Cyclic and square wave voltammetry of chitooligosaccharides modified by osmium(VI) tetramethylethylenediamine
Introduction
Chitosan oligosaccharide (COS) represents a class of natural oligomers that holds therapeutic promise in several diseases due to their favorable physiochemical properties (water-solubility and low viscosity) and pharmacological properties, including good pharmacokinetics, safety profiles and diverse beneficial biological activities. COS can be prepared from chitin via deacetylation [1] followed by degradation by acid hydrolysis or by enzymes [2], [3]. Chitin is one of the most abundant polysaccharides in nature, occurring in some fungi as well as in the bodies of insects and crustaceans [1], [4]. It is a linear N-acetyl-D-glucosamine (GlcNAc) polymer bound with a β-1,4 binding. Partial chitin deacetylation produces chitosan. The degree of deacetylation corresponds to the molar fraction of GlcNAc acetamido groups in the chitosan molecule (commonly 30–10%). Other parameters, which characterize chitosan, are molecular weight distribution (polydispersity) and the sequence or pattern of N-acetylation. These chitosan chemical characteristics strongly influence its physico-chemical and biological properties (such as anti-inflammatory, anti-bacterial and anti-oxidative activities …). The presence of D-glucosamine, with a protonated amino group (NH3+) at physiological pH, makes chitosan a positively charged polymer at neutral pH.
COS is partially degraded chitosan, usually with a degree of polymerization <50–55 and an average molecular weight (MW) <10 kDa. Unlike chitosan, COS are readily soluble in water due to their short chain lengths and free amino groups in D-glucosamine units [5]. An increase in the COS deacetylation degree induces an increase in positive COS molecule charges, correlating with an increase in anti-inflammatory, anti-cancer, anti-bacterial and anti-oxidative activities [6].
Chitosan and its oligomers were characterized (degree of deacetylation, MW distribution, N-acetylation sequence or pattern) by different methods [1], [7], including electrochemical approaches. Palecek et al. showed that chitosan samples produced voltammetric and chronopotentiometric catalytic peak on mercury and solid amalgam electrodes in a wide pH range, allowing chitosan determination at 5 µg/mL level [8], [9] in sodium acetate pH 5.2. Other authors determined the chitosan content in real samples by cathodic stripping voltammetry in phthalate buffer at pH 2.5 using the peak appearing due to a reduction of carbonyl group [10].
Non-electrochemical methods for the determination of chitosan or chitin are mostly based on spectroscopic (colorimetric) approach. They are often laborious, time consuming and reach lower sensitivity than electrochemical methods [1], [7], [11].
In our previous papers, we dealt with the modification of polysaccharides, oligosaccharides, glycoproteins and oligonucleotides with the ligand complexes of sixvalent osmium (Os(VI)L) and their electrochemical analysis [12], [13], [14], [15]. Potassium osmate is a non-volatile, easy to handle compound making the preparation of the Os(VI)L simple. Os(VI)L reacts predominantly with vicinal diols (glycols), namely aliphatic, cyclic cis- and trans- on the six- or seven-membered ring in non-water solvents with pyridine as a ligand [16]. Simple saccharides, like polyhydroxy compounds involving the vicinal diol moieties, could easily be modified with Os(VI)L [17]. The later works proved modifying saccharides containing cis- glycol units on furanose [18], [19] and saccharides containing cis- and trans-glycol on a pyranose ring in aqueous solutions [12], [20]. These saccharides form stable electrochemically active products with Os(VI)L in aqueous solutions [12], [13]. For such purposes, more stable bidentate ligands are often used instead of pyridine, such as N,N,N′,N′- tetramethylethylenediamine (tem) or 2,2′-bipyridine (bipy).
To our knowledge there is no report on the reaction of Os(VI)L and the formation of the stabile Os(VI)L adduct with organic compounds other than glycols. In this work, we modified oligosaccharides derived from chitin (acCOS) and chitosan (COS) with Os(VI)tem followed by electrochemical analysis of obtained products at the mercury and carbon electrodes. Both acCOS and COS contain only one glycol group on the non-reducing end of the molecule, and they should be modified equally. Contrary to expectations, COS was modified with Os(VI)tem substantially more than acCOS. We suggested from the obtained results, that COS produced an adduct with Os(VI)tem, in which an amino group is involved.
Section snippets
Reagents
Potassium osmate dihydrate, min. 99%; pyridine (py), min. 99,7%; and N,N,N′,N′- tetramethylethylenediamine (tem) p.a. were purchased from Merck (USA); N,N',N'',N''',N'''',N'''''-hexaacetylchitohexaose (acCH); N,N',N'',N'''-tetraacetylchitotetraose; N,N',N''-triacetylchitotriose; chitohexaose.6 HCl (CH); chitotetraose.4 HCl and chitotriose.3 HCl from Carbosynth (Compton, Berkshire, UK); dextran (Dextran standard Fluka 50,000); stachyose and 3α,6α-mannopentaose (MPO) from Sigma-Aldrich (USA). All
Results and discussion
Simple saccharides modified by Os(VI)tem are electrochemically active, while their unmodified forms not [12], [25]. The Os(VI)tem modified simple saccharides yielded redox peaks usually appearing in three couples corresponding to Os(VI) reduction/oxidation [27], [28]. These peaks are marked as peaks I–III at HMDE.
Conclusions
Os(VI)tem simple saccharide modification appears useful in determining the content of α(2,3) and α(2,6) regioisomers containing Neu5Ac in their mixture [34], [35]. This simple approach can be applied instead of laborious and expensive mass spectrometry. In this work we show that we can modify not only simple saccharides with Os(VI)tem but also aminosaccharides. Acetylated COS forms modified by Os(VI)tem at HMDE and PGE behaved electrochemically similarly to previously described Os(VI)tem
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the Czech Science Foundation 18-18154S project. We are grateful to L. Havran and H. Černocká for critically reading the manuscript and to L. Fojt for his technical assistance with ICP MS.
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