Institute of Biological Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria.
Interdepartmental Research Unit of Peptide and Protein Chemistry and Biology, Department of Chemistry "Ugo Schiff", University of Florence, via della Lastruccia 13, 50019, Sesto Fiorentino (Florence), Italy.
Angew Chem Int Ed Engl. 2022 Sep 26;61(39):e202206116. doi: 10.1002/anie.202206116. Epub 2022 Aug 25.
Granulocyte colony stimulating factor (G-CSF) is a cytokine used to treat neutropenia. Different glycosylated and non-glycosylated variants of G-CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O-glycoforms of G-CSF, thereby enabling the preparation of selectively and homogeneously post-translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N-terminal segment with a synthetic C-terminal part, transiently equipped with a side-chain-linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G-CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays.
粒细胞集落刺激因子（G-CSF）是一种用于治疗中性粒细胞减少症的细胞因子。目前，通过重组表达产生了用于治疗应用的不同糖基化和非糖基化 G-CSF 变体。在这里，我们描述了建立第一个半合成策略来获得 G-CSF 的糖苷元和 O-糖型的方法，从而能够制备这种重要细胞因子的选择性和均一的翻译后修饰变体。最终，我们通过在低浓度下组合使用重组产生的 N 端片段的硒代半胱氨酸连接与合成的 C 端部分成功实现，该 C 端部分暂时配备了侧链连接的光裂解 PEG 部分。瞬时 PEG 化使碳水化合物在 Thr133 处的酶促延伸成为可能。总的来说，我们能够显著减少与两个蛋白质片段的低溶解度和聚集倾向相关的问题，这使得能够制备四种 G-CSF 变体，这些变体成功折叠并在细胞增殖测定中显示出生物活性。
