l-Glutamic Acid and l-Lysine as Useful Building Blocks for the

Dec 31, 1998 - Bisalkylation of suitably protected l-glutamic acid and l-lysine derivatives with tert-butyl N-(2-bromoethyl)iminodiacetate 2, followed...
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Bioconjugate Chem. 1999, 10, 137−140

137

L-Glutamic

Acid and L-Lysine as Useful Building Blocks for the Preparation of Bifunctional DTPA-like Ligands†

Pier Lucio Anelli,* Franco Fedeli, Ornella Gazzotti, Luciano Lattuada, Giovanna Lux, and Fabrizio Rebasti Bracco spa, Milano Research Centre, via E. Folli 50, 20134 Milano, Italy. Received December 26, 1997; Revised Manuscript Received November 19, 1998

Bisalkylation of suitably protected L-glutamic acid and L-lysine derivatives with tert-butyl N-(2bromoethyl)iminodiacetate 2, followed by deprotection of the ω functional group affords N,N-bis[2[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid 1-(1,1-dimethylethyl) ester 4 and N2,N2-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine 1,1-dimethylethyl ester 7. Such compounds feature a carboxylic or an amino group, respectively, which are available for conjugation with a suitable partner via formation of an amide bond. The conjugates, which can be prepared in this way, contain a chelating subunit in which all five acetic residues of DTPA are available for the complexation of metal ions. Direct bisalkylation of glycine with 2 promptly gives N,N-bis[2-[bis[2(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]glycine 11. The latter allows to achieve conjugates in which the central acetic group of DTPA is selectively converted into an acetamide.

Diethylenetriaminepentaacetic acid (DTPA) is a very efficient chelating agent for the complexation of the gadolinium ion. The corresponding complex, due to its high in vivo stability, has been safely used as contrast agent in MRI over the past decade (2). Conjugation of DTPA-like ligands to macromolecules (3), iodinated synthons (4) or bile acids (5) has been recently pursued to achieve MRI contrast agents featuring improved organ specificity. The most popular way to conjugate DTPA to other molecules involves the conversion of one of the four equivalent carboxylic groups into a carboxamide via DTPA bisanhydride (6). Besides being not selective, due to the concomitant formation of some diamide (7), this methodology has the disadvantage of yielding conjugates containing a ligand weaker than DTPA. Indeed, such decrease in chelating ability of DTPA-like ligands in which one of the carboxylic group has been converted into a carboxamide is well-known (8). Nevertheless, due to its chemical stability, amide bond is one of the preferred choices for the preparation of conjugates containing metal complexes which have to be administered in vivo. Therefore, we became interested in the preparation of DTPA ligands bearing either a carboxylic group or an amino group available for conjugation. Williams and Rapoport recently introduced a simple methodology which is a breakthrough in the synthesis of DTPA-like ligands (9). We have now found that L-glutamic acid and L-lysine, after suitable protections and using such methodology, conveniently yield functionalized DTPA pentaesters which are versatile intermediates for the preparation of conjugates of metal complexes. Since DTPA-like ligands have been proposed for the chelation of radionuclides [e.g., 111In(III), 212Bi(III), and 90Y(III)] (10, 11) we believe that the above intermediates could also be of some importance for the preparation of new radiopharmaceuticals. † Presented in part at the 35th National Organic Chemistry Symposium, San Antonio, TX, June 1997. * To whom correspondence should be addressed. Phone: +39 02 21772353. Fax: +39 02 26410678.

Scheme 1a

a (i) 1 EtOH/H O, pH 8; for 5 MeCN/pH 8 phosphate buffer; 2 (ii) H2, 5% Pd/C, EtOH; (iii) H2, 5% Pd/C, MeOH.

