154
Bloconjugate Chem. lQ92, 3, 154-159
Molecular Amplifiers: Synthesis and Functionalization of a Poly(aminopropy1)dextran Bearing a Uniquely Reactive Terminus for Univalent Attachment to Biomolecules Jeffry S. Mann,+Jin Cheng Huang, and John F. W. Keana' Department of Chemistry, University of Oregon, Eugene, Oregon 97403. Received November 18, 1991
The synthesis and characterization of the versatile dextran-based molecular amplifier 6 is described. Dextran (M, = 40 200) was selectively monofunctionalized in high yield at its reducing terminus via reductive amination with 2-(4-nitrophenyl)ethylamineto give 1. The nitro group in 1 serves as amasked amino group which is eventually converted into a reactive isothiocyanato group used for monovalent attachment of the completed assembly to a target molecule. Cyanoethylation of 1 gave the terminally nitrophenylated poly(cyanoethy1)dextran 5 which was selectively reduced to the corresponding poly(aminopropyl) derivative 6 with BHrTHF, a reagent which preserved the end nitro group. Conjugation of amplifier 6 with the isothiocyanate-derivatizedGd(II1) chelate 7 gave conjugate 9 containing about 22 mol of chelate/mol of amplifier. The TI relaxivity per Gd(II1) ion of 9 in H20 was 15.0 mM-l s-l, about 3-fold higher than that of free Gd(II1)DTPA in HzO. The nitro group of 9 was then selectively reduced to the corresponding amine 10, which was converted into isothiocyanate 11. The reactivity of the single isothiocyanate group in 11 was demonstrated by coupling to 5-aminoeosin, giving conjugate 12. Amplifier 6 was also conjugated with the acid-labile N-cis-aconityl derivative 8 of the potent anticancer agent daunomycin. The nitro group of the resulting conjugate 13 was then reduced and the resulting amine 14 was converted into mono isothiocyanate 15. Compound 15 reacted with a waterinsoluble amine-containing solid support to give 16. Free daunomycin was released from 16 by exposure to citrate-phosphate buffer at pH 4.0.
The conjugation of therapeutically or diagnostically relevant small molecules (agents) to biomolecules with intrinsic tissue specificity provides a means of targeting the agent to a desired tissue. Due to their exquisite selectivity, monoclonal antibodies (mAbs) have been actively studied as carriers for drug molecules (I). Diverse agents such as anthracyclines ( 2 , 3 ) ,methotrexate (41, ricin A (5), and metal chelates (6) have been attached to mAbs. In general, the methods used to achieve agent-antibody conjugation involve the random modification of mAb amino acid side chain amines with drug derivatives and frequently result in an unacceptable reduction in the mAb immunoreactivity at useful levels of drug incorporation (7).Recent approaches have involved the modification of specificsites on the antibody such as the mAb carbohydrate moiety (8) or interchain disulfide bridge (9, 10). While use of site-specific labeling techniques avoids many of the pitfalls of random labeling, site-specific labeling methods have the disadvantage of providing immunoconjugates with low levels of agent incorporation. In order to maximize the amount of the agent borne by the mAb, macromolecular carrier molecules such as poly(L-aminoacids) (11),humanserum albumin (12),andpolyaldehyde dextran (13) have been used. In principle, the macromolecule is capable of carrying a large number of drug molecules, amplifying the amount of drug that the antibody is able to carry to its target while causing minimal loss of the mAb immunoreactivity. Polyaldehyde dextran, produced by periodate oxidation of dextran, has been conjugated with mitomycin C (14), methotrexate (15), daunomycin (161,and the hydrazides of the antineoplastic drugs ellipticine and CI-921 (17). The resulting drugcarrier assemblies have been coupled with a mAb. Drug~~
~
Current Address: Contrast Media Laboratory, Department of Radiology, University of California, San Francisco, CA 94143. +
1043- 180219212903-0154$03.0010
