scholarly journals Successive remodeling of IgG glycans using a solid-phase enzymatic platform

2021 ◽  
Author(s):  
Yen-Pang Hsu ◽  
Deeptak Verma ◽  
Shuwen Sun ◽  
Caroline McGregor ◽  
Ian Mangion ◽  
...  
Keyword(s):  
Solid Phase ◽  
Biological Behavior ◽  
High Yield ◽  
Complex Type ◽  
Biologic Drugs ◽  
Naturally Occurring ◽  
Igg Binding ◽  
Fundamental Understanding ◽  
Enzyme Combination ◽  
Potential Strategy

AbstractThe success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity.SignificanceGlycosylation plays critical functional and structural roles in protein biology. However, our understanding of how discrete glycan structures affect protein behaviors remains extremely limited due to the naturally occurring microheterogeneity. Through the use of characterized glycoengineering enzyme combination, we report a solid-phase glycan remodeling (SPGR) platform that enables efficient IgG glycan harmonization into several glycoforms of interest with high biocompatibility to the substrates. It provides an efficient strategy to screen the biological behavior of distinct glycoforms, building a fundamental understanding of glycosylation.

Polymer-Bound Triphenylphosphine and 4,4′-Dinitroazobenzene as a Coupling Reagents for Chromatography-Free Esterification Reaction

2019 ◽  
Vol 16 (7) ◽  
pp. 1024-1031
Author(s):  
Diparjun Das ◽  
Kalyani Rajkumari ◽  
Lalthazuala Rokhum
Keyword(s):  
Microwave Irradiation ◽  
Solid Phase ◽  
Reaction System ◽  
Triphenylphosphine Oxide ◽  
High Yield ◽  
Sustainable Chemistry ◽  
Esterification Reaction ◽  
Coupling Reagent ◽  
Simple Filtration ◽  
Esterification Reactions

Aim and Objective: Sustainable production of fine chemicals both in industries and pharmaceuticals heavily depends on the application of solid-phase synthesis route coupled with microwave technologies due to their environmentally benign nature. In this report, a microwave-assisted esterification reaction using polymer-bound triphenylphosphine and 4,4′-dinitroazobenzene reagent system was investigated. Materials and Methods: The solvents were obtained from Merck India. Polymer-bound triphenylphosphine (~3 mmol triphenylphosphine moiety/g) was acquired from Sigma-Aldrich. The progress of the reaction was observed by thin-layer chromatography. All the reactions were performed in Milestones StartSYNTH microwave. The NMR spectra were recorded on Bruker Avance III 300, 400, and 500 MHz FT NMR Spectrometers. Using azo compound and polymer-bound triphenyl phosphine as a coupling reagent, esterification of different carboxylic acids with alcohols was performed under microwave irradiation. Results: Esterification of benzoic acid with 1-propanol under microwave irradiation gave a high yield of 92% propyl benzoate in 60 minutes only. Isolation of the ester products was relatively simple as both the byproducts polymer-bound triphenylphosphine oxide and hydrazine could be removed by simple filtration. The rates of reactions were found to be directly proportional to the pKa of the benzoic acids. Conclusion: 4,4′-Dinitroazobenzene was introduced as a novel coupling reagent, in conjugation with polymer-bound triphenylphosphine, for esterification reactions under microwave irradiation. The low moisture sensitivity of the reaction system, easy separation of the byproducts, and column chromatographyfree isolation of esters help our methods with application significance, particularly from the ‘Sustainable Chemistry’ perspective.

Synthesis of O-Phosphotyrosine-Containing Peptides. II. Solution-Phase Synthesis of Asn-Glu-PTyr-Thr-Ala Through Methyl Phosphate Protection

1989 ◽  
Vol 42 (9) ◽  
pp. 1519 ◽  
Author(s):  
RM Valerio ◽  
JW Perich ◽  
EA Kitas ◽  
PF Alewood ◽  
RB Johns
Keyword(s):  
Trifluoroacetic Acid ◽  
Dimethyl Sulfide ◽  
Solution Phase ◽  
High Yield ◽  
Phase Synthesis ◽  
Rous Sarcoma ◽  
Naturally Occurring ◽  
Solution Phase Synthesis ◽  
Sarcoma Virus ◽  
Methyl Phosphate

The PTyr (O-phosphotyrosine) pentapeptide H-Asn-Glu-Tyr(PO3H2)-Thr-Ala-OH.HO2CCF3, which is a naturally occurring sequence from the autophosphorylated Rous sarcoma virus pp60V-SrC, was prepared in high yield by the use of Boc-Tyr(PO3Me2)-OH in the Boc mode of solution-phase peptide synthesis. The protected pentapeptide, Z-Asn-Glu(OBzl)-Tyr(PO3Me2)-Thr(Bzl)-Ala-OBzl, was deprotected by a two-stage procedure which involved initial palladium-catalysed hydrogenolysis followed by the removal of the phosphate methyl groups by the use of one of the following treatments: (A) 10% bromotrimethylsilane/acetonitrile, (B) 1 M bromotrimethylsilane/thioanisole in trifluoroacetic acid, or (C) trifluoromethanesulfonic acid/trifluoroacetic acid/dimethyl sulfide/m-cresol.

