Fig. 2. eDHFR-selective labeling in vitro. (a) SDS-PAGE analysis of the covalent labeling of purified eDHFR with 1, 2, and 3 in a test tube. Reaction conditions: eDHFR (10 μM) and labeling reagents (20 μM) were incubated in the presence or absence of 100 μM MTX in 50 mM HEPES buffer (pH 7.2) at 37 °C. In lane 10, an independently prepared Dc–eDHFR conjugate (60% yield) was used as a standard marker for the determination of the labeling yields. The gel was analyzed by in-gel fluorescence imaging (FL, lower), and stained with Coomassie brilliant blue (CBB, upper). (b) (left) MALDI-TOF MS spectra of eDHFR (10 μM) labeled with compound 6 (20 μM) in 50 mM HEPES buffer (pH 7.2) at 37 °C. denotes native eDHFR and denotes labeled eDHFR. (right) Time profiles of eDHFR (10 μM) labeled with 1 (), 2 (), 3 (), 5 (), 6 () and 7 (LDAI reagent, ) in buffer at 37 °C. The labeling reaction was monitored by MALDI-TOF MS analyses. (c) (left) HPLC charts of labeling reagent 6 () in buffer solution at 37 °C for 0 h (top) or 13 h (bottom) of incubation. denotes decomposed reagent 6. denotes the internal standard. (right) HPLC analyses of the stability of labeling reagents 3, 5 and 6 in the absence (; 3, ; 5, ; 6) or presence (; 3, ; 6) of porcine liver esterase (100 nM) in buffer solution at 37 °C. (d) SDS-PAGE analysis of the labeling of Trx–His6–eDHFR with 6 (10 μM) in E. coli lysates.