26
Bioconjugate Chem. 1991, 2, 26-31
Carboxymethyl-Substituted Bifunctional Chelators: Preparation of Aryl Isothiocyanate Derivatives of 3-(Carboxymethyl)-3-azapentanedioic Acid, 3,12-Bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic Acid, and 1,4,7,1O-Tetraazacyclododecane- N,i”JV”,N’”- tetraacetic Acid for Use as Protein Labels Steven J. Kline, David A. Betebenner, and David K. Johnson’ Abbott Laboratories, Department 90M, Abbott Park, Illinois 60064. Received September 5, 1990
3-(Carboxymethyl)-3-azapentanedioicacid (NTA), 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid (EGTA), and 1,4,7,10-tetraazacyclododecane-N~,N”,N”’-tetraaceticacid (DOTA) structures having a 4-nitrophenyl substituent attached via an alkyl spacer to the methylene carbon atom of one carboxymethyl arm of the chelator were obtained by alkylation of 4-nitrophenylalanine with bromoacetic acid (NTA),by reductive alkylation of l,B-diamino-3,6-dioxaoctane with (4-nitropheny1)pyruvic acid followed by alkylation with bromoacetic acid (EGTA), and by alkylation of the trimethyl acid with the methyl ester of a-brom0-4-(4ester of 1,4,7,10-tetraazacyclododecane-N,N’,N”-triacetic nitropheny1)pentanoic acid and subsequent saponification (DOTA). The nitrophenyl-substituted chelators were converted to the corresponding amines by hydrogenation then reacted with thiophosgene to give the protein-reactive aryl isothiocyanate derivatives.
Labeling of proteins and other biologically active molecules with metal ions is often accomplished with synthetic chelating agents that contain a bioreactive substituent (1, 2). The first such bifunctional chelators to find widespread use were EDTA’ analogues derivatized at a methylene carbon atom of the diamine backbone with phenyl (3),benzyl ( 4 ) , or phenethyl (5) moieties bearing a protein-reactive substituent on the aromatic ring. In recent years, interest in tumor targeting using radiometallabeled antibody-chelator conjugates has prompted the development of alternative bifunctional ligands (6-15). As in vivo stability of the chelate label is of paramount importance in such applications, these efforts have concentrated on the design and synthesis of bifunctional chelating agents that impart enhanced kinetic and/or thermodynamic stability to their chelates with radiometals of interest. Systems of this type include analogues of DTPA (6-8) and HBED (9, 10) and ligands based on tetraaza (11-13) and triaza (14)macrocyclicpolyaminopolycarboxylates and their polyamine precursors (151,once again derivatized at methylene carbon atoms of the polyamine backbone with various protein-reactive side chains. Although the recent focus of bifunctional chelator development has been on high stability, future applications of these potentially versatile agents may emphasize additional properties. For example, metal ion selectivity will be important if bifunctional chelators are to be used to target endogenous metal pools that are implicated in producing tissue damage in some disease states (16). While stability and selectivity are often maximized through the
use of high-denticity ligands, applications in which biomolecules are labeled with metal complexes that possess catalytic activity are likely to require bifunctional chelators that do not completely saturate the coordination sphere, in order to avoid blocking substrate access to the metal ion. Before any such uses can be systematically investigated, a spectrum of bifunctional chelators spanning a range of metal-binding properties will be required, so that structure-activity relationships can be established empirically. To this end, we are expanding a series of bifunctional polyaminopolycarboxylates in which an aryl isothiocyanate side chain is incorporated into one carboxymethyl arm of the chelator. The preparation of EDTA and DTPA systems of this type has been previously described (17). This report presents syntheses of three additional analogues (4, 10, 20; Schemes 1-111).
