Identification of cell culture conditions to control N‐glycosylation site‐occupancy of recombinant glycoproteins expressed in CHO cells

The effect of different cell culture conditions on N‐glycosylation site‐occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t‐PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N‐glycosylation site that is variably occupied. Different environmental factors that affect the site‐occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t‐PA (type I t‐PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site‐occupancy of t‐PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site‐occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site‐occupancy of t‐PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N‐glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site‐occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N‐glycan site‐occupancy within a specific range. An increase in culture pH correlated with a decrease in site‐occupancy. Similarly, delaying the timing for butyrate addition also decreased site‐occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N‐glycan site‐occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

Comparison of sialylated N‐glycopeptide levels in serum of pancreatic cancer patients,acute pancreatitis patients,and healthy controls

Serum protein glycosylation is known to be affected by pathological conditions, including cancer and inflammatory diseases. Pancreatic cancer patients would benefit from early diagnosis, as the disease is often detected in an advanced stage and has poor prognosis. Searching for changes in serum protein site‐specific glycosylation could reveal novel glycoprotein biomarkers. We used Sambucus nigra lectin affinity chromatography to enrich α‐2,6 sialylated tryptic N‐glycopeptides from albumin‐depleted sera of pancreatic cancer patients, acute pancreatitis patients, and healthy individuals, and compared their relative abundance using ultra performance LC‐MS. Relative quantitation was done using the spectrum processing software MZmine. Identification was performed on the web‐based tool GlycopeptideID, developed for in silico analysis of intact N‐glycopeptides. Seventeen high‐abundance serum proteins, mainly acute‐phase proteins, and immunoglobulins, with total 27 N‐glycosylation sites, and 62 glycoforms, were identified. Pancreatitis patient sera contained 38, and pancreatic cancer patients sera contained 13 glycoform changes with statistical significance (p < 0.05). In pancreatitis, up to tenfold changes were found in some glycoforms, and in pancreatic cancer, threefold. Analysis showed that the changes often concerned one or two, but not all, N‐glycosylation sites in a specific glycoprotein. In conclusion, the analysis shows that pancreatic cancer, and acute pancreatitis are associated with changes in concentrations of intact sialylated N‐glycopeptides derived from acute‐phase proteins, and immunoglobulins, and that changes are site specific.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.     

Polymer‐Supported Optically Active fac(S)‐Tris(thiotato)rhodium(III) Complex for Sulfur‐Bridging Reaction With Precious Metal Ions

The optically active mixed‐ligand fac(S)‐tris(thiolato)rhodium(III) complexes, Δ_L‐fac(S)‐[Rh(aet)₂(L‐cys‐N,S)]^? (aet = 2‐aminoethanethiolate, L‐cys = L‐cysteinate) ( 1 ) and Δ_LL‐fac(S)‐[Rh(aet)(L‐cys‐N,S)₂]^2? were newly prepared by the equatorial preference of the carboxyl group in the coordinated L‐cys ligand. The amide formation reaction of 1 with 1,10‐diaminodecane and polyallylamine gave the diamine‐bridged dinuclear Rh(III) complex and the single‐chain polymer‐supported Rh(III) complex with retention of the Δ_L configuration of 1 , respectively. These Rh(III) complexes reacted with Co(III) or Co(II) to give the linear‐type trinuclear structure with the S‐bridged Co(III) center and the two Δ‐Rh(III) terminal moieties. The polymer‐supported Rh(III) complex was applied not only to the CD spectropolarimetric detection and determination of a trace of precious metal ions such as Au(III), Pt(II), and Pd(II) but also to concentration and extraction of these metal ions into the solid polymer phase. Chirality 28:85–91, 2016. © 2015 Wiley Periodicals, Inc.     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

An oligosaccharyltransferase from Leishmania major increases the N‐glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central‐protein complex facilitating the N‐glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N‐glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single‐subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well‐established production platform for recombinant proteins. A fluorescent protein‐tagged LmSTT3D variant was predominately found in the ER and co‐located with plant oligosaccharyltransferase subunits. Co‐expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N‐glycosylation site occupancy on all N‐glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N‐glycosylation efficiency in plants.     

O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc₂, N‐acetyl galactosamine₁, Gal₁). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.     

Regulation of Glucose Transporter SGLT1 by Ubiquitin Ligase Nedd4‐2 and Kinases SGK1, SGK3, and PKB

Objectives : Serum‐ and glucocorticoid‐inducible kinase 1 (SGK1) inhibits the ubiquitin ligase neuronal cell expressed developmentally downregulated 4‐2 (Nedd4‐2), which retards the retrieval of the epithelial Na⁺ channel ENaC. Accordingly, SGK1 enhances ENaC abundance in the cell membrane. The significance of this effect is shown by an association of an E8CC/CT;I6CC polymorphism in the SGK1 gene with increased blood pressure. However, strong expression of SGK1 in enterocytes not expressing ENaC points to further functions of SGK1. This study was performed to test for regulation of Na⁺‐coupled glucose transporter 1 (SGLT1) by Nedd4‐2, SGK1, and/or the related kinases SGK3 and PKB. Additional studies searched for an association of the SGK1 gene with BMI. Research Methods and Procedures : mRNA encoding SGLT1, wild‐type Nedd4‐2, inactive ^C938SNedd4‐2, wild type SGK1, constitutively active ^S422DSGK1 or inactive ^K127NSGK1, wild‐type SGK3, and constitutively active ^T308DS473DPKB or inactive ^T308AS473APKB were injected into Xenopus oocytes, and glucose transport was quantified from glucose‐induced current (I_glc). BMI was determined in individuals with or without the E8CC/CT;I6CC polymorphism. Results: I_glc was significantly decreased by coexpression of Nedd4‐2 but not of ^C938SNedd4‐2. Coexpression of SGK1, ^S422DSGK1, SGK3, or ^T308DS473DPKB, but not of ^K127NSGK1 or ^T308AS473APKB, enhanced I_glc and reversed the effect of Nedd4‐2. SGK1 and SGK3 phosphorylated Nedd4‐2. Deletion of the SGK/PKB phosphorylation sites in Nedd4‐2 blunted the kinase effects. BMI was significantly (p < 0.008) greater in individuals with the E8CC/CT;I6CC polymorphism than in individuals without. Discussion : Overactivity of SGK1 may lead not only to excessive ENaC activity and hypertension but also to enhanced SGLT1 activity and obesity.     

