Methodology for the rapid separation of gentamicin components and regiospecific synthesis of gentamicin conjugates

Tetrahedron Letters

Volumn 53, Issue 50, 2012, Pages 6751-6754

  Grote, Jonathan ^a   Himmelsbach, Richard ^a   Johnson, Don ^a  

^a ABBOTT LABORATORIES   (United States)

Author keywords

Conjugation; Gentamicin; Immunoassay

Indexed keywords

GENTAMICIN;

ARTICLE; CATALYSIS; CONJUGATION; DRUG DECOMPOSITION; DRUG SYNTHESIS; FREEZE DRYING; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY;

EID: 84868602700     PISSN: 00404039     EISSN: 18733581     Source Type: Journal    
DOI: 10.1016/j.tetlet.2012.09.113     Document Type: Article

References (18)

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11. Preparation of the fully protected gentamicin component mixture: Gentamicin sulfate (Sigma; 6.0 g, 8.6 mmol) was dissolved in water (100 mL) with stirring. Sodium carbonate (9.2 g, 85.7 mmol) was added, followed by a solution of benzyl chloroformate (8.0 mL, 56.0 mmol) in acetonitrile (100 mL) slowly via addition funnel. After stirring overnight, no gas evolution was evident. The reaction was diluted with 200 mL water, and extracted 3 × 250 mL with ethyl acetate. The combined extracts were dried over sodium sulfate (30 g), filtered through Celite 521, and concentrated to produce 11.384 g of a colorless gum after 24 h on high vacuum.
12. Initial experimentation utilized a 4.6 × 150 mm YMC ODS-AQ analytical HPLC column employing acetonitrile/dilute aqueous trifluoroacetic acid mixtures, accompanied by a 47 × 300 mm YMC ODS-AQ (compression cartridge) preparative HPLC column employing acetonitrile/aqueous ammonium formate mixtures. Since the 47 × 300 mm preparative column became unavailable, subsequent experimentation was then completed on a 50 × 250 mm YMC ODS-AQ steel column.
13. To isolate fully protected gentamicin C1a, portions of the fully protected gentamicin component mixture were separated by preparative HPLC on a 50 × 250 mm YMC ODS-AQ column using a 70:20:10 mixture of acetonitrile:water:0.01 M ammonium formate as eluent (detection at 215 nm). The combined product fractions were concentrated on a rotovap to remove most of the acetonitrile, frozen, and lyophilized to provide a white solid. To individually isolate all three (C1a, C2/C2a, and C1) of the fully protected gentamicin components, the component mixture was separated on a 47 × 300 mm YMC ODS-AQ column using a 63:27:10 mixture of acetonitrile:water:0.1 M ammonium formate as eluent (detection at 215 nm), followed by concentration and lyophilization to yield the separated components as white solids.
14. +.
15. 3COOH: C 44.56, H 4.73, N 6.54. Found C 44.72, H 4.94, N 6.47.
16. El Kouhen, R.; Hoffman, A.; Baugher, W.; Krishnan, K.; Rygiel, C.; Novotny, M.; Storck-Schattner, D.; Xu, L.; Grote, J.; Merchant, B.; Wang, Y.; Naik, N.; Wong, P.Y.; Ford, K.; Wiesner, D. American Association of Clinical Chemistry Annual Meeting, Anaheim, CA, July 2010, Session 21, Poster E57.
17. Grote, J. U.S. Patent Application 2,011,029,4994.
18. Preliminary communication: Grote, J.; Himmelsbach, R.; Johnson, D.; El Kouhen, R.; Baugher, W. Eleventh Tetrahedron Symposium, Beijing, China, June 2010, Poster PSB13.