Bioconlugete Chem. 1991, 2, 242-253
242
Novel Carboranyl Amino Acids and Peptides: Reagents for Antibody Modification and Subsequent Neutron-Capture Studies Aravamuthan Varadarajan and M. Frederick Hawthorne' Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90024. Received March 8, 1991
A new a-amino acid derivative incorporating the 1,2-dicarba-closo-dodecarborane(l2) cage, namely 5-(2-methyl-1,2-dicarba-closo-dodecarborane(l2)-l-yl)-2-aminopentanoic acid (2),was synthesized by the alkylation of the benzophenone Schiff s base of glycine methyl ester with 3-(2-methyl-1,2-dicabacloso-dodecaborane(l2)-l-yl)propyliodide (8). This amino acid was employed in the synthesis of peptide derivatives such as 19-21 using solid-phase Merrifield methods. Dipeptide 19 was converted to a water-soluble ionic derivative by the pyrrolidine-mediated carborane cage degradation reaction followed by cation exchange to afford sodium salt 22. Dansylation of 22 with dansyl chloride yielded fluorescencelabeled dipeptide 23. Undecapeptide 21 was dansylated while still anchored to the Merrifield resin. Following its cleavage from the resin with hydrogen fluoride, product 25 was acetylated to block the free amino group on the lysine residue and then converted to water-soluble derivative 27. Trial conjugations of dipeptide 23 and undecapeptide 27 to T84.66, an anti-CEA antibody, were carried out by means of carboxyl activation with N-hydroxysulfosuccinimideand NJV-diisopropylcarbodiimide. Studies of the chemical syntheses of these and other peptide derivatives and the conjugation of 23 and 27 to the antibody are described.
INTRODUCTION
The possible utilization of boron-10 labeled antibodies for tumor cell destruction via the binary cytotoxic lOB(n,a)'Li reaction has been extensively investigated (I). The problem associated with this form of therapy has been the lack of a viable method for the selective delivery of the required quantity of boron-10 to the neoplasm (10-30 pg/g tumor). Since a number of studies have previously revealed the limitations associated with attaching large numbers of small boron-containing molecules to the antibody (2-4), attention has recently focused upon the covalent linkage of boron-loaded polymers to the antibody. These methods include the use of boron-labeled PO~Y-DLlysine @-IO), a carborane-linked dextran moiety (oxidized for antibody coupling) (II-I3),and boron-labeled polyL-ornithine (14). While these studies demonstrated that it is possible to attach more that 103boron atoms to each antibody molecule, such heavily boronated antibody conjugates suffered from either significantly reduced immunoreactivity or low tumor uptake. The inherent inhomogeneity of the attached boron-containing polymeric reagent in the conjugate is expected to aggravate the problem since no viable method for purification of the conjugated antibody is available. In order to mitigate these problems we have directed our attention toward Merrifield solid-phase peptide synthesis for the precision syntheses of designed oligomeric peptide molecules which contain a predetermined number of closo-C2BloH11 carborane cages capable of delivering 100-500 boron atoms per oligomer. This methodology removes the problem of oligomeric boron reagent heterogeneity. The hydrophilicity of these peptide structures may be markedly increased by conversion of their hydrophobicneutral carboranecages to anionic [nido-7,8-C2BsH11]- moieties. EXPERIMENTAL PROCEDURES
General. The lH and llB NMR spectra were recorded on a Bruker AM500 Spectrometer operating at 500.137 and 160.463 MHz, respectively, and IR spectra were obtained with a Beckman FT 1100 spectrophotometer. 1043-1802/Q1/2Q02-0242$02.50/0
Mass spectra were obtained from MS-9 and ZAB-SE mass spectrometers. Fluorescence measurements were performed on a Spex fluorimeter using 313 and 515 nm for the wavelengths of excitation and emission, respectively. Melting points were obtained with a Thomas-Hoover capillary melting point apparatus and are uncorrected. 3-(2-Methylcarboranyl)propanol(10)was synthesized from methylcarborane(9)by a previously reported method (15). Peptide syntheses were carried out with a Beckman-990C automated peptide synthesizer. Cleavage of the finished peptide from the resin with HF was accomplished by employing the hydrogen fluoride handling apparatus purchased from Peptide Institute Inc., Osaka, Japan. Dicyclohexylcarbodiimide, NJV-(dimethylamino)pyridine, dansyl chloride, trifluoroacetic acid, Merrifield chloromethyl resin (1% cross-linked, 200-400 mesh, 1mequiv chloride/g) and di-tert-butyldicarbonate were purchased from Aldrich Chemical Co. and were used without purification. Elemental analyses were performed by Galbraith Laboratories, Knoxville, TN. All solvents were dried and distilled by standard methods. Synthesis of 3-(2-Methyl-lf-dicarba-closo-dodecaboran(12)-l-y1)propylIodide (8). To a solution of 3-(2-methylcarboranyl)propanol(10;12.0 g, 56 mmol) in anhydrous pyridine (50 mL) was added p-toluenesulfonyl chloride (21.3 g, 112 mmol) at 0 "C and the solution was stirred at this temperature for 10 h. The reaction mixture was quenched by the addition of ice-cold water (600 mL) and extracted with ether (4 X 60 mL). The combined ether extracts were washed repeatedly with ice-cold solutions of dilute HCl (1 M, 2 X 60 mL) and saturated NaHC03 (2 X 60 mL) and finally with ice-cold water. Drying over anhydrous MgSO4, filtration, and concentration yielded the tosylate as a white solid. Due to its reactivity, tosylate 11, which contained small amounts of tosyl chloride and the alcohol, was used without further purification. 1H NMR (CDCla): 6 7.73 (d, 2 H, Ar-H), 7.32 (d, 2 H, Ar-H), 3.99 (t,2 H, CH2), 2.41 (8, 3 H, tosyl CH3), 2.16 (m, 2 H, CHz), 1.92 (8, 3 H, carboranyl CHa), 1.88 (m, 2 H, CHd. llB NMR (CHzC12): 6 -4.78, -6.24, -9.46, -10.12, -11.03. 0 1991 Amerlcan Chemlcal Society
Novel Carboranyi Amino A c k and Peptldes