L-Glutamic acid 5-benzyl ester (12) was converted, with isobutene in dioxane in the presence of H2SO4, into the 1-tert-butyl 5-benzyl diester 1 (13), which was directly reacted with bromide 2 in EtOH/H2O at pH 8 to afford hexaester 3 in only 31% yield (Scheme 1). The isolation of 3 in fair yields is related to the easy lactamization of both 1 and the monoalkylated intermediate. However, in our hands the dialkylation of 1 in homogeneous phase proved to be the best procedure, even better then the reaction under Rapoport’s two-phase conditions (i.e., MeCN/pH 8 phosphate buffer). Cleavage of the benzyl ester protection of 3 by hydrogenolysis yielded the monoacid pentaester 4. On the other hand, to obtain a DTPA pentaester with a free amino group, commercially available N6-Cbz L-

10.1021/bc970212u CCC: $18.00 © 1999 American Chemical Society Published on Web 12/31/1998

138 Bioconjugate Chem., Vol. 10, No. 1, 1999

Anelli et al.

Scheme 2b

We found that dialkylation of glycine benzyl ester (16) with 2 directly affords pentaester 12 of DTPA which can be hydrogenated in MeOH in the presence of 10% Pd/C to give 11. More surprisingly, we realized that dialkylation of glycine with 2 in EtOH/H2O at pH 10 straightforwardly leads to 11 in 70% yield after flash chromatography. Tetraester 11 is a very convenient intermediate for the synthesis of conjugates containing DTPA monoamide subunits. In this respect, the preparation of 11 appears less troublesome and quicker than that of the unsymmetrical tetraester 13, which has been recently prepared with a six step synthesis (17).

b (i) DEPC, Et N, DMF; (ii) CF COOH, CH Cl ; (iii) NaOH, 3 3 2 2 pH 13.

lysine was esterified in tert-butyl acetate in the presence of HClO4 to afford 5. Subsequent dialkylation with bromide 2 in MeCN/pH 8 phosphate buffer afforded protected pentaester 6. Classical removal of the Cbz moiety by hydrogenolysis yielded the amino pentaester 7 (Scheme 1).1 It must be stressed that Rapoport demonstrated that, under the reaction conditions that they developed for the dialkylation of amino esters, no racemization is observed. Pentaesters 4 and 7 can be easily reacted with substrates containing amino or carboxylic functionalities, respectively, and subsequently, the tertbutyl ester groups can be removed using routine methodologies. According to this strategy, conjugates containing DTPA subunits, in which all five acetic groups are available for complexation, are promptly achieved. Indeed, we synthesized ligand 10 by coupling of 4 to aminocholic methyl ester 8 (14) (Scheme 2). On complexation with Gd(III) ligand 10 gives rise to a complex which will be studied as MRI contrast agent in comparison with other bile acid conjugates (5). We also investigated the preparation of a synthon in which the acetic residue on the central nitrogen atom of DTPA can be selectively activated. Compound 11 has been recently prepared by hydrogenolysis of the corresponding mixed monobenzyl tetra-tert-butyl ester of DTPA 12 in its turn obtained through a synthetic route involving time consuming protection-deprotection of diethylenetriamine (15). 1Similarly, reaction of O-benzyl-L-serine tert-butyl ester with bromide 2 under Rapoport’s conditions, followed by hydrogenolysis of the benzyl group, yielded a DTPA pentaester bearing an hydroxymethyl moiety on the central acetic residue. Such derivative can also be used for the preparation of conjugates.

It is noteworthy that DTPA polyesters 4, 7, and 11 are all prepared using bromide 2, which indeed proved to be a very versatile intermediate. A limit to the direct application of compounds 4, 7, and 11 for the preparation of conjugates is represented by the absence, in the substrate to which they have to be coupled, of chemical bonds labile under conditions of cleavage of the tert-butyl ester groups. This drawback can be circumvented, reacting 4 and 11 with either N-(2aminoethyl)maleimide (17) or S-(2-pyridylthio)cysteamine (18) to give, after deprotection of the tert-butyl ester functions, bifunctional ligands which can be coupled to SH containing substrates (e.g., proteins). A similar strategy could be exploited for the use of 7. EXPERIMENTAL PROCEDURES

