Synthesis of Neomycin Analogs to Investigate Aminoglycoside-RNA Interactions

Abstract

A series of novel aminoglycoside oligosaccharide analogs containing a 2,5-dideoxystreptamine core scaffold was prepared to study aminoglycoside binding to the small subunit of 16S rRNA. A set of monosaccharide building blocks carrying amino groups in different positions and conformations around the pyranose ring was utilized in the assembly of oligosaccharides. These aminoglycoside analogs were analyzed for their RNA-binding capacity using a high throughput mass spectroscopy assay.

aminoglycosides - antibiotics - oligosaccharides - RNA-targeting

References

1a

Present address: Aventis Pharma Deutschland GmbH, 65926 Frankfurt, Germany

1b

Present address: Provid Pharmaceuticals, Piscataway, NJ 08854, USA

2 Pearson ND. Prescott CD. Chem. Biol.  1997,  4:  409 

3 Baumann M. Bischoff H. Schmidt D. Griesinger C. J. Med. Chem.  2001,  44:  2172 

4 Michael K. Tor Y. Chem.-Eur. J.  1998,  4:  2091 

5 Stage TK. Hertel KJ. Uhlenbeck OC. RNA  1995,  1:  95 

6 Sears P. Wong C.-H. Angew. Chem. Int. Ed.  1999,  38:  2300 

7 Hendrix M. Priestley ES. Joyce GF. Wong C.-H. J. Am. Chem. Soc.  1997,  119:  3641 

8 Tor Y. Hermann T. Westhof E. Chem. Biol.  1985,  5:  277 

9 Carter AP. Clemons WM. Brodersen DE. Morgan-Warren RJ. Wimberly BT. Ramakrishnan V. Nature  2000,  407:  340 

10 Alper PB. Hendrix M. Sears P. Wong CH. J. Am. Chem. Soc.  1998,  120:  1965 

11 Kavadias G. Velkof S. Belleau B. Can. J. Chem.  1978,  56:  404 

12a Vasella A. Witzig C. Chiara JL. Martin-Lomas M. Helv. Chim. Acta  1991,  74:  2073 

12b Lemieux R. Cushley S. Can. J. Chem.  1963,  43:  858 

12c Baeschlin DK. Chaperon AR. Green LG. Hahn MG. Ince SJ. Ley SV. Chem.-Eur. J.  2000,  6:  172 

