A Robust and Convergent Synthesis of Dipeptide−DOTAM Conjugates

Bioconjugate Chem. 2007, 18, 1625−1636

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A Robust and Convergent Synthesis of Dipeptide-DOTAM Conjugates as Chelators for Lanthanide Ions: New PARACEST MRI Agents Filip Wojciechowski,† Mojmir Suchy,†,‡ Alex X. Li,‡ Hassan A. Azab,§ Robert Bartha,‡ and Robert H. E. Hudson*,† Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7. Received April 16, 2007; Revised Manuscript Received July 11, 2007

A generally applicable synthetic approach to dipeptide-DOTAM conjugates has been developed which is based on the peralkylation of 1,4,7,10-tetraazacyclododecane (cyclen) with protected N-iodoacetyl dipeptides. Standardized procedures were used for the alkylation, metalation, and purification of the resultant lanthanide complexes. Using this approach, we have been able to rapidly and reliably prepare and screen five different ligands each with up to six lanthanide ions. This preliminary investigation has identified several paramagnetic compounds with strong chemical exchange saturation transfer (PARACEST) properties in water at physiological temperature and pH. Extension of the synthetic approach to a wide variety of amino acids is possible.

INTRODUCTION Over the course of the past decade, there have been dramatic developments in the use of chelated lanthanide metals as contrast agents in magnetic resonance imaging (MRI). The recognition that chemical exchange saturation transfer (CEST) via proton exchange (1) could be exploited for generating MR contrast has led to new ligand designs. Subsequently, it has been shown that paramagnetic lanthanide ions induced a large hyperfine shift of the bound water protons and exchangeable protons in the ligand framework, and that MR signal modulation could be generated if the exchange rate of the spins with bulk water was slow (2). Selective saturation of these exchangeable spins led to a new method of generating MRI contrast now referred to as PARACEST: paramagnetic chemical exchange saturation transfer (2). The PARACEST effect has been exploited to image or otherwise detect biologically important metabolites, such as glucose (3) and lactate (4), and to measure physiologic parameters such as temperature (5) and pH by a ratiometric method (6, 7). A further advantage of employing the PARACEST effect is that one ligand may be endowed with different magnetic properties by the use of different lanthanide(III) ions (7, 8). The syntheses of tetra-substituted 1,4,7,10-tetraazacyclododecanes (cyclens) has been well-documented in the literature. The reaction of the cyclic polyamine with electrophiles is the most common route for the synthesis of tetra-substituted cyclen. Even so, there exists little by way of standard procedures for enabling systematic variation of the periphery of a derivatized cyclen. It has been established previously that the tetraamide derivatives of cyclen are an important structural feature for the slow exchange of bound water with bulk water in the sample and also provide a pool of exchangeable protons in the ligand (6, 7, 9-11). Therefore, a straightforward and reliable synthetic route to a variety of peptide-decorated cyclens may produce interesting ligands with potential to be used as PARACEST MRI agents. * E-mail address: [email protected]. † The University of Western Ontario. ‡ Centre for Functional and Metabolic Mapping, Robarts Research Institute, Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada, N6A 5K8. § Chemistry Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt.

The compounds described herein are structurally related to the lanthanide chelators 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′tetraacetic acid (DOTA) and 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetamide (DOTAM) (12), as shown in Figure 1. To access peptide-decorated cyclens, there are two obvious routes to consider: the coupling of appropriately protected amino acids to DOTA, which has been reported to give a low yield (13), or the alkylation of cyclen with N-terminal haloacetyl amino acids, which can proceed in very good yields (14-18). The latter approach is shown herein to provide a robust and flexible route to a diverse selection of dipeptide-conjugated DOTAM derivatives (Scheme 1). In this synthetic scheme, the DOTAM-ligand framework is preserved while the nature of the periphery is readily varied. This approach has proven to be very efficient and convenient, permitting the synthesis of ligands possessing neutral (N-GlyPhe-C), basic (N-Gly-Lys-C), and acidic (N-Asp-Glu-C) amino acids with minimal protecting group manipulations. We also describe a convenient and reproducible size-exclusion purification method for the isolation of metalated compounds. The MR behavior of the metalated ligands has been determined. It is shown that Eu3+-DOTAM-(Gly-Phe)4 possesses a large CEST effect at physiological temperature and pH while Nd3+DOTAM-(Gly-Lys)4 and Yb3+-DOTAM-(Gly-Lys-OMe)4 possess CEST effect associated with amide protons.

EXPERIMENTAL SECTION General Experimental Procedures. All amino acids (naturally occurring L-isomers) and reagents were commercially available, unless otherwise stated. All solvents were HPLC grade and used as such, except for CH2Cl2 and THF (dried over Al2O3, in a solvent purification system) and water (18.2 MΩ/cm). Organic extracts were dried with Na2SO4, and solvents were removed under reduced pressure by rotary evaporation. Flash column chromatography (FCC) was carried out using silica gel, mesh size 230-400 Å. Size exclusion chromatography (SEC) was carried out on Bio-Gel P2, 45-90 µm mesh resin (20 g, column size 15 × 2 cm per 0.1 mmol of compound). Ten fractions (10 mL each) were collected. Fractions were identified by 5% solution of ninhydrin in AcOH/EtOH (tetraacetyl-GlyLys-OH and Gly-Lys-OMe cyclen derivatives); by I2 vapors (tetraacetyl-Asp-Glu-OH derivatives); or by UV/I2 vapors (tetraacetyl-Gly-Phe-OH and Asp-Phe-OH derivatives). Meta-

10.1021/bc0701287 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/22/2007

1626 Bioconjugate Chem., Vol. 18, No. 5, 2007

Wojciechowski et al.

Figure 1. Structural comparison of the known chelators DOTA and DOTAM to the dipeptide conjugates of DOTAM. Scheme 1. Retrosynthetic Analysis of Lanthanide(III) Complexes of Cyclen-Oligopeptide Conjugates

lation of the ligands was confirmed by inspection of the 1H NMR spectra (Supporting Information), MS (ESI-TOF) spectrograph, or by HPLC analysis of the reaction mixture and fractionated products. Effective separation of the metalated ligand from free metal by SEC was confirmed by MS (ESITOF) analysis of the fractions. Thin layer chromatography (TLC) was carried out on Al-backed silica gel plates; compounds were visualized by UV light or I2 vapors. Melting points were obtained on a Fisher-Johns apparatus and are uncorrected. Specific rotations [R]D were determined at ambient temperature using a 5 mL, 10 cm path length cell; the units are 10-1 deg cm2 g-1 and the concentrations are reported in g/100 mL. NMR spectra of the synthetic intermediates were recorded on 400 MHz spectrometer for 1H; δ values were referenced as follows: CDCl3 (7.23 ppm); DMSO-d6 (2.49 ppm); D2O (4.75 ppm) for 13C (125 MHz) CDCl (77.0 ppm); DMSO-d (39.5 ppm). 3 6 Chemical exchange saturation transfer (CEST) spectra were acquired on a 400 MHz (9.4 T) Varian INOVA AS400 spectrometer. The CEST effect was measured by recording the bulk water signal intensity as function of presaturation frequency. The basic pulse sequence consisted of a 10 s presaturation pulse (B1 ) 14 µT) followed by a hard (13.8 µs) 90° pulse and a 3 s data acquisition (receiver bandwidth ) 6400 Hz, repetition time ) 13 s). All CEST spectra were recorded at a ligand concentration of 10 mM in a NaH2PO4 (10 mM), pH 7.0, buffer at near-physiologic temperature (37 or 38 °C). Mass spectra (MS) were obtained using electron impact (EI) and electrospray (ES) for ionization. Peptide sequences are denoted in the conventional way from left-to-right, N-to-C terminus. Synthesis. N-Chloroacetylglycine (1a). Glycine (30 g, 0.4 mol) was dissolved in 2.5 N NaOH (400 mL, 1.0 mol), and diethyl ether (200 mL) was added. The mixture was cooled (0 °C), and the solution of chloroacetyl chloride (32 mL, 0.4 mol) in 150 mL of ether was added dropwise over 30 min. The mixture was stirred for 45 min at 0 °C, then allowed to gradually warm up to rt while stirring for further 2 h. The organic layer

was separated, and the aqueous layer was extracted with Et2O (100 mL). The aqueous layer was acidified (pH 2) and extracted with EtOAc (4 × 250 mL). The combined ethyl acetate extracts were washed with 100 mL of brine, dried, and concentrated to afford a colorless oil. Hexanes (200 mL) were added to the obtained oil, and the solvent was removed to give a white solid. The crude product was recrystallized from EtOAc to give the title compound in 43% yield. mp 105-106 °C. 1H NMR (DMSO-d6) δ 12.68 (br s, 1H); 8.51 (t, J ) 5.0 Hz, 1H); 4.13 (s, 2H); 3.79 (d, J ) 5.9 Hz, 2H). O-Chloroacetyl-N-hydroxysuccinimide (5). DCC (15.5 g, 75.0 mmol) was dissolved in a minimum amount of THF and added dropwise to a solution (cooled to 0 °C) of NHS (8.7 g, 75.0 mmol) and chloroacetic acid (7.1 g, 75.0 mmol) in THF (200 mL). The reaction was stirred for 5 h at 0 °C, the precipitate (N,N′-dicyclohexylurea) was filtered off, and the filtrand was washed with 50 mL of THF. Hexanes (50 mL) were added and the filtrate was set aside for 12 h at 5 °C. Additional amounts of N,N′-dicyclohexylurea were filtered off, and the filtrate was concentrated in vacuo. The product was triturated with 100 mL of hexanes, set aside at 5 °C for 2 h, and the product collected by filtration. White solid, 11.9 g (83%); mp 109-110 °C (lit. 120 °C (19)). 1H NMR (CDCl3) δ 4.36 (s, 2H); 2.86 (s, 4H). Aspartic Acid β-Methyl Ester Hydrochloride (4). Methanol (MeOH, 100 mL) was placed in a round-bottom flask (I) and cooled to 0 °C. Acetyl chloride (AcCl, 33.4 g, 0.42 mol) was added dropwise over a period of 30 min (caution: the reaction is exothermic and dropwise addition is necessary), and the mixture was stirred for a further 30 min at 0 °C. A solution of aspartic acid (40.0 g, 0.3 mol) in MeOH (100 mL) in a separate round-bottom flask (II) was cooled to 0 °C, and the cold (0 °C) methanolic HCl generated in the flask I was added to the flask II. After 3 h at 0 °C, the ice bath was removed and stirring continued for a further 8 h at rt. The reaction mixture was concentrated to approximately 1/3 of its original volume (bath temperature not exceeding 25 °C). Diethyl ether (Et2O, 500 mL)

