Faculty of Chemistry, Organic and Bioorganic Chemistry, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Cellular and Molecular Biotechnology, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Chembiochem. 2020 Dec 11;21(24):3580-3593. doi: 10.1002/cbic.202000416. Epub 2020 Sep 18.
Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C -formylglycine (FGly). This noncanonical electrophilic amino acid can subsequently be addressed selectively by bioorthogonal hydrazino-iso-Pictet-Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to proteins to obtain a defined payload-to-protein ratio. However, the disadvantages of these conjugation techniques include the need for a large excess of conjugation building block, comparably low reaction rates and limited stability of FGly-containing proteins. Therefore, functionalized clickable HIPS and tandem Knoevenagel building blocks were synthesized, conjugated to small proteins (DARPins) and subsequently linked to strained alkyne-containing payloads for protein labeling. This procedure allowed the selective bioconjugation of one or two DBCO-carrying payloads with nearly stoichiometric amounts at low concentrations. Furthermore, an azide-modified tandem Knoevenagel building block enabled the synthesis of branched PEG linkers and the conjugation of two fluorophores, resulting in an improved signal-to-noise ratio in live-cell fluorescence-imaging experiments targeting the EGF receptor.
甲酰甘氨酸生成酶特异性地氧化共识序列 CxPxR 中的半胱氨酸为 C -甲酰甘氨酸（FGly）。这种非典型的亲电氨基酸随后可以通过生物正交的肼基-异-Pictet-Spengler（HIPS）或 Knoevenagel 连接来选择性地修饰，以将荧光团或药物等有效载荷连接到蛋白质上，从而获得特定的有效载荷与蛋白质的比例。然而，这些缀合技术的缺点包括需要大量的缀合构建块、相对较低的反应速率和 FGly 含量蛋白质的稳定性有限。因此，合成了功能化的可点击的 HIPS 和串联的 Knoevenagel 砌块，并将其与含有张力炔烃的小分子蛋白质（DARPins）缀合，随后连接到含有张力炔烃的小分子蛋白质上，用于蛋白质标记。该程序允许在低浓度下以近乎化学计量的量选择性地连接一个或两个携带 DBCO 的有效载荷。此外，一个叠氮修饰的串联 Knoevenagel 砌块能够合成支化的 PEG 接头，并连接两个荧光团，从而提高了针对表皮生长因子受体的活细胞荧光成像实验的信噪比。