dextran-mAb conjugates have been shown to offer therapeutic advantages in several instances (16, 18, 19). The drug-dextran conjugation is conducted in a manner which leaves several of the dextran aldehyde functions unmodified. The carrier-drug assembly is attached to the mAb through the remaining aldehydes by reductive amination of amino acid side chains of the mAb. A limitation of the polyaldehyde carrier-drug assembly is its ability to act as a mAb cross-linking agent, causing antibody aggregation. A dextran-based carrier which, after labeling with drug molecules, bears one mAb-reactive group would represent a significant improvement over the polyaldehyde dextran carrier. The following account details the synthesis of a poly(aminopropy1)dextran derivative which is monofunctionalized at its reducing terminus with a masked aminereactive functional group. The aminopropyl groups were conjugated to a derivative of the magnetic resonance imaging (MRI) contrast agent Gd-DTPA, which is functionalized to allow its attachment to other molecules at a site distinct from its chelating moieties (20). Previously studied dextran-DTPA-Gd(II1) conjugates were constructed through ester or amide linkages using a DTPA carboxylate and result in a substantial loss of chelate stability (10l8 vs (21, 22). A conjugate was also prepared by functionalizing the amines with daunomycin via the acid-labile N-cis-aconityldaunomycinderivative (23, 24). The release of daunomycin from the resulting conjugate at pH 4 and 8 was studied. EXPERIMENTAL PROCEDURES
Materials. Dextran ( M , = 40 200, Lot No. 15779),cisaconitic anhydride, daunomycin, and l-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI) were purchased from Sigma. Dextran was dried at 80 "C and 0.05 mmHg over P205 for 24 h prior to use. EAH Sepharose 0 1992 American Chemlcal Soclety
Molecular Ampllflers
(7-1 1pmol amine/mL) was purchased from Pharmacia. Acrylonitrile, tetramethylammonium chloride (TMAC), 2-(Cnitrophenyl)ethylamine hydrochloride, BH30THF (1.0 M in THF), and anhydrous DMSO (Gold Label) were purchased from Aldrich. 5-Aminoeosin was purchased from Molecular Probes. N-cis-Aconityldaunomycinwas prepared by the method of Shen (24). The isothiocyanatobenzyl-DTPA-Gd chelate 7 was prepared as described earlier (20). NMR Spectroscopy. Proton NMR spectra were obtained using a G.E. QE 300 spectrometer. The samples were dissolved in DMSO (2.49 ppm) or DzO (4.80 ppm) and the chemical shifts reported in ppm on the 6 scale using the residual proton absorptions of these solvents as references. Proton NMR relaxation measurements were carried out at 10 MHz using a Praxis 2 spectrometer. TI values were measured using the inversion-recoverymethod, 18O0-r-9Oo-fid-delay,where the delay was 5 X 21' . Infrared Spectroscopy. Infrared spectra of dextran derivatives as KBr pellets were obtained on a Nicolet 5DXB FTIR spectrometer. Ultraviolet Spectroscopy. UV absorbance spectra of aqueous solutions were measured using a Beckman DU-7 instrument. The concentrations of free and bound daunomycin were estimated from the height of the absorbance maximum at 475 nm (e = 9860) in the UV spectra of aqueous solutions (24). The concentration of 2-(4-nitropheny1)ethylamine was estimated from the absorbance maximum at 275 nm (e = 9.7 X lo3). Concentrations of the thiourea derivative of 5-aminoeosin were estimated in a similar manner from an absorbance maximum at 525 nm (e = 9.0 x 104). Analyses. Elemental analyses (C, H, N) were performed by Desert Analytics (Tucson, AZ). Metal ion analysis (Gd3+)was performed by Galbraith Laboratories, Inc. (Knoxville, TN), using atomic absorption spectroscopy. Molecular weight analyses were performed by Arro Laboratories, Inc. (Joliet, IL), using gel permeation chromatography. Thin-LayerChromatography. TLC of daunomycin and N-cis-aconityldaunomycinwas performed on silica 0.2-mm thickness) from EM Science using a gel (60 FZM, 17:3:1 acetone-HCCl3-AcOH system as eluent. Gel Permeation Chromatography. Before use the Sephadex G-25 gel was swelled with the aqueous eluting buffer at 25 "C for 16 h or 80 "C for 1 h. The poured columns were equilibrated with the eluting buffer by passing five column volumes of the buffer through the column. Column void volumes were determined using a solution of blue dextran (5 mg/mL). During chromatography the column effluent was monitored continuously at 254 nm with an ISCO UA-4 detector. The chromatograms displayed complete baseline separation of the macromolecular and small molecule components in all cases. 