Facile Synthesis of a Carboxylic Linker on Halofuginone: A Method to Derivatize Haptens Containing Both Amine and Hydroxy Functionalities

References

• Stenorol , T M . 1991 . Handbook of Approved New Animal Drug Applications in the United States, R12–1–1991Hoechst-Roussel Agri-Vet, Somerville, NJ , Edited by: Shepard , M. L. 3 – 9 . Dallas : Shotwell and Carr, Inc. .
   Google Scholar
• Congress , U. S. 1988 . Office of Technology Assessment. Pesticide Residues in Food: Technologies for Detection, OTA-F-398 , 37 Washington, DC : U.S. Government Printing Office .
   Google Scholar
• Golden , C. A. 1991 . J. Am. Vet. Med. Assoc. , 198 : 827
   PubMedGoogle Scholar
• Brunswick , D. J. , Needelman , B. and Mendels , J. 1978 . Life Sciences , 22 : 137
   PubMedGoogle Scholar
• Oliver , G. C. Jr. , Parker , B. M. , Brasfield , D. L. and Parker , C. W. 1968 . J. Clin. Invest. , 47 : 1035
   PubMed Web of Science ®Google Scholar
• Grierson , B. N. , Allen , D. G. , Gare , N. F. and Watson , I. M. 1991 . J. Agric. Food Chem. , 39 : 2327
   Google Scholar
• 1985 . Practice and Theory of Enzyme Immunoassays , 41 – 280 . New York : Elsevier . The succinic acid moiety both links and moves the hapten away from the protein surface to improve its recognition by the immune system. Tijssen P.
   Google Scholar
• Staros , J. V. , Wright , R. W. and Swingle , D. M. 1986 . Anal. Biochem. , 156 : 220
   PubMed Web of Science ®Google Scholar
• Reaction of 1 with succinic anhydride in a molar ratio of 1.1:1 gave a single product, and about 50% of 1 was unreacted. Negative methane CI MS9 of the methylated product gave the dimethyldisuccinate of 1 minus a proton at mz 640 [M-1]−, the loss of a methyl group at m/z 625 [M-1-CH3]−, and the loss of a methoxy group at m/z 609 [M- 1 -OCH3]−
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• All MS measurements were performed on a double-focusing VG 70–250 EHF spectrometer, and the data were recorded on a VG 11–250 data system, Fisons Instruments, Danvers, MA 01923.
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• 1H-NMR (300 MHz, DMSO-d 6) δ 8.79(IH, s, br, NH), 8.37(1H, s, H2), 8.32(1H, s, H5), 8.27(1H, s, H8), 5.72(1H, s, br, OH), 5.16(2H, s, H1′), 3.5(1H, m, H3″), 3.37(1H, m, H2″), 3.26(2H, m, H3′), 2.99(2H, m, H6″), 1.96(2H, m, H4″), 1.63(2H, m, H5″). These assignments were based on COSY, DEPT, and HMQC experiments and comparison with known proton chemical shift values.
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• Separation was done on Pre-Concentration Zone Silica Gel 60 F254 TLC plates (Merck No. 13728) with CHC13/MeOH 1:1 kept over NaHCO3. In addition to quenching of short wave UV light (254 nm), 1, 2, 3, and 4 were visualized by the application of 2,6-dichlorophenol-iodophenol spray reagent which, following the brief application of heat, rendered 1 and 2 dark blue and 3 and 4 pink against a light blue background. Stahl E. Thin-Layer Chromatography: A Laboratory Handbook, Springer-Verlag, New York1969 p 867.
   Google Scholar
• Other significant ions from 1 in negative methane CI MS are m/z 257(78) [M-C5H9NO-C3H5O]−, 259(100), 261(27), 314(4) [M-C5H9NO]−, 316(4.5), 318(1.5), 395(1.8) [M-H2O]−, 397(2.6), 399(1.0), 415(9.5), and 417(2.3).
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• The solution used here is the initial solution of halofuginone·HBr in DMF
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• Horning , M. G. , Moss , A. M. and Horning , E. C. 1967 . Biochim. Biophys. Acta , 148 : 597 N-trimethysilylimidazole reacts smoothly with hydroxyls but not with amines.
   PubMed Web of Science ®Google Scholar
• Horning , M. G. , Moss , A. M. , Boucher , E. A. and Horning , E. C. 1968 . Anal. Lett , 1 : 311
   Google Scholar
• If the amount of 1 is kept near the limit of solubility (100 mg halofuginone·HBr/12.5 mL DMF), the amount of N-trimethylsilylimidazole used does not exceed 0.0135 mL/mg of halofuginone·HBr, and the appropriate amount of H2O (0.65 mL/mg of halofuginone·HBr) is added, then 2 will precipitate in high yield, almost all of which will stick to the walls of the glass container.
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• Other significant ions from 2 in negative methane CI MS are m/z 259(100) 261(74), 316(10.9), 318(3.6), 487(4.6), and 489(1.8).
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• The amount of spontaneous hydrolysis of the trimethylsilyl group was usually small. This reaction is thought to be due to the acid moiety of 3.
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• This and other reactions of this study were unsuccessful when attempted in MeOH resulting in degradation of halofuginone or halofuginone-containing intermediates.
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• Chromatographic separation was performed on Pre-Concentration Zone Silica Gel 60 F254 TLC plates (Merck No. 13728) with CHC13/EtOAc 5:95 kept over NaHCO3. The methyl ester of 3 was visualized via quenching of short wave UV light.
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• Other significant ions from the methyl ester of 3 in negative methane CI MS are m/z 257(77) [M-C5H9NO-C3H5O]−, 259(100), 261(25), 601(1.4), and 603(0.5).
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• Dowex 50X8–100 ion-exchange resin was converted to the H+ form by HCl followed by washing with H2O and then DMF. A modification of Beier R.C., Mundy B.P., Synth. Comm. 1979, 9, 271. Product 4 remains stable for at least 3 months in DMF.
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• FAB MS was conducted with a saddle-field cold cathode ion gun and stainless steel targets (6 × 1.5 mm). Xenon gas was used as the particle source and the ion gun was adjusted to 8 kV and 1 mA current. The sample was applied in DMF (1.5 μl) to the glycerol matrix on the target.
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• Other significant ions from 4 in negative FAB MS are m/z 259(100), 261(28), 514(48), and 516(14).
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• Fifty-percent inhibition of control values for the monoclonal antibodies ranged from 4 to 240 ng halofuginone·HBr/mL in a competition enzyme-linked immunosorbent assay (cELISA).
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