molecules Article A Fluorescent Probe for Glycosaminoglycans Applied to the Detection of Dermatan Sulfate by a Mix-and-Read Assay Melissa Rappold, Ulrich Warttinger and Roland Krämer * Inorganic Chemistry Institute, Heidelberg University, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany; [email protected] (M.R.); [email protected] (U.W.) * Correspondence: [email protected]; Tel.: +49-6221-54-8438; Fax: +49-6221-54-8599 Academic Editors: Giangiacomo Torri and Jawed Fareed Received: 12 April 2017; Accepted: 2 May 2017; Published: 9 May 2017 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. Keywords: perylene diimide dyes; dermatan sulfate; fluorescent probe; Heparin Red; assay; dermatan sulfate; human plasma 1. Introduction Glycosaminoglycans are complex biomolecules of great biological and medical importance. They are composed of linear, polydisperse polysaccharides with a highly variable sulfation pattern (with the exception of hyaluronate, which is not sulfated). This structural heterogeneity makes the quantification of glycosaminoglycans challenging. There is a continuous search for more convenient, precise and sensitive quantification methods, in particular for complex biological matrices. Various molecular probes for the direct optical detection of glycosaminoglycans (mainly of heparin, a widely-used antithrombotic drug) have been described [1]. Such probes enable convenient detection by photometry or fluorimetry, although their performance in complex biological matrices is often limited by interference. The fluorescent probe Heparin Red has recently emerged as a component of commercially available assays for the quantification of heparins in various matrices [2–4]. It is implemented in several drug development projects for the pharmacokinetic monitoring of non-anticoagulant heparins, a promising class of drug candidates [5]. Heparin Red is a polyamine modified, red-emissive perylene diimide fluorophore (Scheme1, right). It forms a supramolecular aggregate with glycosaminoglycans, resulting in contact quenching of fluorescence (Scheme2). The strong binding of the polycationic probe to polyanionic heparin appears to be controlled by both electrostatic and aromatic π-stacking interactions [6]. While electrostatic repulsion between dye molecules prevents spontaneous aggregation in the solution, the charge neutralization Molecules 2017, 22, 768; doi:10.3390/molecules22050768 www.mdpi.com/journal/molecules Molecules 2017, 22, 768 2 of 11 Molecules 2017, 22, 768 2 of 10 byMolecules polyanionic 2017, 22, 768 heparin favors the formation of π-stacked complexes between the hydrophobic2 of 10 hydrophobicchromophore chromophore moieties in the moieties ground in state, the ground leading state, to efficient leading static to efficient quenching. staticHeparin quenching. Red Heparinhas also hydrophobic chromophore moieties in the ground state, leading to efficient static quenching. Heparin Redbeen has applied also been to the applied determination to the determination of polysaccharides, of polysaccharides, such as heparan such sulfate as heparan [7] and sulfate fucoidan [7] and [8], Red has also been applied to the determination of polysaccharides, such as heparan sulfate [7] and fucoidanthat have [8], a lower that have negative a lower charge negative density charge than density heparin, than but heparin, the sensitivity but the sensitivity in a plasma in a matrix plasma is fucoidan [8], that have a lower negative charge density than heparin, but the sensitivity in a plasma matrixsignificantly is significantly lower. An lower. ultrasensitive An ultrasensitive assay with assay a quantification with a quantification limit in thelimit pg/mL in the rangepg/mL for range the matrix is significantly lower. An ultrasensitive assay with a quantification limit in the pg/mL range forhighly the highly sulfated sulfated polysaccharide polysaccharide dextran dextran sulfate sulfate in an in aqueous an aqueous matrix matrix has has also also been been described described [9]. [9]. for the highly sulfated polysaccharide dextran sulfate in an aqueous matrix has also been described [9]. Scheme 1. Molecular structures of the probes PDI-1 and Heparin Red in their fully protonated state Scheme 1. Molecular structures of the probes PDI-1 and Heparin Red in their fully protonated state (charge +10 and +8, respectively). (charge +10 and +8, respectively). respectively). Scheme 2. Schematic representation of fluorescence quenching of polycationic probe molecules in the Scheme 2. SchematicSchematic representation representation of fluorescence fluorescence quen quenchingching of polycationic probe molecules in the presence of dermatan sulfate due to the formation of non-fluorescent aggregates. presence of dermatan sulfate due toto thethe formationformation ofof non-fluorescentnon-fluorescent aggregates.aggregates. Dermatan sulfate is a linear, polydisperse sulfated polysaccharide belonging to the glycosamino- Dermatan sulfate is a linear, polydisperse sulfated polysaccharide belonging to the glycosamino- glycanDermatan family. sulfateIt is built is a of linear, a major polydisperse repeating sulfated disaccharide polysaccharide unit consisting belonging of toiduronic the glycosamino- acid and glycan family. It is built of a major repeating disaccharide unit consisting of iduronic acid and galactosamineglycan family. It (Scheme is built of 3). a major The galactosamine repeating disaccharide may be unit4-O consistingor 6-O-sulfated of iduronic and acidthe iduronic and galactosamine acid is 2- galactosamine (Scheme 3). The galactosamine may be 4-O or 6-O-sulfated and the iduronic acid is 2- O(Scheme-sulfated.3). Typically, The galactosamine the averagemay degree be of 4- sulfationO or 6-O is-sulfated around one and per the disaccharide. iduronic acid Dermatan is 2-O-sulfated. sulfate is O-sulfated. Typically, the average degree of sulfation is around one per disaccharide. Dermatan sulfate is theTypically, predominant the average glycosaminoglycan degree of sulfation expressed is around in the skin one and per is disaccharide. released at high Dermatan concentrations sulfate during is the the predominant glycosaminoglycan expressed in the skin and is released at high concentrations during woundpredominant repair. glycosaminoglycan It is also implicated expressed in other inbiological the skin processes and is released [10], such at high as concentrationsdevelopment, growth, during wound repair. It is also implicated in other biological processes [10], such as development, growth, infection,wound repair. tumorigenesis It is also and implicated coagulation in other [11]. biologicalElevated dermatan processes sulfate [10], suchlevels as in development, plasma or urine growth, have infection, tumorigenesis and coagulation [11]. Elevated dermatan sulfate levels in plasma or urine have beeninfection, suggested tumorigenesis as a biomarker and coagulation for certain [11 ].types Elevated of mucopolysaccharidosis dermatan sulfate levels [12]. in plasma Dermatan or urine sulfate- have been suggested as a biomarker for certain types of mucopolysaccharidosis [12]. Dermatan sulfate- basedbeen suggested drug formulations as a biomarker have forbeen certain launched types for of mucopolysaccharidosisthe prevention of venous [12 thromboembolism]. Dermatan sulfate-based in Italy based drug formulations have been launched for the prevention of venous thromboembolism in Italy (marketeddrug formulations as Mistral) have [13] beenand Portugal launched (marketed for the prevention as Venorix). of It venousis also a thromboembolism component of Sulodexide, in Italy (marketed as Mistral) [13] and Portugal (marketed as Venorix). It is also a component of Sulodexide, a(marketed more widely-used as Mistral) antithrombotic [13] and Portugal agent (marketed [14]. Dermatan as Venorix). sulfate It is is also produced a component by extraction of Sulodexide, from a more widely-used antithrombotic agent [14]. Dermatan sulfate is produced by extraction from animala more widely-usedtissues, including antithrombotic a complex agent multistep [14]. Dermatan purification sulfate process. is produced The bystructural extraction heterogeneity from animal animal tissues, including a complex multistep purification process. The structural heterogeneity makestissues, the including direct aquantification complex multistep of dermatan purification sulfate