EXPERIMENTAL PROCEDURES (4-Nitropheny1)pyruvic acid (5) was synthesized as previously described (17 ) . l,B-Diamino-3,6-dioxaoctane (6) was prepared from 1,B-dichloro-3,6-dioxaoctane via 1,Bdiphthaloyl-3,6-dioxaoctanefollowing the procedure of Sun et al. (18). 1,4,7,10-Tetraazacyclododecane(16) was prepared as described by Richman et al. (19). All other starting materials and reagents were purchased from Aldrich Chemical Co., Milwaukee, WI. ‘H NMR and 13C NMR spectra were recorded on a General Electric QE-300 spectrometer, operating at 300.23 and 75.5 MHz, respectively. Chemical shifts are given in ppm relative to TMS as internal standard, unless otherwise specified, and coupling constants are reported in hertz. Mass spectra The following abbreviations are used: DK, a ,&diketone; were obtained with a Kratos MS-50spectrometer by either DOTA, 1,4,7,10-tetraazacyclododecane-N~~,~”-tetraacetic the fast atom bombardment technique using a thioglycacid; DTPA, diethylenetriaminepentaacetic acid; EDTA, etherol or glycerol/thioglycerol matrix or by direct ionization. ylenediaminetetraaceticacid; EGTA: 3,12-bis(carboxymethyl)Elemental analyses were performed by Galbraith Labo6,9-dioxa-3,12-diazatetradecanedioic acid; HBED, N,N’-bis(2ratories, Knoxville, TN. hydroxybenzy1)ethylenediamine-Nfl-diaceticacid;NTA, 3-(carN,iV-Bis(carboxymethyl)-4-nitrophenylalanineH y boxymethyl)-3-azapentanedioicacid; TMS, trimethylsilane; TSP, 3-(trimethylsilyl)-l-propanesulfonicacid. drochloride (2). A solution of bromoacetic acid (4.99 g, 1043-1802/9 1/2902-0026$02.50/0
0 1991 American Chemical Society
Bioconjtpte Chem., Vol. 2, No. 1, 1991 27
Preparation of Bifunctional Chelators
Scheme I
HozCT NP O z H /
HozC7
/
Nr
CozH
p4 NCS
35 mmol) in H20 (20 mL) was added to a suspension of 4-nitro-dl-phenylalanine (1; 3.02 g, 14 mmol) in H20 (50 mL) and the pH of the resulting solution was raised to 1 2 by addition of 6 M NaOH. The reaction mixture was then stirred at 45 "C for 2 days, the pH being maintained in the range of 11-12 by periodic addition of 6 M NaOH. After cooling to room temperature, the reaction solution was evaporated to dryness under vacuum to give a yellow oil, which was redissolved in H2O (10 mL), adjusted to pH 10 with 6 M NaOH, and applied to a Bio-Rad AGl-X4 anion-exchange column (formate form, column weight 100 g). The column was eluted successively with H2O (1L), 4 M formic acid (500 mL), and 5 M formic acid (500 mL), fractions being evaluated by TLC on silica plates developed in 25% concentratedaqueous NH40H/75% EtOH (95%) (Rf for 1 = 0.60; Rf for 2 = 0.21; Rf for monoalkylation product = 0.38). The desired product, contaminated with some higher Rf material, eluted in the 5 M formic acid fractions. These were combined, evaporated to dryness, redissolved in H20 (10 mL), readjusted to pH 10, and applied to a second Bio-Rad AGl-X4 anion-exchange column (formate form, column weight 70 g) that was eluted successively with HzO (500 mL), 4 M formic acid (500 mL), 4.25 M formic acid (500 mL), 4.5 M formic acid (500 mL), and 4.75 M formic acid (500mL),and 10-mLfractions were collected. The desired product eluted slowly in the 4.75 M formic acid fractions and these