Synthesis of Dipeptide-DOTAM Conjugates for MRI

was added and the solution was set aside for 12 h at 0 °C. The white precipitate was filtered off to give 47.7 g (87%) of methyl ester 4, mp 190-193 °C (lit. 191-193 °C (20)). 1H NMR (D2O) δ 4.30 (t, J ) 5.5 Hz, 1H); 3.67 (s, 3H); 3.05 (m, 2H). N-Chloroacetyl-Asp(OMe)-OH (1b). Aspartic acid β-methyl ester (4) (10.9 g, 50 mmol) was dissolved in dioxane/H2O (1:1, 250 mL), and the solution was cooled to 0 °C. Na2CO3 (5.3 g, 50 mmol) was added to the solution, followed by stirring (at 0 °C) for 30 min. Another portion of Na2CO3 (5.3 g, 50 mmol) was added, followed by portionwise addition of O-chloroacetylN-hydroxysuccinimide (5) (9.58 g, 50 mmol). The mixture was stirred for 2 h at 0 °C, and for 12 h at rt. Organic impurities were removed by extraction with EtOAc (200 mL); the aqueous phase was acidified (pH 2) with HCl (36%) and was extracted with EtOAc (4 × 100 mL). The combined organic extracts (obtained after adjusting the pH to 2) were dried and concentrated to afford a colorless oil (9.10 g, 81%); the product was partially contaminated with NHS (approximately 15 mol %). 1H NMR (CDCl ) δ 10.48 (br s, 1H); 7.68 (br d, J ) 7.5 Hz, 3 1H); 4.87 (m, 1H); 4.10 (m, 1H); 3.71 (s, 3H); 3.10 (dd, J ) 17.5 Hz, 3.5 Hz, 1H); 2.89 (dd, J ) 17.5 Hz, 3.5 Hz, 1H); 2.74 (s, 1H). 13C NMR 173.0, 172.8, 171.4, 167.1, 52.2, 48.7, 42.1, 35.4, 25.3. HRMS (EI) m/z: found 224.0321, calcd 224.0278 for C7H11ClNO5 [M + H]+. LRMS (EI) m/z (rel abundance): 224 [M + H+] (100), 192 (9), 178 (10), 116 (48). Coupling of N-Chloroacetylglycine (1a) with H-Phe-OEt‚HCl and H-Lys(Boc)-OMe‚HCl to GiVe 2a and 2b. General procedure: To a stirred suspension of N-chloroacetylglycine (1a, 455 mg, 3 mmol) in dry CH2Cl2 (25 mL), NHS (345 mg, 3 mmol) and DCC (805 mg, 3.9 mmol) were added (at 0 °C). Separate suspensions of H-Phe-OEt‚HCl (687 mg, 3 mmol) and H-Lys(Boc)-OMe‚HCl (890 mg, 3 mmol) in dry CH2Cl2 (5 mL) were treated with Et3N (840 µL, 6 mmol) and were stirred for 10 min at rt before addition to the mixture containing NHS, DCC, and N-chloroacetylglycine. The mixtures were stirred for 18 h at rt; the precipitate (N,N′-dicyclohexylurea and Et3N‚HCl) was filtered off; the filtrates were consecutively washed with 1 M HCl and brine (30 mL each) and were dried to afford white solids, purified by trituration in Et2O (N-chloroacetyl-Gly-PheOEt, 882 mg, 90%) or by crystallization using CH2Cl2/hexane solution (N-chloroacetyl-Gly-Lys(Boc)-OMe, 1.08 g, 91%). N-Chloroacetyl-Gly-Phe-OEt (2a), amorphous white solid; [R]25D +20 (c 0.50, CH2Cl2). 1H NMR (DMSO-d6) δ 8.44 (br d, D2O exch., J ) 7.5 Hz, 1H); 8.37 (br t, D2O exch., J ) 5.5 Hz, 1H); 7.27 (m, 2H); 7.20 (m, 3H); 4.43 (ddd, J ) 6 Hz, 6 Hz, 1 Hz, 1H); 4.10 (s, 2H); 4.02 (q, J ) 7 Hz, 2H); 3.73 (m, 2H); 2.99 (dd, J ) 6 Hz, 6 Hz, 1H); 2.90 (dd, J ) 8.5 Hz, 8.5 Hz, 1H); 1.08 (t, J ) 7 Hz). 13C NMR (DMSO-d6) δ 171.3, 168.4, 166.1, 137.0, 129.1, 128.3, 126.6, 60.6, 53.7, 42.5, 41.8, 36.8, 13.9. HRMS (EI) m/z: found 326.1035 (326.1033 calcd for C15H19ClN2O4). LRMS (EI) m/z (rel abundance): 326 [M]+ (5), 224 (16), 176 (100), 120 (55), 91 (37). N-Chloroacetyl-Gly-Lys(Boc)-OMe (2b), white crystals, mp 83-84 °C; [R]25D -11 (c 0.47, CH2Cl2). 1H NMR (CDCl3) δ 7.42 (br s, D2O exch., 1H); 6.94 (br d, D2O exch., J ) 7 Hz, 1H); 4.71 (br t, D2O exch., J ) 5.5 Hz, 1H); 4.56 (ddd, J ) 7.5 Hz, 7.5 Hz, 1 Hz, 1H); 4.08 (s, 2H); 4.04 (d, J ) 5.5 Hz, 2H); 3.73 (s, 3H); 3.08 (ddd, 6 Hz, 6 Hz, 1 Hz, 2H); 1.85 (m, 1H); 1.72 (m, 1H); 1.47 (m, 2H); 1.42 (s, 9H); 1.31 (m, 2H). 13C NMR (CDCl3) δ 172.5, 167.9 , 166.6, 156.2, 79.2, 52.5, 52.2, 43.0, 42.3, 39.9, 31.5, 29.5, 28.4, 22.3. HRMS (EI) m/z: found 394.1745 (394.1745 calcd for C16H29ClN3O6). LRMS (EI) m/z (rel abundance): 394 [M + H]+ (53), 360 (27), 338 (60), 294 (100), 225 (51), 100 (30). Coupling of N-Chloroacetyl-Asp(OMe)-OH (1b) with H-Glu(OMe)-OMe‚HCl or H-Phe-OEt‚HCl to giVe 2c and 2d. General procedure: To a stirred suspension of N-chloroacetyl-Asp-

Bioconjugate Chem., Vol. 18, No. 5, 2007 1627

(OMe)-OH (1b, 4.47 g, 20.0 mmol) in dry THF (50 mL), NHS (2.30 g, 20.0 mmol) and DCC (4.54 g, 22.0 mmol) were added (at 0 °C) and the suspension was stirred for 1 h at 0 °C. Separate suspensions of H-Glu(OMe)-OMe‚HCl (4.66 g, 22.0 mmol) or H-Phe-OEt‚HCl (4.30 g, 22.0 mmol) in dry THF (50 mL) were treated with Et3N (3.2 mL, 22.0 mmol) and were stirred for 1 h at 0 °C, followed by the addition to the mixture containing NHS, DCC, and N-chloroacetyl-Asp(OMe)-OH. The ice baths were removed and separate mixtures were stirred for 12 h at rt, and the precipitate (N,N′-dicyclohexylurea and Et3N‚HCl) was filtered off. The filtrates were concentrated and were extracted with EtOAc (150 mL). The organic extracts were consecutively washed with 1 M NaH2PO4 (pH 2.0, adjusted with H3PO4) (2 × 50 mL), a saturated aqueous solution of NaHCO3 (2 × 50 mL), and brine (50 mL); were dried over Na2SO4 and concentrated to give oils. The obtained oils were dissolved in EtOAc (75 mL), the solutions were set aside for 5 h at 5 °C, and more of the precipitated N,N′-dicyclohexylurea was filtered off. The filtrates were concentrated. Compound 2c was purified by trituration with Et2O/hexanes (1:1, 100 mL) to afford an offwhite solid which was further triturated with MeOH/Et2O/ hexanes (1:1:4, 150 mL) to give N-chloroacetyl-Asp(OMe)Glu(OMe)-OMe (2c, 5.8 g, 76%). Compound 2d was triturated with Et2O/hexanes (1:1, 100 mL) to obtain an off-white solid, recrystallized form acetone/hexanes (1:3, 65 mL) to give N-chloroacetyl-Asp(OMe)-Phe-OEt (2d, 6.19 g, 81%). N-Chloroacetyl-Asp(OMe)-Glu(OMe)-OMe (2c), white solid; [R]25D -37 (c 0.54, MeOH). 1H NMR (CDCl3) δ 7.79 (d, J ) 8 Hz, 1H); 7.29 (d, J ) 8 Hz, 1H); 4.82 (m, 1H); 4.51 (m, 1H); 4.08 (m, 2H) 3.69 (s, 3H); 3.69 (s, 3H); 3.64 (s, 3H); 2.99 (dd, J ) 17 Hz, 4.5 Hz, 1H); 2.66 (dd, J ) 17 Hz, 6.5 Hz, 1H); 2.36 (m, 2H); 2.17 (m, 1H); 1.97 (m, 1H). 13C NMR (CDCl3) δ 173.2, 172.2, 171.5, 169.7, 166.3, 52.4, 52.2, 52.0, 51.8, 49.3. 42.4, 35.5, 29.8, 26.6. HRMS (EI) m/z: found 381.1056 [M + H+] (calcd 381.1065 for C14H22ClN2O8). LRMS: m/z (rel. abundance): 381 [M + H]+ (100), 225 (25). N-Chloroacetyl-Asp(OMe)-Phe-OEt (2d), white solid, mp 115-116 °C; [R]25D -18 (c 0.56, MeOH). 1H NMR (CDCl3) δ 7.88; (d, J ) 7.8 Hz, 1H); 7.29 (m, 3H); 7.13; (d, J ) 7.0 Hz, 2H); 6.92; (d, J ) 7.8 Hz, 1H); 4.78 (m, 2H); 4.17 (q, J ) 7.2 Hz, 2H); 3.98 (m, 2H); 3.72 (s, 3H); 3.17 (dd, J ) 13.9 Hz, 5.7 Hz, 1H); 3.17 (dd, J ) 13.9 Hz, 6.6 Hz, 1H); 3.0 (dd, J ) 17.3 Hz, 3.6 Hz, 1H); 2.63 (dd, J ) 17.3 Hz, 6.9 Hz, 1H); 1.25 (t, J ) 7.2 Hz, 3H). 13C NMR (CDCl3) δ 172.0, 170.7, 169.2, 166.1, 135.5, 129.1, 128.3, 126.9, 61.4, 53.3, 52.0, 49.1, 42.1, 37.3, 35.0, 13.9. HRMS (EI) m/z: found 398.1252 [M+] (calcd 398.1243 for C18H23ClN2O6). LRMS (EI) m/z (rel. abundance): 398 [M+] (10), 176 (100), 120 (35), 102 (33). ConVersion of N-Chloroacetyldipeptides 2a and 2b to NIodoacetyldipeptides 3a and 3b. Sodium iodide (2.25 g, 15 mol) was added to separate solutions of N-chloroacetyl-Gly-Phe-OEt (2a, 817 mg, 2.5 mmol) or N-chloroacetyl-Gly-Lys(Boc)-OMe (2b, 985 mg, 2.5 mmol) in acetone (15 mL). The mixtures were stirred for 18 h at rt, concentrated to ca. 1/4 of their original volumes, diluted with EtOAc (25 mL), and finally washed with 10% solution of Na2SO3 (25 mL). The aqueous layers were extracted with EtOAc (20 mL), and the combined organic extracts were dried and concentrated to afford white solids, purified by trituration in Et2O (N-iodoacetyl-Gly-Phe-OEt, 816 mg, 78%) or by crystallization using 1:1 acetone/hexane solution (N-iodoacetyl-Gly-Lys(Boc)-OMe, 1.01 g, 83%). N-Iodoacetyl-Gly-Phe-OEt (3a), amorphous white solid; [R]25D +30 (c 0.50, CH2Cl2). 1H NMR (DMSO-d6) δ 8.42 (br m, D2O exch., 2H); 7.27 (m, 2H); 7.20 (m, 3H); 4.43 (ddd, J ) 8 Hz, 8 Hz, 1 Hz, 1H); 4.02 (q, J ) 7 Hz); 3.70 (m, 2H); 3.68 (s, 2H); 2.98 (dd, J ) 6 Hz, 6 Hz, 1H); 2.90 (dd, J ) 8.5 Hz, 8.5 Hz, 1H); 1.08 (t, J ) 7 Hz). 13C NMR (DMSO-d6) δ 171.3,


