Fmoc-Gly-Pro-Hyp was synthesized from Gly-Pro-Hyp as follows: 3.0 g Gly-Pro-Hyp (10.5 mmol) was dissolved in 54 mL Na2CO3 --H2O (1:9) and stored at 4° C. 4.05 g 9-fluorenylmethyl succinimidyl carbonate (12.0 mmol) was dissolved in 45 mL dimethoxyethane and stirred at 4° C. The aqueous Na2CO3 solution was added slowly to the dimethoxyethane solution, and the reaction proceeded for 2.5 h at 4° C. and 21 h at room temperature. The solution was filtered, and 360 mL H2O was added to the filtrate. The aqueous layer was extracted with 300 mL of diethyl ether, acidified to pH 2 with concentrated HCl, reduced to half volume at 80° C. under reduced pressure, and stored at 4° C. for 24 h. The aqueous layer was decanted from the oily precipitate, reduced to ~30 mL at 81° C. under reduced pressure, and stored at 4° C. for 24 h. The aqueous layer was decanted from the oily precipitate. Both oily precipitates were dissolved in a total of 20 mL methanol, then 250 mL ethyl acetate was added. A white residue was recovered by evaporation at 73° C. for 1 h under reduced pressure; yield 3.23 g (6.39 mmol, 60.6%). The identity of the product as Fmoc-Gly-Pro-Hyp and its homogeneity was verified by thin layer chromatography [chloroform-methanol-HOAc (95:20:3)], scanning UV spectroscopy, and Edman degradation sequence analysis.