were combined, evaporated to dryness under vacuum, and redissolved in 4 M HC1 (100 mL). The resulting solution was again evaporated to dryness under vacuum and the residue was repeatedly redissolved in water and reevaporated to dryness until a solid residue was obtained. This was then dried under vacuum over P205 to afford 2 in the form of a pale yellow powder (3.64 g, 72%): 'H NMR (D20, TSP standard) 6 3.29-3.48 (m, 2 H), 3.95-4.08 (m, 4 H), 4.32 (t, 1H, J = 7.7), 7.54 (d, 2 H, J = 8.4),8.22 (d, 2 H, J = 8.8); mass spectrum (FAB+,thioglycerol/glycerol), m / e 349 (M + Na)+, 327 (M + H)+. N,N-Bis(carboxymethyl)-4-aminophenylalanineDihydrochloride (3). A solution of 2 (0.60 g, 1.65 mmol) in H20 (150 mL) was hydrogenated in a Parr apparatus at room temperature and 35 psi HZover 10% palladium on carbon (60 mg) for 2 h. The catalyst was removed by filtration and the filtrate evaporated to dryness under vacuum. The resulting residue was dissolved in 4 M HC1
(50 mL) and reevaporated to dryness, followed by four cycles of redissolution in H20 (200 mL) and reevaporation to dryness. The oil thus obtained was dissolved in HzO (10 mL), lyophilized, then dried under vacuum over P205 to give 0.55 g (99%) of 3 in the form of a yellow solid: lH NMR (D20, TSP standard) 6 3.28 (dd, 1 H, J = 14.3, 8.1),3.42 (dd, 1H, J = 14.3,7.4), 4.01-4.15 (m, 4 H), 4.36 (t, 1 H, J = 7.7), 7.40 (d, 2 H, J = 8.8), 7.48 (d, 2 H, J = 8.5); mass spectrum (glycerol/thioglycerol),m / e 319 (M + Na)+, 297 (M + H)+. N,N-Bis(carboxymethyl)-4-isot hiocyanatophenylalanine Hydrochloride (4). An 80% (v:v) solution of thiophosgene in CC14 (0.18 mL, 1.87 mmol) was added to a solution of 3 (45 mg, 0.13 mmol) in 3 M HC1 (1.2 mL) and the resulting solution was vigorously stirred at room temperature for 6 h. The solvents and residual thiophosgene were then removed under vacuum in a fume hood, and the residue was dried further over P205 under vacuum to yield 47 mg (97% ) of 4 as a pale beige powder: 'H NMR (DMSO-de, TMS standard) 6 2.89-3.04 (m, 2 H), 3.49-3.62 (m, 4H), 3.72 (t, 1H, J = 7.7),7.33 (s,4H);mass spectrum (glycerol/thioglycerol), m / e 339 (M + H)+. N-( l-Amino-3,6-dioxaoctyl)-3-(4-nitrophenyl)alanine Dihydrochloride (7). A solution of 5 (3.57 g, 17 mmol) in MeOH (70 mL) was added to a solution of 6 (2.53 g, 17 mmol) in H2O (15 mL) and the pH of the resulting dark red solution was adjusted to 6 by addition of 4 M HC1. Sodium cyanoborohydride (1.13 g, 17 mmol) was then added and the reaction mixture stirred at pH 6 and room temperature for 5 days. The resulting yellow solution was then acidified to pH 1 by addition of concentrated HC1 and evaporated to dryness under vacuum. The residue was extracted with a mixture of H2O (250 mL) and EtOAc (200 mL), the organic phase being subsequently discarded. The aqueous phase was extracted with EtOAc (2 X 200 mL) then concentrated to a volume of ca. 5 mL and adjusted to pH 3 by addition of 6 M NaOH. This solution was applied to a Dowex 5OX-2-200 column (H+ form, column weight 300 g) which was eluted sequentially with H z 0 , l M HC1,2 M HC1, and 4 M HC1. Unreacted 6 eluted in the 2 M HC1, whereas the desired product was isolated from the 4 M HC1 