168.5, 167.8, 136.9, 129.1, 128.2, 126.6, 60.5, 53.7, 42.0, 36.9, 33.3, 13.9. HRMS (EI) m/z: found 418.0347 [M+] calcd (418.0390 for C15H19IN2O4). LRMS (EI) m/z (rel abundance): 418 [M]+ (5), 225 (20), 176 (100), 120 (41), 91 (19). N-Iodoacetyl-Gly-Lys(Boc)-OMe (3b), white crystals, mp 101-102 °C; [R]25D -11 (c 0.45, CH2Cl2). 1H NMR (CDCl3) δ 7.12 (br s, D2O exch., 1H); 6.87 (br d, D2O exch., J ) 7.5 Hz, 1H); 4.72 (br t, D2O exch., J ) 6 Hz, 1H); 4.56 (ddd, J ) 7 Hz, 7 Hz, 1 Hz, 1H); 4.01 (m, 2H); 3.75 (s, 2H); 3.74 (s, 3H); 3.10 (ddd, 6 Hz, 6 Hz, 1 Hz, 2H); 1.87 (m, 1H); 1.74 (m, 1H); 1.50 (m, 2H); 1.44 (s, 9H); 1.36 (m, 2H). 13C NMR (CDCl3) δ 172.5, 168.5, 168.2, 156.2, 79.3, 52.5, 52.3, 43.6, 40.0, 39.9, 31.4, 29.5, 28.4, 22.4. HRMS (EI) m/z: found 486.1086 (486.1056 calcd for C16H28IN3O6). LRMS (EI) m/z (rel abundance): 394 [M]+ (5), 385 (36), 187 (21), 142 (100), 128 (14), 84 (36). ConVersion of N-Chloroacetyldipeptides 2c and 2d to NIodoacetyldipeptides 3c and 3d. Sodium iodide (9.0 g, 60 mmol) was added to separate solutions of N-chloroacetyl-Asp(OMe)Glu(OMe)-OMe (2c, 3.81 g, 10.0 mmol) or N-chloroacetyl-Asp(OMe)-Phe-OEt (2d, 3.84 g, 10.0 mmol) in acetone (50 mL). The mixtures were stirred for 1 h at rt and for 2 h at 45 °C, cooled to rt, concentrated to ca. 1/4 of their original volumes, diluted with EtOAc (100 mL), and finally washed with 10% solution of Na2SO3 (4 × 25 mL). The aqueous layers were extracted with EtOAc (25 mL), and the combined organic extracts were dried and concentrated to afford white solids which were purified by trituration in acetone/hexanes (1:10 v/v, 100 mL) to give N-chloroacetyl-Asp(OMe)-Glu(OMe)-OMe (3c, 3.89 g, 82%) or N-chloroacetyl-Asp(OMe)-Phe-OEt (3d, 4.31 g, 90%). N-Iodoacetyl-Asp(OMe)-Glu(OMe)-OMe (3c), white solid; [R]25D -27 (c 0.56, MeOH). 1H NMR (CDCl3) δ 7.57 (d, J ) 7.8 Hz, 1H); 7.32 (d, J ) 7.6 Hz, 1H); 4.81 (m, 1H); 4.48 (m, 1H); 3.70 (m, 2H) 3.64 (s, 6H); 3.59 (s, 3H); 2.85 (dd, J ) 17.0 Hz, 4.7 Hz, 1H); 2.69 (dd, J ) 16.8 Hz, 6.4 Hz, 1H); 2.34 (m, 2H); 2.12 (m, 1H), 1.94 (m, 1H). 13C NMR (CDCl3) δ 173.0, 171.7, 171.4, 170.0, 167.9, 52.3, 52.0, 51.8, 51.7, 49.6, 35.5, 29.8, 26.7. HRMS (EI) found 472.0339 [M+] (calcd 472.0343 for C14H21IN2O8) LRMS m/z (%): 472 [M+] (5), 440 (5), 345 (5), 270 (20), 144 (100). N-Iodoacetyl-Asp(OMe)-Phe-OEt (3d), white solid; [R]25D -19 (c 0.54, MeOH). 1H NMR (CDCl3) δ 7.29 (m, 3H); 7.14; (d, J ) 7.2 Hz, 2H); 6.94; (d, J ) 8.0 Hz, 1H); 4.75 (m, 2H); 4.15 (q, J ) 7.2 Hz, 2H); 3.70 (s, 3H); 3.63 (m, 2H); 3.14 (dd, J ) 13.9 Hz, 6.0 Hz, 1H); 3.07 (dd, J ) 13.9 Hz, 6.6 Hz, 1H); 2.96 (dd, J ) 17.2 Hz, 3.9 Hz, 1H); 2.61 (dd, J ) 17.3 Hz, 6.9 Hz, 1H); 1.25 (t, J ) 7.2 Hz, 3H). 13C NMR (CDCl3) δ 171.4, 170.6, 169.6, 167.7, 135.6, 129.0, 128.2, 126.8, 61.2, 53.5, 51.9, 49.5, 37.4, 35.4, 13.8. HRMS (EI) m/z: found 490.0597 [M+] (calcd 490.0598 for C18H23IN2O6). LRMS (EI) m/z (rel. abundance): 490 [M+] (10), 270 (20), 176 (100), 102 (50). Alkylation of Cyclen with Iodoacetyl Dipeptides 3a, 3b, 3c, and 3d. N-Iodoacetyl-Gly-Phe-OEt (3a, 836 mg, 2 mmol), N-iodoacetyl-Gly-Lys(Boc)-OMe (3b, 971 mg, 2 mmol), Niodoacetyl-Asp(OMe)-Glu(OMe)-OMe (3c, 950 mg, 2 mmol), and N-iodoacetyl-Asp(OMe)-Phe-OEt (3d, 985 mg, 2 mmol) were added (at rt) to a stirred solution of cyclen (86 mg, 0.5 mmol) and diisopropylethylamine (350 µL, 2 mmol) in acetonitrile (7 mL). The mixtures were stirred for 18 h at 50 °C (3a and 3b) or 70 °C (3c and 3d), cooled to rt, diluted with EtOAc (25 mL), and washed with 10% solution of Na2SO3 (25 mL). The aqueous layers were extracted with EtOAc (20 mL), and the combined organic extracts were dried and concentrated to afford white solids, purified by repeated (twice) trituration with hexanes.