scholarly journals COMPOSITION, PROPERTIES AND RECYCLING OF VEGETATIVE POPLAR AFTER REMOVAL OF EXTRAC-TIVES. REPORT 3. OBTAINING BIOIOGICAL PRODUCTS BASED ON FUNGI OF THE GENUS TRIGHODERMA

2020 ◽  
pp. 415-423
Author(s):  
Elena Vladimirovna Isaeva ◽  
Ol'ga Olegovna Mamaeva ◽  
Tat'yana Vasil'yevna Ryazanova
Keyword(s):  
Plant Growth ◽  
Solid Phase ◽  
Liquid Waste ◽  
Raw Material ◽  
High Yield ◽  
Mycelial Fungi ◽  
Biological Product ◽  
Vegetative Part ◽  
Trichoderma Fungi ◽  
Composition Properties

The purpose of this work was to assess the suitability of solid and liquid waste generated during processing of the vegetative part of poplar as substrates for biochemical processing in order to obtain biologics for various purposes. For the study, we used post-extraction residues, as well as a cubic liquid formed after distilling essential oils and extracting alcohol-soluble substances from the vegetative part of the balsamic poplar (Populus balzamifera L.). Siberian strains of fungi of the genus Trichoderma used as a biodestructor. Studies have shown that the vegetative part of poplar and its individual elements are an available substrate for the growth of mycelial fungi. The high yield of spores (4.5×109 spor/g) and the formation of humic substances (11%) used as plant growth stimulators during solid-phase cultivation of the MG-97 strain of Trichoderma fungi gives grounds to use the vegetative part of poplar as a technological raw material for obtaining a biological product of the "Trichodermin" type or soil humification. Depending on the purpose of the preparations, the duration of cultivation can vary: for obtaining agricultural biologics up to 15 days, more – for soil humification. The inclusion of a cubic liquid at the stage of substrate humidification allows to obtain a biological product with a higher spore titer (5×109 spor/g), makes it possible to close the water consumption cycle and make the technology of processing the vegetative part of poplar waste-free.

scholarly journals Processing of RNA Containing 8-Oxo-7,8-Dihydroguanosine (8-oxoG) by the Exoribonuclease Xrn-1

2021 ◽  
Vol 8 ◽  
Author(s):  
Cheyenne N. Phillips ◽  
Shawn Schowe ◽  
Conner J. Langeberg ◽  
Namoos Siddique ◽  
Erich G. Chapman ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Conformational Changes ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Spatial Constraints ◽  
Phase Synthesis ◽  
Naturally Occurring ◽  
Processive Enzyme ◽  
Moving Band ◽  
Hydrolysis Of

Understanding how oxidatively damaged RNA is handled intracellularly is of relevance due to the link between oxidized RNA and the progression/development of some diseases as well as aging. Among the ribonucleases responsible for the decay of modified (chemically or naturally) RNA is the exonuclease Xrn-1, a processive enzyme that catalyzes the hydrolysis of 5′-phosphorylated RNA in a 5′→3′ direction. We set out to explore the reactivity of this exonuclease towards oligonucleotides (ONs, 20-nt to 30-nt long) of RNA containing 8-oxo-7,8-dihydroguanosine (8-oxoG), obtained via solid-phase synthesis. The results show that Xrn-1 stalled at sites containing 8-oxoG, evidenced by the presence of a slower moving band (via electrophoretic analyses) than that observed for the canonical analogue. The observed fragment(s) were characterized via PAGE and MALDI-TOF to confirm that the oligonucleotide fragment(s) contained a 5′-phosphorylated 8-oxoG. Furthermore, the yields for this stalling varied from app. 5–30% with 8-oxoG located at different positions and in different sequences. To gain a better understanding of the decreased nuclease efficiency, we probed: 1) H-bonding and spatial constraints; 2) anti-syn conformational changes; 3) concentration of divalent cation; and 4) secondary structure. This was carried out by introducing methylated or brominated purines (m1G, m6,6A, or 8-BrG), probing varying [Mg2+], and using circular dichroism (CD) to explore the formation of structured RNA. It was determined that spatial constraints imposed by conformational changes around the glycosidic bond may be partially responsible for stalling, however, the results do not fully explain some of the observed higher stalling yields. We hypothesize that altered π-π stacking along with induced H-bonding interactions between 8-oxoG and residues within the binding site may also play a role in the decreased Xrn-1 efficiency. Overall, these observations suggest that other factors, yet to be discovered/established, are likely to contribute to the decay of oxidized RNA. In addition, Xrn-1 degraded RNA containing m1G, and stalled mildly at sites where it encountered m6,6A, or 8-BrG, which is of particular interest given that the former two are naturally occurring modifications.