General Comments. All reagents were purchased from Fluka Chemie AG (Buchs, Switzerland), Aldrich (Milwakee, WI) and Merck KGaA (Darmstadt, Germany). 1H and 13C NMR spectra were recorded at 200 and 50 MHz, respectively, on a Bruker AC 200 spectrometer. Electrospray ionization was performed on a Finnigan TSQ 700 triple quadrupole mass spectrometer fitted with a Finnigan ESI interface, dissolving the samples in MeOH, H2O/MeOH, or H2O/MeCN. Optical rotations (10-1 deg cm2 g-1) were measured with a Perkin-Elmer 341 polarimeter. Melting points were determined with a Bu¨chi 510 apparatus and are uncorrected. Preparative HPLC was performed on a Merck KGaA Prepbar 100 apparatus. N,N-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid 1-(1,1-dimethylethyl) 5-(Phenylmethyl) Diester (3). L-Glutamic acid 5-benzyl ester (12) (171 g, 0.72 mol) was added to a mixture of 98% H2SO4 (50.5 mL, 0.93 mol) in dioxane (500 mL) at 20 °C and the solution was stirred over 6 days under an isobutylene atmosphere (132 kPa). Crushed ice (720 g) was added to the reaction mixture, and then a solution of K2CO3 (128.5 g, 0.93 mol) in water (350 mL) was added dropwise over 1 h. The precipitate, containing the start-

Technical Notes

ing monoester (60 g, 0.25 mol) and salts, was filtered and the solution freeze-dried. L-Glutamic acid 1-tert-butyl 5-benzyl diester 1 thus obtained (137.8 g; 0.47 mol; 65%) was dissolved in EtOH (780 mL) and water (170 mL) and bromoderivative 2 (9) (247 g; 0.7 mol) was added at 5 °C. The mixture was maintained at pH 8 by means of a pH-stat apparatus with 10 N NaOH. After 48 h at 5 °C and 98 h at 25 °C further, bromoderivative 2 was added (106 g, 0.3 mol). After 120 h at 25 °C and 22 h at 50 °C, the mixture was neutralized with 36% HCl and filtered. After evaporation of EtOH, the solution was diluted with water (300 mL) and extracted with n-hexane (1 × 1100 mL, 1 × 300 mL). The combined organic phase was washed with water (250 mL), dried, and evaporated. The residue (322 g) was purified by a first flash chromatography with 1:4 EtOAc/n-hexane followed by a second flash chromatography using CH2Cl2 f 1:12 Et2O/CH2Cl2 as the eluent. Evaporation gave 3 (122.8 g, 0.147 mol, 1 31%) as a yellow oil. [R]20 D : -30.43 (c 5.5, CHCl3). H NMR (CDCl3): δ 1.40 (s, 45H), 1.92 (m, 2H), 2.45 (m, 2H), 2.70 (m, 8H), 3.40 (m, 9H), 5.05 (s, 2H), 7.29 (m, 5H). 