Highly Efficient and Economical One-Pot Two-Step Multienzymatic Synthesis of 6′/3′-Sialyllactosamine from In Situ-Produced <i>N</i>-Acetyllactosamine

Among over 200 human milk oligosaccharides, 6'-SLN (6'-sialyl-<i>N</i>-acetyllactosamine) and 3'-SLN possess potential antiviral function and can potentially enhance brain development and knowledge. So far, no efficient and cost-effective synthetic pathways for 6'-SLN and 3'-SLN have been developed. In this study, efficient enzymatic pathways to synthesize 6'-SLN and 3'-SLN were established involving economic cytidine 5'-triphosphate (CTP) regeneration from commercial <i>N</i>-acetyllactosamine (LacNAc) with yields of >95%. To meet the requirement of industrial production, their synthesis was further optimized starting from in situ-produced LacNAc. Consequently, the cost of LacNAc purification could be saved. Furthermore, conducting synthesis at pH 7.0 and with supplementation of polyP₆ in three batches led to yields of 6'-SLN (95.0%) and 3'-SLN (94.8%), respectively. Under optimized conditions, enzymatic syntheses of 6'-SLN and 3'-SLN were conducted at a 5 L scale with yields of 96.1% (259.2 g) and 92.3% (248.7 g), respectively. This study would lay the foundation for industrial production of 3'-SLN and 6'-SLN in the future and provide a guide for multienzymatic preparation of other sialylated oligosaccharides.