A new synthetic protocol for coumarin amino acid

The hydrochloride of the racemic amino acid (2-(7-hydroxycoumarin-4-yl)ethyl)glycine, which can serve as a fluorescent probe in proteins, and two halogen derivatives of it, were synthesized by using a new synthetic protocol in five steps. It is less costly and relatively easy to prepare this kind of fluorescent amino acid with the new synthetic method. Furthermore, it can be applied to synthesize other derivatives of the coumarin amino acid with some specific properties.


Experimental
General remarks: All reagents were purchased from commercial suppliers and used without further purification. Flash chromatography was carried out with silica gel (200-300 mesh). Analytical TLC was performed with silica gel GF 254 plates, and the products were tested by UV detection. 1 H NMR spectra were recorded at 500 MHz (Varian DD2) and 13 C NMR spectra were recorded at 125 MHz (Varian DD2). Chemical shifts (δ) are reported in ppm with TMS as internal standard, and spin-spin coupling constants (J) are given in Hz. 19 F NMR spectra were recorded at 470 MHz (Varian DD2) and were reported in ppm with TFA as internal standard. HRMS (ESI) were measured on an Agilent Technologies 6110 mass spectrometer.

4-(Chloromethyl)-7-hydroxy-2H-chromen-2-one (3a):
Resorcinol (11.0 g, 100 mmol) was carefully dissolved in H 2 SO 4 solution (95%, 90 mL) at 0 °C with stirring. Ethyl 4-chloroacetoacetate (18.1 g, 110 mmol) was slowly added to the solution and the reaction mixture was stirred at 0 °C to room temperature for 5 h. TLC indicated that resorcinol was completely consumed and a fluorescent product was formed. The solution was poured slowly into an ice/water mixture (700 mL) and a large amount of solid was precipitated. The precipitate was filtered and S3 washed with water several times. It was then dried in an oven to afford a white solid, which is product 3a ( 125.80, 155.17, 155.45, 160.15, 161.14, 167.20, 169.40.
TLC indicated the disappearance of 4-fluoro-1,3-dihydroxybenzene and the formation of a new fluorescent compound. The reaction mixture was poured into ice-water (100 mL), and a large amount of yellow solid was formed immediately. The mixture was filtered, washed with water several times and dried to afford a solid product (

Diethyl (6-chloro-7-hydroxy-2-oxo-2H-chromen-4-yl)
methylphosphonate (4c): Compound 3c (2.83 g, 11.6 mmol) and a catalytic amount of potassium iodide were dissolved in triethyl phosphite (7.64 g, 46.0 mmol) to form a brown solution. It was then heated under reflux at 155 °C for 4 h under N 2 . The reaction mixture was concentrated using an oil pump to afford a brown solid. It was resuspended in ethyl acetate and the mixture was put in a freezer set at −20 °C. The solid product was precipated overnight, which was filtered, washed with ethyl acetate several times and dried to afford brown solid product 4c (2.48 g, 62.0% yield). The product was used in the following reaction without further purification. S11

Protein expression
To determine whether compound 1a is incorporated into the protein with high efficiency and fidelity, an amber stop codon was substituted for Ile38 in E.coli