13C NMR (CDCl3): δ 24.7 (CH2), 28.0 (CH3), 28.1 (CH3), 30.7 (CH2), 50.0 (CH2), 53.7 (CH2), 55.8 (CH2), 63.2 (CH), 65.9 (CH2), 80.6 (C), 80.7 (C), 127.9 (CH), 128 (CH), 128.3 (CH), 136.0 (C), 170.3 (CO), 171.9 (CO), 173.1 (CO). Anal. (C44H73N3O12) C, H, N. MS: m/z 836 [M + H]+, 858 [M + Na]+. N,N-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid 1-(1,1-Dimethylethyl) Ester (4). Pd (5%) on carbon (16 g) was added to a solution of 3 (121 g, 142 mmol) in EtOH (800 mL), and the suspension was stirred over 2 h under a hydrogen atmosphere (111 kPa) at 20 °C. The mixture was filtered over Millipore FH 0.45 µm, the solution evaporated and the residue purified by flash chromatography eluting with 1:2 EtOAc/n-hexane f EtOAc. Evaporation gave 4 (59.3 g, 56%) as a very viscous yellow oil. [R]20 D : -13.49 (c 2.3, CHCl3). 1H NMR (CDCl3): δ 1.36 (s, 45H), 1.95 (m, 2H), 2.65 (m, 2H), 2.85 (m, 8H), 3.42 (s, 8H), 3.85 (dd, 1H). 13C NMR (CDCl3): δ 24.3 (CH2), 27.9 (CH3), 28.0 (CH3), 31.6 (CH2), 40.6 (CH2), 53.0 (CH2), 55.5 (CH2), 63.6 (CH), 80.8 (C), 81.1 (C), 170.3 (CO), 171.1 (CO), 176.9 (CO). Anal. (C37H67N3O12) C, H, N. MS: m/z 746 [M + H]+, 768 [M + Na]+. N6-[(Phenylmethoxy)carbonyl]-L-lysine 1,1-Dimethylethyl Ester (5). HClO4 (70%, 19.5 mL) was added dropwise to a suspension of N6-[(phenylmethoxy)carbonyl]-L-lysine (56 g, 0.2 mol) in tert-butyl acetate (700 mL) to afford a clear solution. After 20 h, 10% Na2CO3(aq) (600 mL) was dripped into the solution obtaining precipitation of the unreacted starting material which was filtered. NaOH (10 N) was added to adjust to pH 10 the aqueous phase then the organic phase was separated, washed with water (5 × 50 mL) and dried (Na2SO4). Evaporation gave 5 (53.5 g, 79%) as a colorless oil. [R]20 D: +8.88 (c 4.9, CHCl3). 1H NMR (CDCl3): δ 1.30-1.80 (m, 15H), 3.10-3.35 (m, 3H), 4.90 (br s, 1H), 5.05 (s, 2H), 7.03 (m, 5H). 13C NMR (CDCl3): δ 22.6 (CH2), 27.9 (CH3), 29.5 (CH2), 34.4 (CH2), 40.7 (CH2), 54.7 (CH), 66.3 (CH2), 80.7 (C), 127.9 (CH), 128.3 (CH), 136.5 (C), 158.3 (CO), 175.2 (CO). Anal. (C18H28N2O4) C, H, N; H, calcd: 8.33. Found: 8.80. MS: m/z 337 [M + H]+, 359 [M + Na]+. N6-[(Phenylmethoxy)carbonyl]-N2,N2-bis[2-[bis[2(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]- Llysine 1,1-Dimethylethyl Ester (6). Phosphate buffer (1 L; 2 M, pH 8) was added to a solution of compound 5 (81 g, 0.24 mol) and bromoderivative 2 (209 g, 0.59 mol) in CH3CN (900 mL), and the mixture was vigorously

