Sensitive, precise fingerprint profiling for monosaccharide analysis of Bacillus Calmette-Guérin polysaccharide and nucleic acid isolates.

A sensitive and precise HPLC-DAD method with pre-column PMP derivatization was established and validated, for analyzing the polysaccharides in Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN) isolates, after acid hydrolysis. And the HPLC fingerprint profiling was used to analyze its monosaccharide composition. The monosaccharide concentration-peak area calibration curve was of good linearity (R2 > 0.99), over the range of 0.016-0.08 mg/mL for mannose or 0.24-1.20 mg/mL for glucose, with high recovery of 93-105 % for quality control samples. The intra-day RSD values of mannose and glucose concentration were less than 2.5 % and 2.1 %, respectively, and their inter-day RSD values were less than 4.3 % and 2.2 %, respectively, and remained stable for up to 14 days. This method also remained durable against changes in chromatographic parameters, but it's susceptible to the flow rate of mobile phase. Additionally, the method was applied to analyze the content of mannose and glucose in 22 batches BCG-PSN powder and 17 batches BCG-PSN injection. The results showed that the HPLC-DAD fingerprint spectra of all the BCG-PSN powder and BCG-PSN injection samples had a high degree of similarity, with the similar indexes up to 0.999 and 0.998, respectively. The HPLC-DAD method with pre-column PMP derivatization is highly rapid, effective, visual, and accurate for determination of monosaccharide contents. The validated method was successfully applied to the analysis of polysaccharide in both BCG-PSN powder and injection.

The structures of two polysaccharides from Lepidium meyenii and their immunomodulatory effects via activating NF-κB signaling pathway.

Lepidium meyenii Walp., also known as the "Peruvian national treasure", is a popular functional food in the daily lives of Peruvian people due to its bioactive with main polysaccharides. However, studies on polysaccharides isolated from Lepidium meyenii were few. Two new highly heterogeneous polysaccharides, MCP-1a and MCP-2b, were isolated and purified from the tuber of Lepidium meyenii. The structure characterization revealed that MCP-1a primarily consisted of D-Glc and had a molecular weight of 6.6 kDa. Its backbone was composed of 1,4,6-α-D-Glc, while branches feature T-α-L-Ara, 1,5-α-L-Ara, and T-α-D-Glc attached to the O-6 positions. MCP-2b was a rare arabinogalactan with a molecular weight of 49.4 kDa. Interestingly, the backbone of MCP-2b was composed of 1,6-ß-D-Gal, 1,3,6-ß-D-Gal with a few 1,3-ß-D-GlcpA-4-OMe units inserted. Side chains of MCP-2b were mainly composed of 1,3-ß-D-Gal, T-ß-D-Gal, T-α-L-Ara, 1,5-α-L-Ara, with trace amounts of 1,4-ß-D-Glc and T-ß-D-Glc. The bioactivity assay results revealed that MCP-1a and MCP-2b increased the release of NO, IL-1ß, TNF-α, and IL-6 from RAW 264.7 cells at concentrations ranging from 50 µg/mL to 400 µg/mL. Furthermore, MCP-1a and MCP-2b could promote the expression of key transcription factors (IκB-α, p-IκB-α, p65, and p-p65) in the NF-κB pathway, indicating that MCP-1a and MCP-2b had potential immunomodulatory activities.

Purification, structural characterization, and immunomodulatory activity of two polysaccharides from Portulaca oleracea L.

