Bioconjugate Chem. 1990, 1, 309-313
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ARTICLES Synthesis and Characterization of Digoxin-Phospholipid Conjugates',$ D. R. Hwang,. M. E. Scott,+ and E. Hedaya Immunology Department, Research and Development Division, Technicon Instruments Corporation, Tarrytown, New York 10591. Received June 27, 1990
The preparation of immunoreactive derivatives of digoxin for analytical applications is most often carried out by periodate cleavage of the terminal sugar ring (digitoxose) followed by reaction with an enzyme, protein, carrier, or related biologicalmolecules. Here we report an improved and more efficient synthesis which was developed to provide digoxin-phospholipid conjugates useful for liposome immunoassay. The approach used involved the linking of the cleaved digitoxose through a carboxymethyl oxime functionality, which provides much improved yields of readily purified products. The synthetic modification should be applicable to the preparation of analogous phospholipid conjugates involving linkage through a sugar ring (digitoxin, ouabain, and related cardiac glycosides) or to those involving steroids (Le., 3-digoxigenone)which can be modified to form oxime derivatives remote from key functionalities important for immunorecognition by specific antibody. The characterization of the digoxinphospholipid conjugates with high-resolution NMR and fast atom bombardment mass spectrophotometry will also be discussed.
Digoxin is a potent cardiac glycoside. Toxic amounts of digoxin exert undesirable and potentially lethal electrophysiological effects (2). Accordingly, various immunoassay methods for cardiac glycosides are now widely used for determination of appropriate dosage schedules for patients receiving these drugs. Because digoxin is too small a molecule to be antigenic by itself, it is necessary to conjugate digoxin covalently as a hapten to an antigenic carrier, for example, human serum albumin (HSA),bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH), in order to elicit digoxin-specific antibodies in experimental animals for use in immunoassay. The preparation of immunoreactive digoxin derivatives is typically carried out by the procedures of Butler and Chen ( 3 )which is based on the work of Erlanger and Beiser ( 4 ) . The reaction sequence involved periodate cleavage of the terminal sugar ring (digitoxose or rhamnose) followed by reaction with an amino functionality from a protein carrier, enzyme, or related biological molecule, and finally reductive amination of the intermediate Schiff base with sodium borohydride. Thus, digoxin-HSA (3),digoxin-BSA (5), mellitin-ouabain (6),and digoxin-dibenzo-18-crown-6(7) conjugates have been prepared by using the aforementioned reaction sequence. We have attempted to adopt the procedure of Butler and Chen (3) to prepare diogoxin-dipalmitoyl phosphatidylethanolamine (digoxin-DPPE) conjugates for use in homogeneous liposome immunoassays for determination of haptens such as digoxin (9). In our hands, following the procedure of Butler ( 3 ) and Cole (8) gave a very complex mixture from the reaction, and thus the reaction
* To whom correspondence should be addressed.
Chemistry Department, University of Akron. Abbreviations: DPPE, dipalmitoyl phosphatidylethanolamine; HSA, human serum albumin; BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; TLC, thin-layer chromatography; IR, infrared spectroscopy;UV, ultraviolet spectroscopy; NMR, nuclear magnetic resonance;FAB,fast atom bombardmenk LPLC, low-pressure liquid chromatography. +
t
sequence via the Schiff base was not useful for our purpose. Here, we report an improved and more efficient synthesis for digoxin-DPPE conjugates which were immunoreactive and useful for liposome immunoassay. RESULTS AND DISCUSSION The synthetic sequence is outlined in Scheme I. The terminal digitoxose in digoxin is cleaved to give dialdehyde in quantitative yield by using sodium periodate under nitrogen atmosphere. The condensation reaction of digoxin dialdehyde and carboxymethoxylamine hemihydrochloride proceeds rapidly in sodium acetatelethanol under a nitrogen atmosphere. A quantitative yield of the bis[O(carboxymethyl)oxime] was obtained (11). The digoxin dioxime derivative was used immediately in the next reaction step, where the carboxy functionalities of the dioxime is reacted