A Fluorescent Probe for Glycosaminoglycans Applied to the Detection of Dermatan Sulfate by a Mix-and-Read Assay

Abstract

Glycosaminoglycans are complex biomolecules of great biological and medical importance. The quantification of glycosaminoglycans, in particular in complex matrices, is challenging due to their inherent structural heterogeneity. Heparin Red, a polycationic, fluorescent perylene diimide derivative, has recently emerged as a commercial probe for the convenient detection of heparins by a mix-and-read fluorescence assay. The probe also detects glycosaminoglycans with a lower negative charge density than heparin, although with lower sensitivity. We describe here the synthesis and characterization of a structurally related molecular probe with a higher positive charge of +10 (vs. +8 of Heparin Red). The superior performance of this probe is exemplified by the quantification of low dermatan sulfate concentrations in an aqueous matrix (quantification limit 1 ng/mL) and the detection of dermatan sulfate in blood plasma in a clinically relevant concentration range. The potential applications of this probe include monitoring the blood levels of dermatan sulfate after administration as an antithrombotic drug in the absence of heparin and other glycosaminoglycans.

Pharmacology of Heparin and Related Drugs: An Update

“…For example, the concentrations of heparin mimetics and derivatives with reduced anticoagulant activity cannot be estimated by their effects on coagulation (Warttinger et al, 2016). However, there is one fluorescent dye assay currently available in a kit formulation (Heparin Red) (Warttinger et al, 2016;Rappold et al, 2017), and a considerable number of heparin-sensing fluorescent systems are still at the development stage and have been reviewed elsewhere (Fan et al, 2021). A few recent examples follow.…”

Fucoidan Characterization: Determination of Purity and Physicochemical and Chemical Properties

“…(C) Representation of polyanionic polysaccharide reaction with fluorescent perylene diimide molecules (e.g., Heparin Red ® ). The reaction electrostatically produces aggregates, followed by fluorescence quenching (modified according to [80]). (D) Calibration curve of Heparin Red ® assay showing crude fucoidan samples deviating from the linear range of the reference sample with no interference from alginate [81].…”

Endogenous Glycosaminoglycans in Various Pathologic Plasma Samples as Measured by a Fluorescent Quenching Method

“…However, it is prone to interference with other endogenous polyanions (eg, HS, hyper-sulfated CS, nucleic acids, polyphosphates, lipid-A) which usually are not present in blood plasma at significant concentrations. [25][26][27][28][29][30] Heparin and HS can be digested by heparinases, a group of bacterial lyases that are widely used as tools for processing and analyzing these polysaccharides. 31 Heparinases degrade heparin and HS GAGs through an eliminative mechanism.…”