eluate. Fractions containing the product were identified by TLC on silica plates developed in 20 % concentrated aqueous NH40H/80 % EtOH (95%)and treatedwith ninhydrin (Rf= 0.5). These were combined, evaporated to dryness under vacuum, redissolved in HzO (250 mL), and again evaporated to dryness. A further four cycles of dissolution in 250-mL aliquots of H2O followed by reevaporation to dryness gave a residue that upon lyophilization afforded 2.68 g (38%) of 7 in the form of apale yellow solid: 'H NMR (D20, TSP standard) 6 3.21 (t, 2 H, J = 5.0), 3.33-3.42 (m, 3 H), 3.50 (dd, 1 H, J = 14.2,6.1), 3.67-3.86 (m, 8 H), 4.31 (dd, 1H, J = 7.5,6.1), 7.55 (d, 2 H, J = 8.4), 8.24 (d, 2 H, J = 8.4); 13CNMR (DzO,TSP standard) 6 37.72,41.99,49.23,63.57, 68.38, 69.33, 72.47, 72.53, 126.97, 133.42, 144.93, 150.01, 173.08; mass spectrum (glycerol/thioglycerol), m / e 342 H)+; high-resolution mass spectrum calcd for (M C16H24N306 342.1665, found 342.1667. Anal. Calcd for C15H2&12N306: C, 43.49; H, 6.08; N, 10.14. Found: C, 43.16; H, 6.07; N, 9.93. 2-(4-Nitrobenzyl)-3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioicAcid Dihydrochloride (8). Solid 7 (1.29 g, 3 mmol) was added to a solution of bromoacetic acid (1.74 g, 12.0 mmol) in HzO (10 mL) and the pH of the resulting yellow solution was adjusted to 12 by addition of 6 M NaOH. The solution was then stirred at 45 "C for 7 days, the pH being maintained at 1 2 by
+
28
Kline et al.
Bioconjugate Chem., Vol. 2, No. 1, 1991
Hozc4 +
Scheme I1
5
NO2
-
Scheme 111 02N-OH
OZN-SOJCH~
11
-OzN-CN
I
12
l3
I
l5
Br
n
i" HN
N> NH
W 16
17
periodic addition of 6 M NaOH. The orange solution thus obtained was applied to a Bio-Rad AGl-X4 anion-exchange column (formate form, column weight 40 g), which was eluted successively with H2O (200 mL), 0.1 M formic acid (350 mL), 0.5 M formic acid (350 mL), and 0.7 M formic acid (1.5 L). The desired product eluted in the 0.7 M formic acid, product-containing fractions being identified by TLC on silica plates developed in 20% concentrated aqueous NH40H/80% EtOH (95%) (Rf for 8 = 0.6).
Fractions containing the product were combined, and 4 M HCl(5 mL) was added, then the solution was evaporated to dryness under vacuum and the residue was lyophilized. Trituration of the lyophilized material with CH2C12 produced a yellow solid that was dried under vacuum to afford 1.20 g (67%) of 8: lH NMR (DzO, TSP standard) 6 3.43-3.73 (m, 10 H), 3.82-3.89 (m, 4 H), 4.12-4.31 (m, 6 H), 4.61 (t, 1 H, J = 7.2), 7.60 (d, 2 H, J = 8.4), 8.25 (d, 2 H, J = 8.4); 13C NMR (D20, TSP standard) 6 35.23,
Bioconjugate Chem., Vol. 2, No. 1, 1991 29
Preparation of Bifunctional Chelators
56.39,58.24,58.33,58.80,67.79,70.22,72.43,72.53,126.97, 133.39, 145.54, 149.91, 170.88, 171.49, 172.04; mass spectrum (thioglycerol), m / e 516 (M H)+; high-resolution mass spectrum calcd for C21H30N3012 516.1830, found 516.1830. Anal. Calcd for C21H31Cl~N3012:C, 42.87; H, 5.31; N, 7.14. Found: C, 42.60; H, 5.13; N, 7.06. 