Galactose as a recognizing motif for asialoglycoprotein receptor (ASGPR) is a widely accepted vector to deliver cytotoxic agents in the therapy of hepatocellular carcinoma (HCC), however, the individual hydroxyl group of galactose (Gal) contributed to recognizing ASGPR is obscure and remains largely unanswered in the design of glycoconjugates. Herein, we designed and synthesized five positional isomers of Gal-anthocyanin Cy5.0 conjugates and three Gal-doxorubicin (Dox) isomers, respectively. The fluorescence intensity of Gal-Cy5.0 conjugates accumulated in cancer cells hinted the optimal modification sites of positions C2 and C6. Comparing to the cytotoxicity of other conjugates, C2-Gal-Dox (11) was the most potent. Moreover, Gal-Dox conjugates significantly the toxicity of Dox. A progressively lower internalization capacity and siRNA technology implied the cellular uptake and cytotoxicity directly related to the ASGPR expression level. Accordingly, position C2 of galactose may be the best substitution site via ASGPR mediation in the design of anti-HCC glycoconjugates.

半乳糖作为脱唾液酸糖蛋白受体（ASGPR）的识别基序，是一种广泛接受的载体，用于在肝细胞癌（HCC）的治疗中递送细胞毒性药物，然而，半乳糖（Gal）的单个羟基对识别ASGPR的贡献尚不清楚，并且在很大程度上仍然存在在糖复合物的设计中尚未得到解答。在此，我们分别设计并合成了半乳糖花青素 Cy5.0 缀合物的五种位置异构体和三种半乳阿霉素（Dox）异构体。 Gal-Cy5.0缀合物在癌细胞中积累的荧光强度暗示了位置C2和C6的最佳修饰位点。与其他缀合物的细胞毒性相比，C2-Gal-Dox ( 11 ) 是最有效的。此外，Gal-Dox 结合显着降低了 Dox 的毒性。逐渐降低的内化能力和siRNA 技术意味着细胞摄取和细胞毒性与 ASGPR 表达水平直接相关。因此，在抗 HCC 糖缀合物的设计中，半乳糖的 C2 位可能是通过ASGPR 介导的最佳取代位点。