Glycosylated Amino Acids Research Articles

Palladium‐Catalyzed Directing‐Group‐Mediated γ‐C(sp3)−H Glycosylation for Synthesis of C‐Alkyl Glycosides

AbstractC‐alkyl glycosides play a crucial role in various bioactive compounds. However, the synthesis of C‐alkyl glycosides poses significant challenges, particularly through C(sp3)−H glycosylation. Here, we report a set of reactions for constructing C‐alkyl glycosides through directing‐group‐mediated functionalization of unactivated γ‐C(sp3)−H bonds under mild conditions. These reactions not only achieve high regioselectivity and stereoselectivity in glycosylation, but also exhibit a wide substrate scope. They are compatible with both arene and alkane substrates, as well as natural and unnatural amino acid substrates. Mechanistic studies have shown that the directing‐group 8‐aminoquinoline (AQ) and picolinamide (PA) may affect the chirality of the β‐carbon of L‐valine through a sterically favorable trans‐palladacycle intermediate, resulting in (R) or (S) configuration of glycosylated amino acid, respectively. These reactions are promising to provide a convenient and powerful tool for constructing C‐alkyl glycosides and carbohydrate‐based drugs in the future.

Combined Peptidomics and Metabolomics Analyses to Characterize the Digestion Properties and Activity of Stropharia rugosoannulata Protein-Peptide-Based Materials.

Protein-peptide-based materials typically possess high nutritional value and various physiological regulatory activities. This study evaluated the digestion, metabolism, and activity of Stropharia rugosoannulata protein-peptide-based materials. After the S. rugosoannulata protein-peptide-based materials were digested (simulated) orally, in the stomach, and in the intestines, the proportions of >10,000 Da, 5000~10,000 Da, and <180 Da in the digestion products increased, and the peptide content was maintained at more than 120 mg/g dry weight. The digestion products of eight test groups with different oral-gastrointestinal digestion-level settings all had suitable ACE inhibitory activity (IC50 range 0.004~0.096 mg/mL). The main metabolite groups were lipid-like molecules, fatty acids, carboxylic acids, their derivatives, amino acids, peptides, and analogs. Bile and glycosylated amino acids were the main compounds that caused differences between groups. KEGG pathways enriched in differentially expressed metabolites included eight significantly upregulated pathways, including valine, leucine, and isoleucine biosynthesis, etc., and six significantly downregulated pathways, including the citric acid cycle (tricarboxylic acid cycle), etc. The arginine and proline metabolism pathways and the aminoacyl-tRNA biosynthesis pathways were upregulation and downregulation pathways that enriched multiple differentially expressed metabolites. Twenty-six metabolites, including bile acids, total bile acids, and the essential amino acids L-isoleucine and L-leucine, were differentially expressed metabolite markers of the protein-peptide-based material oral-gastrointestinal digestion products.

C-Mannosyl tryptophan is a novel biomarker for thrombocytosis of myeloproliferative neoplasms

C-Mannosyl tryptophan (CMW), a unique glycosylated amino acid, is considered to be produced by degradation of C-mannosylated proteins in living organism. Although protein C-mannosylation is involved in the folding and secretion of substrate proteins, the pathophysiological function in the hematological system is still unclear. This study aimed to assess CMW in the human hematological disorders. The serum CMW levels of 94 healthy Japanese workers were quantified using hydrophilic interaction liquid chromatography. Platelet count was positively correlated with serum CMW levels. The clinical significance of CMW in thrombocytosis of myeloproliferative neoplasms (T-MPN) including essential thrombocythemia (ET) were investigated. The serum CMW levels of the 34 patients with T-MPN who presented with thrombocytosis were significantly higher than those of the 52 patients with control who had other hematological disorders. In patients with T-MPN, serum CMW levels were inversely correlated with anemia, which was related to myelofibrosis (MF). Bone marrow biopsy samples were obtained from 18 patients with ET, and serum CMW levels were simultaneously measured. Twelve patients with bone marrow fibrosis had significantly higher CMW levels than 6 patients without bone marrow fibrosis. Collectively, these results suggested that CMW could be a novel biomarker to predict MF progression in T-MPN.

Preparation of Mucin Glycopeptides by Organic Synthesis.