2-(4-Aminobenz yl)-3,12-bis (carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioicAcid Trihydrochloride (9). A solution of 8 (404 mg, 0.69 mmol) in HzO (100 mL) was hydrogenated in a Parr apparatus at room temperature and 35 psi Hz over 10% palladium on carbon (40 mg) for 2 h. The catalyst was removed by filtration and 4 M HCl(5 mL) was added to the filtrate, which was then evaporated to dryness under vacuum. The resulting oil was dissolved in HzO (10 mL) and lyophilized to give 399 mg (97%) of 9 in the form of a hygroscopic white solid: 'H NMR (D20, TSP standard) 6 3.35-3.77 (m, lOH), 3.813.92 (m, 4 H), 4.22 (m, 4 H), 4.25 (s, 4 H), 4.57 (t, 1 H, J = 7.6), 7.43 (d, 2 H, J = 8.31, 7.53 (d, 2 H, J = 8.3); 13C NMR (DzO, TSP standard) 6 35.12, 56.48, 58.00, 58.48, 58.74, 67.71, 67.81, 70.82, 72.50, 72.60, 126.60, 132.21, 133.91,138.84,171.12,171.79,172.58;massspectrum(thioglycerol), m / e 486 (M + H)+; high-resolution mass spectrum calcd for C Z ~ H ~ Z N486.2088, ~ O I O found 486.2084. Anal. Calcd for Cz1H34C13N3010: C, 42.40; H, 5.76; N, 7.06. Found: C, 42.52; H, 5.58; N, 6.71. 2-(4-Isothiocyanatobenzyl)-3,12-bis(carboxymethy1)-6,9-dioxa-3,12-diazatetradecanedioic Acid Dihydrochloride (10). An 80% (v:v) solution of thiophosgene in C c 4 (50 pL, 0.52 mmol) was added to a solution of 9 (22 mg, 0.037 mmol) in 3 M HC1 (330 pL) and the resulting reaction mixture was stirred vigorously at room temperature for 6 h. Evaporation to dryness under vacuum followed by vacuum drying over P206 gave 21.9 mg (99%) of 10 in the form of a light beige powder: 'H NMR (DMSO-&, TSP standard) 6 3.06-3.26 (m, 5 H), 3.40-3.53 (m, 6 H), 3.55-3.64 (m, 2 H), 3.72-3.80 (m, 2 H), 3.88 (br s, 2 H), 4.08 (t, 1 H), 4.21 (s, 3 H), 7.39 (s, 4 H); mass spectrum (thioglycerol),m / e 528 (M + H)+;high-resolution mass spectrum calcd for C Z Z H ~ O N ~528.1652, O ~ O S found 528.1647. Anal. Calcd for C Z Z H ~ ~ C ~ Z NC, ~O 44.00; ~ O SH,: 5.20; N, 7.00. Found: C, 43.81; H, 5.19; N, 6.87. 4-(4-Nitrophenyl)butylMethanesulfonate (12). 444Nitropheny1)-1-butanol (19.44 g, 99.6 mmol) and triethylamine (15.2 g, 151 mmol) were dissolved in dry CHzC12 (250 mL), and the resulting solution was stirred under N2 at 0 "C while a solution of methanesulfonyl chloride (17.1 g, 149 mmol) in CH2C12 (100 mL) was added dropwise over 30 min. After stirring at 0 "C for a further 45 min, the reaction mixture was extracted sequentially with cold HzO (2 X 100 mL), cold 2 M HCl(2 X 100 mL), cold 1M NaOH (2 X 100 mL), and cold HzO (2 X 100 mL). The organic phase was dried over anhydrous NazS04 then evaporated to dryness under vacuum to give a yellow oil that solidified on standing at 0 "C. This crude product was extracted at reflux with Et20 (600 mL), giving a colorless extract and leaving an insoluble orange tar. Evaporation to dryness of the ether extract gave 26.12 g (96%) of 12 in the form of a tan solid: lH NMR (CDC13) 6 1.78-1.85 (m, 4 H), 2.75-2.82 (m, 2 H), 3.01 ( 5 , 3 H), 4.24-4.29 (m, 2 H), 7.34 (d, 2 H, J = 9.0), 8.16 (d, 2 H, J = 9.0);l3CNMR (CDCl3)6 26.67,28.46,34.90,37.19,69.43, 123.50,123.60,129.08,146.25,149.37; mass spectrum (DCI), m / e 291 (M + NH4)+. Anal. Calcd for CllH15N05S: C, 48.34; H, 5.53; N, 5.13. Found: C, 48.25; H, 5.59; N, 5.15. 4-(4-Nitrophenyl)pentanenitrile (13). A mixture of 12 (5.0 g, 18.3 mmol) and NaCN (2.8 g, 57 mmol) in dry DMSO (40 mL) was stirred at 60 "C for 30 min under Nz.
