Acetic Anhydride → Ninhydrin

After removal of the Fmoc group, N-α-Fmoc-O-t-butyl-L-threonine (1.9880 g, 5.00 mmol) was coupled by mixing a solution of the amino acid in 0.5 M HOAt in DMF (10.0 mL, 5.00 mmol) and DIC (0.6360 g, 5.04 mmol) with the deprotected peptidyl-resin for 16 hours. After DMF and DCM washes (both 4×12 mL×1 min.), a sample of wet peptidyl-resin was cleaved using 1 ml of TFA/triisopropylsilane/water (96:2:2) for 1.5 hours and showed, upon HPLC and MS analysis, only a very small amount of uncoupled peptide. The peptide-resin was treated with 10% acetic anhydride in DCM (1×12 mL×1 hr.) and then washed with DCM (6×12 mL×1 min.). After removal of the Fmoc group, N-Fmoc-glycine (0.8941 g, 3.01 mmol) and N-α-Fmoc-L-glutamic acid γ-t-butyl ester (1.2765 g, 3.00 mmol) were successively coupled for 2 hrs as solutions with HOBt monohydrate (0.4604 g, 3.01 mmol) and DIC (0.3885 g, 3.08 mmol) in DMF/DCM (1:1) (20 ml). Negative Kaiser ninhydrin tests were obtained after each coupling step. The two peptidyl-resins thus obtained were each transferred to separate 35 mL syringes fitted with frits and dried in vacuo. The synthesis was then continued with 0.28 mmol of each peptidyl-resin. After Fmoc removal, a solution of N-α-Fmoc-α-methyl-L-proline (0.3943 g, 1.12 mmol) in 0.5 M HOAt in DMF (2.24 mL, 1.12 mmol) and DIC (0.1413 g, 1.12 mmol), diluted with an additional 8 mL of DMF, was divided into two equal volumes, which were added to each of the deprotected peptide-resins. After vortexing for 12 hrs and washing with DMF and DCM (both 3×8 mL×1 min.), 0.02 mmol aliquots of the peptidyl-resins were placed into 3-4 reaction wells on an Advanced ChemTech Ω396 Synthesizer. The Fmoc group was removed as follows:

1. customboth 4×12 mL×1 min.
2. customfor 1.5 hours
3. washwashed with DCM (6×12 mL×1 min.)

ORD record

ord-67804c8913a8442a9f49294adacfae0e

Source dataset

uspto-grants-2011_06

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US07960349B2