Bioconjugate Chem. 1994, 5, 98-100
9a
TECHNICAL NOTES Carboxymethyldextran Lactone: A Preactivated Polymer for Amine Conjugations Ned D. Heindel,' Michael A. Kauffman, Eric K. Akyea, Stephanie A. Engel, Michael F. Frey, C. Jeffrey Lacey, and Roger A. Egolf Department of Chemistry & Institute for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015. Received September 8, 1993@
The linking of amino haptens to carboxymethyldextran (CMD) requires carboxyl activation, for example, via carbodiimides. We have discovered that substantial N-acylurea, derived from these carbodiimides, can be trapped on the CMD backbone. As an alternative, CMD can be conveniently lactonized by heating in inert solvents, and this carboxymethyldextran lactone (CDL) can be employed directly for amine conjugation.
Conjugates of various dextran derivatives (carboxymethyldextran, polyaldehyde dextran, aminopropyl dextran, dextran hydrazide) have proven very useful for drug targeting, controlled release, and immunoconjugate spacer applications (1-4). In most cases the dextran derivative must be prepared de nova just prior to conjugation, requires the use of an activating catalyst (frequently a mixed anhydride or carbodiimide), or requires a postconjugation reduction to stabilize a hydrolytically-labile imine bond. In particular, couplings to carboxymethyldextran (CMD) which frequently use a carbodiimide activation are especially problematic because of the short half-life of the activating reagent in water, a necessary solvent for most such conjugates (5). Also, an equally serious problem during coupling is the established tendency of carbodiimides to undergo 0- to N-acyl shifts to form the more stable N-acylureas thereby leading to nonproductive couplings (6). Interest of our group in tumor delivery of radiotherapeutic levels of l25I on biologically-stable carriers (7) prompted the synthesis of a dextran framework to which would be attached a highly reactive aromatic amine for electrophilic radioiodination. Using established carbodiimide coupling techniques we coupled 4-methoxybenzylamine to carboxymethyldextran (8). We were amazed, however, to observe that the degree of substitution determined by UV quantification ( 4 ) of our methoxybenzylamine chromophore was in substantial disagreement with the degree of substitution determined by another commonly-employed technique which uses the 7% nitrogen analysis to determine drug load (9),in our case 4-methoxybenzylamine content. High resolution lH-NMR studies soon demonstrated that our carboxymethyldextran conjugate contained an appreciable amount of bound N-acylurea derived from the carbodiimide. To address this problem we developed a readilyprepared, shelf-stable, preactivated dextran derivative which permits facile drug coupling without the use of small molecule promoters. This derivative, carboxymethyldexAbstract published in Aduance ACS Abstracts, November
15, 1993.
1043-1802/94/2905-0098$04.50/0
tran lactone (CDL), is obtained in high yield from the thermal cyclodehydration of carboxymethyldextran (Scheme 1). We have shown that CDL can be loaded with 4-methoxybenzylamine, without activating catalysts (Scheme 21, in conversions superior to those obtained by conjugating the parent carboxymethyldextran to small molecules in more traditional fashions. EXPERIMENTAL PROCEDURES
Materials and Methods. The dextran used in these studies was high-purity, clinical-grade 40-kD dextran produced by Pharmachem Corp., Bethlehem, PA. The carboxymethyldextran (CMD) employed herein was prepared by carboxymethylation of 40-kD dextran as described by Novak and analyzed for degree of substitution by the titration procedure described therein (10). The degree of substitution was 1.02 CHzCOOH groups per repeating glucose. Ho's lH-NMR method for determination of carboxymethyl groups per glucose residue (by integration ratios of the grouping of multiple -0CH2COOH's to the C1-H) gave a degree of substitution of 1.06 on this sample (11). Infrared spectra were obtained as 1%disks in potassium bromide on a Mattson Polaris highresolution FTIR. Ultraviolet spectra were obtained in