Tetraacetyl-Gly-Phe-OEt cyclen (6a, 653 mg, 98%), white solid; [R]25D -10 (c 0.50, CH2Cl2). 1H NMR (CDCl3) δ 7.407.08 (m, D2O exch., 7H); 4.74-4.62 (m, 1H); 4.14-3.79 (br m, 4H); 3.30-3.01 (br m, 4H); 2.82-2.26 (br m, 4H); 1.241.11 (m, 3H). 13C NMR (CDCl3) δ 171.9, 171.4, 171.3, 169.4, 167.8, 136.2, 129.3, 128.5, 126.9, 61.6, 61.4, 56.5, 54.6, 54.0, 53.7, 42.8, 42.4, 37.7, 37.6, 14.0. HRMS (ESI) m/z: found 1355.6617 [M + Na]+ (calcd 1355.6652 for C68H92N12O16Na). Tetraacetyl-Gly-Lys(Boc)-OMe cyclen (6b, 777 mg, 97%), white solid, [R]25D -30 (c 0.50, CH2Cl2). 1H NMR (CDCl3) δ 7.79 (br s, D2O exch., 1H); 7.45 (br s, D2O exch., 1H); 4.98 (br s, D2O exch., 1H); 4.46-4.43 (br m, 1H); 4.05-3.95 (br m, 2H); 3.71 (s, 3H); 3.52-2.62 (br m, 4H); 3.07 (ddd, J ) 6.5 Hz, 6.5 Hz, 1 Hz); 2.05-1.98 (br m, 2H); 1.85-1.76 (br m, 2H); 1.51-1.34 (br m, 2H); 1.41 (s, 9H). 13C NMR (CDCl3) δ 173.1, 172.7, 172.0, 156.9, 79.0, 52.5, 52.4, 50.6, 49.0, 41.1, 40.1, 33.8, 29.4, 28.4, 25.6, 24.9. HRMS (ESI) m/z: found 1623.9191 [M + Na]+ (calcd 1623.9185 for C72H128N16O24Na). Tetraacetyl-Asp(OMe)-Glu(OMe)-OMe cyclen (6c, 670 mg, 87%), white solid; [R]25D -7 (c 0.69, CH2Cl2). 1H NMR (CDCl3) δ 7.88 (m, 2H); 7.34 (m, 2H); 7.17 (m, 2H); 4.51 (m, 4H); 4.19 (m, 4H); 3.79 (m, 3H); 3.36 (m, 36H); 2.77 (m, 10H); 2.60 (m, 12H); 2.10 (m, 8H); 1.86 (m, 4H); 1.71 (m, 4H). 13C NMR (CDCl3) δ 173.0, 172.8, 171.8, 171.7, 171.6, 171.4, 171.2, 170.3, 170.1, 52.2, 52.1, 51.7, 51.6, 51.4, 49.5, 49.1, 35.4, 29.6, 26.3. HRMS (ESI) found 1571.6516 [M + Na]+ (calcd 1571.6446 for C64H100N12O32Na). Tetraacetyl-Asp(OMe)-Phe-OEt cyclen (6d, 800 g, 98%), white solid; [R]25D -8 (c 0.61, CH2Cl2). 1H NMR (CDCl3) δ 7.37-7.07 (m, D2O exch., 7H); 4.78-4.66 (m, 1H); 4.20-4.08 (m, 3H); 3.62 (s, 3H); 3.49-2.38 (m, 8H); 1.35-1.18 (m, 3H). 13C NMR (CDCl ) δ 171.9, 171.8, 171.2, 170.1, 135.8, 129.2, 3 128.2, 128.1, 126.6, 61.3, 61.2, 55.7, 53.3, 53.2, 51.8, 51.6, 49.0, 37.0, 34.7, 13.8. HRMS (ESI) m/z: found 1643.7426 [M+Na]+ (calcd 1643.7497 for C80H108N12O24Na). Hydrolysis of Tetraacetyl-Gly-Phe-OEt Cyclen (6a). A solution of NaOH (384 mg, 9.6 mmol) in water (2.5 mL) was added to a stirred solution of tetraacetyl-Gly-Phe-OEt cyclen (6a, 800 mg, 0.6 mmol) in THF (2.5 mL). The mixture was stirred for 2 h at rt, then cooled to 0 °C, and the pH was adjusted to 4 (2.5 M HCl), after which an oil spontaneously separated. The water was decanted and the oil which deposited on the flask walls was triturated with Et2O (twice) to afford tetraacetyl-Gly-PheOH cyclen (7) as a white solid (550 mg, 75%); [R]25D -10 (c 0.50, EtOH). 1H NMR (DMSO-d6) δ 12.80 (br s, D2O exch., 1H); 8.46-8.30 (br m, D2O exch., 2H); 7.26-7.16 (m, 5H); 4.45-4.40 (br m, 1H); 3.80-3.64 (br m, 2H); 3.24-2.65 (br m, 4H); 3.05-3.00 (m, 2H); 2.90-2.84 (m, 2H). 13C NMR (DMSO-d6) δ 172.8, 170.8, 170.7, 137.5, 129.2, 128.3, 126.5, 56.07, 53.7, 41.7, 41.5, 36.8, 33.4. HRMS (ESI) m/z: found 1222.6270 [M + H]+ (calcd 1222.3242 for C60H77N12O16). Deprotection of Tetraacetyl-Gly-Lys(Boc)-OMe Cyclen (6b). Tetraacetyl-Gly-Lys(Boc)-OMe cyclen (6b, 150 mg, 0.094 mmol) was dissolved in TFA (1.5 mL), and the mixture was stirred for 15 min at rt. It was concentrated to afford tetraacetyl Gly-Lys-OMe cyclen tetratrifluoroacetate (8, 155 mg, 100%) as a slightly yellow oil which was used directly for further reactions. Hydrolysis of Tetraacetyl-Gly-Lys-OMe Cyclen Tetratrifluoroacetate (8). A solution of NaOH (2.5 M, 940 µL) was added to a solution of tetraacetyl-Gly-Lys-OMe cyclen tetratrifluoroacetate 8 (155 mg, 0.094 mmol) in MeOH (900 µL). The mixture was stirred for 2 h at 60 °C, MeOH was evaporated, and the mixture was neutralized (using 2 M HCl). It was subjected to size exclusion chromatography; fractions containing the product were combined and were freeze-dried to afford


tetraacetyl-Gly-Lys-OH cyclen (9) as a colorless oil (97 mg, 90%), [R]25D -167 (c 0.27, H2O). 1H NMR (D2O) δ 3.83 (br s, 1H); 3.69 (s, 2H); 3.62-3.58 (m, 2H); 3.13-2.62 (br m, 4H); 2.89-2.83 (m, 2H); 1.90-1.72 (m, 2H); 1.67-1.52 (m, 2H); 1.41-1.20 (m, 2H). 13C NMR (D2O) δ 178.9, 170.5, 55.2, 50.9, 49.3, 42.2, 39.4, 31.3, 26.5, 22.1. HRMS (ESI) m/z: found 1145.6628 [M + H]+ (calcd 1145.6642 for C48H89N16O16). Hydrolysis of Tetraacetyl-Asp(OMe)-Glu(OMe)-OMe Cyclen (6c). A solution of NaOH (2.5 M, 2 mL) was added to a solution of tetraacetyl-Asp(OMe)-Glu(OMe)-OMe cyclen (6c, 155 mg, 0.1 mmol) in MeOH (2 mL). The mixture was stirred for 2 h at 60 °C, MeOH was evaporated, and the mixture was neutralized (using 4 M HCl). It was subjected to size exclusion chromatography; fractions containing the product were combined and were freeze-dried to afford tetraacetyl-Asp-Glu-OH cyclen (10) as a colorless solid (103 mg, 75%); [R]25D +7 (c 0.62, H2O). 1H NMR (D O) δ 4.39-4.36 (m, 1H); 3.98-3.93 (m, 1H); 2 3.71-3.57 (m, 2H); 3.24-3.02 (m, 4H); 2.07-2.03 (m, 2H); 1.89-1.83 (m, 1H); 1.74-1.64 (m, 1H). 13C NMR (D2O) δ 181.9, 181.8, 179.0, 55.4, 52.3, 34.0, 28.8, 28.5. HRMS (ESI) m/z: found 1381.4708 [M + H]+ (calcd 1381.4767 for C52H77N12O32). Hydrolysis of Tetraacetyl-Asp(OMe)-Phe-OEt Cyclen (6d). A solution of NaOH (2.5 M, 7.5 mL) was added to a solution of tetraacetyl-Asp(OMe)-Phe-OEt cyclen (6d, 610 mg, 0.38 mmol) in MeOH (7.5 mL). The mixture was stirred for 20 min at 60 °C, MeOH was evaporated, and the mixture was cooled to 0 °C and was acidified (pH 2, using 4 M HCl). The oily product deposited on the flask wall and the water was decanted. The residue was triturated with Et2O (twice) to afford tetraacetylAsp-Phe-OH cyclen (11) as a colorless solid (530 mg, 95%); [R]25D +7 (c 0.73, DMSO). 1H NMR (DMSO-d6) δ 12.74 (br s, D2O exch., 2H); 8.85 (br s, D2O exch., 1H); 8.42 (br s, D2O exch., 1H); 7.32-7.17 (m, 5H); 4.63-4.37 (m, 2H); 3.78-3.73 (m, 1H); 3.45-2.55 (m, 9H); 13C NMR (DMSO-d6) δ 172.8, 172.6, 172.1, 169.1, 137.6, 129.2, 128.3, 126.5, 65.0, 56.1, 53.8, 48.9, 36.8, 36.5, 36.4, 18.6, 15.2. HRMS (ESI) m/z: found 1453.5862 [M + H]+ (calcd 1453.5800 for C68H85N12O24). Treatment of Tetraacetyl-Gly-Phe-OH Cyclen (7), TetraacetylGly-Lys-OH Cyclen (9), Tetraacetyl-Asp-Glu-OH Cyclen (10), and Tetraacetyl-Asp-Phe Cyclen (11) with Lanthanide(III) Chlorides. Separate solutions of tetraacetyl-Gly-Phe-OH cyclen (7, 114 mg, 0.094 mmol), tetraacetyl-Gly-Lys-OH cyclen (9, 107 mg, 0.094 mmol), tetraacetyl-Asp-Glu-OH cyclen (10, 130 mg, 0.094 mmol), and tetraacetyl-Asp-Phe-OH cyclen (11, 137 mg, 0.094 mmol) in water (3 mL) were treated with lanthanide(III) chlorides (0.094 mmol) as follows: tetraacetyl-Gly-PheOH cyclen (EuCl3‚6H2O, NdCl3‚H2O, TmCl3‚6H2O, and YbCl3‚ 6H2O); tetraacetyl-Gly-Lys-OH cyclen (DyCl3‚6H2O, EuCl3‚ 6H2O, NdCl3‚H2O, TbCl3‚6H2O, TmCl3‚6H2O, and YbCl3‚ 6H2O); tetraacetyl-Asp-Glu-OH cyclen (EuCl3‚6H2O, NdCl3‚ H2O, TmCl3‚6H2O, and YbCl3‚6H2O); tetraacetyl-Asp-Phe-OH cyclen (EuCl3‚6H2O). Reaction mixtures were treated with NaOH (2.5 M solution) to adjust the pH to 9 and were stirred for 18 h at rt followed by size exclusion chromatography. The fractions containing the lanthanide(III) complexes were combined and were isolated as dry solids by lyophilization. Eu3+ tetraacetyl-Gly-Phe-OH cyclen (12), white solid; 1H NMR (D2O) δ 24.44 (s); 21.93 (s); 8.50 (s); 7.83 (s); 7.47 (s); 7.36 (s); 7.10-6.80 (m); 6.59-6.58 (m); 6.38 (s); 6.22 (s); 6.04 (s); 3.57-2.05 (m); 1.50 (s); -3.08 (s); -3.78 (s); -4.49 (s); -5.04 (s); -8.03 (s); -8.91 (s); -9.20 (s); -9.91 (s); -11.66 (s); -12.05 (s); -13.06 (s). HRMS (ESI) m/z: found 1369.4544 [M - 2H]+ (calcd 1369.4597 for C60H74N12O16Eu). Nd3+ tetraacetyl-Gly-Phe-OH cyclen (13), white solid; 1H NMR (D2O) δ 15.00 (s); 13.34 (s); 9.31 (s); 7.44-6.37 (m);


