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Oligosaccharides composed of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and/or 2-amino-2-deoxy-D- glucopyranose (GlcN) were prepd. by chem. degrdn. of chitin or chitosan and sepd. by gel permeation chromatog. Oligosaccharides obtained after enzymic hydrolysis of chitosan [FA 0.19] with a fungal chitinase were derivatized by reductive amination with 2-aminoacridone and sequenced by matrix-assisted laser desorption ionization time-of-flight postsource decay (PSD) mass spectrometry (MS). The sequence of a trimer, D1A2, was established as D-A-A. The compn. of a hexamer D3A3 was .apprx.65% D-A-D-D-A-A and 35% D-D-A-D-A-A. The PSD MS of a nonamer D5A4-amac revealed four isobaric species D-X-Y-D-X-Y-D-A-A, where A is GlcNAc, D is GlcN, and X and Y (X ¹ Y) are mutually either D or A. This structure motif was also obsd. in a dodecamer D7A5 which was composed of eight isobaric sequences of the general formula (D-X-Y)3- D-A-A.
Recent evidences indicating that cellular kinase signaling cascades are triggered by oligomers of N-acetylglucosamine (ChOS) and that condrocytes of human osteoarthritic cartilage secrete the inflammation associated chitolectin YKL-40, prompted us to study the binding affinity of partially acetylated ChOS to YKL-40 and their effect on primary chondrocytes in culture. Extensive chitinase digestion and filtration of partially deacetylated chitin yielded a mixture of ChOS (Oligomin(TM)) and further ultrafiltration produced T-ChOS(TM), with substantially smaller fraction of the smallest sugars. YKL-40 binding affinity was determined for the different sized homologues, revealing micromolar affinities of the larger homologues to YKL-40. The response of osteoarthritic chondrocytes to Oligomin(TM) and T-ChOS(TM) was determined, revealing 2- to 3-fold increases in cell number. About 500 mu g/ml was needed for Oligomin(TM) and around five times lower concentration for T-ChOS(TM), higher concentrations abolished this effect for both products. Addition of chitotriose inhibited cellular responses mediated by larger oligosaccharides. These results, and the fact that the partially acetylated T-ChOS(TM) homologues should resist hydrolysis, point towards a new therapeutic concept for treating inflammatory joint diseases.
Heterochitooligosaccharides possess interesting biol. properties. Isobaric mixts. of such linear heterochitooligosaccharides can be obtained by chem. or enzymic degrdn. of chitosan. However, the sepn. of such mixts. is a challenging anal. problem which is so far unresolved. It is shown that these isobaric mixts. can be sequenced and quantified simultaneously using std. derivatization and multistage tandem mass spectrometric techniques. A linear ion trap mass spectrometer equipped with a vacuum matrix-assisted laser desorption ionization (vMALDI) source is used to perform MS2 as well as MS3 expts. [on SciFinder (R)].