Cross-linkable gelatin methacryloyl (GM) is widely used for the generation of artificial extracellular matrix (ECM) in tissue engineering. However, the quantification of modified groups in GM is still an unsolved issue, although this is the key factor for tailoring the physicochemical material properties. In this contribution, ¹H–¹³C-HSQC NMR spectra are used to gain detailed structural information on GMs and of 2-fold modified gelatin containing methacryloyl and acetyl groups (GMAs). Distinctive identification of methacrylate, methacrylamide, and acetyl groups present in GMs and GMAs revealed an overlap of methacrylamide and modified hydroxyproline signals in the ¹H NMR spectrum. Considering this, we suggest a method to quantify methacrylate and methacrylamide groups in GMs precisely based on simple ¹H NMR spectroscopy with an internal standard. Quantification of acetylation in GMAs is also possible, yet, 2D NMR spectra are necessary. The described methods allow direct quantification of modified groups in gelatin derivatives, making them superior to other, indirect methods known so far.

中文翻译：

---

明胶甲基丙烯酰基取代的定量：最佳实践和当前的陷阱

可交联的明胶甲基丙烯酰基（GM）被广泛用于组织工程中的人工细胞外基质（ECM）的生成。然而，尽管这是定制理化材料性能的关键因素，但GM中修饰基团的定量仍然是一个尚未解决的问题。在这一贡献中，使用了¹ H – ¹³ C-HSQC NMR光谱来获得有关GM以及含有甲基丙烯酰基和乙酰基（GMA）的2倍改性明胶的详细结构信息。对GM和GMA中存在的甲基丙烯酸酯，甲基丙烯酰胺和乙酰基的区别鉴定揭示了甲基丙烯酰胺和修饰的羟脯氨酸信号在¹1 H NMR谱。考虑到这一点，我们建议一种基于内标的简单¹ H NMR光谱法精确定量GM中甲基丙烯酸酯和甲基丙烯酰胺基团的方法。也可以对GMA中的乙酰化进行定量，但是2D NMR光谱是必需的。所描述的方法允许直接定量明胶衍生物中的修饰基团，使其优于目前已知的其他间接方法。