1-[(4-Nitrophenethyl)amino]dihydrodextran (1). The free base of 2-(4-nitrophenyl)ethylamine hydrochloride was obtained by chloroform extraction (4 X 25 mL) of an emulsion produced by basifying a solution of the amine hydrochloride (1.01 g, 5.0 mmol) to pH 12-13 with 2 N NaOH (3 mL). The extracts were pooled, dried (Kzcos), and evaporated to dryness. The resulting dark orange oil (800 mg) was purified by chromatography over silica gel (30 g) and elution with HCCl3-MeOH-concentrated NH40H (4:l:O.l) afforded the free base (750 mg, 90%) as a yellow oil. NMR (CDC13): 6 1.261 (s,2), 2.286 (t, 2), 3.023 (t, 2), 7.349-8.175 (AA'BB', 4). UV (0.5% EtOH): ,A, = 278 nm (e = 8.6 x 103). Dextran (1.10 g, 2.75 X mmol) was dissolved in anhydrous DMSO (5.5 mL) with gentle heating (45 "C) and allowed to return to 25 "C. 2-(CNitrophenyl)eth-
Bloconjugate Chem., Vol. 3, No. 2, 1992
155
ylamine (680 mg, 4.1 mmol) and crushed 4-19 molecular sieves (130 mg) were added. The flask was flushed with Ar for 3 min and then sealed with a serum cap. The reaction mixture was stirred for 24 h at 37 "C. N&H4 (17 mg, 0.44 mmol) was added, the flask was flushed with Ar, and the suspension was stirred for 24 h at 37 "C. The viscous brown mixture was cooled in an ice bath as water (10 mL) was slowly added. The pH was adjusted to 5.5 by the addition of glacial acetic acid (about 0.4 mL). The orange suspension was centrifuged and the supernatant was loaded onto a column of Sephadex G-25 (2.5 cm X 65 cm) and eluted with 0.1 M NH40Ac. The modified polysaccharide eluted off in the void volume (150 mL). It was collected as one fraction (80 mL), evaporated to approximately 15 mL, dialyzed against flowing distilled water for 18 h, and lyophilized to afford 984 mg (90% recovery based on starting dextran) of a cream-colored solid. NMR (DzO): 6 3.477-3.989 (m, 7), 4.970 (8, 1); at increased gain 6 8.205 (d), 7.495 (d). UV (HzO): ,A, = 278.0 nm (e = 8.5 X 103). The yield of functionalization (97-98% ) was calculated from the above absorbance (see discussion in the text). A commercial molecular weight analysis by gpc gave an M,value of -40 200. 1-[ (4- Aminophenethy1)aminoldihydrodextran (2). End-group modified dextran 1(10.2 mg, 2.52 X mmol) was dissolved in HzO (5 mL) and 30% Pd/C (5 mg) was added. The suspension was shaken under 40 psi of Hz for 6 h and then filtered through Celite and lyophilized. The resulting powder was dried at 1mmHg over P z Oa~t 80 "C for 18h, giving 8.8mg (86 % ) of 2 as a white powder. NMR (DzO): 6 3.480-3.968 (m, 7), 4.966 (s, 1);at increased gain 6 7.130 (d), 6.815 (d). 1-[ (4-Isothiocyanatophenethyl)amino]dihydrodextran (3). Dextran 2 (8.8 mg, 2.2 X lo4 mmol) was dissolved in HzO (1mL) and 0.1 M NaHC03 (1mL) was added. The stirred solution was cooled in an ice bath and thiophosgene (100 pL, 1.4 X mmol) was added. The mixture was stirred at 0 "C for 2 h then at 25 "C for 30 min. The mixture was extracted with Et20 (3 X 5 mL), transferred to dialysis tubing, and dialyzed against 1L of distilled H20 (pH = 6.5) at 4 "C for 16 h and then lyophilized. The resulting solid was dried at 1mmHg over P z O at ~ 35 "C for 18 h, giving 5.5 mg (62%) of a creamcolored powder. 5-Aminoeosin Derivative 4 of Dextran. Isothiocyanate-derivatized dextran 3 (5.5 mg, 1.4 X mmol) was dissolved in H20 (1mL), and MeOH (200rL) was added. To this stirred solution were added 0.1 M NaHC03 (1 mL) and 5-aminoeosin (10 mg, 1.4 X mmol). The bright red solution was stirred for 16 h at 25 "C and then loaded onto a Sephadex G-25 column (1.5 X 26 cm) and eluted with 0.1 M NH40Ac. The modified polysaccharide eluted in the void volume (5 mL) as a bright red band and was collected in one fraction (2 mL), dialyzed against 4 L of distilled water at 4 "C for 18 h, and then lyophilized, giving 5.0 mg of a bright red solid. UV Determinationof 5-AminoeosinIncorporation into 4. The extent of 5-aminoeosin incorporation into 4 was estimated from the UV spectrum of solutions of varying concentrations in 4. A blank prepared using unmodified dextran (11.0 mg, 2.7 X lo4 mmol) showed that the extent of noncovalent binding of 5-aminoeosin, estimated from the UV spectrum of the blank, was 2.4 % . The extent of covalent binding of 5-aminoeosin in 4 was 90%. This agreed closely with the extent of incorporation of the 2-(4-aminophenyl)ethylaminegroup estimated from the intensity of the absorbance of the aromatic nitro chromophore at 278.0 nm (e = 8.6 X lo3) in the UV spectrum of 1.
156 Bioconjugate Chem., Vol. 3, No. 2, 1992
Menn et al.