Bioconjugate Chem., Vol. 10, No. 1, 1999 139

stirred for 2 h. The two phases were separated and the aqueous phase replaced with fresh phosphate buffer (800 mL, 2 M, pH 8). After 48 h, the mixture was separated and the organic layer concentrated to dryness. The residue was dissolved in CH2Cl2 (1 L), washed with water (2 × 50 mL), dried (Na2SO4), and evaporated. The residue was purified by flash chromatography with 1:2 EtOAc/ n-hexane to give 6 (190 g, 90%) as a very viscous yellow 1 oil. [R]20 D : -26.40 (c 4.98, CHCl3). H NMR (CDCl3): δ 1.20-1.80 (m, 51H), 2.75 (m, 8H), 3.20 (m, 3H), 3.40 (s, 8H), 5.10 (s + br s, 3H), 7.23 (m, 5H). 13C NMR (CDCl3): δ 23.1 (CH2), 27.9 (CH3), 28.1 (CH3), 29.3 (CH2), 40.6 (CH2), 50.0 (CH2), 53.4 (CH2), 55.8 (CH2), 63.7 (CH), 66.1 (CH2), 80.4 (C), 80.5 (C), 127.7 (CH), 127.8 (CH), 128.2 (CH), 136.6 (C), 156.2 (CO), 170.4 CO), 172.5 (CO). Anal. (C46H78N4O12) C, H, N. MS: m/z 337 [M + H]+, 359 [M + Na]+. N2,N2-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine 1,1-Dimethylethyl Ester (7). Pd (5%) on carbon (9 g) was added to a solution of 6 (180 g, 0.2 mol) in MeOH (1 L) and the suspension was stirred over 2 h under a hydrogen atmosphere at 20 °C. The mixture was filtered over Millipore HA 0.45 µm and evaporated. The residue was dissolved in 0.5 N HCl and the solution maintained under vacuum for 10 min, then 1 N NaOH was added and the product extracted with Et2O. The solution was evaporated and the residue was purified by flash chromatography with MeOH to give 7 (90 g, 60%) as a very viscous yellow oil. [R]20 D : -27.97 (c 5.7, CHCl3). 1H NMR (CDCl3): δ 1.20-1.90 (m, 51H), 2.60-2.85 (m, 10H), 3.12 (m, 1H), 3.40 (s, 8H). 13C NMR (CDCl3): δ 23.3 (CH2), 28.0 (CH3), 29.6 (CH2), 33.4 (CH2), 41.6 (CH2), 50.2 (CH2), 53.5 (CH2), 55.7 (CH2), 64.2 (CH), 80.2 (C), 170.3 (CO), 172.4 (CO). Anal. (C38H72N4O10) C, H, N; H, calcd: 9.74. found: 10.25. MS: m/z 745 [M + H]+, 767 [M + Na]+. [3β(S),5β,7r,12r]-3-[[5-(1,1-Dimethylethoxy)-4-[bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]amino]-1,5-dioxopentyl]amino]-7,12-dihydroxycholan24-oic Acid Methyl Ester (9). Monoacid pentaester 4 (18.6 g, 25 mmol), (3β,5β,7R,12R)-3-amino-7,12-dihydroxycholan-24-oic acid methyl ester (14) (11.6 g, 27.5 mmol) and diethyl cyanophosphonate (DEPC) (4.8 g, 29.6 mmol) were dissolved in DMF (500 mL). The resulting solution was cooled to 0 °C and Et3N (2.7 g, 26.2 mmol) was added dropwise. After 30 min at 0 °C and 1 h at room temperature, the solution was evaporated and the residue dissolved in EtOAc (200 mL). The organic phase was washed with 5% NaHCO3 (aq) (2 × 100 mL) then with brine (2 × 100 mL), dried, and evaporated. The crude product was purified by flash chromatography with EtOAc to give 9 (21 g, 73%) as a yellowish solid: mp 5457 °C. 13C NMR (CDCl3): δ 12.3 (CH3), 17.1 (CH3), 22.8 (CH3) 23.1 (CH2), 24.5 (CH2), 25.4 (CH2), 25.9 (CH), 27.3 (CH2), 28.0 (CH3), 28.3 (CH2), 30.7 (CH2), 30.9 (CH2), 31.0 (CH2), 32.8 (CH2), 33.5 (CH2), 34.5 (CH2), 35.1 (CH), 35.2 (C), 37.0 (CH), 39.2 (CH), 41.6 (CH), 45.1 (CH), 46.4 (C), 47.0 (CH), 49.4 (CH2), 51.3 (CH3), 53.1 (CH2), 55.7 (CH2), 63.2 (CH), 68.2 (CH), 72.9 (CH), 80.6 (C), 80.7 (C), 170.5 (CO),172.2(CO),172.4(CO),174.6(CO).Anal.(C62H108N4O15) C, H, N. MS: m/z 1150 [M + H]+, 1172 [M + Na]+. [3β(S),5β,7r,12r]-3-[4-Carboxy-4-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-1-oxobutyl]amino]7,12-dihydroxycholan-24-oic Acid (10). CF3COOH (399 g, 3.5 mol) was added dropwise in 30 min to a solution of hexaester 9 (96.5 g, 0.084 mol) in CH2Cl2 (360 mL) at 0 °C. The reaction mixture was stirred at room temperature for 18 h and then evaporated, and the