Mucins are sugar-rich glycoproteins. Glycoprotein sugar moieties are structurally diverse, making it difficult to obtain naturally pure glycoproteins. Chemical synthesis is a powerful tool for obtaining target or designed compounds. Automated peptide synthesizers are commercially available, and many use the solid-phase peptide synthesis (SPPS) method. In addition, some of these synthesizers apply microwave irradiation to obtain higher reaction yields, thereby enabling the synthesis of 40 to 50 amino acid residual glycopeptides. Theoretically, glycopeptides can be synthesized using methods similar to those used for peptide synthesis, but glycosylated amino acid synthons are less stable than amino acid synthons and are also very expensive. Therefore, they are not suitable for use in large excess amounts. Many of oligosaccharide-linked amino acid synthons are not commercially available, so they must be specially prepared, and they also require careful handling that demands specific organic synthesis experience and techniques. However, monosaccharide-linked amino acid synthons are commercially available and are relatively easy to handle. Here, as an entry into glycopeptide synthesis, we describe a typical glycopeptide synthesis procedure for a 27 amino acid residual MUC1 repeating unit with monosaccharides.

Inside Back Cover: Fucose Binding Motifs on Mucin Core Glycopeptides Impact Bacterial Lectin Recognition (Angew. Chem. Int. Ed. 32/2023)

O-fucosylated glycan epitopes of membrane-bound mucin glycoproteins are key ligands of many bacterial lectins. In their Research Article (e202302437), Ulrika Westerlind and co-workers describe the synthesis of fucosylated mucin glycopeptides using a chemoenzymatic approach. Starting from simple building blocks, Fmoc-protected glycosylated amino acids were prepared and incorporated into peptides and enzymatically diversified. Bacteria lectin interactions were then elucidated on glycopeptide microarrays.

Innenrücktitelbild: Fucose Binding Motifs on Mucin Core Glycopeptides Impact Bacterial Lectin Recognition (Angew. Chem. 32/2023)

O-fucosylated glycan epitopes of membrane-bound mucin glycoproteins are key ligands of many bacterial lectins. In their Research Article (e202302437), Ulrika Westerlind and co-workers describe the synthesis of fucosylated mucin glycopeptides using a chemoenzymatic approach. Starting from simple building blocks, Fmoc-protected glycosylated amino acids were prepared and incorporated into peptides and enzymatically diversified. Bacteria lectin interactions were then elucidated on glycopeptide microarrays.

Characterization of the Glycans of Equine κ-Casein.

To study the protein-bound glycans of equine κ-casein, equine sodium caseinate was first obtained from raw mare's milk by acid precipitation and then fractionated by cation-exchange chromatography. The oligosaccharides of the obtained equine κ-casein were analyzed by RP-HPLC-UV-HRMS after β-elimination with simultaneous derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). In addition to the acidic tetrasaccharide derivative Neu5Ac-Gal-[Neu5Ac]-GalNAc-2PMP known from bovine κ-casein, the acidic pentasaccharide derivative Neu5Ac-Gal-[Gal-GlcNAc]-GalNAc-2PMP was identified as the most abundant glycan. The glycosylated amino acid residues were identified using a peptide sequencing approach after digestion with trypsin by HRMS. The threonine T109 was experimentally confirmed for the first time as a glycosylation site in equine κ-casein. Therefore, equine κ-casein seems to be more highly glycosylated than previously thought.

Comparative protein analysis of two maize genotypes with contrasting tolerance to low temperature

BackgroundLow temperature (LT) stress is one of the major environmental stress factors affecting the growth and yield of maize (Zea mays L.). Hence, it is important to unravel the molecular mechanisms behind LT stress tolerance to improve molecular breeding in LT tolerant genotypes. In the present study, two maize genotypes viz. Gurez local from Kashmir Himalaya and tropical grown GM6, were dissected for their LT stress response in terms of accumulation of differentially regulated proteins (DRPs). Leaf proteome analysis at three-leaf stage of maize seedlings subjected to LT stress of 6 °C for a total of 12 h duration was performed using two dimensional gel electrophoresis (2D-PAGE) followed by subsequent identification of the proteins involved.ResultsAfter MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) and bioinformatics analysis, 19 proteins were successfully identified in Gurez local, while as 10 proteins were found to get successful identification in GM6. The interesting observations from the present investigation is the identification of three novel proteins viz. threonine dehydratase biosynthetic chloroplastic, thylakoidal processing peptidase 1 chloroplastic, and nodulin-like protein, whose role in abiotic stress tolerance, in general, and LT stress, in particular, has not been reported so far. It is important to highlight here that most of LT responsive proteins including the three novel proteins were identified from Gurez local only, owing to its exceptional LT tolerance. From the protein profiles, obtained in both genotypes immediately after LT stress perception, it was inferred that stress responsive protein accumulation and their expression fashion help the Gurez local in seedling establishment and withstand unfavorable conditions as compared to GM6. This was inferred from the findings of pathway enrichment analysis like regulation of seed growth, timing of floral transition, lipid glycosylation, and aspartate family amino acid catabolic processes, besides other key stress defense mechanisms. However, in GM6, metabolic pathways enriched were found to be involved in more general processes including cell cycle DNA replication and regulation of phenylpropanoid metabolism. Furthermore, majority of the qRT-PCR results of the selected proteins demonstrated positive correlation between protein levels and transcript abundance, thereby strengthening our findings.ConclusionsIn conclusion, our findings reported majority of the identified proteins in Gurez local exhibiting up-regulated pattern under LT stress as compared to GM6. Furthermore, three novel proteins induced by LT stress were found in Gurez local, requiring further functional validation. Therefore, our results offer more insights for elucidating the molecular networks mediating LT stress tolerance in maize.