5.76 (s); 5.19 (s); 4.91 (s); 4.18-2.60 (m); -14.68 (s); -16.55 (s). HRMS (ESI) m/z: found 1360.4401 [M - 2H]+ (calcd 1360.4423 for C60H74N12O16Nd). Tm3+ tetraacetyl-Gly-Phe-OH cyclen (14), white solid; 1H NMR (D2O) δ 29.42 (s); 14.79 (s); 13.18 (s) 7.49 (s); 7.097.00 (m); 5.09 (s); 4.86 (s); 4.20-4.11 (m); 3.54-3.38 (m); 2.94-1.43 (m); 1.16-0.94 (m); -3.41 (s); -4.69 (s); -14.15 (s); -15.76 (s); -16.54 (s). HRMS (ESI) m/z: found 1387.4661 [M - 2H]+ (calcd 1387.4688 for C60H74N12O16Tm). Yb3+ tetraacetyl-Gly-Phe-OH cyclen (15), white solid; 1H NMR (D2O) δ 91.49 (s); 16.96 (s); 14.04 (s); 10.89 (s); 9.23 (s); 7.95 (s); 7.69-6.81 (m); 5.71 (s); 3.93-1.00 (m); 0.52 (s); 0.41 (s); -2.59 (s); -2.91 (s); -24.02 (s); -29.20 (s); -53.19 (s). HRMS (ESI) m/z: found 1392.4300 [M - 2H]+ (calcd 1392.4688 for C60H74N12O16Yb). Dy3+ tetraacetyl-Gly-Lys-OH cyclen (22), colorless oil; 1H NMR (D2O) δ 36.98 (s); 34.01 (s); 13.58 (s); 11.01 (s); 10.70 (s); 10.09 (s); 9.72 (s); 8.11-7.92 (m); 4.29-4.14 (m); 3.602.84 (m); 1.87-1.58 (m). HRMS (ESI) m/z: found 1306.5760 [M - 2H]+ (calcd 1306.5700 for C48H86N16O16Dy). Eu3+ tetraacetyl-Gly-Lys-OH cyclen (23), colorless oil; 1H NMR (D2O) δ 25.10 (s); 15.13 (s); 13.37 (s); 9.36 (s); 6.54 (s); 5.55 (s); 3.21-0.68 (m); -2.89 (s); -5.06 (s); -10.18 (s); -13.14 (s); -16.60 (s). HRMS (ESI) m/z: found 1361.5056 [M - 5H + 3Na]+ (calcd 1361.5078 for C48H83N16O16EuNa3). Nd3+ tetraacetyl-Gly-Lys-OH cyclen (24), colorless oil; 1H NMR (D2O) δ 15.24 (s); 13.40 (s); 9.45 (s); 6.63 (s); 5.58 (s); 3.78 (s); 3.07 (s); 2.84-2.80 (m); 2.63 (s); 2.54 (s); 2.27 (s); 2.02 (s); 1.84-1.51 (m); 1.22-1.16 (m); -16.81 (s). HRMS (ESI) m/z: found 1287.5600 [M - 2H]+ (calcd 1287.5587 for C48H86N16O16Nd). Tb3+ tetraacetyl-Gly-Lys-OH cyclen (25), colorless oil; 1H NMR (D2O) δ 41.68 (s); 31.21 (s); 3.89-3.62 (m); 3.10-2.90 (m); 2.78-2.10 (m); 1.63-1.39 (m); 1.11 (s); -0.46 (s); -0.52 (s); -2.79 (s); -3.26 (s); -4.35 (s); -5.05 (s); -6.09 (s); -14.76 (s); -16.31 (s); -75.61 (s). HRMS (ESI) m/z: found 1301.5658 [M - 2H]+ (calcd 1301.5661 for C48H86N16O16Tb). Tm3+ tetraacetyl-Gly-Lys-OH cyclen (26), colorless oil; 1H NMR (D2O) δ 30.65 (s); 4.66 (s); 3.91-3.38 (m); 3.16-2.11 (m); 1.44-0.84 (m); -0.46 to -0.74 (m); -2.74 to -3.55 (m); -4.30 to -4.57 (m); -4.98 to -5.25 (m); -6.00 to -6.28 (m); -14.63 to -14.93 (m); -16.06 to -16.33 (m). HRMS (ESI) m/z: found 1311.5773 [M - 2H]+ (calcd 1311.5750 for C48H86N16O16Tm). Yb3+ tetraacetyl-Gly-Lys-OH cyclen (27), colorless oil; 1H NMR (D2O) δ 17.29 (s); 14.34 (s); 3.94-3.90 (m); 3.71 (s); 3.32 (s); 2.78-2.74 (m); 2.59 (s); 2.54 (s); 2.48 (s); 1.61-1.41 (m); 1.20-1.10 (m); -0.12 (s); -0.26 (s); -0.60 (s); -0.85 (s); -1.11 (s); -1.20 (s); -3.17 (s); -3.43 (s); -24.97 (s); -30.13 (s); -55.19 (s). HRMS (ESI) m/z: found 1316.5853 [M - 2H]+ (calcd 1316.5796 for C48H86N16O16Yb). Eu3+ tetraacetyl-Asp-Glu-OH cyclen (28), white solid; 1H NMR (D2O) δ 23.75 (s); 3.88-2.57 (m); 1.41-0.72 (m); -2.49 to -3.26 (m); -4.58 to -5.28 (m); -8.69 to -8.93 (m); -12.72 to -13.43 (m). HRMS (ESI) m/z: found 1529.3807 [M - 2H]+ (calcd 1529.3731 for C52H74N12O32Eu). Nd3+ tetraacetyl-Asp-Glu-OH cyclen (29), white solid; 1H NMR (D2O) δ 15.47 (s); 13.65 (s); 8.18-6.81 (m); 4.44-3.73 (m); 3.20-2.05 (m); 1.70-0.70 (m); -2.80 to -3.28 (m); -8.63 to -9.19 (m); -12.78 to -13.40 (m); -16.75 (s). HRMS (ESI) m/z: found 1522.3549 [M - 2H]+ (calcd 1522.3633 for C52H74N12O32Nd). Tm3+ tetraacetyl-Asp-Glu-OH cyclen (30), white solid; 1H NMR (D2O) δ 1.52 (s); -3.32 (s); -7.36 (s); -14.08 (s). HRMS (ESI) m/z: found 1547.3875 [M - 2H]+ (calcd 1547.3874 for C52H74N12O32Tm).


Yb3+ tetraacetyl-Asp-Glu-OH cyclen (31), white solid; 1H NMR (D2O) δ 19.17-13.45 (m); 1.32 to -6.21 (m); -23.84 to -34.64 (m). HRMS (ESI) m/z: found 1552.3845 [M - 2H]+ (calcd 1552.3921 for C52H74N12O32Yb). Eu3+ tetraacetyl-Asp-Phe-OH cyclen (32), white solid; 1H NMR (D2O) δ 24.99-23.14 (m); 7.25-6.58 (m); 4.26-4.09 (m); 3.19-1.09 (m); -2.59 to -5.03 (m); -8.17 to -9.43 (m); -12.07 to -13.13 (m). HRMS (ESI) m/z: found 1604.4856 [M - 2H]+ (calcd 1604.4916 for C68H83N12O24Eu). Treatment of Tetraacetyl-Gly-Lys-OMe Cyclen Tetratrifluoroacetate (8) with Lanthanide(III) Chlorides. Solutions of tetraacetyl-Gly-Lys-OMe cyclen tetratrifluoroacetate (8, 155 mg, 0.094 mmol) in water (3 mL) were treated with lanthanide(III) chlorides (DyCl3‚6H2O, EuCl3‚6H2O, NdCl3‚H2O, TbCl3‚H2O, TmCl3‚6H2O, and YbCl3‚6H2O, 0.094 mmol). Reaction mixtures were treated with NaOH (2.5 M solution) to adjust the pH to 9 and were stirred for 18 h at rt. The reaction mixtures were subjected to size exclusion chromatography. The fractions containing the lanthanide(III) complexes were combined and were freeze-dried to afford colorless oils. Dy3+ tetraacetyl-Gly-Lys-OMe cyclen (16), colorless oil; 1H NMR (D2O) δ 35.31 (s); 26.08 (s); 13.01 (s); 10.42 (s); 9.19 (s); 8.78 (s); 8.61 (s); 6.82 (s); 6.45 (s); 6.29 (s); 4.08-4.00 (m); 3.80 (s); 3.49-3.42 (m); 3.28 (s); 3.14 (s); 3.05-2.66 (m); 2.04-1.69 (m); 1.47-1.32 (m). HRMS (ESI) m/z: found 1362.6381 [M - 2H]+ (calcd 1362.6326 for C52H94N16O16Dy). Eu3+ tetraacetyl-Gly-Lys-OMe cyclen (17), colorless oil; 1H NMR (D2O) δ 24.19 (s); 12.56 (s); 6.06 (s); 3.86 (s); 3.60 (s); 3.11-2.45 (m); 1.74-0.85 (m); -3.17 (s); -4.63 (s); -8.80 (s); -9.62 (s); -12.80 (s). HRMS (ESI) m/z: found 1351.6251 [M - 2H]+ (calcd 1351.6246 for C52H94N16O16Eu). Nd3+ tetraacetyl-Gly-Lys-OMe cyclen (18), colorless oil; 1H NMR (D2O) δ 14.97 (s); 13.25 (s); 9.41-9.01 (m); 6.69 (s); 5.85-5.54 (m); 4.04-3.86 (m); 3.05-3.04 (m); 2.83 (s); 2.64 (s); 2.55 (s); 2.25-1.53 (m); 1.26-1.16 (m); -16.27 (s). HRMS (ESI) m/z: found 1343.5500 [M - 2H]+ (calcd 1343.6213 for C52H94N16O16Nd). Tb3+ tetraacetyl-Gly-Lys-OMe cyclen (19), colorless oil; 1H NMR (D2O) δ 31.81 (s); 22.90 (s); 12.26-11.52 (m); 9.35 (s); 8.15 (s); 7.98 (s); 7.71 (s); 6.88 (s); 6.15 (s); 6.02 (s); 5.71 (s); 4.14 (s); 3.89-3.86 (m); 3.61-3.60 (m); 3.11-3.05 (m); 3.00 (s); 2.90 (s); 2.80 (s) 2.03-1.56 (m). HRMS (ESI) m/z: found 1357.6310 [M - 2H]+ (calcd 1357.6287 for C52H94N16O16Tb). Tm3+ tetraacetyl-Gly-Lys-OMe cyclen (20), colorless oil; 1H NMR (D2O) δ 39.76 (s); 29.66 (s); 3.71 (s); 3.27 (s); 2.51 (s); 1.20-0.40 (m); -0.22 to -0.40 (m); -1.62 (s); -1.94 (s); -2.72 to -3.02 (m); -3.43 to -3.80 (m); -4.29 to -4.61 (m); -5.36 to -5.61 (m); -9.56 to -10.44 (m); -13.56 to -14.81 (m); -72.82 (s); -83.14 (s). HRMS (ESI) m/z: found 1367.6353 [M - 2H]+ (calcd 1367.6376 for C52H94N16O16Tm). Yb3+ tetraacetyl-Gly-Lys-OMe cyclen (21), colorless oil; 1H NMR (D2O) δ 90.91 (s); 16.82 (s); 14.02 (s); 4.20 (s); 3.793.74 (m); 3.52 (s); 3.22-2.49 (m); 1.94 (s); 1.68 (s); 1.551.45 (m); 1.32 (s); 1.20 (s); 0.37-0.26 (m); -0.05 (s); -0.23 (s); -0.44 (s); -0.59 (s); -0.84 (s); -2.59 (s); -24.01 (s); -28.93 (s); -53.35 (s). HRMS (ESI) m/z: found 1371.6310 [M - 2H]+ (calcd 1371.6476 for C52H94N16O16Yb). SelectiVe Deprotection of di-Cbz-His-OMe (33). A suspension of di-Cbz-His-OMe (21) (33, 1.65 g, 3.77 mmol) in 30% HBr in AcOH (25 mL) was stirred until a clear solution was obtained (ca. 90 min) at rt. The mixture obtained was cooled to 0 °C, Et2O (50 mL) was added, and the mixture was set aside for 30 min at 0 °C. Separated precipitate was filtered off, was washed with Et2O, and was dried to afford H-His(Cbz)-OMe‚2HBr (1.42 g, 81%) of sufficient purity for the next reaction. H-His(Cbz)-OMe‚2HBr (34), white solid, mp 166-68 °C (167-167.5 °C lit. (22)); [R]25D +13 (c 0.39, H2O). 1H NMR


