反应详情

Diphenyl N-(6-biotinamidohexanoyl)amino(4-amidinophenyl)methanephosphonate (1) was prepared from diphenyl amino(4-amidinophenyl)methanephosphonate (19,20) and 6-biotinamidohexanoic acid (Anaspec; San Jose, Calif.) by the aid of PyBOP (Novabiochem; San Diego, Calif.). The HPLC-purified material [retension time 20.76 min, purity 95% (220 nm); YMC ODS-AM column (4.6×250 mm), 0.05% TFA in water (A):0.05% TFA in acetonitrile (B) 90:10 to 20:80 in 45 min (1.0 m/min)] was characterized by ESI-MS [Observed m/z 721.3 (MH+; calculated MH+ for C36H45N6O6PS, 721.3)] and stored as 10 mM solution in DMF at −70° C. The active ester 2 was prepared by acylating the same precursor amine with disuccinmidyl suberate (Pierce; Rockford, Ill.) and characterized in the same way [Observed m/z, 635.3 (MH+; calculated MH+ for C32H35N4O8P, 635.2)]. VIP-CRA (3) was synthesized as follows. The VIP sequence with N-terminal biotin was constructed on Rink arnide MBHA resin (0.72 mmol/g; Novabiochem) by the standard 9-fluorenylmethoxycarbonyl protocol (21) except that 4-methyltrityl (22) was used for side-chain protection of Lys20. The peptide-resin was treated with 1% TFA in dichloromethane (5 min×10) to remove the 4-methyltrityl group and the deprotected amino group of Lys20 was acylated with 2 in 1-methyl-2-pyrrolidinone containing 0.1 mM N,N-diisopropylethylamine. The peptide resin was treated with TFA-ethanedithiol-thioanisole-phenol (90:1:1:8) at room temperature for 2 h. After removing the resin by filtration, diethyl ether was added to the solution to afford a precipitate, which was collected by centrifugation and washed with diethyl ether. The HPLC-purified material [retention time 50.25 min, purity 96% (220 nm); Vydac 214TP C4 column (4.6×250 mm); A:B 90:10 to 60:40 in 60 min (1.0 ml/min)] was characterized by ESI-MS [Observed nlz, 4071.4 (MH+; calculated MH+ for C185H282N49O49PS2, 4072.0) and stored as 10 mM solution in DMSO at −70° C.

1. customThe active ester 2 was prepared
2. customto remove the 4-methyltrityl group
3. additionThe peptide resin was treated with TFA-ethanedithiol-thioanisole-phenol (90:1:1:8) at room temperature for 2 h
4. customAfter removing the resin
5. filtrationby filtration, diethyl ether
6. additionwas added to the solution
7. customto afford a precipitate, which
8. customwas collected by centrifugation
9. washwashed with diethyl ether
10. customstored as 10 mM solution in DMSO at −70° C.