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

Abstract

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.

A Click-Flipped Enzyme Substrate Boosts the Performance of the Diagnostic Screening for Hunter Syndrome

“…We have recently developed a building-block approach using click chemistry as a key step for the divergent synthesis of enzyme substrates and the corresponding isotope-labeled internal standards for accurate quantification of the assay products by LC-MS/MS. 23 This method gives access to sulfated O-iduronates similar to previously developed I2S substrates (Fig. 1b).…”

“…Based on the click substrates for the screening of MPS types I and IVa recently developed in our lab, 23 we designed Oand triazole-linked substrates for MPS II, containing a phenylene linker and a tert-butyl carbamoyl (NBoc) moiety to promote ionization in MS analysis. Deuterated isotopologues of the linker were used for the synthesis of internal standards ( Fig.…”

A click-flipped enzyme substrate boosts the performance of the diagnostic screening for Hunter syndrome

“…[17][18][19][20][21][22] We have recently reported on the development of a buildingblock approach using click chemistry as a key step, for the divergent synthesis of substrates to assay the enzymes a-Liduronidase and N-acetylgalactosamine-6-sulfate sulfatase, as well as the corresponding isotope-labeled internal standards for accurate quantication of the assay products by LC-MS/MS. 23 Applying this method gives access to sulfated O-iduronates similar to previously developed I2S substrates (Fig. 1b).…”

“…Based on the click substrates for the screening of MPS types I and IVa recently developed in our lab, 23 we designed O-and triazole-linked substrates for MPS II, containing a phenylene linker and a tert-butyl carbamoyl (NBoc) moiety to promote ionization in MS analysis. Deuterated isotopologues of the linker were used for the synthesis of internal standards ( Fig.…”

“…Starting from intermediate 1, 23 the O-linked click substrate CS O and the corresponding internal standard IS O were prepared using copper-catalyzed azide-alkyne cycloaddition (CuAAC) [24][25][26] with an azido-functionalized click marker and its deuterated isotopologue, respectively (Fig. 2b).…”