An identification method to distinguish monomeric sugar isomers on glycopeptides.

A one-step protocol for the automated flow synthesis of protected glycosylated amino acids is described using pumps with open-source controls in overall yields of 21-50%. The resulting glycosylated amino acids could be used directly in solid-phase peptide synthesis (SPPS) protocols to quickly produce glycopeptide standards. Access to a variety of stereoisomers of the sugar enabled the development of an LC-MS/MS protocol that can distinguish between peptides modified with carbohydrates having the same exact mass. This method could definitively identify fucose in an O-glycosylation site on the transmembrane protein, Notch1.

Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of β-lactoglobulin.

To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

Catalyst- and Base-free One-pot, Multicomponent, De Novo Assembly of Structurally Diverse Morpholine Glycoconjugates.

A catalyst-, metal- and base-free unprecedented one-pot multicomponent synthetic strategy has been established for the construction of various substituted morpholine glycoconjugates through four-component reactions of glycosyl amino alcohol, chloroacetone, acid, and isocyanides. Unexpectedly, this one-pot approach generates only the cyclized morpholine scaffolds rather than normal Ugi adduct without any external base. This reaction proceeds via in situ formations of Schiff-base followed by an intramolecular halogen displacement process, finally the Joullie-Ugi reaction takes place leading to the corresponding products with a mixture of diastereomers.

THE BIOMOLECULAR CHARACTERISTICS OF ANGIOTENSIN II TYPE 1 RECEPTORS AS PARAMETERS IN KIDNEY FIBROSIS: A BIOINFORMATICS ANALYSIS

AGTR1 is a modulator of angiotensin II-induced signal transduction. This article aims to examine the biomolecular characteristics of AGTR1 and the role of AGTR1 in the renal fibrosis pathway in Rattus norvegicus rats. The gene ID NC 051352.1 and protein ID NP 112271.2 for AGTR1 were retrieved from the National Center for Biotechnology Information's website. The 'NCBI' website has details on AGTR1's structure, location, and expression. Protease prediction using 'PEPTIDE CUTTER'; glycosylation index prediction using NETNGLYC; and target protein location evaluated on the 'TARGETP' homepage. AGTR1 in Rattus norvegicus is located on chromosome 17p12, This protein has a stability score of 34.48. The aliphatic index was 114.62. AGTR1 is found inside, outside, and on the transmembrane, as determined by the THMM. Only 21 enzymes were predicted to be capable of cleaving the AGTR1 protein, out of a total of 37 protease. AGTR1 protein has four glycosylated amino acid sequences. AGTR1 protein is primarily at other location 0.850, secretory pathway (0.590), and a little amount in mitochondria (0.018AGTR1/AT1R is a marker for renal fibrosis in the renin angiotensin system, which can lead in vasoconstriction, inflammation, fibrosis, sodium retention, and water retention. The AGTR1 protein's molecular feature is a determinant in the progression of renal fibrosis, revealed to a bioinformatics study.

Protonated α-N-Acetyl Galactose Glycopeptide Dissociation Chemistry.