with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimideto give an active ester. The dioxime active ester is then condensed with a stoichiometric amount of dipalmitoyl phosphatidylethanolamine (DPPE) with gentle heating for 1 2 h. The reaction was monitored closely by TLC. Thin-layer chromatography with the solvent system chloroform/methanol/ water 7512513 by volume showed two major components a t R f 0.20 and 0.13 along with N-hydroxysuccinimide at Rj 0.30. The phospholipid moiety of the conjugates was detected by molybdenum spray (11). The excess N-hydroxysuccinimide was removed from the reaction mixture by preparative LPLC. Pure digoxin-diDPPE conjugate (0.1348 g, 20%; Rf 0.30) and digoxinmono-DPPE conjugate (0.1424 g, 26.5%; R f 0.15) were isolated from crude reaction mixture by preparative TLC in chloroform/methanol/water (7512513 by volume). Two other minor products were also isolated which were not identified. It should be noted that when twice the stoichiometric amount of dipalmitoyl phosphatidylethanolamine was used for condensation reaction with the digoxin dioxime active ester, only digoxin-di-DPPE was
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Bioconlugate Chem., Vol. 1, No. 5, 1990
Scheme I. Conjugates
Improved Synthesis of Digoxin-DPPE 0
Table I. NMR, UV, and IR Spectral Data for Digoxin-Mono-DPPE absorption description NMR (300 MHz, CDC13, ppm) singlet, 3 H, 18 CH3 0.8 singlet, 3 H, 19 CH3 0.96 triplet, 6 H, terminal methyl in 0.90 . group phospholipid 2.23-1.05 comDlex multblet, 83 H. 2 (CHdiz, 3 CHa (digitoxose iing), 3 CH, (digitoxose ring), 8 CH2 (digoxigenin ring), 2 CHz(CHz)1z two overlapping triplets, 4 H, 2 CHzCO 2.32 4.72-3.05 complex multiplet, 27 H, CHzOCOR, 9 CH (digitoxose ring protons), 6 CH (digoxigenin ring proton), glycero CHzOP protons, ethanolamine CHzOP, -OCH2CO-, and CHzNH multiplet, 5 H, CHz in lactone and 3 protons at 4.95 C1, Clg,and C1.f in digitoxose 5.25 multiplet, 1 H, CHOCOR singlet, 1 H, lactone, C=CH 5.95 ~
i
DIGOXIN
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Chart I. Structure of Digoxin-Phospholipid Conjugates DIGOXIN-MONO-DPPE H
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Table 11. NMR, UV, and IR Spectral Data for Digoxin-Di-DPPE absorption description NMR (300 MHz, CDCl3, ppm) singlet 3 H, 18 CH3 0.82 singlet, 3 H, 19 CH3 0.94 triplet, 12 H, terminal methyl group in 0.90 phospholipid multiplet, 8 H, 4 CHzCO 2.32 2.2-1.05 complex multiplet 4.75-2.8 complex multiplet multiplet, 5 H, CHz in lactone and 3 protons at 4.9 C1, Clt, and Cl,,, in digitoxose multiplet, 2 H, 2 CHOCOR 5.25 singlet, 1 H, lactone C=CH 5.95 241 (e 2071) 3427 2923 2853 1781 1743 1668
C H ~ O C O C H ~ C Hc~nI 2 ) p 3
DPPE
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= CH3 ( C H Z ) I 2 C H ~ C H ~ C O O C H I
CHZOP(O)OCH~CH~NH~ OH
formed (45%) along with some unreacted DPPE and degradation products of DPPE. The proof of the structure of the two major conjugates was extracted fro the IR, UV, and high-resolution proton NMR spectra. The structure of diogoxin-mono-DPPE is shown (Chart I) as one of the two possible positional isomers. At the present time, we are unable to distinguish these unambiguously because of the complexity of the
UV (Cay 219, CHC13, nm) maximum
IR (KBr, Perkin-Elmer 1430 Ratio Reading, cm-') broad OH
NMR spectra. The NMR, UV, and IR data are summarized in Tables I and 11. As a further confirmation of the structure, positive ion fast atom bombardment (FAB) mass spectra from pure conjugates in a thioglycerol matrix were obtained with an MS-50 high resolution mass spectrometer. The most intense peak appeared in the molecular ion region, representing the mlz of (M + metal)+ and the isotopically enriched species. The molecular ion for digoxin-mono-DPPE is 1621 (M + Na)+ and the molecular ion for digoxin-di-DPPE is 2311 (M + K)+. EXPERIMENTAL PROCEDURES
Materials and Methods. Digoxin and d,l-dipalmitoy1 phosphatidylethanolamine were obtained from Sigma and Biosynth AG, respectively. N-hydroxysuccinimide, dicyclohexylcarbodiimide,and carboxymethoxylamine hemihydrochloride were obtained from Aldrich. Molyb-
Bioconjugate Chem., Vol. 1, No. 5, 1990
Digoxin-Phospholipid Conjugates
DIGOXINa DIALDEHYDE
10
311
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Figure 2. NMR of (a) digoxin dialdehyde and (b) digoxin dioxime,
Figure 1. Digoxin-mono-DPPE (top) and digoxin-di-DPPE (bottom) are shown.