water on a Perkin-Elmer Lambda 5 UV-vis spectrophotometer. Differential scanning calorimetry (DSC) was performed on a Perkin-Elmer 1020 Series DSC7 thermal analysis system. Combustion analyses were performed on a Perkin-Elmer Model 2400 combustion analyzer by QTI Corporation, Bound Brook, NJ, with pre- and postrun calibration for % N (using high purity acetamide standard) and a standard deviation from the calculated value of f0.02 7% on the calibration standard. Loading levels of agents onto the dextran were determined by published techniques in which spectrophotometric quantification (by reference to a curve obtained from preprepared standard solutions) and combustion analyses for % nitrogen were employed (9, 12, 13). For the UV measurements, conjugates of 4-methoxybenzylamine were read at 272.6 Am=. Preparation of Carboxymethyldextran Lactone (CDL). Lactonization was effected by heating the CMD in a well-stirred suspension at reflux in four different 0 1994 American Chemical Society
Technical Notes
solvent systems, toluene (bp 109-110 "C), mixed xylenes (bp 138-144 "C), diglyme (bp 162 "C), and acetonitrile (bp 82 "C). A typical procedure involved suspending 0.650.75 g of dried carboxymethyldextran in 15-30 mL of the anhydrous solvent in a round-bottomed flask. The contents were magnetically stirred at reflux for 5 h, cooled to ambient temperature, and filtered. The white solid thus obtained possessed diminished water solubility and was vacuum dried to constant weight. DSC analysis showed no distinct phase transitions between 30 and 290 "C but evidenced a marked exothermic sample decomposition between 292 and 335 "C. Infrared analysis of the product showed a barely detectable absorption from the original carboxymethyldextran carboxylic acid C=O stretch at 1734 cm-l constituting less than 10% of the integrated intensity of the entire C=O envelope. The major absorption, the lactone carbonyl, appears a t 1755 cm-' in CDL. Condensation with 4-Methoxybenzylamine. Standard conditions for lactone-opening (Scheme 2) with 4methoxybenzylamine in toluene, xylene, diglyme, and acetonitrile were 6- to 10-fold mole excess of the amine per mole of available lactone residues in the dextran. Reactants were stirred at reflux in a volume of solvent 10-15 times the mass of the reactants for 5-20 h. This technique gave polyamides whose maximum degrees of substitutions (UV and 74 N analysis) fell between 60 and 90 3' 5 of the originally available carboxyl concentrations. Specifically, when 0.62 g of CDL, 4.2 g (31 mmol) of 4-methoxybenzylamine, and 50 mL of xylene were heated and stirred a t 90 "C for 20 h, the resulting yellow conjugate precipitate could be isolated by filtration, taken up in distilled water, and subjected to exhaustive dialysis to remove unbound drug (Spectrapor membrane dialysis bag, MW cutoff 12 000-14 000, dialyzed against distilled water for 5 days, two changedday). The contents of the bag were evaporated in uucuo to 20 mL and lyophilized to produce a fluffy white solid. By the ultraviolet measurement method, the degree of substitution of the methoxybenzylamine on the dextran obtained on a sample which had been lyophilized and vacuum-dried to constant weight was 0.78 mol/mol of glucose units for the toluene-derived product, 0.62 mol/mol of glucose for the xylene-derived material, and 0.60 mol/mol for the diglyme-derived substance. The values for degree of substitution obtained by %N (combustion) analysis were within * 8 % of the ultraviolet-quantification results. These conjugates decomposed above 250 "C and displayed characteristic infrared spectra: the amide I band at 1652 cm-l, the amide I1band at 1562cm-l, and the residual carboxyl absorptions at 1734 cm-l. There was a complete absence of the lactone absorption. Direct in Situ Lactonization-Conjugation. A 25mL round-bottom flask was charged with 0.279 g of CMD (1.28 mmol of carboxyl residues), 1.00 g (8.02 mmol) of 4-methoxybenzylamine, and 10 mL of anhydrous xylene. The reaction mixture was stirred and heated to 90 "C and maintained at that temperature for 20 h. Cooling the reaction mixture to room temperature was followed by the addition of 20 mL of distilled water which partitioned the product-conjugate into the aqueous layer. That layer was dialyzed as noted and lyophilized and a sample dried to constant weight. The degree of substitution was determined to be 0.56 amines/glucose residue by ultraviolet quantification. Carbodiimide-ActivatedConjugation of CMD with pMethoxybenzylamine. To a solution of CMD (0.125 g) dissolved in 50 mL of distilled water was added a single
