Fig. 3.

Synthesis of hSHMT fluorescent probe 1 and NMR probe 2. Reagents and conditions for synthesis of probe 1: a) N-Dibenzylglycine tert-butyl ester, LDA, dry THF, –60 °C, 10 min, then 6-(dimethylamino)-2-naphthaldehyde, –60 °C, 30 min; b) N-(p-Toluenesulfonyl)-l-phenylalanyl chloride, DMAP, dry THF, r.t., 14 h; c) 5 M NaOH aq., THF, EtOH, r.t., 5 min; d) Pd/C, H₂, MeOH, r.t., 28 h; e) 4 M HCl/EtOAc, r.t. reagents and conditions for synthesis of probe 2: a) N-Dibenzylglycine tert-butyl ester, LDA, dry THF, –78 °C, 10 min, then 4-fluorobenzaldehyde, –78 °C, 30 min; b) N-(p-Toluenesulfonyl)-l-phenylalanyl chloride, pyridine, dry THF, 60 °C, 19 h; c) 5 M KOH aq., EtOH, 40 °C, 2 h; d) Pd/C, H₂, MeOH, r.t., 16 h; e) TFA, DCM, r.t., 17 h. The lower inset indicates the X-ray crystal structure of probe 1 l-erythro intermediate. Color code: oxygen: red; nitrogen: blue; sulfur: yellow; carbon: black; hydrogen: white