Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Development of a rapid simultaneous assay of two urinary tetrasaccharide metabolites using differential ion mobility and tandem mass spectrometry and its application to patients with glycogen storage disease (type Ib and II).

Glucose tetrasaccharide (Glc4) and maltotetraose (M4) are important biomarkers for Pompe disease and other glycogen storage diseases (GSDs). With the development of new treatments for GSDs, more specific and sensitive bioanalytical methods are needed to determine biomarkers. In recent years, differential mobility spectrometry (DMS) has become an effective analytical technique with high selectivity and specificity. This study aimed to develop an efficient analytical method for the two urinary tetrasaccharide metabolites using DMS and apply it to patients with GSDs (type Ib and II). Urine samples were directly diluted and injected into liquid chromatography-differential mobility spectrometry tandem mass spectrometry (LC-DMS-MS/MS). Chromatographic separation was performed on an Acquity™ UPLC BEH Amide column (2.1 × 50 mm, 1.7 µm) with a short gradient elution of 2.6 min. DMS-MS/MS was used to detect two urinary tetrasaccharide metabolites in a negative multiple reaction monitoring mode with isopropanol as a modifier. A total of 20 urine samples from 6 healthy volunteers and 10 patients with GSDs (type Ib and II) were collected for analysis. The method was linear over a concentration range of 0.5~100.0 µg/mL for each urinary tetrasaccharide (r≥0.99). The intra- and inter-day precision RSD% were less than 14.3%, and the accuracy RE% were in the range of -14.3~13.4%. The relative matrix effect was between 86.6 and 114.3%. No carryover or interference was observed. Patients with GSDs (type Ib and II) had significantly higher median urinary Glc4 (P=0.001) and M4 (P=0.012) excretion than healthy subjects. The developed method was simple, rapid, sensitive, and specific. It was successfully applied to healthy volunteers and patients with GSDs (type Ib and II). DMS technology greatly improved analysis efficiency and provided high sensitivity and specificity.

Sulfated modification of hyaluronan tetrasaccharide enhances its antitumor activity on human lung adenocarcinoma A549 cells in vitro and in vivo.

Hyaluronan (HA) is a glycosaminoglycan polymer involved in cell phenotype change, inflammation modulation, and tumor metastasis progression. HA oligosaccharides have a higher solubility and drug-forming ability than polysaccharides. HA tetrasaccharide was reported as the smallest fragment required for inhibiting triple-negative breast cancer, but the anti-tumor activity of HA tetrasaccharide (HA4) and its sulfated derivatives in lung cancer is still unknown. In this study, HA4 was prepared via HA degradation by chondroitinase ABC (CSABC), while its sulfated derivatives were prepared by sulfur pyridine trioxide complex in N, N-dimethylformamide (DMF). Then, the anti-tumor activity was detected via MTT assay and xenograft tumor experiments, while the expression level change of apoptosis genes was analyzed by qRT-PCR. Electrospray mass spectrometry (ESI-MS) analysis showed several HA4 sulfated derivatives, GlcA2GlcNAc2 (SO3H)n contains 0-6 sulfation groups, which mainly contain 3-6, 2-3, and 0-1 sulfation groups were classified as HA4S1, HA4S2, and HA4S3, respectively. After the addition of 1.82 mg/mL HA4, HA4S1, HA4S2, and HA4S3, the cell viability of A549 cells was reduced to 81.2 %, 62.1 %, 50.3 %, and 65.9 %, respectively. Thus, HA4S2 was chosen for further measurement, the qRT-PCR results showed it significantly up-regulated the expression of genes in the apoptosis pathway. Moreover, HA4S2 exhibited stronger antitumor activity than HA4 in vivo and the tumor inhibition rate reached 36.90 %. In summary, this study indicated that the CSABC enzyme could effectively degrade HA into oligosaccharides, and sulfation modification was an effective method to enhance the antitumor activity of HA tetrasaccharides.

Serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies.

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.