denum spray was prepared according to the procedure of Dittmer and Lester ( I I ) . FT NMR spectra were obtained with the 7 T spectrometer at the Rockefeller University. FAB mass spectra were obtained with the MS-50 highresolution mass spectrometer a t the Middle Atlantic Regional Mass Spectrometer Center at Johns Hopkins University. Preparative low-pressure liquid chromatography (LC) was carried out on a home-built LC system using a Kieselgel60 (200 g, 0.040-0.063 mm) glass column (2.5 X 50 cm). Digoxin Dialdehyde. Digoxin (0.4985 g, 0.64 mmol) is dissolved in 10 mL of chloroform/methanol(3/1.5)and placed into a 100-mL two-necked flask. Sodium periodate (0.3102 g, 1.4 mmol) is dissolved in a 4 mL of distilled water and placed into a pressure-equalized addition funnel. The periodate solution is slowly added to the flask while the reaction mixture is stirred under nitrogen. A white precipitate is immediately formed and the reaction is complete within 15 min after addition of the periodate. Reaction progress is monitored by TLC (E. M. Merck, precoated TLC sheets, silica gel 60 F254,0.2 mm thickness) in chloroform/methanol (10/1 by volume, Rf 0.16 = dialdehyde, one homogeneous spot; Rf 0.07 = digoxin). Both spots became dark brownish when the TLC plate is sprayed with methanol/concentrated sulfuric acid (9/ 1by volume) and placed in a 100 "C oven for 5 min. The reaction mixture is evaporated on a rotary evaporator and dissolved in 30 mL of chloroform and 3 mL of water. The cloudy solution is extracted and the aqueous layer is washed three times with 10 mL of chloroform. The organic phases are combined (60 mL) and dried over magnesium sulfate. The organic solvents are evaporated to dryness. A light yellow brownish, oily material is left. Satisfactory NMR spectra have been obtained. The aldehydic protons appear as two complex multiplets near 9.68 and 9.77 ppm (Figure 2a). The J values are relatively small. This material is used
immediately in the next reaction. No attempt was made to isolate the dialdehyde because of its lability. Digoxin Bis[ 0-(carboxymethyl)oxime] Carboxymethoxylamine hemihydrochloride (0.3119 g, 1.4 mmol) and sodium acetate (0.2260 g, 1.6 mmol) are dissolved in 3 mL of water and placed into a 50-mL two-necked flask. The digoxin dialdehyde, dissolved in 1.3 mL of methanol, is placed into a pressure-equalized funnel and slowly added to the flask while the reaction mixture is stirred under nitrogen. The reaction is completed within 10 min (TLC chloroform/methanol, 6/ 1by volume, Rf 0.09-0.13). The reaction mixture is evaporated to dryness and dissolved in 20 mL of ethyl acetate and 3 mL of water. The organic layer is separated and the aqueous layer is washed three times with 5.0 mL of ethyl acetate. The organic layers are combined and dried over anhydrous magnesium sulfate. The solution is filtered and evaporated to dryness. The residue is dried for 30 min under high vacuum (0.1 mmHg) and used immediately for the next step. Satisfactory elemental analysis and NMR spectra were obtained for the residual product. Elemental anal. for C45H~8018 