To a solution of tosylate 11 (17.4 g, 47 mmol) in acetone (150 mL) was added sodium iodide (14.1 g, 94 mmol) and the solution refluxed under nitrogen for 20 h. After cooling to ambient temperature, the reaction mixture was filtered and the solid washed with acetone. The filtrate and washings were combined and concentrated. The residue was dissolved in ether and washed with sodium thiosulfate solution and water. Drying (anhydrous MgSO4) and concentration produced the crude iodide which was purified by column chromatography on silica gel using petroleum ether as the solvent. Yield 14.3 g (93%). Mp: 34-35 OC. 'H NMR (CDCl3): 6 3.19 (t,2 H, CHz), 2.33 (m, 2 H, CHz), 2.06 (m, 2 H, CHz), 2.05 (8, 3 H, CH3). 13C NMR (CDCl3): 64.51,22.18,32.59,35.87,75.01,76.67. "B NMR (EhO): 6 -4.14, -5.61, -9.21, -9.72, -10.43, -10.75. IR (Nujol): 2587,1239,1172and 729cm-l. FAB MS (m/e) calcd for C611B10HlgI: 328. Observed: cluster of peaks centered near 325. Anal. Calcd for C&oHgI: C, 22.09; B, 33.14; H, 5.87; I, 38.90. Found: C, 21.85; B, 32.80; H, 5.62; I, 38.42. Akylation of Methyl N-(Dipheny1methylidene)glycinate (6) with 3-(2-Methyl-l,2-dicarba-clos~dodecaboran( 12)-l-yl)propyl Iodide (8). (i) Homogeneous Alkylation in Solution. To a solution of diisopropylamine (5.6 mL, 39.6 mmol) in a mixture of tetrahydrofuran (110 mL) and hexamethylphosphoramide (25 mL) at -78 OC was added n-butyllithium (2.4 M in hexane, 16.5 mL, 39.6 mmol). The solution was stirred a t this temperature for 1 h, after which a solution of Schiff's base 6 (10.0 g, 39.6 mmol) in THF (30 mL) was added. The solution passed througha series of color changes from blood red to brownish yellow. After stirring for 1h a t -78 "C, a solution of iodide 8 (12.9 g, 39.6 mmol) in THF (20 mL) was added. The color of the solution changed from brownish yellow to orange yellow. After stirring at this temperature for 2 h, the reaction mixture was warmed to ambient temperature and stirred for a further 20 h and the solution then quenched with aqueous ammonium chloride. The organic layer was separated, washed with water, dried, and concentrated. Crude imine 12, containing some solvent (18.7 g, 41.3 mmol), was obtained as a viscous yellow material. (Due to its hydrolytic instability, it was not purified and was directly converted to amino ester 13).'H NMR (CDC13): 6 7.82-7.17 (m, 10 H, Ar-H), 4.07 (dd, 1 H,CH), 3.75 (m, 2 H,CHz), 3.72, (s,3 H, COOCH3), 2.12 (t, 2 H, CHz), 1.93 (8, 3 H, CH3), 1.85 (m, 2 H, CH2). llB NMR (CH2C12+ EtzO): 6-439,416,-9.66,-10.15,-11.08. 13CNMR(CDCl3): 6 172.33,137.68,132.40,130.62,130.06, 128.90,128.83,128.71,128.43,128.29,128.19,127.78,67.98, 64.72, 52.22, 36.86, 35.11, 33.00, 25.19, 23.10. ( i i ) Alkylation by Phase-Transfer Catalysis. To a solution of Schiff s base 6 (96 mg, 0.38 mmol) and iodide 8 (124 mg, 0.38 mmol) in acetonitrile (7 mL) were added K2CO3 (315 mg, 2.28 mmol) and tetra-n-butylammonium bromide (36.8 mg, 0.114 mmol), and the solution was refluxed under nitrogen for 3 days. The reaction mixture was cooled to ambient temperature and filtered to remove the solid which was washed with CHzClz. The filtrate and the washings were combined and concentrated. Crude imine 12 (126 mg, 0.28 mmol, 74%) was obtained as a yellow oil. Partial Hydrolysis of Alkylated Schiff's Base 12. The crude alkylated Schiff s base (13.8 g, 30.5 mmol) was dissolved in ether (40 mL) and treated with dilute HCl(1 M, 40 mL). Upon stirring the mixture at ambient temperature, a white solid began to separate within a short time. The solution was stirred for 9 hand filtered to collect amino ester 13, which was purified by recrystallization
Bbconjlugete (.%em., Vol. 2, No. 4, 1991 249
from methanol (7.50 g, 23.2 mmol, 76%). Mp: 224-225 OC. 'H NMR (CD30D): 6 4.10 (t, 1H, a-CH), 3.85 ( ~ , 3 H, COOCH3),2.36 (t,2 H, 6-CHz), 2.09 ( ~ , H, 3 COOCHa), 1.98 (m, 1 H, fl-CHz), 1.93 (m, 1H, @-CHz),1.78 (m, 1H, T-CH~), 1.67 (m, 1H,yCH2). '3c NMR (CH30D): 6 170.33 (ester CO), 79.02 (carboranyl C), 76.64 (carboranyl C), 53.52 (ester CH3), 53.23 (CH), 35.09 ( ~ C H Z 30.49, ), (aCHz), 26.08 (fl-CHz),23.30 (CH3). "B NMR (CH30H): 6 -4.53, -5.93, -8.97, -9.70, -10.73. IR (Nujol): 3387 (br), 2604,1757,1529,1516,1268 cm-l. FAB MS (m/e) calcd for Cg11BloH~N02: 290. Observed: cluster of peaks centered around 288. Anal. Calcd for CsBloHdOzCk C, 33.38; H, 8.09; N, 4.32; B, 33.38: C1 10.95. Found C, 33.08; H, 8.05; N, 4.15; B, 32.16; C1, 10.49. Hydrolysis of Amino Ester 13. Carboranyl amino ester derivative 13 (2.0 g, 6.2 mmol) was suspended in 6 M HC1 (50 mL) and the mixture was refluxed for 24 h. Cooling and filtration of the mixture yielded amino acid derivative 14 as a colorless solid which was recrystallized from methanol (1.80 g, 5.8 mmol,95 % 1. Mp: 260-262 OC. 'H NMR (CDsOD): 6 3.98 (t, 1 H, a-CH), 2.36 (t, 2 H, CH2), 2.08 (s, 3 H, CH3), 1.96 (m, 1 H, 8-CH), 1.89 (m, 1 H, r-CH), 1.70 (m, 1 H, yCH). llB NMR (CHsOH): 6 -4.51, -5.92, -8.97, -9.70, -10.73. IR (Nujol): 3411 (br), 2597,1740, 1602, 1514, 1209, 730 cm-l. FAB MS ( m / e ) calcd for CsllBloHuNOz: 276. Observed: cluster of peaks centered around 274. Anal. Calcd for CeBloHz4NO2Cl: C, 31.01; H, 7.81; N, 4.52; B, 34.89; C1, 11.44. Found C, 30.80; H, 8.11; N, 4.29; B, 35.15; C1, 10.82. Synthesis of Free Base 2. Amino acid hydrochloride 14 (1.8g, 5.8 mmol) was dissolved in methanol and treated with propylene oxide (11mL). After stirring a t ambient temperature overnight, crude product 2 was obtained by concentration and was purified by recrystallization from methanol (1.4 g, 5.1 mmol, 88%). Mp: 255-256 OC. 'H NMR (CDsOD): 6 3.54 (t, 1H, a-CH), 2.32 (t,2 H, 6-CHz), 2.07 (s, 3 H, CH3), 1.84 (m, 2 H, fl-CHz), 1.74 (m, 2 H, 7-CH2). "B NMR (CH30H): 6-4.22,-5.59,-8.63,-9.38, -10.38; IR (Nujol): 3492-3216 (br), 2579,1635,1500,1023, 730 cm-l. FAB MS (m/e) calcd for CS~~BIOHZ~NOZ: 275. Observed: cluster of peaks centered around 275. Anal. Calcd for C&H23N02: C, 35.15; H, 8.48; N, 5.12; B, 39.54. Found: C, 35.32; H, 8.47; N, 4.75; B, 37.20. Preparation of 17, the t-BOC Derivative of Amino Acid 2. Amino acid 2 (1.6 g, 5.8 mmol) in absolute alcohol (30mL) was treated with di-tert-butyldicarbonate(2 mL, 9 mmol) and triethylamine (0.8mL, 6 mmol). The mixture was heated to reflux under nitrogen for 12 h. The solvent was removed and the residue was cooled in ice prior to treatment with CHzClz and HCl(1 M, 25 mL). After 30 min the aqueous layer was removed and the organic layer was washed with water. The solution was dried and concentrated. t-BOC-protected amino acid 17 was obtained as a fluffy white solid and was recrystallized from CHzCl2/petroleum ether (2.0g, 5.3 mmol, 90%). Mp: 84 OC (dec). 1H NMR (CDC13): 6 5.02 (d, 1H, NH), 4.33 (m, 1H, CH), 2.27 (m, 1H, CH2), 2.18 (m, 1H, CHn), 2.01 (a, 3 H, CHs), 1.90 (m, 1 H, CHz), 1.68 (m, 3 H, CHz), 1.46 (8, 9 H, t-BOC-CH3). "B NMR (CDC13): 6 -5.23, -6.60, -9.82, -10.48, -11.39. IR (KBr): 3432-2874 (br), 2596, 1723-1694 (br), 1509,1477,1455,1405,1396,1369,1284, 1250,1156,1060,1025,728 cm-l. MS: no parent ion was observed, but a cluster of peaks centered around m / e 273.31 corresponding to M+- (t-BOC) was observed. Anal. Calcd for C13BloH3lNO4: C, 41.81; H, 8.37; N, 3.75; B, 28.94. Found: C, 41.34; H, 8.83; N, 3.54; B, 28.67. Conversion of Closo Amino Acid 2 to Its Anionic Nido Derivative 15. A mixture of amino acid 2 (0.81 g,