+
After cooling to room temperature, the reaction mixture was poured into Et20 (300 mL) and the resulting BUSpension was filtered. The filtrate was extracted with HzO (4 X 150 mL), dried over anhydrous NazS04, and evaporated to dryness. The resulting residue was chromatographed on a silica gel column eluted with CHzC12 to give 3.31 g (89%) of 13 in the form of a yellow oil: 'H NMR (CDCl3) 6 1.66-1.90 (m, 4 H), 2.40 (t, 2 H, J = 7.5), 2.79 (t, 2 H, J = 7.5), 7.35 (d, 2 H, J = 9.0), 8.17 (d, 2 H, J = 9.0); 13CNMR (CDCl3) 6 17.04,24.80,29.77,34.89,119.43, 123.71,129.18,146.45,149.18; mass spectrum (DEI), m / e 204 (M)+. 4-(4-Nitrophenyl)pentanoicAcid (14). A solution of 13 (10.26 g, 50.2 mmol) in 6 M HCl (500 mL) was refluxed for 60 h. After cooling to room temperature, the reaction mixture was extracted with CHzC12 (2 X 150 mL), and the organic extracts were combined and extracted with 5% aqueous NaHC03 (10 X 100 mL). The aqueous extracts were combined, acidified to pH 2 by addition of concentrated HC1 at 0 "C, then extracted with CHzClz (4 X 150 mL). The combined organic extracts were dried over anhydrous Na2S04 then evaporated to dryness to give a white solid. Recrystallization from H2O gave 10.24 g (91%) of 14 in the form of colorless prisms: 'H NMR (CDC13)6 1.62-1.79 (m, 4H), 2.35-2.45 (m, 2 H), 2.70-2.80 (m, 2 H), 7.33 (d, 2 H, J = 9.0), 8.15 (d, 2 H, J = 9.0); 13C NMR (CDCl3) 6 24.04,30.13,33.67,35.38,123.61, 129.11, 146.30,149.78,179.80;mass spectrum (DEI),m / e 223 (M)+. Anal. Calcd for CllH13N04: C, 59.19; H, 5.87; N, 6.27. Found: C, 58.96; H, 5.98; N, 6.20. ru-Bromo-4-(4-nitrophenyl)pentanoic Acid Methyl Ester (15). A mixture of 14 (3.97 g, 17.8 mmol) and thionyl chloride (16.3 g, 137 mmol) in dry cc14 (40 mL) was refluxed for 45 min under Nz. The reaction mixture was cooled to room temperature and N-bromosuccinimide (3.97 g, 22.3 mmol) and HBr (10 drops of a 47-49??, aqueous solution) were added, then the mixture was stirred at reflux for a further 2 h. After cooling to room temperature, the reaction mixture was evaporated to dryness, redissolved in MeOH (100 mL), and refluxed under Nz for a further 1.5 h. After again cooling to room temperature, the reaction mixture was evaporated to dryness under vacuum and the residue was dissolved in CHzClz (150 mL) and extracted with HzO (2 X 75 mL). The organic phase was dried over anhydrous Na~S04and evaporated to dryness to give a dark brown oil which was chromatographed on a silica gel column eluted with CHzClz. The eluate was evaporated to dryness and the residue recrystallized from hexane to give 2.2 g (39%) of 15 as colorless prisms: lH NMR (CDCl3) 6 1.65-2.19 (m, 4 H), 2.77 (t, 2 H, J = 7.5), 3.78 (s, 3 H), 4.24 (m, 1 H), 7.34 (d, 2 H, J = 9.0),8.16 (d, 2 H, J = 9.0); mass spectrum (DCI), m / e 333 (M + NH4)+. Anal. Calcd for C12H14N04Br: C, 45.59; H, 4.46; N, 4.43. Found: C, 45.63; H, 4.49; N, 4.38. 1,4,7,10-Tetraazacyclododecane-N,iV',~'-triacetic Acid Trimethyl Ester (17). A mixture of 1,4,7,10-tetraazacyclododecane (1.0 g, 5.8 mmol), methyl bromoacetate (3.55 g, 23.2 mmol), and triethylamine (1.69 g, 16.7 mmol) in dry MeOH (25 mL) was refluxed for 16 h under N2. After cooling to room temperature, the reaction mixture was evaporated to dryness and the residue was triturated with 2-propanol (5 mL). The resulting white solid was filtered off and washed with additional 2-propanol (5 mL), then the filtrate plus washings were evaporated to dryness. The resulting oil was further purified by preparative TLC on silica gel plates developed in 10% i-PrOH/SO% CHzClz to give 1.21 g (54%) of 17 in the form of a pale brown oil: 'H NMR (CDC13) 6 2.72-
30 Bioconjugate Chem., Vol. 2, No. 1, 1991
Kline et al.