1-[(4-Nitrophenethyl)amino]dihydropoly(2-cyano- of a solution of 9 in water at 10 MHz and 37 "C was 330.3 mM-ls-l, corresponding to a relaxivity per Gd(II1) ion of ethy1)dextran (5). (Nitropheny1)dextran 1(744mg, 1.85 15.0 mM-' s-l. X loe2mmol) was slurried in HzO (1mL) containing tetConjugate 10. End group modified dextran conjugate ramethylammonium chloride (30 mg, 0.3 mmol) with 9 (100.6 mg, 6.37 X mmol) was dissolved in H20 (10 stirring. The slurry was stirred to produce a clear solution mL) and 30% Pd/C (30 mg) was added. The suspension (-20 min) and then freshly distilled acrylonitrile (15 mL) was shaken under 30 psi of Hz for 8 h and then filtered was added. NaOH (12%, 750 pL, 2.2 mmol) was added through Celite; the filtrate was lyophilized, giving 90 mg and the resulting mixture was stirred for 1h. At the end (90%) of conjugate 10 as a white powder. of this time 2-propanol (40 mL) was added to the reaction Isothiocyanate-DerivatizedConjugate 11. Conjumixture and the resulting viscous mixture was triturated mmol) was dissolved in HzO gate 10 (90.0 mg, 6.2 X with fresh portions of 2-propanol (3 X 40 mL) until a free(8 mL) and 0.1 M NaHC03 (2 mL) was added. The flowing suspension was produced. The white solid was solution was cooled in an ice bath and a 0.2 M solution isolated by filtration, washed with EtOH (2 X 25 mL) and of thiophosgene (500 pL, 0.1 mmol) in HCCl3 was added. Et20 (2 X 25), and dried a t 40 "C over PzO5 for 16 h at The mixture was stirred at 0 "C for 3h and then evaporated 1 mmHg, giving 825 mg (85% based on degree of to dryness. The residual solid was dissolved in HzO (2 substitution = 0.5) of a white powder. IR: 2254 cm-l. mL) and dialyzed against flowing distilled water for 16 h. NMR (DzO): 6 2.811 (s, 2), 3.497-4.020 (m, 71, 4.956 (s, The contents of the dialysis tubing were concentrated to 1). Anal. Calcd for (C6H905)n(C3H4N)~.5n(H~0)~.5n: C, about 1mL and the product was precipitated with acetone. 47.87; H, 5.85; N, 3.72. Found: C, 47.68; H, 5.85; N, 3.72. The precipitate was isolated by centrifugation, washed 1-[(4-Nitrophenethyl)amino]dihydropoly(3-aminowith acetone (3 X 5 mL), and dried at 0.05 mmHg at 25 propy1)dextran (6). Dextran derivative 5 (668 mg, 1.4 "C for 24 h, giving 88mg (90%)of aslightly yellow powder. x 10-2 mmol) was suspended with stirring in anhydrous 5-Aminoeosin Derivative 12 of Conjugate 11. THF (20 mL) and cooled to 0 "C. BHrTHF (20 mL, 20 Isothiocyanate-derivatizeddextran conjugate 11 (10 mg, mmol) was added via syringe and the suspension was remmol) was dissolved in HzO (1mL) and MeOH 6.33 X fluxed for 18h. At the end of this time the suspension was (200 pL) was added. To this stirred solution were added cooled to 0 "C and MeOH (25 mL) was added slowly via 0.1 M NaHC03 (500 pL) and 5-aminoeosin (2.8 mg, 4 X syringe. The volatiles were removed by rotary evaporation 10-3 mmol). The bright red solution was stirred for 16 h and the resulting white solid was treated again with MeOH at 25 "C then loaded onto a Sephadex G-25 column (1.5 (15 mL). The suspension was evaporated to dryness and cm X 26 cm) and elutedwith 0.1 M NH40Ac. The modified the solid was slurried in dioxane (10 mL) with stirring. A polysaccharide eluted in the void volume (5mL) as a bright mixture of Et3N (1mL) and HzO (9 mL) was added slowly red band and was collected in one fraction (2.5 mL), to the stirring suspension. The reaction mixture was dialyzed against 4 L of distilled water at 4 "C for 18h, and stirred for 2 h at 25 "C, during which time the solid then lyophilized to provide 9.6 mg (96%) of conjugate 12 dissolved completely. The resulting solution was evapas a bright red powder. UV (HzO): ,A, = 525 nm (e = orated to near dryness, redissolved in water (5mL), loaded 9.0 x 104). onto a Sephadex G-25column (2.5 cm X 50 cm), and eluted UV Determination of 5-Aminoeosin Incorporation withO.l M NH40Ac. The modified polysaccharide eluted into 12. The extent of 5-aminoeosin incorporation into in the void volume (23 mL), was collected as one fraction 12 was estimated from the UV spectra of solutions varying (10 mL), dialyzed against distilled water (4 L) at 4 "C for in concentrations of 12. A blank was prepared as a control 18 h, and lyophilized. The resulting white solid was dried by submitting a sample (3.1 mg, 5.5 X mmol) of the at 0.05 mmHg over PzO5 