In present study, two water-soluble polysaccharides designated as POL-1 and POL-2 were purified from purslane and their structural characteristics as well as immunomodulatory activity were investigated. The weight-average molecular weight (Mw) of POL-1 and POL-2 were determined to be 64,100 Da and 21,000 Da, respectively. Comprehensive techniques including UV, IR, GC-MS, and NMR were applied to deduced that POL-1 was a pectin polysaccharide homogalacturonan (HG) consisting of →4)-α-GalpA-(1→ with methyl ester degree of 9.71 % and acetylation degree of 0.34 %, while POL-2 was composed of a 1, 4-linked ß-Galp backbone substituted by short side chain →4)-α-Glcp-(1→ and →6)-α-Glcp-(1→. The →4)-α-Glcp-(1→ was attached at the O-6 position of →4)-ß-Galp-(1→. TEM further revealed that POL-1 was non-branched single chains, while POL-2 was entangled microstructure with side chains. Moreover, POL-2 significantly promoted macrophage phagocytosis as well as the secretion of NO and cytokines (TNF-α, IL-6) through activating NF-κB signaling pathway, thus demonstrating potential immunomodulatory activity. These findings suggested that purslane may be exploited as a potential adjuvant and dietary supplement with immunostimulatory purpose.

Long-term outcomes of single-incision plus one-port laparoscopic surgery versus conventional laparoscopic surgery for rectosigmoid cancer: a randomized controlled trial.

BACKGROUND: Though our previous study has demonstrated that the single-incision plus one-port laparoscopic surgery (SILS + 1) is safe and feasible for sigmoid colon and upper rectal cancer and has better short-term outcomes compared with conventional laparoscopic surgery (CLS), the long-term outcomes of SILS + 1 remains uncertain and are needed to evaluated by an RCT. METHODS: Patients with clinical stage T1-4aN0-2M0 rectosigmoid cancer were enrolled. The participants were randomly assigned to either SILS + 1 (n = 99) or CLS (n = 99). The 3-year DFS, 5-year OS, and recurrence patterns were analyzed. RESULTS: Between April 2014 and July 2016, 198 patients were randomly assigned to either the SILS + 1 group (n = 99) or CLS group (n = 99). The median follow-up in the SILS + 1 group was 64.0 months and in CLS group was 65.0 months. The 3-year DFS was 87.8% (95% CI, 81.6-94.8%) in SILS + 1 group and 86.9% (95% CI, 81.3-94.5%) in CLS group (hazard ratio: 1.09 (95% CI, 0.48-2.47; P = 0.84)). The 5-year OS was 86.7% (95% CI,79.6-93.8%) in the SILS + 1 group and 80.5% (95% CI,72.5-88.5%) in the CLS group (hazard ratio: 1.53 (95% CI, 0.74-3.18; P = 0.25)). There were no significant differences in the recurrence patterns between the two groups. CONCLUSIONS: We found no significant difference in 3-year DFS and 5-year OS of patients with sigmoid colon and upper rectal cancer treated with SILS + 1 vs. CLS. SILS + 1 is noninferior to CLS when performed by expert surgeons. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02117557 (registered on 21/04/2014).

RNF43 mutation as a predictor of immunotherapeutic efficacy in colorectal cancer.

RNF43 is a tumor suppressor for various cancers and is considered to drive carcinogenesis when mutated. However, the correlation between RNF43 mutation and colorectal cancer (CRC) immunotherapy remains unreported. We evaluated the role of RNF43 using publicly available data from the Cancer Genome Atlas (TCGA) and Memorial Sloan Kettering Cancer Center (MSKCC). In addition, further analysis was performed on an internal validation cohort (hcohort). The mutant profiles of RNF43 were analyzed in 873 Chinese CRC patients. The relationship between clinical pathologic features and RNF43 were analyzed using the two-sided chi-squared test or the Fisher exact test. Clinicopathologic characteristics were associated with overall survival using Cox regression and the Kaplan-Meier method. We found that RNF43 mutation was significantly associated with high TMB and high MSI score (all p-values < 0.05) in the MSKCC cohort. Additionally, RNF43 mutation was found to be enriched in MSI instability. Kaplan-Meier survival analysis revealed that patients with RNF43 mutation had better OS compared to RNF43 wild-type (not reached vs. 13 months, HR, 0.12; 95% CI 0.03 to 0.49; P = 0.0034). However, no association was observed between RNF43 and OS in the TCGA cohort (HR, 1.83; 95% CI 0.66 to 5.07; P = 0.2479). Our CRC hcohort confirmed the significance of RNF43 mutation in predicting better clinical outcomes, including ORR (45% vs. 21%, P = 0.0468). RNF43 mutation correlated with a high tumor mutation burden (P < 0.001). The mutation frequency of RNF43 in CRC patients was 8.4% (73/873); RNF43 G659Vfs*41 was found to be the most frequent mutation site. In patients with RNF43 mutations, TP53, KRAS, and TGFBR2 were genes with a high frequency of mutations. Compared with RNF43 wild-type patients, those with RNF43 mutations had a higher TMB score and a greater proportion of MSI-H, but no difference in PD-L1 expression. Moreover, the content of immune-related B cells, CD8+ T cells, neutrophils, and dendritic cells was higher in the RNF43 mutant group than in the wild-type group. Our results suggest that RNF43 mutation may correlate with better OS in CRC patients receiving PD-1/PD-L1 inhibitors. The exact mechanisms underlying RNF43 require further investigation.