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Bkconlugete Chem., Vol. 2, No. 4, 1991
Varadarajan and Hawthorne
(16). The basic procedure involved the deprotection of 3.0 mmol) and sodium hydroxide (40 g, 8.9 mmol) in the t-BOC group with TFA (25% in CHzC12,0.5 h), washing absolute alcohol (30mL) was refluxed under nitrogen for with triethylamine (10% in CHzClz), coupling with the 24 h. The excess base was neutralized by gassing CO2 next t-BOC amino acid mediated by DCC and DMAP, through the reaction mixture and separation of Na2C03 washing with solvent (CHzClz), and recoupling, if necesby filtration. The solvent was removed under vacuum sary. llB NMR spectra of the resin-bound peptides were and the sodium salt of nido-carboranyl amino acid 15 was obtained by suspending the loaded resin (prior to HF obtained as a sticky yellow paste. The solid was dried cleavage) in a minimal amount of CH2Clz and employing under vacuum (0.82 g, 2.8 mmol). The material was the spectral acquisition parameters normally used for dissolved in water, acidified with 1 M HC1, filtered, and solution spectra. treated with an excess of a solution of tetrabutylammonium bromide in water. The precipitation was not The completed peptides were cleaved from the solid complete owing to the partial solubility of the tetrabusupport by anhydrous HF using standard procedures (16). tylammonium salt in water. The yield was 0.63 g (1.2 The resin was treated with anisole (1 mL/g of resin) and mmol, 40%). 1H NMR disodium salt (CD30D): 6 3.82 HF was condensed into the reaction vessel at -78 OC (ca. (br, 1 H, a-CH); 1.64 (m, 3 H, CHd, 1.56 (m, 3 H, CHd, 5 mL/g of resin). The mixture was stirred at 0 OC for 1.38 (9, 3 H, CH3). 1lB NMR (CH3CN): 6 -9.20,-10.61, 30-60 min and HF was removed under vacuum at this -18.11,-34.63,-37.09. IR (Nujol): 3358 (br), 3287 (br), temperature. Anisole was removed under high vacuum 2510,1637,1598,1873, 736 cm-l. IR (KBr): 3483-3103 and the peptide was extracted with TFA (3X 10mL). The (br), 3054,3032,2608,1727,1676,1624,1557,1519,1458, TFA extracts were concentrated and the peptide was 1206, 1180, 1120, 1084-1034 (br), 730 cm-l. FAB MS precipitated by the addition of dry ether. The solid was separated by filtration, washed with ether, and dried under (negative ion) calcd for CsllBgH~02N:264. Observed: cluster of peaks centered around m / e 263. vacuum. Preparation of 18, the PBOC Derivative of nide Synthesis of (closeCB)2 (19). Starting with 1.06 g of Carboranyl Amino Acid 15. Crude sodium salt 15 the Merrifield resin loaded with the t-BOC-protectedclosoobtained (0.23g, 0.78 mmol) from the cage degradation carboranyl amino acid (1.06g, ca. 1 mequiv/g), the product dipeptide was obtained as an off-white solid (130mg, 0.25 reaction was dissolved in absolute alcohol (7 mL) and mmol) after purification by flash column chromatogratreated withdi-tert-butyldicarbonate(0.35mL, 1.6 "01). phy through a reverse-phase CIS column employing an After heating at 80 "C for 12h, under nitrogen, the solvent was removed under vacuum. The residue was dissolved acetonitrile/water solvent gradient. 'H NMR (CDsOD): 6 7.57 (br, NH), 4.37 (m, CH), 3.92 (m, CHI, 2.32 (m, CHz), in water and the filtered solution cooled in ice and treated 2.06 (s, CH3), 1.89 (m, CH2), 1.67 (m, CH2). 13C NMR with dilute HC1 (1 M, 2 mL). Addition of tetrameth(CD30D): 6 79.67,79.11,76.97,76.88,55.58,54.17,53.87, ylammonium chlorideyielded the tetramethylammonium 35.85,35.74,35.65,35.57,34.18,32.61,32.27,32.06,31.97, salt of the nido t-BOC-amino acid 18 (140mg, 0.32mmol, 30.10,27.33,27.25, 26.31,25.67,23.52, 23.47. 1lB NMR 41%). 1H NMR (CD30D): 6 4.02 (br, 1 H, a-CH), 3.2 (s, (CH30H): -4.24, -5.59,-8.65,-9.36, -10.38. IR (Nujol): 12 H, N(CH&), 1.66 (br, H, CHd, 1.53 (br, H, CHd, 1.44 3360 (br), 2576,1675 (br), 1065 (br), 728 cm-l. FAB MS ( ~ ,H, 9 t-BOC CH3), 1.38 ( ~ ,H, 3 CH3). "B NMR (CH3( m / e )calcd for ClellB~H~N203: 532. Observed: cluster OH): 6 -8.64,-10.16,-17.59,-34.10,-36.56. IR (Nujol): of peaks centered around 530. 3356 (br), 3217 (br), 2522, 1709,1694,1653,1635,1627, 1311,1234,1167,725cm-l; FAB MS (negative ion, m / e ) Synthesis of (closeCB)a.Lys (20). Chloromethyl calcd for C13l1BgH3104N: 364. Observed: cluster of peaks resin (Log)loaded with Na-t-BOC-N,-2-chloroCBZ-lysine centered around 362. The triphenylmethylphosphonium (0.8 mequiv/g) was employed in the peptide synthesis salt was similarly prepared. Anal. Calcd for CszBgHrgwhich involved the successive coupling of five residues of N04P: C, 60.06;H, 7.72;N, 2.19;B, 15.20;P, 4.84. Found the protected carboranylamino acid 17. After HF cleavage C, 59.94;H, 7.75;N, 2.12;B, 13.23;P, 4.91. the crude peptide, which was obtained as an off-white solid (1.04g), was subjected to flash chromatography on Stepwise Synthesis of Carboranyl Peptides. The reverse-phase ((218) silica gel using an acetonitrile/water protected amino acids Na-t-BOC-N,-2-chloro-CBZ-lysine, solvent gradient (0.91g). 1H NMR (CD3OD): 6 7.03 (br, t-BOC-glycine, t-BOC-y-aminobutyric acid, and t-BOCCOOH), 4.37 (br, CH), 2.30 (br, CHz), 2.07 (8, CHa), 2.06 carboranyl amino acid 17were loaded as their cesium salts (8, CHs), 1.88 (br, CHz), 1.69(br m, CHz). llB NMR (CH3in DMF on the chloromethyl resin by following the OH): 6 -6.05 (br), -10.88 (br). IR (KBr): 3233 (br), 3057 reported procedure (16). The loading efficiency was (br), 2944,2604,1706,1640,1560,1548,1510,1460,1440, monitored by the increase in weight of the resin, as well 1247,1202,1136,1030,802,746,725cm-1. FAB MS ( m / e ) as by the amount of amino acid recovered after HF cleavage calcd for C&Bdl1807N7: 1432. Observed: cluster of of an aliquot of the resin. The loading efficiency was 45peaks centered around 1424. 