Efficient chemoenzymatic synthesis of biotinylated human serum albumin-sialoglycoside conjugates containing O-acetylated sialic acids

Abstract

Sialyl Tn (STn) and sialyl lactoside derivatives containing O-acetylated sialic acid residues have been chemoenzymatically synthesized using a one-pot three-enzyme system and conjugated to biotinylated human serum albumin (HSA) using an adipic acid para-nitrophenyl ester coupling reagent. This approach provides an efficient and general protocol for preparing carbohydrate-protein conjugates containing base-sensitive groups.

Scheme 1

Chemoenzymatic synthesis of STn antigen analogs using a one-pot three-enzyme system. a) 3-chloropropanol, acetyl chloride, 70 °C, 87%; b) NaN₃, NaI, DMF, 60 °C, 95%; c) 3 (20 mM), 4, 5, 6, or 7 (30 mM), pyruvate (150 mM), CTP (30 mM), MgCl₂ (20 mM), a sialic acid aldolase, a CMP-sialic acid synthetase, Pd2,6ST, Tris-HCl (100 mM, pH 8.5 for 4 and 5; pH 7.5 for 6 and 7).

Scheme 2

Conjugation of STn antigens to human serum albumin (HSA) using adipic acid p-nitrophenyl diester coupling reagent. a) Pd/C, H₂, H₂O; b) adipic acid p-nitrophenyl diester, DMF, rt, > 90%; c) phosphate buffer (100 mM, pH 7.4). Average numbers of sialoside molecules incorporated were determined by MAlDI-TOF.

Scheme 3

Conjugation of sialyllactosides to human serum albumin (HSA) using adipic acid p-nitrophenyl diester coupling reagent. a) H₂, Pd/C, H₂O; b) adipic acid p-nitrophenyl diester, DMF, rt, > 90%; c) phosphate buffer (100 mM, pH 7.4). Average numbers of sialoside molecules incorporated were determined by MAlDI-TOF.