Short-term and long-term outcomes of single-incision plus one-port laparoscopic surgery for colorectal cancer: a propensity-matched cohort study with conventional laparoscopic surgery.

BACKGROUND: Single-incision plus one-port laparoscopic surgery (SILS + 1) has been demonstrated to be minimally invasive while possessing better cosmesis and less pain compared with conventional laparoscopic surgery (CLS). However, SILS + 1 as an alternative to CLS for colorectal cancer is still controversial. METHODS: A total of 1071 patients who underwent curative laparoscopic surgery for colon cancer between 2015 and 2018 were included. Of these patients, 258 SILS + 1 cases and 516 CLS cases were analyzed using propensity score matching. The baseline characteristics, surgical outcomes, pathologic findings and recovery course, morbidity and mortality within postoperative 30 days and 3-year disease-free and overall survival were compared. RESULTS: Baseline characteristics were balanced between the groups. The mean operating time was significantly shorter in SILS + 1 group, with less estimated blood loss. Tumor size, tumor differentiation, number of harvested lymph nodes, resection margin and pathologic T, N, TNM stage was similar between the groups. There was no significant difference in overall perioperative complications. Uni- and multivariate analyses revealed that SILS + 1 was not a risk factor for complications. Postoperatively, SILS + 1 group showed faster recovery than CLS group in terms of ambulation, bowel function, oral intake and discharge. The 3-year disease-free survival rates of SILS + 1 and CLS groups were 90.1% and 87.3%(p = 0.59), respectively and the 3-year overall survival rates were 93.3% vs. 89.8%(p = 0.172). DISCUSSION: Our study revealed that SILS + 1 is safe, feasible, oncologically efficient, and may be considered as a surgical option for selected patients with colorectal cancer.

Snail-inspired AFG/GelMA hydrogel accelerates diabetic wound healing via inflammatory cytokines suppression and macrophage polarization.

Diabetic foot ulcers (DFUs) are a severe and rapidly growing diabetic complication, but treating DFUs remains a challenge for the existing therapies are expensive and highly non-responsive. Recently, we discovered that a natural adhesive from snail mucus can promote skin wound healing. Herein, inspired by the finding, we developed a double-network hydrogel biomaterial that composed of snail glycosaminoglycan (AFG) and methacrylated gelatin (GelMA), in which AFG is the main bioactive component of snail mucus and GelMA provides a scaffold mimicking the proteins in snail mucus. The biomimetic hydrogel exhibited strong tissue adhesion, potent anti-inflammatory activity, and excellent biocompatibility. The biodegradable AFG/GelMA hydrogel markedly promoted chronic wound healing in both STZ-induced type 1 diabetic rat and db/db mouse models after a single treatment. Further mechanistic research showed that the hydrogel significantly attenuated inflammation by sequestrating pro-inflammatory cytokines, as well as downregulated their expression by inhibiting NF-ĸB signaling pathway, and it can also promote macrophage polarization to M2 phenotype. Taken together, the bioinspired hydrogel can effectively promote the transition of chronic wounds from inflammation to proliferation stage. These data suggest that the AFG/GelMA hydrogel is a promising therapeutic biomaterial for the treatment of chronic diabetic wounds.