Synthesis of the tetrasaccharide repeating unit of the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T using linear and one-pot iterative glycosylations.

A straightforward synthetic strategy was developed for the synthesis of the tetrasaccharide repeating unit corresponding to the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T in a very good yield adopting sequential glycosylation followed by removal of the p-methoxybenzyl (PMB) group in the same pot. Further, the synthetic strategy was modified by carrying out three stereoselective iterative glycosylations followed by in situ removal of the PMB group in one pot. The stereochemical outcome of the newly formed glycosidic linkages was excellent using thioglycoside derivatives as glycosyl donors and a combination of N-iodosuccinimide (NIS) and perchloric acid supported on silica (HClO4-SiO2) as the glycosyl activator.

Liver fibrosis during clinical ascertainment of glycogen storage disease type III: a need for improved and systematic monitoring.

PURPOSE: In glycogen storage disease type III (GSD III), liver aminotransferases tend to normalize with age giving an impression that hepatic manifestations improve with age. However, despite dietary treatment, long-term liver complications emerge. We present a GSD III liver natural history study in children to better understand changes in hepatic parameters with age. METHODS: We reviewed clinical, biochemical, histological, and radiological data in pediatric patients with GSD III, and performed a literature review of GSD III hepatic findings. RESULTS: Twenty-six patients (median age 12.5 years, range 2-22) with GSD IIIa (n = 23) and IIIb (n = 3) were enrolled in the study. Six of seven pediatric patients showed severe fibrosis on liver biopsy (median [range] age: 1.25 [0.75-7] years). Markers of liver injury (aminotransferases), dysfunction (cholesterol, triglycerides), and glycogen storage (glucose tetrasaccharide, Glc4) were elevated at an early age, and decreased significantly thereafter (p < 0.001). Creatine phosphokinase was also elevated with no significant correlation with age (p = 0.4). CONCLUSION: Liver fibrosis can occur at an early age, and may explain the decrease in aminotransferases and Glc4 with age. Our data outlines the need for systematic follow-up and specific biochemical and radiological tools to monitor the silent course of the liver disease process.

Carbohydrate Specificity and Isotypes of Monoclonal and Polyclonal Antibodies to Conjugated Tetrasaccharide, a Synthetic Analogue of Repeating Unit of Capsular Polysaccharide of Streptococcus Pneumoniae Serotype 14.

We studied carbohydrate specificity and isotypes of antibodies to BSA-conjugated tetrasaccharide, a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 14, in mouse polyclonal sera and hybridoma-synthesized products. Natural IgM antibodies to the tetrasaccharide containing epitopes similar to surface carbohydrate structures of mammalian and human cells in low titers were determined in native mouse serum by ELISA using biotinylated tetrasaccharide and synthetic capsular polysaccharide as the solid-phase antigens. Polyclonal sera to the conjugated tetrasaccharide contained IgM and all subclasses of IgG antibodies, which were detected in a higher titer when the biotinylated tetrasaccharide was used as a solid phase antigen compared to synthetic capsular polysaccharide. Monoclonal antibodies to S. pneumoniae serotype 14 tetrasaccharide were identified in an equivalent titer using either biotinylated tetrasaccharide or synthetic capsular polysaccharide. Monoclonal antibodies obtained in vitro belonged to IgM isotype and cross-reacted with secondary full-size IgG antibodies. In the serum of mice inoculated with hybridoma, IgM and IgG2a antibodies recognizing the tetrasaccharide epitope in the structure of synthetic capsular polysaccharide were simultaneously determined.

Solution Conformation of Heparin Tetrasaccharide. DFT Analysis of Structure and Spin⁻Spin Coupling Constants.