50% in the case of 17 and greater than 90% in the case of other amino acids. The coupling reactions were carried Synthesis of (cZoso-CB)lo*Lys(21). Chloromethyl out in the Beckman solid-phase automated peptide resin (1.0g) loaded withN,-t-BOC-N,-2-chloroCBZ-lysine synthesizer employing standard coupling programs proas the first amino acid (0.8 mequiv/g) was employed in vided by the manufacturer. The amino acids and DCC the peptide synthesis and the t-BOC protected carborawere used in 2.5-fold excess, and 0.5 equiv of DMAP was nyl amino acid 17 was again employed as the repeating used as an acylation catalyst. The progress of coupling reagent. After coupling 10 monomer residues, the pepwas monitored by the ninhydrin test applied to the resin. tide loaded resin (2.66g) was recovered. A portion of the In the case of the carboranyl amino acids, coupling was resin (1.0g) was subjected to treatment with HF and unallowed to proceed for 2 days and followed by recoupling decapeptide 21 was isolated as a colorless solid (0.72g) for a further period of 2 days, when the initial coupling after purification by flash chromatography through a reaction did not proceed to completion. Except in the reverse-phase (CIS)silica gel column (acetonitrile/water solvent gradient). lH NMR (DMSO-de): 6 8.11 (br, NH), case of the ionic amino acid derivatives, dichloromethane 7.68 (br, NH), 4.36 (br, a-CH), 2.72 (br, Lys-CHz), 2.06 (8, was used as the solvent. The synthetic methods followed CH3), 1.64 (br, CH2), 1.52 (br, CHz). llB NMR (CD3OD were taken from those reported for normal amino acids
Novel Carboranyl Amlno Aclda and Peptides
+
CDsCN, CDCls): 6-6.21,-10.87. IR (Nujol): 3322,3273, 2586,1661,1547,1534,1200,1179,1026,726 cm-l. FAB MS ( m / e )calcd for CssllBl&zuOlzNlz: 2719. Observed: cluster of peaks centered around m / e 2698. Preparationof (mideCB)z (22) by the PyrrolidineMediated Cage Degradation of ( c h e C B ) z (19). Dipeptide 19 (130 mg, 0.24 mmol) was treated with pyrrolidine (1mL, 12 mmol) under nitrogen. After stirring a t ambient temperature for 1h, the residue was treated with CHzClz (1mL) and stirred for a further period of 0.5 h. The reaction mixture was concentrated under vacuum and the residue was washed repeatedly with 1M HCl(5 X 5 mL) and then with ether. The product was subjected to cation exchange on Bio-Rad AG-X2 (100-200 mesh) cation-exchange resin (Na+ form) preequilibrated with water/acetonitrile/methanol(541) solvent mixture which was used as the eluent. After removal of the solvents, the product was dried under vacuum (127 mg, 0.23 mmol, 96%). lH NMR (CD3OD): 6 4.12 (m, a-CH), 1.83 (m, CHz), 1.73 (m, CH~),1.40(s,CH3), 1.39 (5, CH3). llB NMR (HzO + CH30H): 6 -10.46, -11.83, -18.45, -20.07, -35.06, -38.03. IR (pyrrolidinium salt,thin film): 3411-3083 (br), 2955,2524,1673,1622,1456,1200,1077, 1025,911,843, 735 cm-l. FAB MS (negative ion, m / e ) calcd for C#B18H~Nz03: 511. Observed: cluster of peaks centered around 511. Dansylation of (nidcFCB)z (22) with Dansyl Chloride. A mixture of (nido-CB)~(22; 127 mg, 0.23 mmol) and dansyl chloride (72 mg, 0.27 mmol) in a mixture of CH3CN (3 mL) and THF (1mL) was stirred at ambient temperature under nitrogen for 66 h. The solvent mixture was removed under vacuum and the solid was washed with CHzC12. The crude product (73 mg) was dried under vacuum. A portion of the crude product (41 mg) was purified by passage through a cation-exchange resin (BioRad AG-X2 resin, 100-200 mesh, Na+ form, 5:41, HzO/ CH3CN/CH30H solvent system), decolorization with charcoal and bypassage through a reverse-phase (CU) flash chromatography column (1:lCHsCN/HzO solvent system) twice. Product 23 was obtained as a yellow solid (31.3 mg). lH NMR (CDsOD + CD&N + DzO): 6 8.82 (t,ArH), 8.35 (d, Ar-H), 8.25 (d, Ar-H), 7.70 (m, Ar-H), 4.35 (br, a-CH), 3.23 (s, N(CH&), 1.68 (m, CHz), 1.60 (m, CH2), 1.37 (8, CH3). IIB NMR (CD3CN + DzO): 6 -9.47, -12.01, -17.96, -34.74, -37.96. IR (Nujol): 3324-3181 (br), 2526, 1674,1532,1189cm-l. Rf(C18silicage1,l:l CH&N/HzO): 0.84. Synthesis of Dansyl-GABA-(closeCB)s-GABA (24). Merrifield resin loaded with GABA as the initial amino acid residue (1.0 g, 1.0 mequiv/g) was employed for the coupling of five residues of 17 and a final t-BOCsGABA residue. The t-BOC-protecting group was cleaved in the synthesizer using the standard program. The resin (2.46 g) was treated with a solution of dansyl chloride (0.67 g, 2.5 mmol) in CHzClz (10 mL) and triethylamine (0.35 mL, 2.5 mmol). After stirring for 48 h a t ambient temperature, the solution was removed by filtration and the resin washed with dichloromethane. After testing for completion of dansylation with ninhydrin, peptide 24 was cleaved from the resin with HF. Crude product 24 (1.13 g) was purified by column chromatography on reverse-phasesilica gel (acetonitrile/water solvent gradient) to yield 0.90 g (0.63 mmol) of dansyl~GABA~(closo-CB)~~G~A. lH NMR (CDCls + CDsOD): 6 9.00 (d, Ar-H), 8.54 (br, Ar-H), 8.37 (d, Ar-H), 8.22 (d, Ar-H), 8.16 (br, Ar-H), 8.06 (d, Ar-H), 7.81 (d, Ar-H), 7.62 (d, Ar-H), 4.16 (br, a-CH), 3.20 (s, N(CH3)2),2.84 (br, CHz), 2.24 (br, CHz), 2.14 (br, CHZ), 1.91 (8, CHa), 1.70 (br, CHz), 1.50 (br, CHz). llB NMR
B&nJt&wte
Chem,, Vol. 2, No. 4, 1SSl 245
(CHzC12 + CHsOH): 6-6.31, -11.09. IR (KBr): 3413 (br), 3309 (br), 2445,2577,1629,1529,1457,1321,1206,1179, 1076, 1024, 789, 744 cm-l. FAB MS ( m / e ) calcd for C & B M H ~ ~ ~ O ~ O1706. N ~ S :Observed cluster of peaks centered around 1710. Dansylation of Resin-Bound (closeCB)lo-Lys, Cleavage from Solid Support, Acetylation, and Degradation. A sample of the resin with the undecapeptide (closo-CB)lwLys(0.48 g, 0.38 mequiv) was subjected to the deprotection of the terminal amino group in the synthesizer employing the standard program. The resin was treated with a solution of dansyl chloride (0.26 g, 0.96 mmol) in CHzClz (12 mL) and triethylamine (53 pL, 0.38 mmol). After stirring for 46 h, the solution was removed by filtration and the resin was washed repeatedly with dichloromethane, methanol, acetonitrile, and dichloromethane. The resin was tested with ninhydrin for completion of dansylation and subjected to HF treatment as previously described. The cleaved, dansylated peptide was extracted with methanol and concentration of the solution yielded crude product 