Site-directed transformants with E407K substitution in Bcmdl1 possesses different fitness from field anilinopyrimidine resistant isolates with E407K mutation in Botrytis cinerea.

Botrytis cinerea is the causal agent of devastating disease gray mold on numerous crops worldwide. To control gray mold, anilinopyrimidine (AP) fungicides have been widely applied since the 1990s. However, the development of resistance in B. cinerea brought a new challenge to this disease control. Due to the unknown mode of action, the mechanism of AP resistance is still ambiguous. In our previous study, mutation E407K in Bcmdl1 was identified to be associated with AP resistance. Since this mutation is the major mechanism of AP resistance in our cases, it is essential to investigate the fitness of E407K strains before designing anti-resistance management strategies. Besides using field-resistant isolates with the E407K mutation, strains with E407K substitution obtained by site-directed mutagenesis were also used to estimate the specific effect of this mutation or substitution on fitness. The fitness of E407K strains were evaluated by determining mycelial growth, sporulation, conidial germination, virulence, acid production, osmotic and oxidative sensitivity, and sclerotial production and viability. Field resistant isolates with E407K mutation produced fewer sclerotia on intermediate medium (IM) but more conidia on PDA when compared with sensitive isolates, whereas site-directed transformants with E407K substitution did not show any fitness costs. The competitive ability of E407K strains was also evaluated on apple fruit using conidial mixtures at three initial ratios of resistant and sensitive isolates at 1:9, 1:1, and 9:1, respectively. Similar with fitness, impaired competitive ability was observed in field resistant isolates but not site-directed transformants at all initial ratios tested. These results indicated that field strains associated with AP resistance suffer a fitness penalty not linked directly to the E407K substitution in Bcmdl1.

A natural biological adhesive from snail mucus for wound repair.

The discovery of natural adhesion phenomena and mechanisms has advanced the development of a new generation of tissue adhesives in recent decades. In this study, we develop a natural biological adhesive from snail mucus gel, which consists a network of positively charged protein and polyanionic glycosaminoglycan. The malleable bulk adhesive matrix can adhere to wet tissue through multiple interactions. The biomaterial exhibits excellent haemostatic activity, biocompatibility and biodegradability, and it is effective in accelerating the healing of full-thickness skin wounds in both normal and diabetic male rats. Further mechanistic study shows it effectively promotes the polarization of macrophages towards the anti-inflammatory phenotype, alleviates inflammation in chronic wounds, and significantly improves epithelial regeneration and angiogenesis. Its abundant heparin-like glycosaminoglycan component is the main active ingredient. These findings provide theoretical and material insights into bio-inspired tissue adhesives and bioengineered scaffold designs.

Detailed Structural Analysis of the Immunoregulatory Polysaccharides from the Mycobacterium Bovis BCG.

Bacillus Calmette-Guérin polysaccharide and nucleic acid (BCG-PSN), extracted from Mycobacterium bovis, is an immunoregulatory medicine commonly used in clinic. However, the structural characteristics and potential pharmacological efficacy of the polysaccharides from BCG-PSN remain unclear. Herein, two polysaccharides (BCG-1 and BCG-2) were purified and their structures were characterized. Monosaccharide composition analysis combined with methylation analysis and NMR data indicated that BCG-1 and BCG-2 were an α-D-(1→4)-mannan with (1→2)-linked branches, and an α-D-(1→4)-glucan with (1→6)-linked branches, respectively. Herein, the mannan from BCG-PSN was first reported. Bioactivity assays showed that BCG-1 and BCG-2 dose-dependently and potently increased the production of inflammatory mediators (NO, TNF-α, IL-6, IL-1ß, and IL-10), as well as their mRNA expressions in RAW264.7 cells; both have similar or stronger effects compared with BCG-PSN injection. These data suggest that BCG-1 and BCG-2 are very likely the active ingredients of BCG-PSN.

