Novel biotin-specific monoclonal antibody and use thereof
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example 1
Derivatization of Biotin at the C5 Atom of the Thiophene Ring and Synthesis Steps to Obtain an Immunogen
[0178]1.1
From biotin [1] to compound [2]; see FIG. 2 A
(S)-5-((1S,6R)-2-Oxo-hexahydro-thieno[3,4-d]imidazol-6-yl)-pentanoic Acid Methyl Ester [2]
[0179](S. A. Sundberg, R. W. Barrett, M. Pirrung, A. L. Lu, B. Kiangsoontra, C. P. Holmes J. Am. Chem. Soc. 1995, 117(49), 12050-12057) Acetyl chloride (7.50 mL) was slowly added to stirred dry methanol (160 mL) at 0° C. under an argon gas atmosphere. The resulting mixture was stirred for 30 min at room temperature. Then, biotin ([1], 6.00 g, 24.6 mmol) was added in one portion and stirring was continued overnight. After concentrating in vacuo, the crude product was re-dissolved in a mixture of CH2Cl2 and methanol (200 mL, v / v=95:5) and washed with saturated aqueous NaHCO3 solution. The aqueous phase was then re-extracted with a mixture of CH2Cl2 and methanol (100 mL, v / v=95:5, 3 times). The combined organic layers were dried over Na2SO4 a...
example 2
Synthesis of a Reagent for Screening Monoclonal Antibodies In Vitro
[0303]2.1
[0304]The screening reagent [30] was synthesized by using standard reaction conditions, by reacting the NHS ester [24] described above with an amino derivative of digoxigenine like Card-20(22)-enolide, 3-[2-[[2-[2-(2-aminoethoxy)ethoxy]ethyl] amino]-2-oxoethoxy]-12,14-dihydroxy-, (3β,5β,12β)-(9CI), CAS Registry Number 185523-10-8, which was synthesized as described in EP 0747447, with the NHS ester described above in DMF for 2 h at room temperature. Afterwards the solvent was removed and the product isolated by preparative HPLC chromatography. The structure of [30] is also shown in FIG. 2L.
2.2
5-[(3aS,4S,6R,6aR)-6-(2-{2-[2-(2-Amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-2-oxo-hexahydro-thieno[3,4-d]imidazol-4-yl]-pentanoic Acid
[0305]Biotin derivative [16] (5.00 mg, 11.4 μmol, 1.00 eq.) was dissolved in methanol (1.0 mL) under an argon gas atmosphere. Thereto, Pd / C (17 mg, 1.50 eq., 10 wt-% Pd) was added and the resu...
example 3
Derivatization of Biotin at the N′1 Atom and Synthesis of a Screening Reagent
(1) FMOC-Beta-Alanine-Acid Chloride
[0313]To 4.7 g FMOC-beta-alanine (IRIS, FAA1300) in a flask under dry conditions ca. 20 mL thionyl chloride were added slowly. The mixture was stirred at room temperature for 50 min. and afterwards heated to reflux for 10 min. After cooling, the mixture was evaporated, the residue was dissolved in absolute toluene three times and evaporated each time.
[0314]The yield was 5.1 g.
(2) FMOC-Beta-Alanyl-Biotin
[0315]The carboxylic acid of biotin was protected by reacting biotin (2.48 g) with t-butylchlorodiphenylsilane (6.5 ml) in 15 mL dry pyridine in the presence of DMAP (0.63 g) overnight at room temperature in an inert atmosphere of argon as described by Fang and Bergstrom in Nucleic Acids Research, 2003, Vol. 31, No. 2, 708.
[0316]4.9 g FMOC-beta-alanine-acid chloride dissolved in 10 mL dichloromethane were added and stirred for 3.5 h at room temp. The mixture was evaporated, ...
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