25 as a yellow solid (0.29 g, 0.10 mmol). lH NMR (CD30D + CDzClz): 6 8.48 (m, Ar-H), 8.27 (m, Ar-H), 8.08 (m, Ar-H), 7.44 (m, Ar-H), 4.11 (br, a-CH), 3.18 (s,N(CH&), 2.80 (br,CH2),2.16 (br, CHz), 2.03 (8, CHs), 1.78 (br, CHz), 1.37 (br, CH2). llB NMR (CD3CN + CDZClz): 6 -6.74,-11.37. IR (KBr): 3310 (br), 2944,2607,1661,1548,1458,1202,1181,1141,1023,796, 728 cm-l. FAB MS ( m / e )calcd for Cs811BlmH~~OlrN13S: 2949. Observed: cluster of peaks centered around 2949. The crude dansylated peptide obtained from the resin by HF cleavage (288 mg, 0.10 mmol) was treated with acetic anhydride (2 mL, 20 mmol) and triethylamine (40 pL, 0.29 mmol). The mixture was stirred a t ambient temperature under nitrogen for 12h, when the solution turned brown. The solution was concentrated in uacuo and was washed with petroleum ether. The brown solid material was dried under vacuum (0.29 g, 0.1 mmol). This crude product (26) was used in the subsequent reaction without purification. lH NMR (CDZCW: 6 8.50 (d, Ar-H), 8.38 (d, Ar-H), 8.12 (d, Ar-H), 8.05 (d, Ar-H), 7.48 (m, Ar-H), 7.37 (m, Ar-H), 7.18 (m, Ar-H), 7.15 (m, Ar-H), 6.38 (m, Ar-H), 3.78, (br, a-CH), 3.04 (q,N(CzH&H), 2.87 (e, N(CH&), 2.28 (s, CH3CO), 2.19 (br, CHz), 2.01 (br, CH3), 1.96 (8, CH3), 1.95 (br, CHz), 1.65 (br, CH2), 1.27 (t, N(C2Hs13H). "B NMR (CDzClz): 6 -6.95, -11.14. Crude acetylated peptide 26 obtained from above (0.29 g, 0.090 mmol) was treated with pyrrolidine (2 mL) and the mixture was stirred under nitrogen for 1 h a t 25 "C. The solvent was removed under vacuum and the product was washed twice with 1 M HC1, once with water, and once with petroleum ether. The brownish solid was dried under vacuum (0.18 g). The pyrrolidinium cation was exchanged for sodium with Bio-Rad AG-X2 cationexchangeresin (mesh 100-200, Na+ form) and acetonitrile/ water mixture (1:l)as the solvent. Product 27 was further purified by flash chromatography on reverse-phase (CIS) silica gel column (1:l acetonitrile/water) and decolorized with charcoal. The product was obtained as a pale yellow solid (95 mg, 0.030 mmol). lH NMR (CD3OD + CDsCN): 6 8.53 (d, Ar-H), 8.34 (d, Ar-H), 8.11 (d, Ar-H), 7.66 (d, Ar-H), 7.49 (m, Ar-H), 7.27 (d, Ar-H), 5.44 (br, Ar-H), 4.08 (br, a-CH), 2.85 (s,N(CH&), 1.68 (br, CHz), 1.55 (br, CHz), 1.38 (8, CH3). llB NMR (Hz0): 6 -8.82, -17.00, -33.23, -35.72. IR (Nujol): 3219 (br), 2512, 1658, 1192 cm-l. Rf (Cl8 silica gel, 1:l CH&N/HzO): 0.63. Activation of Dansyl*(nideCB)z(23) and Conjugation to T84.66. A solution of 23 (6.36 mM, 80 pL) in phosphate buffer (0.1 M, pH 4.35) was treated with
246
"te
Varadarajan and Hawthorne
Chem., Vol. 2, No. 4, 1991
Table I. Conjugation of Daneyl-(nido-CB)r (23) to T84.66 Antibody conjugation of 1 mg of T84.86 in 1.1 mL of buffer, 60 min, 25 O C expt no. 1 2
peptide activation reaction buffer (pH) time, min 0.1 M PO, (4.4) 60
buffer (pH) 0.05 M PO, (7.4)
0.1 M PO4 (4.4)
0.10 M COs/HC03 (9.3)
60
[peptide]/ [T84.66Ia 10.6 10.5 (control) 10.6 10.5 (control)
bound peptide/T84.66 molecule 8.5 (6.1)b 2.3 9.4 (7.1)b 1.3
% protein recovery 77.6 86.2 78.9 92.9
%
HMW' 7.3 3.5 6.1 5.0
a High molecular weight material (HMW) comprised leea than 4% of T84.66 protein. b Ratio observed after purification of conjugate by gel filtration HPLC. High molecular weight species determined by HPLC analysis of the conjugate.
solutions ofN-hydroxysuccinimide(55mM, 18pL)in water and NJV-diisopropylcarbodiimide(65 mM, 8 pL) in dimethylformamide. The mixture was incubated a t ambient temperature for 1h. An aliquot of the active ester solution (40 pL) was added to the T84.66 antibody solution (0.12 mM, 50 pL) diluted with phosphate buffer (0.05 M, 1mL, pH 7.43). Another solution of the antibody (0.12 mM, 50 pL) diluted with carbonate/bicarbonate buffer (0.1 M, 1 mL, pH 9.30) was subjected to identical treatment. Two control experiments were run by treating the same amount of the antibody in the respective buffers with unactivated peptide 23 (6.3 mM, 10 pL) in phosphate buffer (0.1 M, pH 4.35). The molar ratio of the activated or unactivated peptide to the antibody was 1 0 5 in all cases. After incubating for 1h at ambient temperature, the controls and the conjugates were subjected to ultrafiltration using Centricon-30 concentrators. The concentrates were treated with the respective conjugation buffers (2 X 2 mL) and water (1X 2 mL) and concentrated after each dilution. The respective volumes of the final concentrates were measured and aliquota taken for Bio-Rad protein assay, fluorescence measurements, and gel filtration HPLC analysis. The fluorescence intensities of a series of aqueous solutions of peptide 23 of different concentrations (in the range 1.27-12.7 mM) were measured and a linear plot of the fluorescence intensity vs concentration was obtained. A known volume (- 5-10 pL) of the concentrate was diluted to 5 mL and the fluorescence intensity was measured. Neglecting the contribution of the antibody protein to the fluorescence and/or the quenching of fluorescence by the peptide, if any, the concentration of the peptide in the antibody concentrate was determined from the above data. The ratio of the peptide concentration measured from the fluorescence intensity and the protein concentration measured from the Bio-Rad protein assay revealed the average number of peptide units attached to each of the antibody molecules. HPLC analysis of the controls and the conjugates was carried out on a Du Pont Zorbax GF250 column using 0.1 M Tris-HC1containing 0.15 M NaCl and 0.005% NaNa) as solvent with a flow rate of 1 mL/ min and monitored by UV detection at 280 nm. The results are reported in Table I. Activation of Daneyl*(nido-CB)lo.Lys.Ac(27) and Conjugation to T84.66. To a solution of 27 (6.5 mM, 45 pL) in phosphate buffer (0.1 M, pH 4.35) were added solutions of N-hydroxysulfosuccinimide(46 mM, 13 pL) in water and N,N-diisopropylcarbodiimide (65 pM, 4.5 pL) in dimethylformamide, and the mixture was incubated at ambient temperature for 45 min. Different amounts of the active ester solution (6.5,13, and 19.5 pL) were added to T84.66 antibody solutions (0.12 mM, 50 pL) diluted with phosphate buffer (0.05 My1mL, pH 7.43). A control experiment was carried out by treating unactivated peptide 27 (6.46 mM, 5 pL) with the same amount of the antibody in phosphate buffer under identical conditions.