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residue was taken up with fresh CF3COOH (114 g, 1 mol). After an additional 24 h, the reaction mixture was diluted with Et2O (700 mL) to afford a precipitate that was filtered and suspended in H2O. The mixture was basified to pH 13 with 10 N NaOH to give a clear solution. After 1 h, MeCN (300 mL) was added then the solution was acidified to pH 2 with 37% HCl. Slow evaporation under vacuum of the organic solvent gave precipitation of a white solid that was filtered off. The crude product was purified by preparative reversed-phase HPLC [Lichroprep RP-18 column, 50 × 250 mm, 25-40 µm, Merck KGaA; with 80% 0.01 M KH2PO4/20% MeCN for 105 min then 50% 0.01 M KH2PO4/50% MeCN for 20 min; flow rate 90 mL/min, monitored on-line by UV at 200 nm]. Fractions containing the product were combined, neutralized to pH 7 with 10 N NaOH, and evaporated under reduced pressure to give a solid (product mixed with inorganic salts) that was dissolved in H2O (790 mL). The solution was heated at 50 °C and, while maintained under vigorous stirring, slowly acidified to pH 2.1 with 37% HCl (48 mL) to yield a white precipitate. The suspension was allowed to cool spontaneously to room temperature, and the precipitate was filtered, washed with H2O (2 × 25 mL), and dried to give 10 (33.1 g, 46%) as a white solid. mp 193-195 °C. 13C NMR (D2O + KOD): δ 15.0 (CH3), 19.6 (CH3), 25.2 (CH3), 25.8 (CH2), 26.6 (CH2), 28.4 (CH2), 28.7 (CH), 30.1 (CH2), 30.7 (CH2), 33.2 (CH2), 35.0 (CH2), 35.6 (CH2), 36.2 (CH2), 37.0 (CH2), 37.3 (C), 38.3 (CH), 39.1 (CH), 41.9 (CH), 44.3 (CH), 48.6 (CH), 49.0 (C), 50.4 (CH2), 54.9 (CH2), 60.5 (CH2), 68.7 (CH), 71.0 (CH), 75.7 (CH), 177.5 (CO), 177.7 (CO), 177.8 (CO), 180.4 (CO), 186.8 (CO). Anal. (C41H66N4O15) C, H, N. MS: m/z 853 [M - H]-. N,N-Bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]glycine (11). Method a. Phosphate buffer (50 mL, 2 M, pH 8) was added to a solution of glycine benzyl ester (16) (2.44 g, 14.8 mmol) and bromoderivative 2 (11.5 g, 32.6 mmol) in CH3CN (100 mL), and the mixture was vigorously stirred for 24 h. The mixture was separated, the organic phase evaporated, and the residue flash chromatographed with CHCl3. The yellow oil obtained (6.5 g) was dissolved in MeOH (50 mL), and 10% Pd on carbon (0.7 g) was added. The suspension was stirred over 24 h under a hydrogen atmosphere at 20 °C. The mixture was filtered over Millipore FH 0.45 µm and evaporated to give 11 (4.87 g, 53%) as a yellow oil. Method b. NaOH (10 M) (10 mL) was added to a solution of glycine (12 g, 0.160 mol) in water (100 mL) and 95% EtOH (100 mL) until pH 10 was reached. A solution of bromoderivative 2 (120 g, 0.341 mol) in 95% EtOH (100 mL) was added dropwise, maintaining at pH 10 by means of a pH-stat apparatus. After 18 h, EtOH was removed under reduced pressure, and the solution was acidified to pH 3.5 with 12 N HCl and extracted with CH2Cl2 (2 × 300 mL). The combined organic phases were washed with water (2 × 100 mL), dried (Na2SO4), and evaporated. The residue was purified by flash chromatography using CH2Cl2 f CH2Cl2/MeOH/25% NH4OH(ag) 150:13:1 as the eluent. Evaporation of the solvent gave a pale yellow oil which was treated with petroleum benzine (boiling range 60-80 °C; 3 × 400 mL) until 11 (69 g, 70%) was obtained as a white crystalline solid. mp 93 °C. 1H NMR (CDCl3): δ, 1.46 (s, 36H), 2.99 (t, 4H), 3.10 (br t, 4H), 3.45(s, 8H), 3.56 (s, 2H). 13C NMR (CDCl3): δ, 28.5 (CH3), 51.2 (CH2), 53.9 (CH2), 56.4, (CH2), 57.0 (CH2), 82.0 (C), 170.8 (CO). Anal. (C30H55N3O10) C, H, N. MS: m/z 618 [M + H]+, 640 [M + Na]+.