Density functional theory (DFT) has provided detailed information on the molecular structure and spin⁻spin coupling constants of heparin tetrasaccharide (GlcNS,6S-IdoA2S-GlcNS,6S-IdoA2S-OMe) representing the predominant heparin repeating-sequence. The fully optimised molecular structures of two tetrasaccharide conformations (differing from each other in the conformational form of the sulphated iduronic acid residue⁻one ¹C4 and the other ²S0) were obtained using the B3LYP/6-311+G(d,p) level of theory and applying explicit water molecules to simulate the presence of a solvent. The theoretical data provided insight into variations of the bond lengths, bond angles and torsion angles, formations of intra- and intermolecular hydrogen bonds and ionic interactions. Optimised molecular structures indicated the formation of a complex hydrogen bond network, including interresidue and intraresidue bonds. The ionic interactions strongly influence the first hydration shell and, together with hydrogen bonds, play an important role in shaping the 3D tetrasaccharide structure. DFT-derived indirect three⁻bond proton⁻proton coupling constants (³JH-C-C-H) showed that the best agreement with experiment was obtained with a weighted average of 67:33 (¹C4:²S0) of the IdoA2S forms. Detailed analysis of Fermi-contact contributions to ³JH-C-C-H showed that important contributions arise from the oxygen lone pairs of neighbouring oxygen atoms. The analysis also showed that the magnitude of diamagnetic spin⁻orbit contributions are sufficiently large to determine the magnitude of some proton⁻proton coupling constants. The data highlight the need to use appropriate quantum-chemical calculations for a detailed understanding of the solution properties of heparin oligosaccharides.

Rigidity and flexibility in the tetrasaccharide linker of proteoglycans from atomic-resolution molecular simulation.

Proteoglycans (PGs) are covalent conjugates between protein and carbohydrate (glycosaminoglycans). Certain classes of glycosaminoglycans such as chondroitin sulfate/dermatan sulfate and heparan sulfate utilize a specific tetrasaccharide linker for attachment to the protein component: GlcAß1-3Galß1-3Galß1-4Xylß1-O-Ser. Toward understanding the conformational preferences of this linker, the present work used all-atom explicit-solvent molecular dynamics (MD) simulations combined with Adaptive Biasing Force (ABF) sampling to determine high-resolution, high-precision conformational free energy maps ΔG(φ, ψ) for each glycosidic linkage between constituent disaccharides, including the variant where GlcA is substituted with IdoA. These linkages are characterized by single, predominant (> 97% occupancy), and broad (45° × 60° for ΔG(φ, ψ) < 1 kcal/mol) free-energy minima, while the Xyl-Ser linkage has two such minima similar in free-energy, and additional flexibility from the Ser sidechain dihedral. Conformational analysis of microsecond-scale standard MD on the complete tetrasaccharide-O-Ser conjugate is consistent with ABF data, suggesting (φ, ψ) probabilities are independent of the linker context, and that the tetrasaccharide acts as a relatively rigid unit whereas significant conformational heterogeneity exists with respect to rotation about bonds connecting Xyl to Ser. © 2017 Wiley Periodicals, Inc.

ALG1-CDG: Clinical and Molecular Characterization of 39 Unreported Patients.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over 100 genes leading to impaired protein or lipid glycosylation. ALG1 encodes a ß1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date 13 mutations in 18 patients from 14 families have been described with varying degrees of clinical severity. We identified and characterized 39 previously unreported cases of ALG1-CDG from 32 families and add 26 new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2 , was seen in all 27 patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Actinotetraose L, a new tetrasaccharide derivative isolated from the mycelia of Amycolatopsis sp. HCa1.

A new tetrasaccharide derivative, actinotetraose L (1), was isolated from the methanol extract of the mycelia of the grasshopper-associated rare actinobacterium Amycolatopsis sp. HCa1. The structure of the new compound was elucidated by a combination of 1D, 2D NMR (HMQC, HMBC, COSY, and NOESY), and HR-ESI-MS analyses as O-{3,4-di-O-[(E)-2-ethyl-2-butenoyl]- ß-D-glucopyranosyl-(1 â†’ 2)}-O-{3,4-di-O-[(E)-2-methyl-2-butenoyl]-ß-D-glucopyranosyl-(1 â†’ 2)}-6,6-di-O-[(E)-2-methyl-2-butenoyl]-α-D-glucopyranosyl-(1 â†’ 1)-α-D-glucopyranoside. The immunosuppressive activity and cytotoxicity of the compound were evaluated by T-cell viability and MTT assays. Nonetheless, no significant activity was observed.

