Skrinjar Philipp, Schwarz Markus, Lexmüller Stefan, Mechtler Thomas P, Zeyda Maximilian, Greber-Platzer Susanne, Trometer Joe, Kasper David C, Mikula Hannes
Institute of Applied Synthetic Chemistry, Vienna University of Technology (TU Wien), 1060 Vienna, Austria.
ARCHIMED Life Science GmbH, 1110 Vienna, Austria.
ACS Cent Sci. 2018 Dec 26;4(12):1688-1696. doi: 10.1021/acscentsci.8b00668. Epub 2018 Dec 6.
Synthetic substrates play a pivotal role in the development of enzyme assays for medical diagnostics. However, the preparation of these chemical tools often requires multistep synthetic procedures complicating structural optimization and limiting versatility. In particular, substrates for enzyme assays based on tandem mass spectrometry need to be designed and optimized to fulfill the requirements to finally enable the development of robust diagnostic assays. In addition, isotope-labeled standards need to be prepared to facilitate accurate quantification of enzyme assay products. Here we report the development of a building block strategy for rapid and modular assembly of enzyme substrates using click chemistry as a key step. These click substrates are made up of a sugar moiety as enzyme responsive unit, a linker that can easily be isotope-labeled for the synthesis of internal standards, and a modifier compound that can readily be exchanged for structural optimization and analytical/diagnostic tuning. Moreover, the building block assembly eliminates the need for extensive optimization of different glycosylation reactions as it enables the divergent synthesis of substrates using a clickable enzyme responsive unit. The outlined strategy has been applied to obtain a series of synthetic α-l-iduronates and sulfated β-d-galactosides as substrates for assaying α-l-iduronidase and -acetylgalactosamine-6-sulfate sulfatase, enzymes related to the lysosomal storage disorders mucopolysaccharidosis type I and type IVa, respectively. Selected click substrates were finally shown to be suitable to assay enzyme activities in dried blood spot samples from affected patients and random newborns.
合成底物在医学诊断酶分析方法的开发中起着关键作用。然而，这些化学工具的制备通常需要多步合成程序，这使得结构优化变得复杂，并限制了通用性。特别是，基于串联质谱的酶分析底物需要进行设计和优化，以满足最终开发稳健诊断分析方法的要求。此外，还需要制备同位素标记的标准品，以促进酶分析产物的准确定量。在此，我们报告了一种使用点击化学作为关键步骤的酶底物快速模块化组装的构建块策略。这些点击底物由作为酶响应单元的糖部分、可轻松进行同位素标记以合成内标的连接子以及可随时交换以进行结构优化和分析/诊断调整的修饰化合物组成。此外，构建块组装消除了对不同糖基化反应进行广泛优化的需要，因为它能够使用可点击的酶响应单元进行底物的发散合成。所概述的策略已被应用于获得一系列合成的α-L-艾杜糖醛酸酯和硫酸化β-D-半乳糖苷，作为分别用于检测与溶酶体贮积症I型和IVa型相关的酶α-L-艾杜糖苷酶和N-乙酰半乳糖胺-6-硫酸酯硫酸酯酶的底物。最终表明，选定的点击底物适用于检测来自受影响患者和随机新生儿的干血斑样本中的酶活性。
