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peptide synthesis

Fmoc-ACA-OH is a useful tool for the SPPS of AMC fluorogenic protease substrates. Fmoc-ACA-OH [1] must be first loaded onto a Wang-type resin. Any unreacted linker hydroxyls must be capped using acetic anhydride. Following Fmoc removal, the first amino acid should be coupled withy HATU/collidine [2]. Insertion of ACA into the middle of a peptide provides fluorogenic substrates for endopeptidases [3]. Treatment of the ACC peptide obtained after TFA cleavage with aqueous base causes facile decarboxylation and formation of the AMC peptide.

reaction type: Fmoc solid-phase peptide synthesis

The general procedure for the synthesis of (7-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-oxo-2H-chromen-4-yl)acetic acid from methyl chloroformate-9-fluorenyl and 7-amino-2-oxo-2H-1-chromen-4-acetic acid was as follows: 7-amino-2-oxo-2H-1-chromen-4-acetic acid (1.60 g, 5.6 mmol), NaOH (2.24 g, 56 mmol) and H2O (80 mL) were added to a 250 mL round bottom flask. The mixture was heated to reflux and kept overnight. Upon completion of the reaction, the reaction was quenched with water. Subsequently, the pH of the reaction mixture was adjusted to 2 with 4 M HCl and extracted three times with a solvent mixture of THF:EtOAc (5:1). The organic phases were combined and concentrated to afford the crude product 2-(7-amino-2-oxo-2H-chromen-4-yl)acetic acid (0.96 g, 4.38 mmol), which could be used directly in the next step of the reaction. 2-(7-Amino-2-oxo-2H-chromen-4-yl)acetic acid (1.00 g, 4.5 mmol) was dissolved in CH2Cl2 and TMSCl (1.45 g, 13.5 mmol) and DIPEA (1.74 g, 13.5 mmol) were added sequentially. The reaction mixture was heated to reflux for 3 hours. After completion of the reaction, the mixture was cooled in an ice bath. Fmoc-Cl (1.4 g, 5.4 mmol) was then added and stirred overnight at room temperature. At the end of the reaction, the reaction was quenched with 2M HCl (pH adjusted to 2) and extracted three times with a solvent mixture of THF:EtOAc (5:1). The organic phases were combined and concentrated, and the crude product was purified by column chromatography (THF:PE = 2:1 with 1.5% AcOH, Rf = 0.4) to afford the target compound Fmoc-ACC-OH (1.43 g, 3.24 mmol, 58% yield in two steps) as a white solid. The structure of the product was confirmed by 1H NMR, 13C NMR and HRMS.

[1] Journal of Organic Chemistry, 2002, vol. 67, # 3, p. 910 - 915
[2] Patent: US2002/22243, 2002, A1
[3] Patent: WO2005/110453, 2005, A2. Location in patent: Page/Page column 52
[4] Tetrahedron, 2016, vol. 72, # 27-28, p. 4085 - 4090