The molar ratios of the activated peptide to the antibody in the conjugates were 51,10:1, and 151,and in the case of the control it was 5.351. After 1h, the conjugates and the control were subjected to ultrafiltration using Centricon-30 concentrators. The concentrates and the conjugates were treated with phosphate buffer used for conjugation (2 X 2 mL) and water (2 X 2 mL) and were concentrated after each dilution. Fluorescence measurementa, Bio-Rad protein assay, and gel filtration HPLC analysis were carried out on the concentrates as described above. Table I1 lists the results obtained from the conjugation studies performed under a variety of experimental conditions. RESULTS AND DISCUSSION
Synthesis of Amino Acids. The synthesis of the homogeneous macromolecular peptide derivatives described here required the availability of significant quantities of amino acids containing the carborane cage. Although such an amino acid, carboranylalanine (l),had been previously synthesized by Leuckart and co-workers (17-20) and employed in the preparation of short-chain peptides such as bradykinin, etc., it was desirable to prepare the carboranyl a-amino acid to be used here in higher yields than those available for 1. Instead of attempting the unattractive carborane-forming reaction of acetylenic amino acid derivatives with decaborane, we initially chose to attach the carborane cage to a suitable a-amino acid derivative. The nucleophilic closo cageopening reactions of closo-1,8-dicarbaundecaborane(ll) (21, 22) with derivatives such as N,-t-BOC-lysine and anionic derivatives of N-t-BOC-tyrosine and N-t-BOCserine did not proceed satisfactorily, and the products were found to be unstable. Our second approach, which attempted to attach the carborane cage toN,-t-BOC-lysine and pentalysine employingN-succinimidyl esters of several carborane carboxylic acids, was also unsuccessful due to the instability of the active esters and solvent incompatibility problems (23).To this end, we chose to synthesize a different amino acid, namely, 5-(2-methyl-l,2-dicarbadodecaborane(l2)-l-yl)-a-aminopentanoic acid (2; Scheme
I). The alkylation of glycine ester Schiff s base is a widely used method for a-amino acid (24,25) synthesis. While it is conceivable to use an alkyl halide containing the carborane as a substituent, the simplest halide, namely (bromomethy1)carborane (31, is unreactive to nucleophilic substitution (26). The other halides such as the ethyl and propyl iodide derivatives 4 and 5 afforded only the starting materials upon attempted alkylation of the carbanion derived from methyl N-(diphenylmethy1idene)glycinate (6). The outcome of the reaction did not change when leaving groups such as chlorides, bromides, and tosylaks were employed and suggests that the hydrogen on the carboranyl carbon atom is sufficiently acidic to protonate the Schiff s base anion. While the incorporation of a me-
Bloconlclgete Chem., Vol. 2, No. 4, lQQl
Novel Carbaranyl Amino Aclds and Peptides
247
Table 11. Conjugation of Dansyl.(n~dPCB)lo.Lys.Ac(27) to T84.66 Antibody conjugation of 1 mg of T84.66 in 1.05 mL of buffer, 60 min, 25 OC peptide activation bound % expt reaction peptide/T84.66 protein % no. buffer (pH) time, min buffer (pH) [peptide]/ [T84.66Ia molecule recovery HMWb 37.9 12.4 1 H20 (7.0) 30 0.1 M COs/HCOs (9.3) 4.8 2.2 30.8 17.3 9.7 2.7 1.4 10.6 18.3 8.0 15.3 22.0 17.6 (control) 6.8 56.8 17.0 2 0.1 M Po4 (4.4) 60 0.1 M COs/HCOs (9.3) 5.2 5.5 29.9 26.6 10.4 9.8 4.1 15.6 15.3 0.0 9.6 (control) 6.1 64.8 20.0 2.9 59.9 14.6 3 0.1 M Po4 (4.4) 40 0.1 M HEPES (8.2) 5.0 4.2 10.0 46.6 21.8 6.9 15.0 38.4 21.0 4.8 (control) 37.6 2.3 12.3 2.8 66.3 16.2 4 0.1 M Po4 (4.4) 30 0.1 M HCOs (8.5) 5.3 4.4 10.6 54.0 18.4 9.1 15.9 30.0 20.9 2.2 4.8 (control) 55.3 12.3 5.9 16.2 5 0.1 M PO4 (4.4) 45 0.05 M Po4 (7.4) 5.0 55.0 10.0 8.5 23.8 35.8 15.4 15.0 15.4 14.0 4.1 14.9 5.4 (control) 78.9 a High molecular weight material (HMW) comprised less than 4% of T84.66 protein. * High molecular weight species determined by HPLC analysis of the conjugah.
Scheme I HC-MH+N-COOH
H s M - C(CHz)&H--COOH
NH, I
‘%io
I NH,
%LHl0
CbsO-CB
1
2 HC--C-(CH2)2I \&IO
a
9
z NOW Throughout HC-CH this text
‘i:, H,
6
B 0 BH C.
thy1 substituent on the carborane carbon would be expected to alleviate this problem, the requirement remained that the leaving group be located on the y-carbon of the alkyl chain for this substitution to occur. This is undoubtedly due to the unique stereoelectronic characteristics of the carborane cage. Thus, 2-(2-methyl-1,2dicarbadodecaborane(12)-l-yl)ethyliodide (7)seemingly yielded a product mixture which contained the abnormal N-alkylated product (27),but ita homologue 3-(2-methyl1,2-dicarbadodecaborane(l2)-l-yl)propyliodide (a), formed the desired alkylate. The starting propyl iodide derivative 8 was prepared from methyl carborane (9, Scheme 11)and the Schiff s base alkylation proceeded smoothly to give monoalkylated product 12 (Scheme 111). The alkylation could also be carried out under phase-transfer conditions (281, but it proceeded at a considerably slower rate in this medium. The alkylated Schiffs base was partially hydrolyzed to amino ester derivative 13 with dilute HC1. Complete hydrolysis of 13 yielded 14 from which free base 2 was obtained upon treatment with propylene oxide. No attempt was made to resolve the enantiomers of this chiral a-amino acid. In order to prepare the water-soluble nido anion derived from this amino acid, we have subjected closo derivative 2 to base degradation in ethanolic medium. Sodium salt
15 was isolated as tetra-n-butylammonium salt 16a or triphenylmethylphosphonium salt 16b,since the tetramethylammonium salt proved to be water soluble. It is to be noted that amino ester 13 could be directly converted to the nido derivative 15 without the isolation of closo amino acid 2 (Scheme IV). (Note: Throughout this text, the neutral amino acid residue 2 and the anionic amino acid residue 15 are referred to as closo-CB and nido-CB, respectively, for convenience.) Preparation of t-BOC Derivatives. Amino acid 2 is exceptionally hydrophobic and it was for this reason that the preparation of t-BOC derivative 17by the conventional method in aqueous medium, did not proceed in high yield. However, this problem was circumvented by carrying out the reaction in ethanol solution (Scheme V). A similar method which employs either DMF or methanol as the solvent has recently been reported in the literature (29). t-BOC derivative 18 of the nido amino acid derivative 15 was similarly prepared via ita sodium salt (SchemeV). All of the simple amino acid derivatives mentioned here were characterized by both spectroscopic methods and elemental analyses. As is well-known in the case of boron compounds which contain nitrogen, boron analyses are frequently difficult and reveal less than the theoretical value for boron. Reactions of t-BOC Amino Acids with Merrifield’s Chloromethyl Resin. t-BOC derivatives 17 and 18 were converted to their cesium salts for loading on the Merrifield resin. Although closo derivative 17was loaded with reasonable efficiency (40-50%), loading of the ionic derivative was poor. Initially, for the synthesis of simple dipeptide derivatives, 17 was directly loaded on the resin. However, for the synthesis of longer peptide sequences, common amino acids such as lysine, glycine, or y-aminobutyric acid were employed as the initial amino acid of the series. These amino acids led to a considerably higher degree of loading (ca. 90-99%) and the lower loading efficiency for 17 and 18 could therefore be attributed to the presence of steric contrainta imposed by the carborane cage. The ionic nature of 18 presumably further aggravated the resin-reactivity problem.