We recently provided mass spectrometric, H/D labeling, and computational evidence of pyranose to furanose N-acetylated ion isomerization reactions that occurred prior to glycosidic bond cleavage in both O- and N-linked glycosylated amino acid model systems (Guan et al. Phys. Chem. Chem. Phys., 2021, 23, 23256-23266). These reactions occurred irrespective of the glycosidic linkage stereochemistry (α or β) and the N-acetylated hexose structure (GlcNAc or GalNAc). In the present article, we test the generality of the preceding findings by examining threonyl α-GalNAc-glycosylated peptides. We utilize computational chemistry to compare the various dissociation and isomerization pathways accessible with collisional activation. We then interrogate the structure(s) of the resulting charged glycan and peptide fragments with infrared "action" spectroscopy. Isomerization of the original pyranose, the protonated glycopeptide [AT(GalNAc)A+H]+, is predicted to be facile compared to direct dissociation, as is the glycosidic bond cleavage of the newly formed furanose form, i.e., furanose oxazolinium ion structures are predicted to predominate. IR action spectra for the m/z 204, C8H14N1O5+, glycan fragment population support this prediction. The IR action spectra of the complementary m/z 262 peptide fragment were assigned as a mixture of the lowest-energy structures of [ATA+H]+ consistent with the literature. If general, the change to a furanose m/z 204 product ion structure fundamentally alters the ion population available for MS3 dissociation and glycopeptide sequence identification.

Expeditious Synthesis of a Glycopeptide Library

AbstractShort proteoglycan fragments are of great importance for biochemical research. The solid‐phase synthesis of such glycopeptides relies on excessive use of glycosylated amino acids, extended reaction times, and additional post‐assembly deprotection protocols. We employed high‐shear mixing for expedient and equimolar O‐glycopeptide assembly. We further developed a stirring‐based deprotection on the solid support, thus completing the synthesis of a glycopeptide library in a minimal amount of time and purification hurdles.

Determination of Insecticidal Potential of Lentil (Lens culinaris L.) Lectin through Bio-computational Tools

Plant and animals are the chief source of lectins playing important role in defense system against various pathogens. Lentil (Lens culinaris) lectin (LL) binds to polysaccharides and other glycoconjugates in a reversibly manner which contain glucose or mannose type sugars. In present investigation, the insecticidal potential of LL is determined through In-silico study by using various bio-computational tools against mustard aphid. The physico-chemical property of LL also exhibited theoretical pI is 5.22, GRAVY value is -0.086, aliphatic index is 78.33 and instability index is 27.51. The functional domain exposure of LL showed metal interacting and N-linked glycosylated amino acid residues. The structural behavior and biological activity of LL protein is determined by secondary structure prediction and its other attributes. Present investigation explores the potential of insecticidal activity of lentil lectin against mustard aphid by using numerous In silico approaches. This probably helps in understanding of LL three dimensional protein structures.

Nickel-Catalyzed Glycosylation of Amino Acids or Peptides

Nickel-Catalyzed Glycosylation of Amino Acids or Peptides

Defining the Cell Surface Proteome of MLL Rearranged Leukemia

Introduction: B-acute lymphoblastic leukemia (B-ALL) patients that harbor rearrangements of the Mixed-lineage leukemia gene (MLLr ) have a particularly dismal clinical outcome. Although it is known that this oncoprotein radically alters epigenetic regulation, the precise molecular mechanisms that define the cellular phenotype of these cancers remain poorly understood and new therapeutic strategies are sorely needed. Furthermore, although flow cytometric immunophenotyping has been performed for decades on B-ALL blasts, relatively little is known about the cell surface proteome of these cancers outside of canonical CD markers. Due to its role in modulating cellular behavior as well as accessibility to immunotherapy, the cell surface represents an attractive target for unbiased proteomic profiling in order to gain insight into cellular behavior as well as enabling the development of targeted therapies. Here, we aim to resolve the cell surface proteomic landscapes of MLLr and non- MLLr B-ALL cellular models. Our goal is to generate new mechanistic insights into the molecular re-wiring that occurs in these forms of cancer and identify novel candidates for targeted immunotherapy.Methods: We examined 10 B-ALL cell lines that harbor different oncogenic fusions including the MLL-AF4, BCR-ABL, and ETV6-RUNX1 translocations, respectively, using both “classical” lines as well as low-passage patient-derived lines, in addition to non-malignant B-cells as a control. We performed unbiased labeling of cell surface proteins by biotinylation of glycosylated amino acids (3 biological replicates each) followed by affinity pull-down with streptavidin. After on-bead trypsin digestion, peptides from bound glycoproteins were analyzed on a Thermo Q-Exactive Plus Mass spectrometer using 4-hour LC runs, data-dependent acquisition, and label-free quantification to identify and quantify members of the cell surfaceome for each cell line. To confirm our proteomic findings we performed flow cytometry using a BD CytoFLEX flow cytometer on select cell lines with fluorescently conjugated monoclonal antibodies, in order to assess presence and qualitative abundance of particular cell surface targets.Results: We quantified on average >900 high-confidence membrane proteins (FDR=0.05) per cell type, comprising ~40% of all proteins identified in each experiments. Principal component analysis identified unique cell surfaceome signatures that distinguish several of the leukemia subtypes and normal B-cells from each other, implying different cell-surface phenotypes associated with specific B-ALL genetic alterations (Figure 1A). As positive controls, we found several proteins differentially expressed in MLLr that were also previously identified based on transcriptomic profiling of MLLr patient samples, such as FLT3 and PROM1. In our data, the MLLr surfaceome was surprisingly enriched for Ephrin receptors, matrix metalloproteases, and other proteins that play a role in cell motility, invasion of tissues, and modulation of the tumor microenvironment (significantly enriched membrane proteins colored in red, Figure 1B). Using flow cytometry with monoclonal antibodies specific for select cell surface markers we found excellent agreement with our proteomic data. We will next move to patient-derived xenografts and tissues for further validation. These findings suggest these novel markers may be candidates for downstream immunotherapy development, potentially specific to MLLr B-ALL, where there is particularly high clinical need.Summary and Conclusions: In this work, we profiled the cell surface proteomes of multiple B-ALL cell models, identifying cell surfaceome signatures unique to different B-ALL subtypes. Overall, our work demonstrates the power of performing un-biased interrogation of the tumor surfaceome to identify new therapeutic targets for immunotherapy, as well as provide a resource to advance our phenotypic understanding of these aggressive cancer subtypes. [Display omitted] DisclosuresWiita:TeneoBio, Inc.: Research Funding; Sutro Biopharma: Research Funding.