N202H20 (calcd): C, 56.65 (56.22); N, 7.39 (7.56);0 33.22 (33.70); H, 2.91 (2.91). The aldehydic protons of the dioxime appeared as two multiplets near 6.94 and 7.5 ppm (Figure 2b). N-Hydroxysuccinimide Ester of Digoxin Bis[ 0(carboxymethyl)oxime]. Dicyclohexylcarbodiimide (DCC, 0.2805 g, 1.3 mmol) is dissolved in 6 mL of dry DMF and placed into a 50-mL two-necked flask. The solution is cooled in an ice-water bath (4 "C). Digoxin (bis[O(carboxymethyl)oxime], dissolved in 80 mL of DMF, is slowly added while the reaction mixture is stirred under nitrogen. Immediately afterward, N-hydroxysuccinimide (NHS) solution (0.1500 g, 1.3 mmol, in 6 mL of DMF) is likewise added. Reaction progress is monitored by TLC [chloroform/methanol/water, 75/25/3 by volume, Rf 1.0 (DCC), 0.75 (dioxime NHS active ester), 0.34 (NHS), 0.1 (dioxime)]. The reaction continues at 4 "C under nitrogen for 18 h. The white solid precipitate in the mixture is removed by filtration. The filtrate is then evaporated with a rotary evaporator under vacuum (0.1 mmHg). An oil material is left. The desired product possesses the following TLC characteristics: (1)homogeneous UV detectable spot (short wavelength), (2) the homogeneous spot turns brownish when the TLC plate is sprayed with methanol/concentrated sulfuric acid (9/ 1by volume) and warmed briefly in a 100 "C oven. Digoxin-DPPE Conjugates. The crude dioxime active ester (17 mL of reaction mixture) is placed into a 100-
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Bioconjugate Chem., Vol. 1, No. 5, 1990
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Figure 4. FAB MS of digoxin-di-DPPE.
mL two-necked flask. A suspension of DPPE (0.4431 g, 0.64 mmol, dispersed in 30 mL of dry chloroform and 0.87 mL of triethylamine) is placed into an addition funnel and slowly added to the flask while the reaction mixture is stirred under nitrogen and protected from light. The mixture was heated gently (40-50 "C) and this continues for 72 h. The reaction was monitored by TLC [solvent system chloroform/methanol/water, 7512513 by volume, Rf 0.75 (active ester), 0.52 (unknown l ) , 0.45 (unknown 2), 0.30 (NHS),0.21 (DPPE),0.20 (disubstituted conjugate), 0.13 (monosubstituted conjugate)]. The phospholipid moiety of the conjugates was detected by molybdate blue spray. The reaction mixture was evaporated and brought up in 10 mL of chloroform/methanol/water (21811). The N-hydroxysuccinimide was removed from the mixture by LPLC [Kieselgel, 200 g, glass column (2.5 cm X 50 cm), solvent system chloroform/methanol/water (21811 by volume)]. Pure mono- and disubstituted conjugates can be obtained by preparative TLC in chloforom/methanol/ water [75/25/3 by volume: Rf 0.30 (disubstituted), 0.15 (monosubstituted)]. Pure digoxin-di-DPPE conjugate (0.1349 g, 20 76) and digoxin-mono-DPPE conjugate (0.1424 g, 26.5%) were obtained. Two other minor products were also isolated. The structures of the minor products were not identified. The molecular ion for digoxin-monoDPPE is 1621 (M + Na)+ (Figure 3) and the molecular ion for digoxin-di-DPPE is 2311 (M K)+ (Figure 4).