A Comprehensive One-Pot Synthesis of Protected Cysteine and Selenocysteine SPPS Derivatives

2014 ◽  
Vol 21 (12) ◽  
pp. 1257-1264
Author(s):  
Stevenson Flemer
Keyword(s):  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Reduction Process ◽  
Building Blocks ◽  
High Yield ◽  
One Pot ◽  
Peptide Models ◽  
Direct Use ◽  
Proof Of Principle ◽  
Subsequent Incorporation

A proof-of-principle methodology is presented in which all commercially-available cysteine (Cys) and selenocysteine (Sec) solid phase peptide synthesis (SPPS) derivatives are synthesized in high yield from easily prepared protected dichalcogenide precursors. A Zn-mediated biphasic reduction process applied to a series of four bis-Nα-protected dichalcogenide compounds allows facile conversion to their corresponding thiol and selenol intermediates followed by insitu S- or Se-alkylation with various electrophiles to directly access twenty one known Cys and Sec SPPS derivatives. Most of these derivatives were able to be precipitated in crude form out of petroleum ether in sufficient purity for direct use as peptide building blocks. Subsequent incorporation of these derivatives into peptide models nicely illustrates their viability and applicability toward SPPS.

Laminin-521 is a Novel Target of Autoantibodies Associated with Lung Hemorrhage in Anti-GBM Disease

2021 ◽  
pp. ASN.2020101431
Author(s):  
Cong-rong Shen ◽  
Xiao-yu Jia ◽  
Wentian Luo ◽  
Florina Olaru ◽  
Zhao Cui ◽  
...  
Keyword(s):  
Solid Phase ◽  
Pulmonary Hemorrhage ◽  
Basement Membranes ◽  
Glomerular Diseases ◽  
Igg Binding ◽  
Lung Hemorrhage ◽  
Pathogenic Antibodies ◽  
Novel Target ◽  
Circulating Autoantibodies ◽  
Cryptic Epitopes

BackgroundAntiglomerular basement membrane (anti-GBM) disease is characterized by GN and often pulmonary hemorrhage, mediated by autoantibodies that typically recognize cryptic epitopes within α345(IV) collagen—a major component of the glomerular and alveolar basement membranes. Laminin-521 is another major GBM component and a proven target of pathogenic antibodies mediating GN in animal models. Whether laminin-521 is a target of autoimmunity in human anti-GBM disease is not yet known.MethodsA retrospective study of circulating autoantibodies from 101 patients with anti-GBM/Goodpasture’s disease and 85 controls used a solid-phase immunoassay to measure IgG binding to human recombinant laminin-521 with native-like structure and activity.ResultsCirculating IgG autoantibodies binding to laminin-521 were found in about one third of patients with anti-GBM antibody GN, but were not detected in healthy controls or in patients with other glomerular diseases. Autoreactivity toward laminin-521 was significantly more common in patients with anti-GBM GN and lung hemorrhage, compared with those with kidney-limited disease (51.5% versus 23.5%, P=0.005). Antilaminin-521 autoantibodies were predominantly of IgG1 and IgG4 subclasses and significantly associated with lung hemorrhage (P=0.005), hemoptysis (P=0.008), and smoking (P=0.01), although not with proteinuria or serum creatinine at diagnosis.ConclusionsBesides α345(IV) collagen, laminin-521 is another major autoantigen targeted in anti-GBM disease. Autoantibodies to laminin-521 may have the potential to promote lung injury in anti-GBM disease by increasing the total amount of IgG bound to the alveolar basement membranes.

scholarly journals Development and Validation of a UHPLC-ESI-MS/MS Method for Quantification of Oleandrin and Other Cardiac Glycosides and Evaluation of Their Levels in Herbs and Spices from the Belgian Market

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 243 ◽  
Author(s):  
Svetlana V. Malysheva ◽  
Patrick P. J. Mulder ◽  
Julien Masquelier
Keyword(s):  
Cardiac Glycosides ◽  
Solid Phase ◽  
Plant Secondary Metabolites ◽  
Naturally Occurring ◽  
Relative Standard ◽  
Liquid Chromatography Tandem Mass ◽  
Repeatability And Reproducibility ◽  
Culinary Herbs ◽  
The Mean ◽  
Development And Validation

