Summary: | <i>Apostichopus japonicus</i> is one of the most economically important species in sea cucumber aquaculture in China. Fucosylated glycosaminoglycan from <i>A. japonicus</i> (AjFG) has shown multiple pharmacological activities. However, results from studies on the structure of AjFG are still controversial. In this study, the deaminative depolymerization method that is glycosidic bond-selective was used to prepare the depolymerized products from AjFG (dAjFG), and then a series of purified oligosaccharide fragments such as tri-, hexa-, nona-, and dodecasaccharides were obtained from dAjFG by gel permeation chromatography. The 1D/2D NMR and ESI-MS spectrometry analyses showed that these oligosaccharides had the structural formula of <span style="font-variant: small-caps;">l</span>-FucS-α1,3-<span style="font-variant: small-caps;">d</span>-GlcA-β1,3-{<span style="font-variant: small-caps;">d</span>-GalNAc<sub>4S6S</sub>-β1,4-[<span style="font-variant: small-caps;">l</span>-FucS-α1,3-]<span style="font-variant: small-caps;">d</span>-GlcA-β1,3-}<i><sub>n</sub></i>-<span style="font-variant: small-caps;">d</span>-anTal-diol<sub>4S6S</sub> (<i>n</i> = 0, 1, 2, 3; FucS represents Fuc<sub>2S4S</sub>, Fuc<sub>3S4S</sub>, or Fuc<sub>4S</sub>). Thus, the unambiguous structure of native AjFG can be rationally deduced: it had the backbone of {-4-<span style="font-variant: small-caps;">d</span>-GlcA-β1,3-<span style="font-variant: small-caps;">d</span>-GalNAc<sub>4S6S</sub>-β1-}<i><sub>n</sub></i>, which is similar to chondroitin sulfate E, and each <span style="font-variant: small-caps;">d</span>-GlcA residue in the backbone was branched with a <span style="font-variant: small-caps;">l</span>-FucS monosaccharide at <i>O</i>-3. Bioactivity assays confirmed that dAjFG and nonasaccharides and dodecasaccharides from AjFG had potent anticoagulant activity by intrinsic FXase inhibition while avoiding side effects such as FXII activation and platelet aggregation.
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