Bioconjugate Chem., Vol. 5, No. 1, 1994
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portion of 4-methoxybenzylamine (0.60 g, 4.37 mmol). These reactant quantities represent a 6.2/1 molar ratio of amine to dextran-pendant carboxyls in the CMD. The same ratio of amine to lactone was employed in the standard coupling to CDL. The pH in the well-stirred solution was adjusted and maintained at 4.5 f 0.5 with 6 N HCl or saturated aqueous sodium bicarbonate while a total of 206 mg (1.07 mmol) of N-[3-(dimethylamino)propyl]-N'- ethylcarbodiimide hydrochloride (EDC) was added in five equal portions over a 3.5-h period. When the addition of the carbodiimide was complete the reaction mixture was stirred for 18 hours a t ambient temperature, transferred to a Spectrapor membrane dialysis bag (MW cutoff 12 000-14 000), and dialyzed against distilled water (5 days, two changes/day). The contents of the bag were evaporated in uucuo to 20 mL and lyophilized to produce a fluffy white solid whose degree of amide substitution was 0.24 by UV measurement at 272.6 nm. Three duplicate nitrogen combustion analyses gave values of 3.60 f 0.05 % . A IH-NMR obtained in DzO on a 360-MHz Bruker spectrometer revealed unexpected resonances a t 0.95 ppm consistent with pendant methyl moieties. RESULTS AND DISCUSSION
The carboxymethyldextran lactone synthesized herein is structurally related to the previously reported so-called Sephadex lactone prepared by carbodiimide-induced dehydration of the highly cross-linked carboxymethyldextran (Sephadex) beads (14). Akanuma reports an absorption of 1735 cm-l for C=O in carboxymethyl Sephadex and 1760 cm-l for the C=O in carboxymethyl Sephadex lactone (14). A structurally similar &lactone was generated on ganglioside GM3 by acid-catalyzed internal cyclization of the sialic acid carboxyl with a C-2 hydroxyl in the galactosyl residue; its carbonyl was assigned a t 1750 cm-l (15). While the main lactone absorption in CDL is at 1755 cm-l, expansion of the C=O spectral envelope reveals at least eight distinct IR bands between 1760 and 1744 cm-l. The parent carboxymethyldextran displays at least three carboxyl bands between 1730 and 1734 cm-I. Carboxymethyldextran with a degree of substitution of 1.06 carboxymethyls/glucose would, of course, be expected to be a mixture of 0-substituted CH2COOH isomers. By careful lH-NMR peak assignments to the methylene resonances at 4.0-4.5 ppm in carboxymethylcellulose, Ho was able to distinguish substitution occurring a t the C-2 OH from the C-3 OH and from the C-6 OH (11). He observed a slight kinetic bias to carboxymethylation at the C-2 hydroxyl but with all possible isomers being formed, and he further reported that the ratio of the integrated methyleneoxy region to the C-1 H provided a reliable technique to determine degree of substitution. The carboxymethyldextran employed in our studies (which had a degree of substitution of 1.02 by titration) did display at least three resolvable methyleneoxy resonances between 4.0 and 4.5 ppm and a degree of substitution (by NMR) of 1.06. With multiple sites of carboxymethylation many possible internal lactones can result and the structure given in Scheme 1is simply one illustration. Likewise, the ringopening amide structure in Scheme 2 is just one of several alternative representations. While infrared spectra of lactonized product always showed a trace of carboxylic acid absorption, titration proved an unreliable way of determining the degree of lactonization since both lactone functions and nonlactonized carboxyl groups responded. The water-insoluble CDL reacted and dissolved upon mixing with standardized
Helndel et al.