(DMSO-d6) δ 8.37 (s, 1H); 8.30 (br s, D2O exch., 2H); 7.48 (m, 3H); 7.40 (m, 3H); 5.43 (s, 2H); 4.33 (m, 1H); 3.71 (s, 3H); 3.04 (m, 2H). 13C NMR (DMSO-d6) δ 168.5, 164.7, 137.4, 134.5, 129.2, 128.6, 128.4, 126.5, 118.2, 53.2, 52.8, 51.1, 34.5. HRMS (EI) m/z: found 303.1223 (free amine) (calcd 303.1219 for C15H17N3O4). LRMS (EI) m/z (rel abundance): 303 [M]+ (10), 152 (23), 110 (24). Chloroacetyl-Gly-His(Cbz)-OMe (35). To a stirred solution of N-chloroacetylglycine (277 mg, 1.5 mmol) in dry THF (15 mL) were added NHS (173 mg, 1.5 mmol) and EDC‚HCl (863 mg, 4.5 mmol). The mixture was stirred for 18 h at rt and then was concentrated. The residue was dissolved in water (30 mL) and was extracted with EtOAc (30 mL + 3 × 20 mL). The combined organic extracts were dried and concentrated to afford 257 mg (70%) of O-hydroxysuccinimidyl N-chloroacetylglycinate. This was dissolved in dry THF (10 mL), H-His(Cbz)OMe‚2HBr (481 mg, 1.03 mmol) and Et3N (580 µL, 4.13 mmol) were added, and the mixture was stirred for 2 h at rt. The solvent was evaporated; the residue was dissolved in EtOAc (30 mL) and was washed with water (30 mL). The aqueous phase was extracted with EtOAc (20 mL), combined organic extract was dried and was concentrated, and the residue was subjected to flash column chromatography (FCC) on 20 g SiO2 (hexane/ acetone, 1:1) to afford 203 mg (45%, based on O-hydroxysuccinimidyl N-chloroacetylglycinate, 31%, based on N-chloroacetylglycine) of chloroacetyl-Gly-His(Cbz)-OMe (35). Chloroacetyl-Gly-His(Cbz)-OMe, colorless oil; [R]25D -35 (c 0.29, DMSO). 1H NMR (CDCl3) δ 8.03 (s, 1H); 7.58 (br d, D2O exch., J ) 7.5 Hz, 1H); 7.41 (m, 5H); 7.34 (br m, D2O exch., 1H); 7.20 (s, 1H); 5.38 (s, 2H); 4.84 (m, 1H); 4.07 (d, J ) 1.5 Hz, 2H); 4.03 (m, 2H); 3.68 (s, 3H); 3.08 (dd, J ) 9.5 Hz, 5.5 Hz, 1H); 3.00 (dd, J ) 11 Hz, 4.5 Hz, 1H). 13C NMR (CDCl3) δ 171.2, 167.8, 166.3, 148.2, 138.8, 137.0, 133.8, 129.2, 128.8, 128.7, 114.7, 70.0, 52.5, 51.9, 42.9, 42.4, 29.2. HRMS (ESI) m/z: found 459.1057 [M + Na]+ (calcd 459.1047 for C19H21ClN4O6Na). Iodoacetyl-Gly-His(Cbz)-OMe (36). Sodium iodide (415 mg, 2.79 mmol) was added to a solution of chloroacetyl-Gly-His(Cbz)-OMe (35, 203 mg, 0.46 mmol) in acetone (6 mL). The mixture was stirred for 18 h at rt, the solvent was then evaporated, and the residue was dissolved in EtOAc (30 mL). The organic phase was washed with 10% NaHSO3 solution (30 mL) and the aqueous wash was back-extracted with EtOAc (20 mL). The combined organic extracts were dried and concentrated. The residue was subjected to FCC on 25 g SiO2 (hexane/ acetone, 1:1) to afford 204 mg (83%) of unstable iodoacetylGly-His(Cbz)-OMe (36). Iodoacetyl-Gly-His(Cbz)-OMe, colorless solid; 1H NMR (CDCl3) δ 8.05 (s, 1H); 7.42 (br m, D2O exch., 6H); 7.21 (s, 1H); 6.90 (br m, D2O exch., 1H); 5.40 (s, 2H); 4.85 (m, 1H); 4.01 (d, J ) 5 Hz, 2H); 3.72 (s, 2H); 3.69 (s, 3H); 3.11 (dd, J ) 10 Hz, 5.5 Hz, 1H); 3.02 (dd, J ) 10.5 Hz, 4.5 Hz, 1H). HRMS (ESI) m/z: found 551.0419 [M + Na]+ (calcd 551.0404 for C19H21IN4O6Na). Iodoacetyl-Gly-Arg(NO2)-OMe (39). To a solution of Nchloroacetylglycine (1a, 303 mg, 2 mmol) in dry THF (20 mL) was added NHS (230 mg, 2 mmol). The mixture was cooled to 0 °C, DCC (536 mg, 2.6 mmol) was added, and the mixture was stirred for 1 h at 0 °C, followed by the addition of H-Arg(NO2)-OMe‚HCl (37, 539 mg, 2 mmol) and Et3N (560 µL, 4 mmol). The cooling bath was removed and the mixture was stirred for 18 h at rt. The solvent was evaporated and the residue was subjected to FCC on 75 g SiO2 (CH2Cl2/MeOH/NH4OH, 80:20:1). Evaporation of the eluate afforded 615 mg of chloroacetyl-Gly-Arg(NO2)-OMe (38), containing ca. 20% of N,N′-dicyclohexylurea, as determined by analysis of the 1H NMR spectrum. The impure N-chloroacetyl-dipeptide 38 was



Scheme 2. Synthesis of N-Iodoacetyl-Gly-Phe-OEt (3a) and N-Iodoacetyl-Gly-Lys(Boc)-OMe (3b)a

a

Reagents and conditions: (i) NHS/DCC, Et3N, CH2Cl2, 18 h, rt; (ii) NaI (6.0 equiv), acetone, 18 h, rt.

dissolved in acetone (15 mL), and NaI (736 mg, 4.91 mmol) was added. The mixture was stirred for 18 h at rt, a small amount of SiO2 was added, the solvent was evaporated, and the residue was subjected to FCC on 50 g SiO2 (CH2Cl2/MeOH/NH4OH, 80:20:1). Evaporation of the eluate afforded 583 mg (64%, based on N-chloroacetylglycine) of iodoacetyl-Gly-Arg(NO2)-OMe (39). Iodoacetyl-Gly-Arg(NO2)-OMe, colorless solid; [R]25D -12 (c 0.41, MeOH). 1H NMR (DMSO-d6) δ 8.46 (br m, D2O exch., 2H); 8.32 (br d, D2O exch., J ) 7.5 Hz, 1H); 7.81 (br d, D2O exch., J ) 7.5 Hz, 2H); 4.25 (m, 1H); 3.73 (m, 2H); 3.68 (s, 2H); 3.61 (s, 3H); 3.13 (m, 2H); 1.60 (m, 4H). 13C NMR (DMSO-d6) δ 172.2, 172.1, 168.6, 168.0, 56.7, 52.0, 51.6, 42.1, 28.1. HRMS (ESI) m/z: found 481.0285 [M + Na]+ (481.0309 calcd for C11H19IN6O6Na). Alkylation of Cyclen with Iodoacetyl-Gly-Arg(NO2)-OMe (39). To a solution of cyclen (15 mg, 0.086 mmol) in acetonitrile (6 mL), ethyldiisopropylamine (60 µL, 0.34 mmol) was added. The mixture was stirred for 30 min at rt followed by the addition of iodoacetyl-Gly-Arg(NO2)-OMe (39, 158 mg, 0.34 mmol). The stirring continued for 18 h at rt, at which time the product had separated from solution. Conveniently, the solvent was decanted and the residue was washed with acetonitrile (2 × 5 mL) and was dried to afford 46 mg (46%) of triacetyl-Gly-Arg(NO2)OMe cyclen (40). Triacetyl-Gly-Arg(NO2)-OMe cyclen (40), colorless solid; [R]25D -53 (c 0.38, MeOH). 1H NMR (D2O) δ 4.40 (m, 3H); 3.92 (m, 6H); 3.68 (3, 9H); 3.29-3.06 (m, 12H); 2.98-2.67 (m, 16H); 1.87 (m, 3H); 1.72-1.56 (m, 9H). 13C NMR (DMSOd6) δ 172.3, 172.2, 171.7, 171.2, 170.8, 169.6, 169.3, 168.9, 159.3, 52.0, 51.7, 49.2, 41.5, 41.4, 40.3, 38.8, 38.5, 33.4, 28.2, 25.3, 24.9, 24.8, 24.7, 24.6, 24.5. HRMS (ESI) m/z: found 1163.5588 [M + H]+ (calcd 1163.5630 for C41H75N22O18).