The structures of two glucomannans from Bletilla formosana and their protective effect on inflammation via inhibiting NF-κB pathway.

Bletilla formosana is a traditional Chinese herbal medicine and is widely consumed as foods and medicines in China. However, the chemical structure and bioactivity of its polysaccharides remain unknown. Herein, two new polysaccharides, BFP60 and BFP80, with molecular weights of 3.99 kDa and 10.07 kDa, respectively, were isolated and purified from dried tuber of B. formosana. Structural analysis suggested that BFP60 and BFP80 may have backbone consisted of →4)-ß-d-Man-(1→,→4)-ß-d-Glc-(1→,→4)-2-O-acetyl-ß-d-Man-(1→, and →4)-3-O-acetyl-ß-d-Man-(1→. Inflammation assay in LPS-induced RAW264.7 cells showed that the productions of NO and pro-inflammatory cytokines including IL-6, IL-1ß, TNF-α, and IFN-γ were significantly reduced, and the expression of iNOS, COX-2, and target proteins in the NF-κB pathway were suppressed after BFP60 and BFP80 pretreatment. These findings indicated that this novel polysaccharide had significant inflammatory protective effects in vitro.

Chemical synthesis and pharmacological properties of heparin pentasaccharide analogues.

The pentasaccharide fondaparinux is a synthetic anticoagulant based on heparin antithrombin-binding sequence. Fondaparinux improves safety and predictable pharmacodynamics compared with heparins; however, it requires a complicate synthesis process which contain more than 50 steps of synthesis. Herein, we designed and synthesized four fondaparinux analogues (compounds 1, 2, 3, 4) using a [2+3] convergent synthetic method, which greatly simplified the synthetic process, improved the product yield, and curtailed the expenditures. These synthesized compounds showed stronger anticoagulant activities by factor Xa inhibition (IC50 725-1126 nM vs. 1909 nM for fondaparinux) in the AT-dependent manner. After subcutaneous (s.c.) administration to rats, the compounds displayed long-lasting anti-factor Xa activities and inhibition of thrombin generation ex vivo. Compared with fondaparinux, these compounds were slowly eliminated after s.c. administration to rats, the half-lies (t1/2) were more than 2-fold of that of fondaparinux. These results suggested the pentasaccharide analogues may exhibit better pharmacokinetic and predictable pharmacodynamic characteristics.

Structures of fructan and galactan from Polygonatum cyrtonema and their utilization by probiotic bacteria.

Polygonatum cyrtonema is a known tonic herb in Chinese Materia Medica, extensively consumed in China, but the structure and activity of its polysaccharide components remain to be clarified. Herein, two new polysaccharides (a fructan and a galactan) were purified from the dried and the processed P. cyrtonema rhizome, respectively. Structural analysis suggested that the fructan consisted of a (2 â†’ 6) linked ß-d-Fruf residues backbone with an internal α-d-Glcp residue and two (2 â†’ 1) linked ß-d-Fruf residues branches, and that the galactan was a (1 â†’ 4)-ß-d-galactan branched with a single ß-d-galactose at C-6 at about every nine residues in its main chain. The bioactive assay showed that the fructan and the galactan remarkably promoted growth of Bifidobacterium and Lactobacillus strains, indicating that they possess prebiotic activity. These findings may help expand the application of the polysaccharides from the tonic herb P. cyrtonema as functional ingredients in food products.

Effect of single-incision plus one port laparoscopic surgery assisted with enhanced recovery after surgery on colorectal cancer: study protocol for a single-arm trial.