100 Bioconjugate Chem., Vol. 5, No. 1, 1994
Scheme I
ACKNOWLEDGMENT
1
t f " l
1
This research was supported by NIH grant ES 03647, the Brady Cancer Research Institute, and the W. W. Smith Charitable Trust. M.A.K. and S.A.E. were supported by an NSF-REU Program Grant. LITERATURE CITED
I
Jn
1
'n
(1) Schechter,B.,Pauzner, R., Wilchek,M.,andArnon,R. (1986)
Scheme I1
t T a
1
I
Jn
I
Jn
aqueous base. Inference of the number of accessible lactones could be deduced from the ring-opening experiments for which the maximum observed degrees of substitution reflected the maximum lactone concentration on the CDL. With 4-methoxybenzylamine and CDL by the conjugation method employed herein one could obtain up to 90% incorporation of amine with respect to original carboxylic content. Furthermore, while CMD and amine do not form amide in aqueous media without an activation promoter, we have found that they do couple in boiling xylene, presumably via in situ generation of lactone. Comparison of two methods for conjugating methoxybenzylamine to carboxymethylated dextrans, direct coupling to CDL versus EDC mediated coupling to CMD, shows EDC to be inferior both in maximum load and in ease of quantification. Unproductive linking on the part of EDC led to the well-known, unreactive, N-acylrearranged products (5,6)which gave disparate analytical results for UV and % N analysis. UV analysis of the chromophore of the EDC product revealed a degree of substitution of 0.24 and predicts nitrogen content to be 1.34% of the total mass, while combustion analysis shows % N to be 3.60%. These data can be reconciled only if a second, nonchromaphoric source of nitrogen was coupled to the dextran. 'H-NMR analysis of the conjugate evidenced the presence of the urea at levels high enough to account for the increased nitrogen content. We believe that this novel reactive dextran lactone can be a useful polymeric drug carrier for clean, facile conjugations without the need for carbodiimide promoters which may subsequently leave N-acylurea residues. Applications to couplings in aqueous media and to the conjugation of pharmaceutical entities are under study.
Cis-Platinum (11) Complexes of Carboxymethyldextran as Potential Antitumor Agents. CancerBiochem.Biophys. 8,289298. (2) Heindel, N. D., Van Dongen, J. M. A. M., Fitzpatrick, D. A., Mease, B. A., and Schray, K. J. (1987) Macromolecular Attachment as a Metabolic Stabilizer for a Labile Radiosensitizer. J.Pharm. Sei. 76, 384-386. (3) Heindel, N. D., Zhao, H., Leiby, J., VanDongen, J., Lacey, C. J., Lima, D. A., Shabsoug, B., and Buzby, J. H. (1990) Hydrazide Pharmaceuticals as Conjugates to Polyaldehyde Dextran. Bioconjugate Chem. 1 , 77-81. (4) Shih, L. B., Xuan, H., Sharkey, R. M., and Goldenberg, D. M. (1990) A Fluorouridine-Anti-CEA Immunoconjugate is Therapeutically Effective in a Human Colonic Cancer Xenograft Model. Int. J. Cancer 46, 1101-1106. ( 5 ) Gilles, M. A., Hudson, A. Q., and Borders, C. L., Jr. (1990) Stability of Water-SolubleCarbodiimides in Aqueous Solution. Anal. Biochem. 184, 244-248. (6) Wong, S. S. (1991) Chemistry of Protein Conjugation and Cross-Linking, pp 122-123, CRC Press, Boca Raton. (7) Heindel, N. D., Frey, M. F., Emrich, J. G., and Bender, H. (1992) Synthesis and Use of Tyraminyl-Cellobiose for Glioma Cell *%I-Radioimmunotherapy.Pharm. Res. 9, 101s. (8) Rosemeyer, H., and Seela, F. (1984) Polymer-linked Acycloguanosine. Makromol. Chem. 185, 687-695. (9) Hurwitz, E., Kashi, R., Arnon, R., Wilchek, M. and Sela, M. (1985) The Covalent Linking of Two Nucleotide Analogues to Antibodies. J. Med. Chem. 28, 137-140. (10) Berger, C., and Novak, L. J. (1958)Ferrous Carboxymethyl Dextran. U. S. Patent 2,862,920. (11) Ho, F. F.-L., and Klosiewicz, D. W. (1980) Proton Nuclear Magnetic Resonance Spectrometry for Determination of Substituents and their Distribution in Carboxymethylcellulose. Anal. Chem. 52,913-916. (12) Wilchek, M., and Bayer, E. A. (1987) Labeling Glycoconjugate8 with Hydrazine. Methods Enzymol. 138,429-442 and references cited therein. (13) Zhao, H. and Heindel, N. D. (1991)Determination of Degree of Substitution of Formyl Groups in Polyaldehyde Dextran. Pharm Res. 8,400-402. (14) Akanuma, H. and Yamasaki, M. (1978) Simple Hydrazidation Method for Carboxymethyl Groups on Cross-Linked Dextran. J. Biochem. 84, 1357-1362. (15) Yu, R. K., Koerner, T. A. W., Ando, S., Yohe, H. C., and Prestegard, J. H. (1985) High-ResolutionProton NMR Studies of Gangliosides. 111.Elucidation of the Structureof Ganglioside GM3 Lactone. J. Biochem. 98, 1367-1373.
