反应详情

Biotin (1.0 g, 4.1. mmol) was placed in a 100 mL round-bottom flask and dissolved in 25 mL of anhydrous dimethyl formamide (DMF) (dried by storing over heat activated (120° C. overnight) molecular sieves 4A, 8-12 mesh for 48 hours). To this solution was added N-hydroxy succinimide (0.71 g, 6.2 mmol) and dicyclohexyl carbodiimide (1.0 g, 4.9 mmol). The reaction mixture was stirred at room temperature for 48 hours under nitrogen. Analytical TLC (85:15 chloroform:methanol) of the reaction showed nearly complete conversion of biotin to a less polar biotin derivative (detection of biotin and biotin derivatives on TLC plates was carried out using a spray reagent consisting of 0.2% (w/w) 4-dimethylaminocinnamaldehyde in acidified ethanol available from Sigma Chemical Company, Product No. D-3588) with Rf =0.68, presumed to be the N-hydroxy succinimidyl ester of biotin. A solution of ethyl N-[(4-aminobutyl)oxy]acetimidate (3) (1.08 g, 6.2 immol) in 15 mL of dry DMF was added and the reaction was stirred under nitrogen at room temperature for 24 hours. Analytical TLC (85:15 chloroform:methanol) showed two main products containing biotin, Rf =0.60 and Rf =0.56. The solvent was removed on a rotary evaporator and absolute ethanol (50 mL) was added to the residue. Not all of the material dissolved, and the solution was filtered from a white solid, dicyclohexyl urea, which was discarded. The ethanolic solution was diluted with 50 mL of deionized water and the resulting white precipitate was dissolved by heating the solution briefly. Upon cooling to room temperature, white crystals formed and the solution was filtered. The crystals were discarded and the filtrate was evaporated to about half the original volume on a rotary evaporator and filtered again. The solid was discarded and the filtrate was further reduced to one fourth of the original volume and filtered again. Finally, the remaining solvent was evaporated to yield ethyl N-[(4-biotinamidobutyl)oxy]acetimidate (4) (0.431 g, 1.07 mmol, 26%). Single spot on TLC, Rf =0.56. 1H NMR (DMSO-d6): δ7.7 (m, 1H), 6.5 (s, 1H), 6.4 (s, 1H), 4.3 (m, 1H), 4.1 (m, 1H), 3.9 (q, 2H), 3.7 (m, 2H), 3.1 (m, 4H), 2.7 (dd, 2H), 2.5 (d, 2H), 2.1 (t, 2H), 1.7 (s, 3H), 1.3-1.5 (m, 8H), 1.2 (t, 2H).

1. stirringthe reaction was stirred under nitrogen at room temperature for 24 hours
2. customThe solvent was removed on a rotary evaporator and absolute ethanol (50 mL)
3. additionwas added to the residue
4. dissolutionNot all of the material dissolved
5. filtrationthe solution was filtered from a white solid, dicyclohexyl urea, which
6. additionThe ethanolic solution was diluted with 50 mL of deionized water
7. dissolutionthe resulting white precipitate was dissolved
8. temperatureby heating the solution briefly
9. temperatureUpon cooling to room temperature
10. customwhite crystals formed
11. filtrationthe solution was filtered
12. customthe filtrate was evaporated to about half the original volume
13. customon a rotary evaporator
14. filtrationfiltered again
15. filtrationfiltered again
16. customFinally, the remaining solvent was evaporated