240 Bkconlugete Chem., Vol. 2, No. 4, 1991
Varadarajan and Hawthorne
Scheme I1 1. n-BuLi, THF H3Gc-
-78°C
HaC--C-CH
\o/
e,
\o/
9
c
0°C
l!2
H,C-C-C-(CH,),OTs
HaGC- G(CH&J
Nal
\o/
b
\o/
BlOHlO
acetone, A
BIOHlO
TsCl/pyridine
G(cH2)30H
BlOHlO
25°C
2.
BioH1o
b
Scheme I11 i.LDA/THF/HMPA -7wc
(C,H&C=NCH2COOCH3
2. -78°C 3. 25%/20 h
6
1. K,COJCH,CN n-BuANOH
(C6H&C=NCH2COOCH,
La
2. &, A 172 h
B
2
14 Scheme IV NaOH, EtOH, AI24 h
Y=,,,WCY)q-H BiOHiO
/ /
c
NH2
2 HCI MBr
COONa
Ls
nido-CB
e
@
' ' 16B M = n-BudN 1Bp M IPh,PMe
Stepwise Synthesis of Carboranyl Peptides. A. Peptide Synthesis Involving the closo- Carboranyl Amino Acid. The subsequent coupling of the closo-carboranyl amino acid residues to the resin-bound, first amino acid of the peptide sequence was carried out by using the dicyclohexylcarbodiimide-mediated coupling of 17 with NJV(dimethy1amino)pyridineas the acylation catalyst. Although various methods such as the use of the N-succinimide active ester and the N-hydroxybenzotriazolemediated coupling were attempted, these methods did not
lead to significant improvement in coupling efficiency. As observed in the case of carboranylalanine (19),coupling reactions using 2 were sluggish and frequently required a second coupling sequence to reach completion. Steric hindrance to coupling has been observed even in the case of such relatively less encumbered amino acids as leucine. Due to the slowness of the reaction, coupling was carried out over a period of 48 h with the same time period allotted for the recoupling process, as well. With this procedure each residue was coupled to the previous residue in a quantitative manner. Since the carboranyl amino acid employed was initially racemic, racemization due to longer coupling times gave no cause for concern. Using this standard procedure we have coupled 2,5, and 10 residues of 2 in separate resin-supported peptide syntheses. In each case, cleavage of the peptide from the solid support was accomplished in the conventional manner employing hydrogen fluoride and anisole and with complete integrity of the closo-carborane cage. Subsequently, we found it convenient to carry out dansylation of the terminal amino group with the peptide still anchored to the resin. This procedurehas recently been reported in the literature (30). We have synthesized the peptides (closo-CB)z(191, (closoCB)vLys (20), and (closo-CB)lo-Lys(21) by this procedure (SchemeVI). A typical example of the stepwise synthesis is presented in Scheme VII. B. Peptide Synthesis Involving the nido-Carboranyl Amino Acid. In order to prepare water-soluble peptide derivatives, nido-carboranyl amino acid t-BOC derivative 18 was employed in coupling reactions with resin-bound glycine. As already mentioned, direct attachment of 18 to the resin was inefficient. It was found to be necessary to employ DMF as the reaction solvent to dissolve tat-
Novel Carboranyl Amino Acids and Peptides
Bloconlugate Chem.,Vol. 2, No. 4, 1991 248
Scheme V
1. (t-BOC)zO EtOH, A
&A~z)r-(f+COOH
NH (t-BOC)
L
2.
HCI/Me,NCI or Ph3PMeBr
e
'MqN or PbPMe
0°C
l4
Scheme VI NH-CH-COOH I
(CH2)4 I
NH2
4
or (clossCB),*Lye
2a
J9
ramethylammonium salt 18 in the coupling reactions. The DCC-mediated coupling reactions of the nido species proceeded much more slowly than those of the closo derivative and did not reach completion, even after recoupling. The inefficiency of this reaction can be attributed to the polar medium (DMF)which has previously been reported to increase the rate of rearrangement of the active 0-acylisourea intermediate to the inactive N-acylurea intermediate (26). It has also been reported that the presence of ionic materials accelerates this unwanted rearrangement. The reaction efficiency did not improve when an active ester such as N-succinimidyl (preformed) or HOBT (in situ)was utilized. Furthermore, the product peptides contained triethylammonium cation rather than the counterion (tetramethylammonium or triphenylmethylphosphonium) present in the starting materials. The cleavage of the peptide from the resin with HF did not present problems as indicated by the stability of model nido-carborane derivatives such as [nido-7,8-C2BsHlzlwith anhydrous HF. Due to the low yield of the coupling reactions, the anionic carboranyl amino acid was not employed in subsequent solid-phase syntheses. In order to obtain water-soluble peptides it was therefore necessary to convert the closo cages of the peptides derived from 2 to their anionic nido counterparts. This was accomplished without peptide bond disruption by the use of the pyrrolidine degradation (21) of the carborane cage. The resulting pyrrolidinium salts of the peptides were washed with dilute hydrochloric acid both to remove the excess pyrrolidine and to regenerate the free carboxyl function.
1.25% TFA 2.10% TEA
0
3. DCC,DMAP NH(FBOC)
II
1. 25%TFA 2. 10%TEA 3. DCC,DMAP CH~CEC-OH NH(I-BOC) Y O H l0
I
iT
Repeat Sequence The pyrrolidinium counterion of the nido-carboranecages was exchanged for sodium with a cation-exchange resin.
250
Varadarajan and Hawthome
B(oC0nlugete Chem., Vol. 2, No. 4, 1991
Scheme VI11 1.Q.254:
H
(c~oso-CB)~
*
2. H30+
L9
3. Na+/lcfl-xchg
8
Me2N
0 S02Cl 0
22
*
CH&N
Scheme IX 1.
Solid Support
Me,N
2. HF
0 S0,Cl 0
8
*
A
(C/OS*CB)
3. N ,25OC H 4. Na'l Ion-xchg
The synthesis of (nido-CB)z (22) from 19 is outlined in Scheme VIII. Fluorescent Labeling of thePeptides. The peptides with two functionally active end groups provide access to labeling for in vitro assays at one terminal group and activation for antibody conjugation at the other. Since we had previously demonstrated the carboxyl activation and subsequent conjugation of a radiometallacarborane sandwich to an anti-CEA antibody (22),we chose to label the terminal amino function with a fluorescent chromophore. Of all the fluorescent labels investigated, the dansyl group provided the most efficient method of introducing a sensitive fluorescent label and eliminated the alternative of a radiolabel for peptide assays prior to antibody conjugation. The dansylation process which leads to the synthesis of dansyl*(nido-CB)z(23) is depicted in Scheme VIII. It was later observed that the dansylation procedure could be better carried out with the resin-bound peptide itself, as exemplified in Scheme IX. When labeling the peptides attached to the resin, the t-BOC protecting group was first removed by treatment with trifluoroacetic acid followed by triethylamine. Further exhaustive treatment of the resin with dansyl chloride and triethylamine completed the labeling process. Labeled peptides 24 and 25 were obtained by this method followed by cleavage from the resin (Scheme X). Dansylated undecapeptide 25 was acetylated to block the free amino group on the lysine residue and the product, dansyl*(closo-CB)lo*Lys.Ac(27), was treated with pyrrolidine to accomplish quantitative carborane cage degradation. Following acidification and sodium cationexchange, the desired product, dansyl*(nido-CB)lo.Lys.Ac (27), was obtained as its sodium salt (Scheme XI). All the peptides were purified by flash chromatography on reverse-phase silica gel and characterized by spectroscopic means. Peptides which contained more than two closo-CB or nido-CBunits often exhibited aliphatic proton resonances in the lH NMR spectra as broad multiplets,
Scheme X I
Dansyl*GABA*(closo-CB)5*GABA
24 -NH-CH-COOH I
(CHd4 I
NHZ
9
Dansyl*(clos+CB)lo*Lys
2s
due to the reduction of their molecular tumbling motion in solution. The llB resonances of these same peptides were also broader than normal. Further, dansyl fluorescence intensity vs concentration curves were established for peptides 23 and 27 by measuring the fluorescence intensity at various concentrations using 313 and 515 nm as the excitation and emission wavelengths, respectively. 1lB N M R of Resin-Bound Peptides. A novel feature of the carborane cages attached to the resin is the fact that it was possible to obtain 1lB NMR spectra of the peptideloaded resins in dichloromethane medium. The spectra so obtained strongly resembled those of the resin-free peptides. It is reasonable to assume that there is considerable
Bloconlugete Chem., Vol. 2,
Novel Carboranyl Amino Ac#s and pept#es
No. 4, 1991 251
Scheme X I
a9
c
1. pymlidine 2. bo’ 3. Na+catknexchange
Dansyl.(M&B)1o*LpAc
22
60
SO
40
30
20
10
0
.lO
.20
-30 -40 -50 .60
.70
PPM
A 60
50
40
30
20
10
0
B
.IO -20 4 0 .40 4 0 4 0 .70 PPM
Figure 1. 11BIlH) FT NMR spectra of (A) dansyl+loeoCB)lrLys-reein suspended in CHnClS and (B)dansyl.(closoCB)irLya (26) as a eolution CDsOD.