12d Contour MO. Deaye J. Little M. Wong E. Carbohydr. Res.  1989,  193:  283 

13

7: R_f = 0.81 (hexanes-EtOAc, 1:1); ¹H NMR (300 MHz, CDCl₃): δ = 7.40-7.28 (m, 10 H), 5.25 (d, J = 3.0 Hz, 1 H), 4.83 (dd, J = 10.1, 10.1 Hz, 2 H), 4.79 (dd, J = 10.2, 10.2 Hz, 2 H), 4.51 (d, J = 9.4 Hz, 1 H), 4.44 (dd, J = 6.3, 10.2 Hz, 1 H), 4.27 (dd, J = 6.8, 10.4 Hz, 1 H), 3.91 (dd, J = 6.6, 6.6 Hz, 1 H), 3.69 (dd, J = 3.0, 9.3 Hz, 1 H), 3.58 (dd, J = 9.4, 9.4 Hz, 1 H), 3.07 (s, 3 H), 3.03 (s, 3 H), 2.75 (m, 2 H), 1.32 (t, J = 7.4 Hz, 3 H); ¹³C NMR (75 MHz, CD₃Cl): δ = 137.7, 137.0, 128.8, 128.6, 128.5, 128.4, 128.2, 85.9, 80.5, 76.1, 76.0, 74.1, 73.6, 67.1, 39.4, 37.8, 25.5, 15.5; HRMS (FAB⁺) Calcd for m/z C₂₄H₃₂O₉S₃Na [(M + Na)⁺] 583.1106, found 583.1112. 9: R_f = 0.38 (hexanes-EtOAc, 1:1); ¹H NMR (300 MHz, CDCl₃): δ = 7.41 -7.30 (m, 10 H), 4.90 (dd, J = 11.1, 11.1 Hz, 2 H), 4.57 (dd, J = 11.1,11.1 Hz, 2 H), 4.32 (d, J = 9.5 Hz, 1 H), 3.74 (dd, J = 2.3, 4.5 Hz, 1 H), 3.78-3.65 (m, 2 H), 3.44 (dd, J = 4.7, 9.6 Hz, 1 H), 3.38 (dd, J = 6.7, 6.7 Hz, 1 H), 3.36 (m, 1 H), 2.82 (br s, 1 H), 2.75 (m, 2 H), 1.32 (t, J = 7.4 Hz, 3 H); ¹³C NMR (75 MHz, CD₃Cl): δ = 138.2, 137.8, 128.8, 128.7, 128.4, 128.3, 128.1, 128.0, 84.7, 84.5, 78.0, 75.5, 73.9, 72.6, 70.7, 65.6, 24.9, 15.3; HRMS (FAB⁺) Calcd for m/z C₂₂H₂₇N₃O₄SNa [(M + Na)⁺] 452.1619, found 552.1624. 10: R_f = 0.53 (hexanes-Et₂O, 1:2); ¹H NMR (300 MHz, CDCl₃): δ = 7.40-7.32 (m, 10 H), 4.72 (dd, J = 11.6, 11.6 Hz, 2 H), 4.57 (s, 2 H), 4.24 (d, J = 10.3 Hz, 1 H), 4.08 (d, J = 3.2 Hz, 1 H), 3.77-3.67 (m, 2 H), 3.54 (dd, J ₁ = 7.1, 7.1 Hz, 1 H), 3.40 (dd, J = 3.1, 9.4 Hz, 1 H), 2.75 (m, 2 H), 1.31 (t, J = 7.2 Hz, 3 H); ¹³C NMR (75 MHz, CD₃Cl): δ =138.0, 137.2, 128.8, 128.7, 128.5, 128.3, 128.1, 128.0, 84.2, 81.3, 77.2, 73.9, 72.1, 69.4, 65.8, 62.1, 24.6, 15.2; HRMS (FAB⁺) Calcd for m/z C₂₂H₂₇N₃O₄SNa [(M + Na)⁺] 452.1619, found 552.1634.

14 Greenberg WA. Priestley ES. Sears PS. Alper PB. Rodenbohm C. Hendrix M. Hung S.-C. Wong C. J. Am. Chem. Soc.  1999,  121:  6527 

15 Garegg PJ. Adv. Carbohydr. Chem. Biochem.  1997,  52:  179 

16

Glycosylation. General procedure. Thioethyl glycoside (0.1 mmol) and acceptor 2 (0.1 mmol) were coevaporated with toluene and dried under vacuum. Et₂O (1.5 mL), CH₂Cl₂ (0.5 mL), toluene (0.2 mL), and 4 Å MS (40 mg) were added and the mixture was cooled to -40 °C. Addition of NIS (0.071 mmol) and AgOTf (0.020 mmol) followed. The mixture was stirred for 5 h at -40 °C, EtOAc (20 mL) was added and the filtrate was washed with Na₂S₂O₃ (5% aqueous, 5 mL) and dried over MgSO₄. After removal of the solvents, the residue was purified by silica gel column chromatography to afford the disaccharides as two diastereomers.

17 Deprotection. General procedure. Protected di- or trisaccharide (0.052 mmol) was dried 48 h at high vacuum, dissolved in MeOH (5 mL) and stirred at room temperature with Pd(OH₂)/C (60 mg) while bubbling hydrogen through the mixture. After 5 h AcOH (2 mL, 50% aqueous) was added to the solution and the mixture was hydrogenated for an additional 2 h. The catalyst was filtered off and the solvent was removed under reduced pressure. The residue was dissolved in water (5 mL) and freeze dried to afford the deprotected aminoglycoside.

18 Hofstadler SA. Griffey RH. Chem. Rev.  2001,  101:  377