tivatable, oxime intermediate. This intermediate overcomes the disadvantages inherent in the Butler et al. procedure discussed earlier. These include the propensity for the dialdehyde intermediate to undergo deleterious side reactions, particularly in the presence of amine derivatives of lesser reactivities such as phospholipids. An additional advantage is t h a t the improved procedure provides products which can be readily isolated, characterized, and purified, in contrast to that of Butler, which, to our knowledge, yields a sufficientlycomplex mixture, thwarting the desired product characterization. The objective of this work has been to provide digoxinphospholipid conjugates which are ultimately incorporated in liposome diagnostic reagents. It is interesting t o compare the digoxin-mono- and -di-DPPE conjugates prepared here with respect to their incorporation within a liposome bilayer membrane was well as their comparative impact on diagnostic utility, even though these considerations go beyond the scope of this preparative report. CPK models of the digoxin-mono- and -di-DPPE derivatives (see Figure 1)suggest that the di-DPPE derivatives should be more effectively incorporated in a bilayer membrane, owing its four fatty acid legs. Data bearing on this and related diagnostic applications will be reported separately. The synthetic method described here is also applicable to the preparation of analogous phospholipid conjugates involving linkage through a sugar ring such as digitoxin, gitoxin, ouabain, digitonin, and related cardiac glycosides, or those involving steroids (i.e., 3-ketodigoxigenin) which can be modified to form oxime derivatives
+
CONCLUSION An important advantage of the above synthetic procedure is the use of the relatively stable, storable, yet ac-
Digoxin-Phospholipid Conjugates
remote from key functionalities important for immunorecognition by specific antibodies. For example, digoxigenin-DPPE was prepared efficiently from digoxigenin 3- [ (carboxymethyl)oxime] and dipalmitoyl phosphatidylethanolamine via a NHS active ester. The digoxigenin 3-[(carboxymethyl)oxime] was prepared quantitatively from the reaction of 3-digoxigenone and carboxymethoxylamine hemihydrochloride. The preparation of 3-digoxigenone from digoxigenin was performed by previous methods (12). ACKNOWLEDGMENT
The technical assistance from Francis Picart of Rockefeller University is highly appreciated. D.H. extends his appreciation to Drs. Robert Cotler and David Heller of the Middle Atlantic Regional Mass Spectrometry Center a t Johns Hopkins University for the FAB mass spectra. LITERATURE CITED (1) Preliminary account of this study was present a t the 189th ACS National Meeting a t Miami Beach, Florida, 1985; Bioorganic section paper 229. (2) Hoffman, B. F., and Bigger, J. T., J r . (1980) in T h e Pharmacological Basis of Therapeutics, 6th ed. p 729, (A. G. Gilman, L. S. Goodman, and A. Gilman, Eds.) McMillian, New
York. (3) (a) Butler, V. P., Jr., and Chen, J. P. (1967) Digoxinspecific antibodies. Proc. Natl. Acad. Sci. U.S.A. 57, 71-78. (b) Butler, V. P., Jr., and Tse-Eng, D. (1982) In immunoassay of digoxin and other cardiac glycosides. Methods Enzymol. 84, 558-577. (4) Erlanger, B. F., and Beiser, S. M. (1964) Antibodies specific for ribonucleosides and ribonucleotides and their reaction with deoxyribonucleic acid (DNA). Proc. Natl. Acad. Sci. U.S.A. 52,68-74.
Bioconjugate Chem., Vol. 1, No. 5, 1990 313
(5) Smith, T. W., Butler, V. P., Jr., and Huber, E. (1970) Characterization of antibodies of high affinity and specificity for the ditigalis glycoside digoxin. Biochemistry 9, 331-337. (6) Freytag, J. W. and Litchfield, J. (1984) Liposome-mediated immunoassays for small haptens (digoxin) independent of complement. J . Immunol. Method 70, 133-140. (7) Keating, M. Y., and Rechnitz, G. A. (1984) Potentiometric digoxin antibody measurements with antigen-ionophore based membrane electrodes. Anal. Chem. 56, 801-806. (8) Cole Francis, X. U S . Patent 4,342,826, Aug. 3, 1982. [immunoassay products and methods (see examples IX and X) and references cited therein]. (9) Adolfsen, R., Hedaya, E., Mark, C., and Schwarzberg, M. US. patent 4,839,276, June 13, 1989 (interference-resistant lipsome specific binding assay). (10) There was only one product according to extensive TLC studies. Whereas, the NMR spectra of digoxin dioxime showed two multiplets (6.94 and 7.5 ppm) separated by 0.56 ppm which were assignable to the aldehydic proton. (Phillips, W. D. (1958) Studies of hindered internal rotation in organic molecules by nuclear magnetic resonance. Ann. N . Y . Acad. Sci. 70, 817832.) The existence of two separated proton resonances could be explained by the simultaneous existence of syn and anti isomers. Area ratio of proton resonances provided the efficient determination of the equilibrium concentration of the isomers. The isomer ratio (syn/anti) was calculated to be 2. (11) Ditter, J. D., and Lester, R. L. (1964)A simple, specific spray for the detection of phospholipids on thin-layer chromatograms. J . Lipid Res. 24, 126. (12) (a) Shimiju, Y., and Mitusuhashi, M. (1968) Studies on C-NOR-D homosteroids-1 X solvolysis of 14B-hydroxy-12Btosyloxy steroids. Tetrahedron 24, 4207. (b) Tamm, Ch., and Gubler, A. (1959) Microbiological conversion of cardiac active glycosides and aglycones. Chimia 13, 116-117.