BACKGROUND: Studies have proved that the enhanced recovery after surgery (ERAS) protocol can significantly improve the recovery course of patients during the perioperative period. The application of minimally invasive surgery is a critical component of ERAS protocol. Single-incision plus one port laparoscopic surgery (SILS plus one) could achieve further minimally invasive surgical results than conventional laparoscopic surgery (CLS). The objective of this trial is to evaluate the safety and feasibility of SILS plus one with ERAS protocol in colorectal cancer. METHODS: This is a prospective, single-center, open-label, single-arm trial. A total of 120 eligible patients with colorectal cancer will receive SILS plus one followed by the ERAS management during the perioperative period. The primary endpoint is postoperative hospital stay. The secondary endpoints include rehabilitative rate of the fourth postoperative day, postoperative medical cost, postoperative pain score, postoperative recovery indexes, inflammatory immune response indexes, compliance with ERAS measures, 6 min postoperative walking test (6MWT), hospital readmissions, and early postoperative complications. DISCUSSION: This trial will be the first to evaluate the short-term outcomes of SILS plus one assisted with ERAS protocol for patients with colorectal cancer and will provide valuable clinical evidence on the benefit of the combination of these two techniques, hopefully, to provide patients with more safe, economic, feasible, and rapid surgery and perioperative strategies. TRIAL REGISTRATION: Clinical Trial Registry, NCT0426829. Registered February 15, 2020 (https://clinicaltrials.gov/ct2/show/NCT04268290).

A non-anticoagulant heparin-like snail glycosaminoglycan promotes healing of diabetic wound.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

Relationship between motor function and psychotic symptomatology in young-adult patients with schizophrenia.

Motor abnormalities have been indicated to be a core manifestation of schizophrenia and not just motor side-effects of antipsychotics. However, little is known about whether all of the complete motor function, including fine motor function, muscle strength, and balance is linked to psychotic symptoms. Therefore, this study was to investigate association between complete motor function and psychotic symptoms in young-adult schizophrenia patients who had no extrapyramidal motor symptoms, which were assessed using the Extrapyramidal Symptom Rating Scale. Seventy schizophrenia patients were recruited. Fine motor function, muscle strength, and balance were assessed using The McCarron Assessment of Neuromuscular Development. Psychotic symptoms were assessed using the Positive and Negative Syndrome Scale. Given gender differences in muscle power, the correlation between muscle strength and psychotic symptoms was analyzed by gender separately. Partial correlation controlling for effects of the chlorpromazine equivalent dosage of antipsychotics was conducted. Better fine motor function was correlated with less-severe negative symptoms (r = - 0.49, p < 0.001) in the total sample. In men, better muscle strength was correlated with more severe positive symptoms and less-severe negative symptoms (r = 0.41, p = 0.008; r = - 0.55, p < 0.001). The link between motor function and psychotic symptoms may support the cerebellar and basal ganglia hypotheses of schizophrenia, proposing that diverse schizophrenia symptoms may share the same neural deficiency, that is, dysfunction of cerebellum or basal ganglia. Considering the moderate-to-strong association between muscle strength and psychotic symptoms, muscle strength might be a powerful physical predictor of psychotic progression.

Nonasaccharide Inhibits Intrinsic Factor Xase Complex by Binding to Factor IXa and Disrupting Factor IXa-Factor VIIIa Interactions.

A nonasaccharide (FG9) derived from natural fucosylated glycosaminoglycan (FG) is identified as a selective intrinsic factor Xase complex (FIXa-FVIIIa-Ca2+-phospholipid, FXase) inhibitor that possesses potential inhibition of venous thrombus in rats and shows negligible bleeding risk. The mechanism and molecular target of the nonasaccharide for intrinsic FXase inhibition were systematically investigated and compared with low molecular weight heparin (LMWH). Our results showed that FG9 dose-dependently inhibited FX activation by intrinsic FXase complex in a noncompetitive inhibition pattern, where the apparent affinity for FG9 was approximately 1.8-fold higher than that for LMWH. FG9 displayed no inhibitory effect on the activity of FIXa/phospholipid, and did not affect the decay rate of FVIIIa activity. FG9 reduced the apparent affinity of FIXa for FVIIIa in a dose-dependent manner, and accelerated the decay of intrinsic FXase complex activity. FG9 bound to FIXa with high affinity and the FIXa binding sites of FG9 were overlapped with that of LMWH, and the ability of FG-derived oligosaccharides to bind FIXa required the minimum 9 degrees of polymerization. FG9 derivatives were prepared and their structures were confirmed by one-dimensional/two-dimensional nuclear magnetic resonance. Structure-activity relationship studies showed that carboxy reduction significantly weakened its anti-FXase activity and binding affinity to FIXa, while the effects of carboxyl ethyl esterification and deacetylation were relatively weaker. Overall, our results suggest that the nonasaccharide FG9 strongly inhibits intrinsic FXase complex activity via binding to FIXa and disrupting FIXa-FVIIIa interactions, and the free carboxyl groups of FG9 are required for its potent anti-FXase activity.