at 60 "C for 24 h, giving 570 mg Gd(II1)-derivatized (nitropheny1)dextran conjugate 9 to (85%) of 6 as a white powder. An aqueous solution (2 the conditions of the 5-aminoeosin incorporation reaction mg/mL) of 6 gave a positive reaction when treated with used to produce 12. The extent of noncovalent binding a 5% solution of ninhydrin in EtOH and heated to 40 "C of 5-aminoeosin was estimated at 4.0% from the blank. for 1min. NMR (DzO): 6 4.980 ( 8 , l),3.291-4.223 (m, 101, 2.730-2.950 (m, 1). Anal. Calcd for ( C ~ H 9 0 5 ) ~ - The extent of covalent binding of 5-aminoeosin in 11was determined to be 88%. ( C ~ H ~ N ) O . S ~ ( H ZC, O 45.22; ) O . ~ ~H, : 7.03; N, 3.52. Found: Conjugation of N-cis-Aconityldaunomycin(8) with C, 45.31; H, 6.87; N, 3.38. Poly(aminopropy1)dextran 6 Giving Conjugate 13.NCouplingof Isothiocyanatobenzyl-DTPA-Gd7 and cis-Aconityldaunomycin (8) was conjugated to dextran 6 Poly(aminopropy1)dextran 6 To Give Conjugate 9. using the procedure of Shen (24). The resulting red End-modified poly(aminopropy1)dextran6 (202 mg, 4.21 solution was stirred in the dark for 17 h. The solution X mmol, 0.826 mequiv of amine) was dissolved in 0.1 gave a positive ninhydrin test. To "cap" the remaining M NaHC03 (5 mL) with stirring. A solution of isothioamines the solution was taken to pH 8 with 0.1 M NaHC03 cyanatobenzyl-DTPA-Gd chelate 7 (820 mg, 1.11mmol) (2 mL) and treated at 0 "C with AczO (500 pL, 5.0 X in HzO (8 mL) was added and the resulting solution was mmol). The resulting mixture was stirred until it gave a stirred for 16 h at 25 "C. A t the end of this time the negative ninhydrin test (2 h). The reaction mixture was mixture was centrifuged to remove a small amount of a loaded onto a Sephadex G-25 column eluted with 0.1 M white solid and the supernatant was drawn off. The NH40Ac (pH 8). The modified polysaccharide eluted in supernatant did not give a positive reaction to 5% ninthe void volume (7.5 mL) and was collected in one fraction hydrin in EtOH. To ensure that all of the available amines (2.5 mL). An aliquot (50 pL) of this solution was diluted were "capped", the supernatant was cooled to 0 "C and to 3.0 mL with H20 and the extent of coupling was taken to pH 9 with saturated NaHC03, and acetic estimated from the UV spectrum to be 48 mol of 8/mol anhydride (500 pL, 5.23 mmol) was added. The resulting of 6. The two solutions were recombined, dialyzed against solution was stirred for 2 h, dialyzed against 4 L of distilled distilled HzO (4 L) at 4 "C for 24 h, and lyophilized, giving H20 at 4 "C for 16 h, lyophilized, and dried at 0.05 mmHg 2.3 mg of 13 as a pink solid. IR (KBr): cm-' 1554.7 (carover PzO5 at 60 OC for 16 h, giving 233 mg (75% recovery, boxylate, cis-aconityl group). see below) of 9 as a white powder. A commercial Gd(II1) A control was prepared by submitting 6 (1.6 mg, 3.5 X analysis by atomic absorption spectroscopy showed that 10-5 mmol) and 8 to the above reaction conditions without 9 contained 7.45% Gd. The Gd(II1) analysis corresponds the addition of EDCI. The control was purified as above, to a conjugate with 22 mol of 7/mol of 6. The relaxivity
Molecular Ampliflers
lyophilized,and redissolved in H2O (2.00 mL). The extent of coupling was estimated from the A475 of this solution mol of 8/mol of 6. to be 1.0 X N-cieAconityldaunomycin-DextranConjugate 14. The nitro group of 13 was reduced to amine 14 by a modification of the procedure of Avery et al. (25). To a 5-mL round-bottomed flask equipped with a magnetic stirrer were added PtO2 (5 mg) and water (3 mL). H2 gas was bubbled through the suspension for 3 min, and then an aliquot (1.0 mL, 1.5 X mmol) of a solution of conjugate 13 in water was added. The flask was flushed with H2 for 3 min and sealed. The suspension was stirred at 1atm of H2 a t 25 "C for 4 h and then filtered through Celite. The filtrate containing 14 was used for the next experiment without further purification. Isothiocyanate-DerivatizedConjugate 15. The filtrate containing 14 was taken to pH 8.5 with 0.1 M NaHC03. The solution was cooled in an ice bath and mmol) was added. The thiophosgene (50 pL, 7.0 X reaction mixture was stirred at 0 "C for 2 h and at 25 "C for 30 min and then dialyzed against 1L of distilled H2O (pH = 6.8) at 4 "C for 16 h. The resulting solution was diluted to 5.00 mL with distilled H2O. An