Cardiac glycosides (CGs) are naturally occurring plant secondary metabolites that can be toxic to humans and animals. The aim of this work was to develop a targeted analytical method utilizing liquid chromatography—tandem mass spectrometry (LC-MS/MS) for quantification of these plant toxins in a herbal-based food and human urine. The method included oleandrin, digoxin, digitoxin, convallatoxin, and ouabain. Samples of culinary herbs were extracted with acetonitrile and cleaned using Oasis® MAX solid-phase extraction (SPE), while samples of urine were diluted with acidified water and purified on Oasis® HLB SPE cartridges. Limits of quantification were in the range of 1.5–15 ng/g for herbs and 0.025–1 ng/mL for urine. The mean recovery of the method complied with the acceptable range of 70–120% for most CGs, and relative standard deviations were at maximum 14% and 19% for repeatability and reproducibility, respectively. Method linearity was good with calculated R² values above 0.997. The expanded measurement uncertainty was estimated to be in the range of 7–37%. The LC-MS/MS method was used to examine 65 samples of culinary herbs and herb and spice mixtures collected in Belgium, from supermarkets and local stores. The samples were found to be free from the analyzed CGs.

Synthesis of Casein Related Peptides and Phosphopeptides. XIV. Solid Phase Synthesis of Glu-Ser(P)-Leu Through the Use of Protected Boc-Ser(PO3R2)-OH Derivatives

1991 ◽  
Vol 44 (6) ◽  
pp. 771 ◽  
Author(s):  
JW Perich ◽  
RM Valerio ◽  
PF Alewood ◽  
RB Johns
Keyword(s):  
High Pressure ◽  
Peptide Synthesis ◽  
Solid Phase ◽  
Phenyl Group ◽  
High Yield ◽  
Palladium Acetate ◽  
Phase Method ◽  
Phenyl Phosphate ◽  
One Step ◽  
The One

A solid phase method is described for the synthesis of O- phosphoseryl-containing peptides by the use of polystyrene resin (Merrifield) as the peptide support and protected Boc-Ser(PO3R2)-OH derivatives for the incorporation of the phosphorylated seryl residue. The viability of this solid phase approach was demonstrated by the synthesis of HBr.H-Glu-Ser (PO3Et2)-Leu-OH in high yield by the use of Bo -Ser(PO3Et2)-OH in peptide synthesis and subsequent use of HBr/CF3CO2H for cleavage of the Ser(PO3Et2)-containing tripeptide from the resin support. Similarly, the dipeptide, CF3CO2H.H-Ser(P)- Leu -OH, was prepared in high yield by using Boc -Ser(PO3But2)-OH in peptide synthesis followed by the one-step deprotection of the Ser(PO3But2)- dipeptide resin by treatment with HBr/CF3CO2H (90 min). Alternatively, the O-phosphoseryl tripeptide , CF3CO2H.H-Glu-Ser(P)- Leu -OH was prepared by using either Ppoc -Ser(PO3Bzl2)-OH or Boc-Ser(PO3Ph2)-OH in peptide synthesis. The one-step deprotection of the Ser(PO3Bzl2)-containing tripeptide and cleavage of the peptide from the resin support was effected by high-pressure hydrogenolysis (palladium acetate). In the case of phenyl phosphate protection, the Ser(PO3Ph2)-containing peptide was cleaved from the resin support by high-pressure hydrogenolysis (palladium acetate) followed by cleavage of the phenyl phosphate groups by platinum-mediated hydrogenolysis (1.0 equiv. PtO2/phenyl group) in 50% CF3CO2H/AcOH.

Use of Radioimmunoassay to Determine the Nature, Quantity and Source of Allergenic Contamination of Sunflower Butter

1983 ◽  
Vol 46 (7) ◽  
pp. 625-628 ◽  
Author(s):  
JOHN W. YUNGINGER ◽  
MARY B. GAUERKE ◽  
RICHARD T. JONES ◽  
MARY JO E. DAHLBERG ◽  
STEVEN J. ACKERMAN
Keyword(s):  
Allergic Reaction ◽  
Food Processing ◽  
Immunoglobulin E ◽  
Solid Phase ◽  
Peanut Butter ◽  
Manufacturing Plant ◽  
Ige Antibodies ◽  
Naturally Occurring ◽  
Solid Phase Radioimmunoassay ◽  
Butter Production

A girl known to be allergic to peanuts experienced a mild allergic reaction after eating sunflower butter, a peanut butter facsimile prepared from sunflower nuts. After learning that the same manufacturing plant also produced peanut butter, we examined several sunflower butter preparations for peanut butter contamination by a solid-phase radioimmunoassay which used naturally-occurring immunoglobulin E (IgE) antibodies from known peanut-sensitive individuals. Peanut butter contamination, ranging from 0.3 to 3.3%, was found in six of eight sunflower butter samples, including both creamy and chunky varieties, and including lots prepared with either peanut oil or palm oil. We concluded that inadequate cleaning of food processing machines following peanut butter production permitted the contamination by peanut butter of subsequent jars of sunflower butter in amounts which could pose risks to peanut-sensitive individuals.

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