2.99 (m, 17 H), 3.40-3.49 (m, 6 H), 3.66-3.75 (m, 9 H); of the protein-reactive side chain was achieved by direct mass spectrum (DCI), m / e 389 (M + H)+. Anal. Calcd alkylation rather than reductive alkylation, as the polyfor C ~ ~ H ~ Z H ~ O & H ZC,C 45.67; ~ Z : H, 7.24; N, 11.84. amine precursor contained only secondary amino groups. Found: C, 45.91; H, 7.43; N, 11.93. The synthetic strategy that was adopted required conN-[4-(4-Nitrophenyl)-l-carboxybutyl]-1,4,7,lO-tet- trolled carboxymethylation of this polyamine, 16, such that trisubstituted species 17 was the major product. raazacyclododecane-N’,N”,N”’-triacetic Acid (18). A Reaction conditions were optimized empirically and it was mixture of 17 (2.43 g, 6.26 mmol), 15 (3.94 g, 12.5 mmol) determined that an input stoichiometry of methyl bromoand triethylamine (636 mg, 6.28 mmol) in dry MeOH (21 acetate:16 of 4:l provided a maximal yield of 17 (54%), mL) was refluxed under N2 for 6 days. After cooling to with the tetrasubstituted side product being isolated in room temperature, the reaction mixture was evaporated yields of 9-20%. The remainder of the synthetic route to to dryness and the residue was dissolved in CHzClz (100 20 was unremarkable. mL) and extracted with saturated aqueous NaHC03 (75 Formation constants for 1:l trivalent metal complexes mL) and HzO (75 mL). The organic phase was then dried of NTA (typically 10*-1012) are inadequate for in vivo over anhydrous Na2S04 and evaporated to dryness under applications, but an ability to form protein-NTA-metalvacuum to give an oil. This was chromatographed on a NTA-protein linkages may provide a nondenaturing and silica gel column eluted first with CHzC12 then with 20% readily reversible means for cross-linking two proteins, MeOH/80% CH2Cl2, fractions eluting in the latter being such as an antibody and an enzyme, for in vitro use. The combined and evaporated to dryness to give a brown oil, bifunctional NTA ligand may also prove useful for which was used in the next step without further purifitethering ternary metal complexes to proteins, e.g. an cation. The oil was dissolved in EtOH (40 mL) and the [ (NTA)Eu(DK)] label might allow direct observation of resulting solution stirred at room temperature while 0.5 europium fluorescencewithout needing to release the metal M NaOH (80 mL) was slowly added. After stirring at from the protein with excess DK, as is presently done room temperature for a further 18 h, the reaction mixture (20). EGTA is an octadentate chelator best known for its was applied directly to a Bio-Rad AGl-X4 anion-exchange selectivity for Ca2+relative to Mg2+,which exceeds that column (formate form, 50-mequiv capacity). The column of most calcium-binding proteins (21). Applications was eluted successivelywith HzO (500 mL), 0.05 M formic ranging from novel chromatographic matrices for the acid (500 mL), 0.1 M formic acid (500 mL), and 0.2 M removal of CaZ+,through selective probes of intracellular formic acid (1L). The 0.2 M formic acid fractions were calcium metabolism, to constructs for intervening in combined and evaporated to dryness to give 0.80 g (36%) calcium-dependent processes in vivo can be envisioned of 18 in the form of a tan solid: lH NMR (DzO) 6 1.55-1.63 with use of the bifunctional version of this ligand. DOTA (m, 4 H), 2.62-4.01 (m, 25 H), 7.45 (d, 2 H, J = 9.0), 8.17 is a recently described macrocyclic chelator that forms (d, 2 H, J = 9.0); mass spectrum (FAB), m / e 568 (M + very stable complexes with radioactive z1zBi2+) and H)+. Anal. Calcd for C25H37N501rH20: C, 51.28; H, 6.71; paramagnetic (Gd3+) metals that are of interest for N, 11.96. Found: C, 50.90; H, 6.53; N, 11.71. radiotherapy (12, 13, 22) and MRI contrast (23) appliN-[4-(4-Aminopheny1)- 1-carboxybutylJ-1,4,7,10-tetcations, respectively. All of these potential uses remain raazacyclododecane-l,N”,M”-triacetic Acid (19). A speculative at this point, but access to appropriate mixture of 18 (199 mg, 0.35 mmol) and 10% palladium on bifunctional ligand systems is an essential first step in carbon (20 mg) in H2O (50 mL) was hydrogenated in a evaluating such possibilities. The ability to introduce a Parr apparatus at room temperature and 35 psi HZfor 3 protein-reactive substituent into any polyaminopolycarh. The catalyst was removed by filtration and the filtrate boxylate chelator, either by reductive alkylation with 5 evaporated to dryness to give 179 mg (95%) of 19 in the (Scheme I1and ref 17),in the case of polyamine precursors form of a white solid: ‘H NMR (D20) 6 1.55-1.90 (m, 4 that contain a primary amine, or by direct alkylation with H), 2.57-4.08 (m, 25 H), 7.36 (m, 4 H); mass spectrum 15 (Scheme 1111,for polyamine frameworks that contain (FAB), m / e 538 (M + H)+. only secondary amino groups, should facilitate the idenN-[4- (4-Isothiocyanatopheny1)-1-carboxybutyll1,4,7,10-tetraazacyclododecane-~,~’,I\p’’-triacetic tification of additional areas where an interface between coordination chemistry and protein biochemistry may bear Acid (20). An 85% (v:v) solution of thiophosgene (223 fruit. mg, 1.94 mmol) in CCld was added to a solution of 19 (81 mg, 0.15 mmol) in 3 M HC1 (3.5 mL) and the mixture LITERATURE CITED stirred a t room temperature for 6 h. The residue obtained on evaporation of the reaction mixture to dryness was (1) Meares, C. F., and Wensel, T. G. (1984) Metal chelates as dissolved in 4 M HCl(l0 mL) and reevaporated to dryness probes of biological systems. Acc. Chem. Res. 17, 209-214. under vacuum to give 88 mg (89%) of 20 in the form of (2) Meares, C. F., and Goodwin, D. A. (1984) Linking radioa tan powder: ‘H NMR (D20) 6 1.55-1.89 (m, 4 H), 2.57metals to proteins with bifunctional chelating agents. J. 4.04 (m, 25 H), 7.28 (s, 4 H); mass spectrum (FAB), m / e Protein Chem. 3, 215-228. 580 (M + H)+. (3) Sundberg, M. W., Meares, C. F., Goodwin, D. A., and DiaRESULTS AND DISCUSSION
Syntheses of these bifunctional chelators were for the most part straightforward. Carboxymethylation of 4-nitrophenylalanine and subsequent conversion to 4 by standard methods was a simple, high-yield procedure. The absence of competing lactam-forming reactions, which had been a major obstacle in preparing the EDTA and DTPA systems (17), facilitated carboxymethylation of 7 and permitted synthesis of the EGTA analogue 10 in relatively high overall yield [ 24 70 based on (4-nitropheny1)pyruvic acid]. In the case of the macrocyclic system, introduction
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Preparation of Bifunctional Chelators
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