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac₅c^dOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and ¹H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac₅c^dOM]‐(Aib)₄‐OEt ( 1) and Cbz‐(Aib)₄‐[(S,S)‐Ac₅c^dOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 3₁₀‐helical structure was present in 1 and that a right‐handed (P) 3₁₀‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Chiral syntheses of methyl (R)‐2‐Sulfanylcarboxylic esters and acids with optical purity determination using HPLC

Accessible chiral syntheses of 3 types of (R)‐2‐sulfanylcarboxylic esters and acids were performed: (R)‐2‐sulfanylpropanoic (thiolactic) ester (53%, 98%ee) and acid (39%, 96%ee), (R)‐2‐sulfanylsucciinic diester (59%, 96%ee), and (R)‐2‐mandelic ester (78%, 90%ee) and acid (59%, 96%ee). The present practical and robust method involves (i) clean S_N2 displacement of methanesulfonates of (S)‐2‐hydroxyesters by using commercially available AcSK with tris(2‐[2‐methoxyethoxy])ethylamine and (ii) sufficiently mild deacetylation. The optical purity was determined by the corresponding (2R,5R)‐trans‐thiazolidin‐4‐one and (2S,5R)‐cis‐thiazolidin‐4‐one derivatives based on accurate high‐performance liquid chromatography analysis with high‐resolution efficiency. Compared with the reported method utilizing AcSCs (generated from AcSH and CsCO₃), the present method has several advantages, that is, the use of odorless AcCOSK reagent, reasonable reaction velocity, isolation procedure, and accurate, reliable optical purity determination. The use of accessible AcSK has advantages because of easy‐to‐handle odorless and hygroscopic solid that can be used in a bench‐top procedure. The Ti(OiPr)₄ catalyst promoted smooth trans‐cyclo‐condensation to afford (2R,5R)‐trans‐thiazolidin‐4‐one formation of (R)‐2‐sulfanylcarboxylic esters with available N‐(benzylidene)methylamine under neutral conditions without any racemization, whereas (2S,5R)‐cis‐thiazollidin‐4‐ones were obtained via cis‐cyclo‐condensation and no catalysts. Direct high‐performance liquid chromatography analysis of methyl (R)‐mandelate was also performed; however, the resolution efficiency was inferior to that of the thaizolidin‐4‐one derivatizations.     

Complete structure determination of N‐acetyl‐D‐galactosamine‐binding mistletoe lectin‐3 from Viscum album L. album

The primary structure of the B chain of the N‐acetyl‐D ‐galactosamine‐recognizing mistletoe lectin‐3 (ML‐3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC‐separation and Edman degradation and confirmation of the peptide sequences by MALDI‐MS. ML‐3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N‐glycosylation sites at positions Asn⁹⁵ and Asn¹³⁵ of the lectin, were determined by a combination of glycosidase treatment and MALDI‐MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type‐II RIPs, although there are remarkable differences in the D ‐galactose‐specific mistletoe lectin‐1B chain. The recently published primary structure of the mistletoe lectin‐3A chain 1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML‐3. The model demonstrates, unequivocally, that ML‐3 is a member of the type‐II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML‐1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin‐3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

Enantioselective synthesis and antioxidant activity of 3‐(3,4‐dihydroxyphenyl)‐glyceric acid—Basic monomeric moiety of a biologically active polyether from Symphytum asperum and S. caucasicum

The racemic and enantioselective synthesis of a novel glyceric acid derivative, namely, 2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid as well as the antioxidant activities is described. The virtually pure enantiomers, (+)‐(2R,3S)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid and (?)‐(2S,3R)‐2,3‐dihydroxy‐3‐(3,4‐dihydroxyphenyl)‐propionic acid were synthesized for the first time via Sharpless asymmetric dihydroxylation of trans‐caffeic acid derivatives using the enantiocomplementary catalysts, (DHQD)₂‐PHAL and (DHQ)₂‐PHAL. The determination of enantiomeric purity of the novel chiral glyceric acid derivatives was performed by high‐performance liquid chromatographic techniques on the stage of their alkylated precursors. The novel glyceric acid derivatives show strong antioxidant activity against hypochlorite and N,N‐diphenyl‐N‐picryl‐hydrazyl free radical. Their antioxidant activity is about 40‐fold higher than that of the corresponding natural polyether and three‐fold higher of trans‐caffeic acid itself. Chirality, 2010. © 2010 Wiley‐Liss, Inc.