aliquot of this solution was used in the next experiment. Reaction of Conjugate 15 with EAH Sepharose To Give Water-InsolubleConjugate 16. A 1.7-mL aliquot of EAH Sepharose (7-11 pmol of amino groups/mL) was centrifuged and the pellet was washed with water (3 X 2 mL). A 250-pL aliquot of the solution containing 15 from the above experiment was diluted to 1.00 mL with 0.1 M NaHC03 and the UV spectrum was recorded (A475 = 0.2337). This solution of compound 15 was added to the gel pellet and the suspension was stirred for 4 h. The mixture was centrifuged and the supernatant wasremoved. The amount of 15 bound to the EAH Sepharose, estimated from the UV spectrum (A475 = 0.0684) of the supernatant, was 71 7% of the amount originally in the supernatant. The pellet of 16 was washed with water (3 X 3 mL) to remove any unbound 15 and then suspended in citrate-phosphate buffer (pH 4.0) with stirring for 8 h. The suspension was centrifuged every 2 h during the course of the reaction and the extent of daunomycin release was estimated from the UV spectrum of the supernatant. A plateau was reached at 8 h (A475 = 0.1107) that corresponded to release of 65% of the total bound daunomycin. TLC (17:3:1 acetone-CHCl3-AcOH) of the supernatant against a known sample of daunomycin gave identical R f values (0.16) for the two solutions. The Supernatant was adjusted to pH 7.5 with 1 N NaOH (1 drop) and then extracted with HCCl3 (3 X 10 mL). The organic layers were pooled and evaporated to dryness, affording 0.6 mg of a pale pink solid. The IR spectrum (KBr) of this material was identical to that of daunomycin. A second sample of conjugate 16 was prepared in an identical manner and suspended at 25 "C in citratephosphate buffer (pH 7.0) with stirring. After 48 h there was no significant release of daunomycin in the supernatant detected by UV spectroscopy. RESULTS AND DISCUSSION
Synthesis of the Dextran Amplifier. The synthesis of amplifier 6 is detailed in Scheme I. The reducing terminus of dextran (40 200) was derivatized with a masked reactive group by reductive amination with 2-(4-nitrophenyllethylamine in DMSO under anhydrous conditions. The macromolecular component was separated from excess 2-(Cnitrophenyl)ethylamine by chromatography over Sephadex G-25. A molecular weight analysis by gel permeation chromatography (gpc)revealed that the degree
Bioconjugate Chem., Vol. 3, No. 2, 19Q2 157
Scheme I** Mr40200 Dearan
ab
HOia*$"
n=- 2 ~ )
i
-
3. R = H , X = N = C = S
4, R - H , X =
1. R = (CH&CN, X E NO2
p.hC
r
6. R E (CHz),NHz, X I NO2
Ar
Ar
(a) 2-(4-Nitrophenyl)ethylamine/DMSO/molecular sieves; (b) NaBH4; (c) Pd/C, Hz; (d) C(S)CldNaHCO3(aq); (e) fi-aminoeosin/NaHC03(aq); (f) 12 76 aqueous NaOH/Me4N+C1-/ acrylonitrile; (8)BH3.THF; (h) 9:l HzO:Et3N/dioxane. b For convenience the R group is shown attached to 0 - 4 although the actual point of attachment likely varies along the dextran backbone. R = H for many of the glucose residues. (I
of polymerization of the product, dextran 1, was identical to that of the starting dextran. The extent of derivatization was estimated a t 9&99% from the absorbance of the 2-(4-nitrophenyl)ethylaminechromophore at 278 nm (t = 8.6 X lo3). The protons of the p-nitrophenyl group were readily observed in the high-gain 'H NMR spectrum of 1. The extent of end-group derivatization in 1 was confirmed by the conversion of 1 into 5-aminoeosin derivative 4 via amine 2 and isothiocyanate 3. The observed absorbance a t 525nm (e = 9.0 X lo4)of an aqueous solution of 4, adjusted for a 2.4% background binding of 5-aminoeosin to unmodified dextran, corresponded to a 90% yield of covalent incorporation. This reaction sequence also served as a model for the introduction of an isothiocyanate group into the new derivatized dextrans described below. End-modified dextran 1was next cyanoethylated to give 5 by adapting the procedure of Daly (26). The appearance of a broad singlet at 2.811 ppm in the IH NMR (D2O) spectrum of 5 was indicative of cyanoethylation. The extent of cyanoethylation could be estimated by comparing the integrals of the dextran anomeric protons (4.956 ppm) and the peak at 2.811 ppm in the IH spectrum of 5 in D20. Further confirmation of the presence of the cyanoethyl groups came from the IR spectrum (KBr) which showed a sharp peak at 2254 cm-1. A series of dextrans differing