motion of the attached peptide moiety within the swollen resin and that this process, along with the inherent quadrupole-broadening of the boron resonances seen in homogeneous solutions, gave rise to the solutionlike spectra. Representative llB NMR spectra are illustrated in Figure 1. Terminal Carboxyl Group Activation for Antibody Conjugation. As representative peptides prepared in this study which contain anionic nido-CB residues, 23 and 27 were chosen for antibody conjugation. A variety of attempts were made to prepare the N-succinimidyl ester
derivatives by the insitu activation of the peptide terminal carboxyl group prior to antibody conjugation. In each case the carboxyl group was activated at pH 4.4 in order to ensure that this group was protonated. When watersoluble ionic carbodiimides such as EDC were employed, the anionic nido-carborane cages present in the peptide precipitated with the added ammonium counterion. Furthermore, DCC was found to be unsuitable due to its separation from the aqueous phase when added as a solution in DMF. However, a solution of diisopropylcarbodiimide proved to be the reagent of choice since no solid separation was observed in the aqueous peptide phase. Both N-hydroxysuccinimide and N-hydroxysulfosuccinimide in water were equally efficient in forming the active ester. The conjugation of dipeptide 23 and undecapeptide 27 toT84.66 antibody was carried out by using the conditions described in Tables I and 11. The conjugates were purified by ultrafiltration using Centricon-30 membrane microfilters. The concentrates were repeatedly washed with appropriate buffers and with water until the filtrates exhibited no detectable fluorescence. In the case of the conjugates of dipeptide 23, further purification was performed by HPLC on a Dupont GF-250 gel filtration column. Control experiments were also carried out employing peptides 23 and 27 alone in the absence of the activating reagents. The activation and conjugation of the peptides to the antibody are depicted in Scheme XII. Measurement of the fluorescence intensities of standard solutions of the conjugates and comparisonof these values with the standard calibration curves for the unconjugated peptides provided the concentration of the peptides in the solutions of the conjugates. These data, when taken in conjunction with the concentrations of the antibody protein in the corresponding solutions (measured by absorbance at 280 nm or by Bio-Rad protein assay) revealed the average number of peptide molecules attached to each antibody molecule of the conjugate samples. The results of the conjugation studies are reported in Tables I and 11. In the case of dipeptide 23, control experiments (Table I) showed that the nonspecific binding of peptide to antibody, though present to a measurable extent, was not prohibitively large. Gel-filtration HPLC removed at least
Varadarajen and Hawthorne
252 Bloconlugte Chem., Vol. 2, No. 4, 1991
Scheme XI1 ,CO-CH,
Dansyl-HN(nido~B),,-COOH
+ HGN,
nNa e
0
Dansyl-HN(nido-CB)n--COO-N nNa@
1
Buffer I
CO-CH ‘S0,Na
?-THz
CO-CH ‘S0,Na
DIC
Buffer Mab-NH,
.
a detectable portion of the noncovalently bound peptide. Furthermore, there was only a slight increase in the proportion of the high molecular weight (HMW) species after conjugation and the conjugated protein was recovered in good yields. As can be seen from Table 11, the conjugation of the higher oligomer, undecapeptide 27, revealed aspects of the peptide-antibody interaction that require attention. The control experiments clearly showed a significant increase in nonspecific binding of peptide 27 to T84.66 compared to that of dipeptide 23. The proportion of this noncovalent binding increased with an increase in the proportion of starting peptide reagents to antibody and the recovery of the antibody protein decreased with an increase in the ratio of 27 to the antibody. For the undecapeptide, the proportion of the high molecular weight species (HMW)formedupon conjugation was considerably higher than that observed in the case of dipeptide 23. Again, this proportion of the HMW species increased with an increase in the starting ratios of peptide 27 to the antibody. It is clear from the foregoing study that with the increase in the average number of the anionic [nido-7,8-CzBeH111cages attached to the antibody through increased conjugation of the nido-carboranyl amino acid residues, noncovalent bonding of peptide molecules to antibody also increases. This type of binding could conceivably result from the enhancement of the hydrophobic character of the peptide reagent which results from an increase in the number of such ambiphilic nido-carborane residues that constitute the peptide molecule. It is interesting to note, in this regard, that on the basis of partition coefficient values, carboranylalanine (1) has been empirically calculated to be the most lipophilic amino acid known (32). The strong hydrophobic interaction between the ambiphilic nido peptide and the hydrophobic regions of the antibody would be expected to lead to the entrapment of the peptide reagent inside the antibody structure in such a manner that traditional gel-filtration techniques would not be able to separate the nonspecifically bound peptide. This idea is further supported by the fact that the use of nonionic surfactants, such as Tween-20,in the gel-filtration buffer medium employed in our subsequent studies allowed the successful separation of the unbound peptide. Further observations also indicated that the HMW species, which were associated with disproportionately high fluorescence intensities, are likely to be heavily conjugated antibody molecules which result from a repetitive cascade conjugation process rather than an aggregate (dimer) of the antibody. Details of these conjugation studies will be reported elsewhere. In conclusion, the foregoing study illustrates the feasibility of the precision synthesis of boron-rich oligomeric materials that circumvent the problems associated with oligomer heterogeneity in the antibody conjugates. However, with an increase in the monomer residues which
constitute the oligomer,noncovalent hydrophobicbinding of the oligomer to the antibody and the formation of HMW materials both undergo a significant increase. While the noncovalently bound materials can be removed by employing HPLC with nonionic detergents, it is also possible to incorporate amino acid residues which carry hydrophilic substituents, such as sugar residues (15,33),in the oligomeric peptide species. Such structural alteration should minimize or completely eliminate the hydrophobic interaction between the boron-rich oligomer and the antibody. In this regard, it is important to note that our method for the precision synthesis of oligomers also has the flexibility necessary for incorporating structural diversity in the product oligomer. Thus, in principle, it is possible to employ monomer residues which bear substituents that deter hydrophobic and other noncovalent interactions of the peptide with the antibody during conjugation. The problems associated with the formation of HMW materials during conjugation will be discussed elsewhere since further work in this direction is currently in progress. ACKNOWLEDGMENT
This research was supported by National Institutes of Health Grant CA 31753for which we express our gratitude. LITERATURE CITED (1) Barth, R. F., Soloway, A. H., and Fairchild, R. A. (1990) Boron
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