RESULTS AND DISCUSSION Our synthetic scheme required the alkylation of cyclen with the appropriate reactive dipeptide derivatives. Our initial approach to N-haloacetyl-dipeptides involved the synthesis of fully protected, side chain and C-terminus, R-N-Boc-dipeptides. After t-butyloxycarbonyl (Boc) removal, the N-terminus of an otherwise fully protected dipeptide was further reacted with bromoacetyl bromide to give the required N-bromoacetyldipeptide. This approach proved to be difficult to execute as the success of the reaction was highly dependent on the dipeptide sequence. Additionally, we also attempted, without much success, to couple partially protected (free N-terminus) dipeptides to DOTA. Although these approaches may be successful in certain cases, we did not consider either of them to be a generally applicable route to cyclen-tetra(oligopeptide) conjugates. We decided to pursue an alternative convergent strategy in order to circumvent the above-mentioned problems and also avoid the tedious synthesis of individual R-N-Boc-dipeptides and eliminate a step that involved a protecting group manipulation. Our strategy involved first preparing N-chloroacetylglycine as a convenient precursor to a variety of N-chloroacetylglycyl(amino acid) compounds. Schotten-Baumann acylation of gly-

cine with chloroacetyl chloride gave N-chloroacetylglycine (1a) in 43% yield after recrystallization from ethyl acetate, similar to the reported procedure (23). Unlike more reactive R-haloacetamides that may not tolerate the presence of nucleophiles, the N-chloroacetylglycine was stable to handling, storage, and amide bond forming reactions. Therefore, next, a straightforward N-hydroxysuccinimide (NHS)/dicyclohexylcarbodiimide (DCC) promoted coupling (24) of N-chloroacetylglycine (1a) with H-Phe-OEt‚HCl or H-Lys(Boc)-OMe‚HCl afforded N-chloroacetyl-Gly-Phe-OEt (2a) and N-chloroacetyl-Gly-Lys(Boc)-OMe (2b) in excellent yield (Scheme 2). In our experience, the alkylation of cyclen proceeded quite slowly with the N-chloroacetylamino acids. The alkylation reaction generally required halide exchange to a more active derivative to be high-yielding. Although cyclen may be reacted in a one-pot procedure with N-chloroacetyl-dipeptides 2a and 2b in the presence of excess NaI and K2CO3, we have found that the success of peralkylation was variable. Therefore, we decided to perform the Finkelstein reaction and isolate the N-iodoacetyl-dipeptides prior to their use. This permitted us to conveniently monitor the consumption of the N-iodoacetyldipeptides during its use by TLC analysis of the reaction mixture. Thus, treatment of 2a and 2b with NaI in acetone proceeded smoothly, and N-iodoacetyl-dipeptides 3a and 3b were obtained in approximately 80% yield (Scheme 2). An upfield shift of the methylene peak in the 1H NMR is observed upon conversion of N-chloroacetyl-dipeptides 2 to N-iodoacetyldipeptides 3. The N-iodoacetyl-dipeptides are stable and easily isolable solids that may be stored for prolonged periods without deterioration. The above strategy was also extended to the synthesis of cyclen containing aspartic acid as the second amino acid; the key compound toward the synthesis of such molecules is N-chloroacetyl-Asp(OMe)-OH (1b). This compound was prepared by first monoesterification of aspartic acid to its β-methyl ester (4) with methanolic HCl (20). Initial attempts at acylating 4 involved neutralization of the hydrochloride salt with 1 equiv of sodium hydroxide followed by the addition of 1 equiv of sodium carbonate and dropwise addition of chloroacetyl chloride. Although this procedure gave 1b in 56% yield as an oil, it was contaminated with the side product chloroacetic acid, which proved difficult to remove. In order to improve the acylation yield and to avoid hydrolysis of the β-methyl ester, we decided to react 4 with O-chloroacetyl-N-hydroxysuccinimide (5). Using sodium carbonate and a 1:1 mixture of dioxane/H2O, this reaction gave 1b in a crude yield of 81%, slightly contaminated with NHS as judged by 1H NMR. Without purification, 1b was condensed with H-Glu(OMe)-OMe‚HCl or H-Phe-OEt‚HCl to give Nchloroacetyl-Asp(OMe)-Glu(OMe)-OMe (2c) and N-chloroacetyl-Asp(OMe)-Phe-OEt (2d), using NHS/DCC. At this point, NHS was easily eliminated by extraction against aqueous sodium bicarbonate. Treatment of 2c and 2d with NaI in acetone gave N-iodoacetyl-Asp(OMe)-Glu(OMe)-OMe (3c), and Niodoacetyl-Asp(OMe)-Phe-OEt (3d) in very good yields (Scheme 3).



Scheme 3. Synthesis of N-Iodoacetyl-Asp(OMe)-Glu(OMe)-OMe (3c) and N-Iodoacetyl-Asp(OMe)-Phe-OEt (3d)a

a Reagents and conditions: (i) ClCH CONHS (5), Na CO , dioxane/H O, 14 h, (ii) NHS/DCC, Et N, THF, 12 h, (iii) NaI (6.0 equiv), acetone, 2 2 3 2 3 rt 1 h.

Scheme 4. Synthesis of Cyclen-Oligopeptide Conjugates 7-11 and Their Treatment with Lanthanide(III) Chloridesa

a Reagents and Conditions. 6a f 7: NaOH, THF/H2O. 6b f 8: TFA. 6b f 9: (i) TFA, (ii) NaOH, MeOH/H2O. 6c f 10: NaOH, MeOH/H2O. 6d f 11: NaOH, MeOH/H2O.

With N-iodoacetyl-dipeptides 3a-3d in hand, we investigated the alkylation of cyclen (Scheme 4). The use of potassium carbonate did not give satisfactory conversion as judged by TLC, and we turned to ethyldiisopropylamine (EDIPA, Hu¨nig’s base), which gave acceptable results. The alkylation reactions were carried out at 50 °C in MeCN, using 4 equiv of N-iodoacetyldipeptides 3a or 3b and EDIPA. Slightly higher temperatures (70 °C) were used for the alkylation of cyclen with the more bulky compounds 3c and 3d. Tetraalkylated cyclen-oligopeptide conjugates 6 were obtained in excellent yields (>90%). To obtain the desired ligands for the complexation with lanthanides, protecting groups were removed as described in Scheme 4. Saponification of 6a, 6c, and 6d afforded compounds 7 (Gly-Phe-OH), 10 (Asp-Glu-OH), and 11 (Asp-Phe-OH), respectively. Treatment of compound 6b with TFA gave 8 (GlyLys-OMe tetratrifluoroacetate), which without further purification under the conditions of basic hydrolysis gave 9 (Gly-LysOH). Cyclen-oligopeptide conjugates 7 and 11 were purified by trituration with Et2O, while conjugates 9 and 10 were purified by size exclusion chromatography on desalting gel with an exclusion limit of 1800 Da. Currently, there are numerous literature reports describing the synthesis of lanthanide(III) complexes derived from cyclen. Various reaction conditions (elevated temperatures (25, 26), slightly acidic pH (27-29), and slightly basic pH (6)) have been employed in order to prepare lanthanide(III) cyclen complexes. As described in the literature, the workup usually involves filtration of insoluble particles followed by the concentration of the filtrate without any further purification (30, 31). Alternatively, the reaction mixtures are sometimes centrifuged, and the supernatant is concentrated as described above (32). Less water-soluble complexes are purified by precipitation of their methanolic solutions with Et2O (25) or by trituration with Et2O (33). Oftentimes, the crude material obtained after concentration is used directly, without any form of purification (7, 16). To the best of our knowledge, a general procedure for the purification of cyclen derived lanthanide(III) complexes has not been described. Taking into account this lack of consistent purification of lanthanide(III) complexes, and the fact that compounds 12-32 are very water-soluble and resist precipita-

tion, we have decided to establish a widely applicable method for the purification of lanthanide(III) complexes of cyclenoligopeptide conjugates and test its scope. Ligands 7-11 have been treated with 1 equiv of various lanthanide(III) chlorides in water under slightly basic conditions (pH 9) (Scheme 4). The conditions for introducing the metal are gentle enough to retain the C-terminal ester, e.g., 16-21. This was also true in the case of DOTAM-(Gly-Phe-OEt)4 (6a), which was metalated; however, the complexes were not sufficiently soluble in water and they were abandoned. Once the ligands were metalated, the resulting complexes were isolated and purified by size exclusion chromatography (SEC) in deionized water (34). The SEC gel that was used had an exclusion limit of 1800 Da and was found to be widely applicable for the purification of water-soluble lanthanide(III) complexes with a molecular weight of approximately 10001800 Dasi.e., most conceivable dipeptide DOTAM conjugates. Compounds with a molecular weight well in excess of 1800 Da would be better purified using a desalting gel with a higher exclusion limit. The fractions containing the compounds were identified by UV/I2 vapors (Gly-Phe-OH and Asp-Phe-OH complexes), ninhydrin test (Gly-Lys-OH and Gly-Lys-OMe complexes), or I2 vapors (Asp-Glu-OH complex). Chemical yields for the purified complexes varied from 44% to 98%, as listed in Table 1. All the prepared complexes were characterized by HR MS (ESI- TOF) and 1H NMR spectroscopy. The synthetic approach reported herein is convenient and robust because it reliably works for a number of diverse dipeptides without modification and utilizes known or commercially available amino acid derivatives. Two notable exceptions were found in the attempted preparation of tetraacetylGly-His-OMe and tetraacetyl-Gly-Arg-OMe. These compounds are potentially interesting targets due to the presence of additional exchangeable protons in the ligand periphery and the nature of the side chain functional groups. We approached the tetraacetyl-Gly-His-OMetarget by first preparing the bis(Cbz)-protected-histidine methyl ester (33) as described in the literature (21). Treatment of 33 with 30% HBr in AcOH (22) resulted in selective removal of Cbz group from the amino group, while the protecting group on the imidazole



Table 1. Yields and HRMS ESI Data of Lanthanide(III) Complexes of Cyclen-Oligopeptide Conjugates 12-32a compound number yield (mass, percent)a metal Dy(III) mass (yield) Eu(III) mass (yield) Nd(III) mass (yield) Tb(III) mass (yield) Tm(III) mass (yield) Yb(III) mass (yield) a

12-15 R ) Gly-Phe-OH

(12) 57 mg, 44% (13) 56 mg, 44% (14) 55 mg, 42% (15) 64 mg, 49%

16-21 R ) Gly-Lys-OMe

22-27 R ) Gly-Lys-OH

(16) 94 mg, 73% (17) 120 mg, 94% (18) 112 mg, 96% (19) 100 mg, 79% (20) 118 mg, 96% (21) 104 mg, 81%

(22) 120 mg, 98% (23) 119 mg, 98% (24) 102 mg, 85% (25) 92 mg, 75% (26) 88 mg, 72% (27) 107 mg, 86%

28-31 R ) Asp-Glu-OH

(28) 115 mg, 80% (29) 71 mg, 49% (30) 125 mg, 86% (31) 89 mg, 59%

Eu(III) Asp-Phe-OH (32) was achieved in 71 mg, 47%.