in their degree of cyanoethylation was synthesized by varying the amount of 12% NaOH(aq) added to the reaction mixture. Poly(cyanoethy1)dextrans of degree of substitution (DS) > 1.0 cyanoethyl groups per subunit formed hazy gels with water. A poly(cyanoethy1)dextran which was soluble in water to at least 50 mg/mL was produced by lowering the DS to 0.5 cyanoethyl groups per glucose subunit as determined by elemental (C, H, N) and NMR analysis. Cyanoethylation by the above procedure was reproducible to within f O . l cyanoethyl group/subunit. Selective reduction of poly(cyanoethy1)dextran 5 to the corresponding poly(aminopropy1)dextran 6 was effected by refluxing a suspension of the cyanoethylated dextran in THF containing excess BH3oTHF for 18 h followed by hydrolysisof the intermediate boratriazine (27)using dilute aqueous triethylamine. Reduction was confirmed by the disappearance of the nitrile absorbance at 2254 cm-l. A solution of 6 in water (5 mg/mL) had a pH of 8.8and gave a positive reaction with 5% ninhydrin in EtOH, indicating the presence of primary amines. A molecular weight analysis of 6 by gpc indicated that the product had approximately the same average molecular weight as the
Mann et al.
150 Bioconlugete Chem., Vol. 3, No. 2, 1992
Scheme 11.
L
Gd3+ 7 Hoz?02H a
IR= ,
dH
3
L
HNR
9. X = NO2
10, X=NH2
-In
&R
5 .
&R
13, XrNO2 14, XI NHz
11, X=N=C=S 12. X=NH I
7-S NH
-0OC
Na*z -0OC-l
Gd3' a
J
See footnote b, Scheme I.
starting material. The chromatogram generated by this analysis displayed a tailing peak for the analyte which was most likely indicative of nonideal interactions between the polyamino analyte and the column (28). The increased retention of the analyte caused by these nonideal interactions gave the appearance of a decrease in average molecular weight to approximately 37 000. Although the possibility of degradation cannot be ruled out, it is unlikely that this would occur under the mild conditions used. An elemental analysis of this compound gave results consistent with a DS = 0.5 aminopropyl groups/subunit. The UV and 'H NMR spectra of 6 confirmed that the nitrophenyl group survived the reductive conditions necessary to convert the cyanoethyl groups to propylamines. As a control a sample dextran l was treated with BHaTHF in a manner identical to that used to convert5 to 6. The starting nitro compound was recovered in nearly quantitative yield. Attachment of Agents to Dextran Amplifier 6. The thiourea-linked conjugate 9 between amplifier 6 and GdDTPA derivative 7 was produced by mixing the two components under basic conditions (Scheme 11). The remaining amines were "capped" by acetylation with acetic anhydride to ensure that they would not interfere with the subsequent conversion of the nitro group to an isothiocyanate or react with this group once it was in place (see below). The success of the conjugation reaction was confirmed by the appearance of a strong phenylthiourea absorbance at 246 nm in the UV spectrum of 9. A Gd(II1) analysis by atomic absorption spectroscopy was consistent with a derivative containing 22 mol of chelate 7/mol of amplifier 6. The moderate degree of chelate incorporation is likely due to the steric requirements of the bulky che-
late or repulsions due to the buildup of negative charge on the dextran with the binding of the chelate. As conjugate 9 contains multiple Gd(II1) chelates and is of interest as a new agent for MRI contrast enhancement, ' 2 relaxation of water is of its ability to affect the 1 considerable interest. The 21' of an aqueous solution of this conjugate was measured at a field strength of 0.25 T by the inversion-recovery method (180°-r-900). The 21' relaxivity of 9 in H20 was 15.0 mM-' s-l per Gd(II1). For comparison the 2'1 relaxivity of Gd(DTPA) in H2O is 4.9 mM-1 s-l (29). The aromatic nitro group of 9 was reduced with Hz and 30% Pd/C to give the corresponding amine 10 which was subsequently converted to isothiocyanate 11 by the action of C(S)Clp. In order to determine the extent of the ability of 11 to react with amino groups on other target molecules, the isothiocyanate was allowed to react with 5-aminoeosin to form 12 in a manner identical to that used in the preparation of 4. Calculations based on the UV absor= 525 nm, e = 9.0 X lo4) gave bance maxima of 12 (A, a value of 95 % for the incorporation of 5-aminoeosin. As a control, 5-aminoeosin was stirred with nitro dextran 1 under conditions identical to those used to produce 4. Calculations based on the UV spectrum of the control gave a value of 4.0% for the extent of noncovalent incorporation of the 5-aminoeosin. The corrected yield of covalent binding involving the isothiocyanate group of 11 was therefore 91 7%. The next series of experiments demonstrates the attachment and release of the anthracycline daunomycin, a potent cytotoxin widely used in cancer chemotherapy (30). The previously reported N-cis-aconityl derivative 8 of daunomycin was synthesized in 80 % yield following