Convergent synthesis of a tetrasaccharide repeating unit of the O-specific polysaccharide from the cell wall lipopolysaccharide of Azospirillum brasilense strain Sp7.

A straightforward convergent synthesis has been carried out for the tetrasaccharide repeating unit of the O-specific cell wall lipopolysaccharide of the strain Sp7 of Azospirillum brasilense. The target tetrasaccharide has been synthesized from suitably protected monosaccharide intermediates in 42% overall yield in seven steps by using a [2 + 2] block glycosylation approach.

Straightforward synthesis of a tetrasaccharide repeating unit corresponding to the O-antigen of Escherichia coli O16.

A straightforward synthesis of the tetrasaccharide repeating unit of the O-antigen of Escherichia coli O16 has been achieved following a sequential glycosylation strategy. A minimum number of steps was used for the synthesis of the target compound involving a one-pot glycosylation and a protecting group manipulation. All intermediate reactions afford their products in high yield, and the glycosylation steps are stereoselective.

Molecular docking analysis of a dermatan sulfate tetra-saccharide to human alpha-L-iduronidase.

Human alpha-L-iduronidase (IDUA) is a 653 amino acid protein involved in the sequential degradation of glycos-amino-glycans (GAG), heparan sulfate (HS), and dermatan sulfate (DS). Some variants in the IDUA gene produce a deficient enzyme that causes un-degraded DS and HS to accumulate in multiple tissues, leading to an organ dysfunction known as muco-poly-saccharidosis type I (MPS I). Molecular and catalytic activity assays of new or rare variants of IDUA do not predict the phenotype that a patient will develop. Therefore, it is of interest to describe the molecular docking analysis, to locate binding regions of DS to IDUA to better understand the effect of a variant on MPS I development. The results presented herein demonstrate the presence of a polar/acidic catalytic site and a basic region in the putative binding site of DS to IDUA. Further, synthetic substrate docking with the enzyme could help in the predictions of the MPS I phenotype.

Development of high sustained anti-drug antibody titers and corresponding clinical decline in a late-onset Pompe disease patient after 11+ years on enzyme replacement therapy.

A late-onset Pompe disease patient developed high sustained antibody titers (HSAT) of ≥51,200 after 11+ years on alglucosidase alfa and previous tolerance. There was a corresponding worsening of motor function and rise in urinary glucose tetrasaccharide (Glc4). Following immunomodulation therapy, HSAT were eliminated with improved clinical outcomes and biomarker trends. This report highlights the importance of continued surveillance of antibody titers and biomarkers, the negative impact of HSAT, and improved outcomes with immunomodulation therapy.

Convergent synthesis of the tetrasaccharide repeating unit of the cell wall lipopolysaccharide of Escherichia coli O40.

A tetrasaccharide repeating unit corresponding to the cell-wall lipopolysaccharide of E. coli O40 was synthesized by using a convergent block glycosylation strategy. A disaccharide donor was coupled to a disaccharide acceptor by a stereoselective glycosylation. A 2-aminoethyl linker was chosen as the anomeric protecting group at the reducing end of the tetrasaccharide. All glycosylation steps are significantly high yielding and stereoselective.

Biosynthesis of non-sulfated high-molecular-weight glycosaminoglycans and specific-sized oligosaccharides.