Scheme 5. Synthesis of Haloacetyl-Gly-His(Cbz)-OMe 35 and 36

Scheme 6. Synthesis of Triacetyl-Gly-Arg(NO2)-OMe Cyclen (40)

ring remained intact (Scheme 5). The possibility of coupling of dihydrobromide 34 with N-chloroacetylglycine was then investigated. The reaction was troublesome and low-yielding. The best conditions proved to be EDC‚HCl and NHS in the presence of Et3N (Scheme 5), which afforded chloroacetyl-GlyHis(Cbz)-OMe (35) in 40% yield. Application of the Finkelstein reaction gave the rather unstable iodoacetyl-Gly-His(Cbz)-OMe (36) (Scheme 5). This compound underwent massive decomposition in solution in less than 1 h, as observed by 1H NMR in CDCl3 or DMSO-d6. Nonetheless, the alkylation of cyclen was attempted; however, no product of alkylation was detected which was likely due to instability of N-iodoacetyl-dipeptide 36. Attempts to prepare N-iodoacetyl-dipeptide 36 in situ were unsuccessful. The reactivity of chloroacetyl-Gly-His(Cbz)-OMe (35) toward the alkylation of cyclen appeared to be insufficient even at elevated temperatures. The synthesis of the required dipeptide electrophile to approach tetraacetyl-Gly-Arg-OMe was achieved much more readily. The NHS/DCC mediated coupling of N-chloroacetylglycine with H-Arg(NO2)-OMe (37) afforded chloroacetyl-GlyArg(NO2)-OMe (38) (Scheme 6), containing a small amount of N,N′-dicyclohexylurea. The Finkelstein reaction of 38 proceeded smoothly, and iodoacetyl-Gly-Arg(NO2)-OMe (39) was isolated in 64% overall yield (Scheme 6). Alkylation of cyclen was carried out in acetonitrile using EDIPA as a base. Surprisingly, the trialkylated cyclen 40 formed exclusively and was found to deposit on the flask walls at room temperature.

Metalation of 40 (using EuCl3‚6H2O and NdCl3‚H2O) could not be achieved even at elevated temperatures and slightly basic pH. The origin of the lack of chelation by this ligand is not understood at this time. Since ligand 40 could not be metalated, no studies were carried out toward the tetraalkylation of cyclen with iodoacetyl-Gly-Arg(NO2)-OMe (39) nor on removal of guanidine protecting group. With the intention of the discovery of new MRI PARACEST agents that generate signal variation by the CEST effect, we have acquired CEST spectra (2) of the metalated ligands under conditions of near-physiological temperature and pH. The results of this survey are presented in Figure 2. The observed CEST effects are the result of the intrinsic properties of the compound as well as the experimental conditions. Since all CEST spectra were measured under identical experimental conditions, the differences in the observed CEST effects were likely due to alterations in the exchangeable proton lifetimes. For example, a CEST effect was observed from bound water protons when the exchange rate of bound water with bulk water was slow (M ) Eu3+, Nd3+). Conversely, no bound water CEST effect was observed for metal complexes when the exchange between bound and bulk water was relatively fast (M ) Dy3+, Tb3+, Tm3+, Yb3+). However, for the latter metals, it is possible in some instances to observe the CEST effect from the protons bound to the amide nitrogen. This phenomenon may be exploited to measure the pH of the environment if the signal intensity is observably sensitive to pH and reasonably resolved from the



Figure 2. CEST spectra for compounds 12-32 determined at 10 mM concentration, pH 7.0. All spectra were collected using a 9.4 T NMR spectrometer with saturation power of 14 µT (saturation time ) 10 s) at the temperature indicated. CEST signals due to bound water and amide protons are marked by an arrowhead and denoted (w) or (a), respectively.

bulk water signal, e.g., 24. In limited circumstances, it is possible to simultaneously observe the signal due to bound water and amide protons under conditions close to physiological pH and temperature (M ) Nd3+). One compound did not produce an observable CEST effect (31), while another compound could not be measured under the conditions employed due to limited solubility (15). Our methodology can be used to readily prepare a number of ligated metals to examine their potential utility as MRI PARACEST agents. The most strongly bound water signals are found for ligands containing Eu3+. For example, the Eu3+DOTAM-Gly-Phe-OH complex 12 clearly exhibits bound water saturation at approximately 43 ppm from bulk water. This compound has a large CEST effect (∼45%) at physiological temperature and pH (Figure 2). The chemical shift of the bound water is also sensitive to temperature (0.3 ppm/°C, data not shown), and will be investigated for its ability to measure

temperature in ViVo. The CEST spectrum of Eu3+-DOTAMAsp-Glu-OH 28 is qualitatively similar to that of Eu3+-DOTAMGly-Phe-OH 12, but with a slightly smaller CEST effect. The bound water signals for the Eu3+-containing complexes 17 and 23 are evident at approximately the same chemical shift (43 ppm) but with weaker intensity, as compared to the aforementioned compounds. Since both Eu3+-DOTAM-Gly-Phe-OH 12 and the Eu3+-DOTAM-Asp-Glu-OH 28 exhibited a large CEST effect from the bound water, we were interested in the effect of combining the nature of the dipeptide; thus, Eu3+-DOTAMAsp-Phe-OH 32 was prepared and studied. Rather than increasing the CEST effect, this complex only showed a small CEST effect due to the bound water at 43 ppm. Many of the complexes, especially those containing Tm3+, Tb3+, or Dy3+, exhibit very small CEST effects due to the amide protons (e.g., 14, 16, 19, 20, 22, 25, 30), yet this is not always the casesa clear CEST signal is observed for 26 (cf. 20 and


26). The two complexes, Tm3+-DOTAM-Gly-Lys-OMe 20 and Tm3+-DOTAM-Gly-Lys-OH 26, highlight the impact of subtle structural changes on the MR properties of the complex. Ligands 8 and 9 differ by the nature of the peripheral groups, the former being a neutral methyl ester and the latter an anionic carboxylate (under the conditions of measurement), but curiously the CEST effect contributed by the amide protons is greater for complexes of 9 for M ) Tm3+, Tb3+, and Dy3+. The ytterbium (Yb3+) complex of both 8 and 9, i.e., Yb3+-DOTAM-Gly-Lys-OMe 21 and Yb3+-DOTAM-Gly-Lys-OH 27, possesses amide protons that produce a CEST effect at -15 ppm and -18 ppm, respectively. For these complexes, the amide proton saturation is better resolved from the bulk water saturation compared to Nd3+-DOTAM-Gly-Lys-OME 18 and Nd3+-DOTAM-Gly-LysOH 24, making the observation of changes with pH easier. However, saturation of the bound water pool was not detected in the Yb3+-containing complexes. Simultaneous observation of the CEST effect produced by the amide and bound water protons may be an advantage for pH measurement using ratiometric methods (6, 7). A CEST effect was observed simultaneously for bound water and amide protons in several compounds. The Nd3+-DOTAM-Gly-Lys-OMe complex 18 showed a broad CEST signal due to bound water at approximately -35 ppm and an amide CEST effect a 11 ppm, not fully resolved from the bulk water signal. Upon hydrolysis of the terminal ester groups of the ligand and subsequent metalation, the Nd3+-DOTAM-Gly-Lys-OH complex 24 exhibited more clearly resolved CEST effects from bound water (-34 ppm) and amide protons (11 ppm). Both signals are clearly observed under the same experimental conditions. The CEST effect from amide protons is very sensitive to pH (data not shown), which potentially may be used to measure the pH of the environment of the complex (5, 6). In summary, we have demonstrated a reliable and convergent synthesis of several new dipeptide-DOTAM conjugates. This approach relies largely on available commercial reagents and a minimum of protecting group manipulations. It is easy to imagine extending this approach of diversification of the ligand periphery by incorporating other natural or unnatural amino acids. Each ligand was used to chelate a variety of lanthanide(III) ions, and the resulting complexes were examined for MRCEST effects. A number of the compounds produced interesting CEST spectra, validating this approach for the discovery of new lead compounds. Currently, we are extending this synthetic approach to the screening of a variety of amino acids for useful PARACEST properties.

ACKNOWLEDGMENT The authors thank the Natural Sciences and Research Council of Canada (RHEH), the Ontario Research and Development Challenge Fund, and the Canadian Institutes of Health Research Strategic Training Program in Cancer Research and Technology Transfer for supporting this work. Ms. Paula Pittock of the Biological Mass Spectrometry Laboratory at the University of Western Ontario and Mr. Doug Hairsine of the Department of Chemistry Mass Spectral Facility are thanked for expert assistance with collection and analysis of mass spectral data. Supporting Information Available: Selected molecular characterization is provided by way of 1H NMR spectra for compounds 1b, 2, 3, 6, 7, 9-32, 34-36, 39, and 40 and 13C NMR spectra of compounds 1b, 2, 3, 34, 35, and 39. This material is available free of charge via the Internet at http:// pubs.acs.org.


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A Robust and Convergent Synthesis of Dipeptide−DOTAM Conjugates

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