Synthesis and anticoagulation studies of "short-armed" fucosylated chondroitin sulfate glycoclusters.

Fucosylated chondroitin sulfate (FuCS) is a structurally complex glycosaminoglycan found in sea cucumbers with a wide spectrum of biological activities, among which anticoagulant activity is particularly attractive for the development of alternative anticoagulant drugs with decreased adverse effects and risks of bleeding. Previous studies show that FuCS glycomimetics bearing several trisaccharide epitopes displayed promising anticoagulant activity and did not change the mode of action of FuCS. To simplify synthetic difficulty of high valent glycoclusters and obtain candidate compounds with relatively low molecular weights, here we report the synthesis of two FuCS glycoclusters with low valence and more compact structures. Anticoagulation studies showed that these simplified "short-armed" glycoclusters demonstrated comparable potency with "long-armed" high valent glycoclusters, offering a concise approach for the development of novel anticoagulant agents.

Synthesis of Fucosylated Chondroitin Sulfate Nonasaccharide as a Novel Anticoagulant Targeting Intrinsic Factor Xase Complex.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan, and its oligosaccharides exhibit excellent anticoagulant activity with lower risks of adverse effects and bleeding. Herein we report a facile approach to the synthesis of FuCS hexa- and nonasaccharides on the basis of the enzymatic degradation of chondroitin over 12 linear steps. As compared with a clinical low-molecular-weight heparin drug (enoxaparin), the nonasaccharide synthesized in this study displayed similar APTT activity and selective intrinsic factor Xase complex inhibitory activity ((12.9±0.83) nm) by binding to factor IXa with high affinity, thus offering promise for the development of new anticoagulant agents targeting the intrinsic coagulation pathway.

Oligosaccharides from depolymerized fucosylated glycosaminoglycan: Structures and minimum size for intrinsic factor Xase complex inhibition.

Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan found up to now exclusively in sea cucumbers, has distinct anticoagulant properties, notably a strong inhibitory activity of intrinsic factor Xase complex (FXase). Knowledge of the FG structures could facilitate the development of a clinically effective intrinsic FXase inhibitor for anticoagulant drugs. Here, a new fucosylated glycosaminoglycan was obtained from the widely traded sea cucumber Bohadschia argus The precise structure was deduced as {→4)-[l-Fuc3S4S-α-(1→3)-]-d-GlcA-ß-(1→3)-d-GalNAc4S6S-ß-(1} through analysis of its chemical properties and homogeneous oligosaccharides purified from its ß-eliminative depolymerized products. The B. argus FG with mostly 3,4-di-O-sulfated fucoses expands our knowledge on FG structural types. This ß-elimination process, producing oligosaccharides with well-defined structures, is a powerful tool for analyzing the structure of complex FGs. Among these oligosaccharides, an octasaccharide displayed potent FXase inhibitory activity. Compared with oligosaccharides with various degrees of polymerization (3n and 3n - 1), our analyses reveal that the purified octasaccharide is the minimum structural unit responsible for the potent selective FXase inhibition, because the d-talitol in the nonsaccharide is unnecessary. The octasaccharide with 2,4-di-O-sulfated fucoses is more potent than that of one with 3,4-di-O-sulfated fucoses. Thus, sulfation patterns can play an important role in the inhibition of intrinsic factor Xase complex.