Non-sulfated forms of glycosaminoglycans (NSGAGs) including hyaluronan, chondroitin and heparosan with high-molecular-weight (HMW) are extensively used biomaterials, while NSGAGs oligosaccharides display strong bioactivities. However, microbial production of HMW-NSGAGs and oligosaccharides with specific size are always challenging. Here, a membrane shield strategy was developed to produce HMW-NSGAGs by recruiting type II NSGAG synthases in Corynebacterium glutamicum. By enhancing precursor supplies and reinforcing cell membrane, the MWs of hyaluronan, heparosan and chondroitin reached 4100 kDa, 3000 kDa and 2400 kDa, respectively. In parallel, a synchronized depolymerization-polymerization strategy was developed by co-expressing NSGAGs synthases and lyases. With cell membrane as a filter, we achieved the direct biosynthesis of NSGAGs tetrasaccharides and disaccharides. The titers of chondroitin, hyaluronan and heparosan tetrasaccharides and disaccharides reached 10.9 g L-1, 12.1 g L-1 and 5.8 g L-1, respectively. The strategies developed here should also be applicable to the biosynthesis of other polysaccharides.

Kaempferol tetrasaccharides restore skin atrophy via PDK1 inhibition in human skin cells and tissues: Bench and clinical studies.

Skin aging is a major risk factor for the dermal diseases, and interventions to attenuate cellular senescence are expected to reduce the risk for age-related diseases involving skin atrophy. However, blocking cell death or extending proliferation causally results in side effects and an increased cancer risk. For identification of a safer approach, we focused on PDK1 inhibition, which could revert cellular senescence and reduce senescence factors in skin in vitro, in a human skin equivalent model and in an exploratory, placebo-controlled, interventional trial. Natural phytochemical kaempferol tetrasaccharides resulted in a significant reduction in cellular senescence, and an increase in collagen fiber was observed in the skin cell and human skin equivalent. Clinical enhancement in skin appearance was noted in multiple participants, and an immunohistochemical study revealed improvement in the histological appearance of skin tissue and extracellular matrix. This change was associated with relative improvement in histological markers of senescence and clinical appearance of the aged skin and an increase in collagen fiber, an essential factor for preventing skin atrophy and consistency of the basement membrane. These results indicate that PDK1 inhibition is a potentially effective antiaging intervention, suggesting a diagnostic role and preventive actions of PDK1 in senescence-associated skin atrophy.

Synthesis of a cyclic tetramer of 3-amino-3-deoxyallose with axially oriented amino groups.

A linear tetramer of ß-(1 â†’ 6)-linked 3-azido-3-deoxy-d-allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-d-allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy-d-allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

Newborn screening for Pompe disease in Italy: Long-term results and future challenges.

Pompe disease (PD) is a progressive neuromuscular disorder caused by a lysosomal acid α-glucosidase (GAA) deficiency. Enzymatic replacement therapy is available, but early diagnosis by newborn screening (NBS) is essential for early treatment and better outcomes, especially with more severe forms. We present results from 7 years of NBS for PD and the management of infantile-onset (IOPD) and late-onset (LOPD) patients, during which we sought candidate predictive parameters of phenotype severity at baseline and during follow-up. We used a tandem mass spectrometry assay for α-glucosidase activity to screen 206,741 newborns and identified 39 positive neonates (0.019%). Eleven had two pathogenic variants of the GAA gene (3 IOPD, 8 LOPD); six carried variants of uncertain significance (VUS). IOPD patients were treated promptly and had good outcomes. LOPD and infants with VUS were followed; all were asymptomatic at the last visit (mean age 3.4 years, range 0.5-5.5). Urinary glucose tetrasaccharide was a useful and biomarker for rapidly differentiating IOPD from LOPD and monitoring response to therapy during follow-up. Our study, the largest reported to date in Europe, presents data from longstanding NBS for PD, revealing an incidence in North East Italy of 1/18,795 (IOPD 1/68,914; LOPD 1/25,843), and the absence of mortality in IOPD treated from birth. In LOPD, rigorous long-term follow-up is needed to evaluate the best time to start therapy. The high pseudodeficiency frequency, ethical issues with early LOPD diagnosis, and difficulty predicting phenotypes based on biochemical parameters and genotypes, especially in LOPD, need further study.