Fig. 4.

Late-stage tagging and isolation of analytically pure tagged proteins. a Design elements of a symmetric bis-aldehyde. b Insulin is labeled through aminoalcohol formation with reagent 2g. The labeled insulin 9 can be treated with derivatives of O-hydroxylamine 10a–10c for tagging them with ¹⁹F-NMR probe, biotin, and coumarin. Alternatively, it can be immobilized on hydrazide functionalized resin through single-site to deliver 13. The unreacted insulin 6d is recovered and recycled. Subsequent treatment of 13 with 10a delivers analytically pure 11a after removing excess 10a through spin concentration. The hydrazide functionalized resin 12 is recovered and recycled multiple (5–8) times without loss of activity. Similar protocol renders pure 11b and 11c. For single-step late-stage installation of a probe, please see Supplementary Fig. 74