Quantification of serum C-mannosyl tryptophan by novel assay to evaluate renal function and vascular complications in patients with type 2 diabetes

C-Mannosyl tryptophan (CMW) is a unique glycosylated amino acid, and a candidate novel biomarker of renal function. In type 2 diabetes (T2D), a combination of metabolites including CMW has recently been the focus of novel biomarkers for the evaluation of renal function and prediction of its decline. However, previous quantification methods for serum CMW have several limitations. We recently established a novel assay for quantifying serum CMW. Serum CMW from 99 Japanese patients with T2D was quantified by this assay using hydrophilic interaction liquid chromatography. The serum CMW levels were cross-sectionally characterized in relation to clinical features, including renal function and vascular complications. Serum CMW level was more strongly correlated with serum creatinine and cystatin C levels and with eGFR than with albumin urea level. The ROC curve to detect eGFR < 60 ml/min/1.73 m2 revealed that the cutoff serum CMW level was 337.5 nM (AUC 0.883). Serum CMW levels were higher in patients with a history of macroangiopathy than in those without history. They correlated with ankle-brachial pressure index, whereas cystatin C did not. Serum CMW levels quantified by the novel assay could be useful in evaluation of glomerular filtration of renal function and peripheral arterial disease in T2D.

Facile Access to C-Glycosyl Amino Acids and Peptides via Ni-Catalyzed Reductive Hydroglycosylation of Alkynes

Facile Access to <i>C</i>-Glycosyl Amino Acids and Peptides via Ni-Catalyzed Reductive Hydroglycosylation of Alkynes

Development of Strategies for Glycopeptide Synthesis: An Overview on the Glycosidic Linkage

Glycoproteins and glycopeptides are an interesting focus of research, because of their potential use as therapeutic agents, since they are related to carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions, which are commonly involved in biological processes. It has been established that natural glycoconjugates could be an important source of templates for the design and development of molecules with therapeutic applications. However, isolating large quantities of glycoconjugates from biological sources with the required purity is extremely complex, because these molecules are found in heterogeneous environments and in very low concentrations. As an alternative to solving this problem, the chemical synthesis of glycoconjugates has been developed. In this context, several methods for the synthesis of glycopeptides in solution and/or solid-phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for both solution and solid-phase synthesis. The synthetic viability of glycoconjugates is a critical parameter for allowing their use as drugs to mitigate the impact of microbial resistance and/or cancer. However, the chemical synthesis of glycoconjugates is a challenge, because these molecules possess multiple reaction sites and have a very specific stereochemistry. Therefore, it is necessary to design and implement synthetic routes, which may involve various protection schemes but can be stereoselective, environmentally friendly, and high-yielding. This review focuses on glycopeptide synthesis by recapitulating the progress made over the last 15 years.