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Rapid and Modular Assembly of Click Substrates To Assay Enzyme Activity in the Newborn Screening of Lysosomal Storage Disorders

[Image: see text] 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, substra...

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Detalles Bibliográficos
Autores principales: Skrinjar, Philipp, Schwarz, Markus, Lexmüller, Stefan, Mechtler, Thomas P., Zeyda, Maximilian, Greber-Platzer, Susanne, Trometer, Joe, Kasper, David C., Mikula, Hannes
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2018
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311692/
https://www.ncbi.nlm.nih.gov/pubmed/30648152
http://dx.doi.org/10.1021/acscentsci.8b00668
Descripción
Sumario:[Image: see text] 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 N-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.