Molecular Amplifiers
the procedure of Shen (24). The acid-labile derivative was attached to poly(aminopropy1)dextran6 in aqueous solution at pH 6.0 using EDCI. Conjugate 13 was purified by chromatography over a column of Sephadex G-25eluted with citrate-phosphate buffer (pH 8) (31). The solution of 13 which eluted off the column gave a positive test toward the ninhydrin reagent, indicating the presence of unreacted amine groups. To ensure that the remaining amines would not interfere with the formation of the isothiocyanate group the unreacted amino groups were “capped” by acetylation with acetic anhydride at pH 8 and 0 OC. The resulting solution was purified on a column of Sephadex G-25 eluted with citrate-phosphate buffer (pH 8). Recovery of a dextran from the Sephadex G-25 column was shown to be quantitative using underivatized 6. Quantitative recovery of the applied capped dextran 13 is assumed in the following calculations. The extent of the attachment of 8 to 6 was estimated at 48 mol of daunomycin/mol of dextran from the UV absorbance of the cis-aconityldaunomycin chromophore e = 9860) (24) of an aqueous solution of at 475 nm A(,, 13. To confirm that the binding in 13 was covalent, a control was performed by mixing an excess of 8 with dextran 6 without the addition of EDCI. After a workup identical to that used in the preparation of 13 the UV spectrum of an aqueous solution of the control was measured. No significant amount of binding of 8 to 6 was observed. The procedure of Avery (25) allowed the selective reduction of the nitro group of 13 to the corresponding amine 14 with H2 (1 atm) and PtO2. Amine 14 was converted to its isothiocyanate derivative 15 by the action of C(S)C12 at pH 8.5. The presence of the isothiocyanate moiety of 15 was confirmed and quantified by reaction of 15 at pH 8.5with EAH Sepharose (EAH),a water-insoluble amine-containing solid support, to form 16. The extent of the binding of 15 to EAH was estimated to be 71 % by measuring the decrease in the absorbance a t 475 nm of the supernatant of the reaction suspension. Free daunomycin could be released from the insoluble conjugate 16 by suspending a sample of 16 in citratephosphate buffer at pH 4.0. The extent of daunomycin released at pH 4.0 was estimated from the absorbance of the supernatant at 475 nm to be 65% of the total amount of daunomycin bound to the EAH Sepharose. The TLC and IR behavior of an HCC4 extract of the supernatant corresponded to that of authentic daunomycin. A suspension of conjugate 16 in citrate-phosphate buffer at pH 7.0 was stable toward release of daumomycin for 48 h at 25 “C. The preceding has detailed the synthesis and characterization of a versatile dextran-based homopolyfunctionalized “amplifier” molecule 6 derivatized selectively at its reducing terminus for attachment to biomolecules. Poly(aminopropy1)dextran 6 is stable and can be prepared in high yield from inexpensive precursors. The utility and versatility of this compound was demonstrated by its conjugation to a modifier Gd-DTPA chelate, a useful contrast-enhancing agent for MRI and to an acid-labile prodrug of the potent cytotoxin daunomycin. An efficient two-step conversion of the aromatic nitro group to an isothiocyanate allows for the monovalent attachment of the assembly to other amine-containing molecules.
Bioconjugate Chem., Vol. 3, No. 2, 1992
159
LITERATURE CITED
ACKNOWLEDGMENT
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This work was supported by NIH Grant GM-27137. We thank Drs. Johannes Volwerk, Bruce Birrell, and Olivier Clement for helpful discussions.
Registry No. 2-(4-Nitrophenyl)ethylamine,24954-67-4;5aminoeosin, 75900-75-3; acrylonitrile, 107-13-1.
