Rapid and Modular Assembly of Click Substrates To
Assay Enzyme Activity in the Newborn Screening of Lysosomal Storage
Disorders
Version 3 2018-12-26, 05:19
Version 2 2018-12-06, 16:22
Version 1 2018-12-06, 14:54
Posted on 2018-12-26 - 05:19
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.
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Skrinjar, Philipp; Schwarz, Markus; Lexmüller, Stefan; Mechtler, Thomas P.; Zeyda, Maximilian; Greber-Platzer, Susanne; et al. (2018). Rapid and Modular Assembly of Click Substrates To
Assay Enzyme Activity in the Newborn Screening of Lysosomal Storage
Disorders. ACS Publications. Collection. https://doi.org/10.1021/acscentsci.8b00668
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AUTHORS (9)
PS
Philipp Skrinjar
MS
Markus Schwarz
SL
Stefan Lexmüller
TM
Thomas P. Mechtler
MZ
Maximilian Zeyda
SG
Susanne Greber-Platzer
JT
Joe Trometer
DK
David C. Kasper
HM
Hannes Mikula
KEYWORDS
tandem mass spectrometry needLysosomal Storage Disordersenzyme assaysassay enzyme activitiesenzyme assay productslysosomal storage disorders mucopolysaccharidosis typebuilding block strategySelected click substratesoptimizationbuilding block assemblyacetylgalactosamine -6-sulfate sulfataseAssay Enzyme Activityblood spot samplessulfated β- d-galactosidesisotope-labeled standards need