); 3.15 (1H, m); 2.85 (1H, m); 2.72 (2H, d, J 4.3); 2.58 (1H, m); 2.44 (2H, t, J 7.3); 1.82 (2H, m); 1.69 (2H, m). ESI-TOF-MS for C62H76N5O17 (M+H)+: calcd