Anelli et al. LITERATURE CITED (1) Anelli, P. L., Fedeli, F., Gazzotti, O., Lattuada, L., and Rebasti, F. Presented in part at 35th National Organic Chemistry Symposium, San Antonio, TX, June 1997, paper T166. (2) Weinmann, H. J. (1994) Characteristics of Gd-DTPA dimeglumine in Magnevist monograph (R. Felix, A. Heshiki, N. Hosten, and H. Hricak, Eds.) pp 5-14, Blackwell Scientific Publications, Oxford. (3) Weiner, E. C., Brechbiel, M. W., Brothers, H., Magin, R. L., Gansow, O. A., Tomalia, D. A., and Lauterbur, P. C. (1994) Dendrimer-based metal chelates: a new class of magnetic resonance imaging contrast agents. Magn. Reson. Med. 31, 1-8. (4) Anelli, P. L., Calabi, L., de Hae¨n, C., Fedeli, F., Losi, P., Murru, M., and Uggeri, F. (1995) A new approach to hepatospecific MRI contrast agents: gadolinium complexes conjugated to iodinated synthons. Gazz. Chim. Ital. 126, 89-97. (5) Anelli, P. L., Calabi, L., de Hae¨n, C., Lattuada, L., Lorusso, V., Maiocchi, A., Morosini, P., and Uggeri, F. (1997) Hepatocyte-directed MR contrast agents. Can we take advantage of bile acids? Acta Radiol. 38 (Suppl. 412), 125-133. (6) Hnatowich, D. J., Layne, W. W., and Childs, R. L. (1982) The preparation and labeling of DTPA-coupled albumin. Int. J. Appl. Radiat. Isot. 33, 327-332. (7) Maisano, F., Gozzini, L., and de Hae¨n, C. (1992) Coupling of DTPA to proteins: a critical analysis of the cyclic dianhydride method in the case of insulin modification. Bioconjugate Chem. 3, 212-217. (8) Sherry, A. D., Cacheris, W. P., and Kuan, K.-T. (1988) Stability constants for Gd3+ binding to model DTPA-conjugates and DTPA-proteins: implications for their use as magnetic resonance contrast agents. Magn. Reson. Med. 8, 180-190. (9) Williams, M. A., and Rapoport, H. (1993) Synthesis of enantiomerically pure diethylenetriaminepentaacetic acid analogues. L-Phenylalanine as the educt for substitution at the central acetic acid. J. Org. Chem. 58, 1151-1158. (10) Gansow, O. A., Brechbiel, M. W., Mirzadeh, S., Colcher, D., and Roselli, M. (1990) Chelates and antibodies: current methods and new directions. in Cancer imaging and radiolabeled antibodies (D. M. Goldenberg, Ed.) pp 153-171, Kluwer Academic Publishers, Dordrecht, The Netherlands. (11) Cummins, C. H., Rutter, E. W., Jr., and Fordyce, W. A. (1991) A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaacetic acid and their coordination chemistry with yttrium(III). Bioconjugate Chem. 2, 180-186. (12) Guttmann, St., and Boissonnas, R. A. (1958) Synthe`se du N-ace´tyl-L-se´ryl-L-tyrosyl-L-se´ryl-L-me´thionyl-γ-L-glutamate de benzyle et de peptides apparente´s. Helv. Chim. Acta 41, 1852-1867. (13) Roeske, R. (1963) Preparation of tert-butyl esters of free amino acids. J. Org. Chem. 28, 1251-1253. (14) Anelli, P. L., Lattuada, L., and Uggeri, F. (1998) One-pot Mitsunobu-Staudinger preparation of 3-aminocholan-24-oic acid esters from 3-hydroxycholan-24-oic acid esters. Synth. Commun. 28, 109-117. (15) Platzek, J., Niedballa, U., and Raduchel, B. (1996) U.S. Patent 5,514,810, Chem. Abstr. 125, 87212. (16) Miller, H. K., and Warlsch, H. (1952) Benzyl esters of amino acids. J. Am. Chem. Soc. 74, 1092-1093. (17) Arano, Y., Uezono, T., Akizawa, H., Ono, M., Wakisaka, K., Nakayama, M., Sakahara, H., Konishi, J., and Yokoyama A. (1996) Reassessment of diethylenetriaminepentaacetic acid (DTPA) as a chelating agent for indium-III labeling of polypeptides using a newly synthesized monoreactive DTPA derivative. J. Med. Chem. 39, 3451-3460. (18) Hayward, M. M., Adrian, J. C. and Schepartz, A. (1995) Convenient synthesis of bifunctional metal chelates. J. Org. Chem. 60, 3924-3927.

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