Synthesis of polypeptides from activated urethane derivatives of α‐amino acids

A series of activated urethane‐type derivatives of α‐amino acids were synthesized and applied to polypeptide synthesis. The urethane used herein, N‐(4‐nitrophenoxycarbonyl)‐α‐amino acids 1 , were synthesized by N‐carbamoylation of γ‐benzyl‐L ‐glutamate, β‐benzyl‐L ‐aspartate, L ‐leucine, L ‐phenylalanine, and L ‐proline, with 4‐nitrophenyl chloroformate. When 1 was dissolved in N,N‐dimethylacetamide (DMAc) and heated at 60 °C, it was smoothly converted into the corresponding polypeptides with releasing 4‐nitrophenol and carbon dioxide. Spectroscopic analyses of the obtained polypeptides revealed that they were comparable with the authentic polypeptides synthesized by the ring‐opening polymerizations of amino acid N‐carboxyanhydrides (NCAs). Besides the successful polycondensations of a series of 1 , their polycondensations of 1a and other 1 were also successfully carried out to obtain the corresponding statistic copolypeptides. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2525–2535, 2008     

Boronic acid functionalized fibrous cellulose for the selective enrichment of glycopeptides

Enrichment of glycoproteins has been important because of their dynamicity and role in biological systems. Study of glycoproteins is complex because of the simultaneous glycosylation and deglycosylation inside the body. Often employed affinities for glycopeptides are hydrazide, boronic acid, or physiosorbed lectin on support materials. Cellulose, a natural polysaccharide, has rich surface chemistry, stable structure, low cost and availability in different variants. In present study, fibrous cellulose is oxidized using periodate to modify with boronic acid. Attachment of boronic acid is confirmed by Fourier transform infrared spectroscopy. Particle size and morphology of boronic acid@fibrous cellulose is studied by scanning electron microscopy. The enrichment efficiency is evaluated by using horseradish peroxidase as model protein. Boronic acid@fibrous cellulose is selective up to 1:250 for spiked horseradish peroxidase in bovine serum albumin digest, sensitive down to 0.1 femtomol and recovering 88.15% glycopeptides. Moreover, protein binding capacity is determined as 213 mg/g and 41% sequence coverage of horseradish peroxidase protein with all eight glycosylation sites detected. Total of 18 glycopeptides are enriched from immunoglobulin digest showing ability of boronic acid@fibrous cellulose to enrich glycoproteins from multiglycoforms. Enrichment from human serum recovers 18% extracellular and 72% secreted glycoproteins via bottom‐up approach and online tools.     

Non-natural amino acids as modulating agents of the conformational space of model glycopeptides

The synthesis and conformational analysis in aqueous solution of different alpha-methyl-alpha-amino acid diamides, derived from serine, threonine, beta-hydroxycyclobutane-alpha-amino acids, and their corresponding model beta-O-glucopeptides, are reported. The study reveals that the presence of an alpha-methyl group forces the model peptides to adopt helix-like conformations. These folded conformations are especially significant for cyclobutane derivatives. Interestingly, this feature was also observed in the corresponding model glucopeptides, thus indicating that the alpha-methyl group and not the beta-O-glucosylation process largely determines the conformational preference of the backbone in these structures. On the other hand, atypical conformations of the glycosidic linkage were experimentally determined. Therefore, when a methyl group was located at the Cbeta atom with an R configuration, the glycosidic linkage was rather rigid. Nevertheless, when the S configuration was displayed, a significant degree of flexibility was observed for the glycosidic linkage, thus showing both alternate and eclipsed conformations of the psi(s) dihedral angle. In addition, some derivatives exhibited an unusual value for the phi(s) angle, which was far from a value of -60 degrees expected for a conventional beta-O-glycosidic linkage. In this sense, the different conformations exhibited by these molecules could be a useful tool in obtaining systems with conformational preferences "à la carte".     

Living polymerization of α‐amino acid‐N‐carboxyanhydrides

This article reviews recent developments in the polymerization of α‐amino acid‐ N‐carboxyanhydrides (NCAs) to form polypeptides. Traditional methods used to polymerize these monomers are described, and limitations in the utility of these systems for the preparation of polypeptides with controlled molecular weights and narrow molecular weight distributions are discussed. The development of transition‐metal‐based initiators, which activate the monomers to form covalent active species, permits the formation of polypeptides via the living polymerization of NCAs. In these systems, polymer molecular weights are controlled by monomer‐to‐initiator stoichiometry, polydispersities are low, and block copolypeptides can be prepared. The scope and limitations of these initiators and their key features and mode of operation are described in detail in this highlight. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3011–3018, 2000     

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

Synthesis of homopolypeptides with PPII structure

Polyprolines are attractive polymers because of their folding property into polyproline II (PPII) structure, their significance in protein/protein interactions, and their potential as new therapeutic targets. Silaproline (Sip) is an analogue of proline, which exhibits similar conformational properties. The presence of dimethylsilyl group confers to Sip a higher lipophilicity as well as an improved resistance to biodegradation. Enantiomerically pure Sip was available in gram quantities from resolution of the enantiomers by chiral high performance liquid chromatography. This study describes the first synthesis of Sip N‐carboxyanhydride (NCA) and shows preliminary results on comparison of polymerization of (l )Pro‐NCA and (d )Sip‐NCA to obtain homopolypeptides with PPII structure, polyproline, and polysilaproline polymers. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 3103–3109     

Facile Synthesis of Glycopolypeptides by Combination of Ring‐Opening Polymerization of an Alkyne‐Substituted N‐carboxyanhydride and Click “Glycosylation”

Synthetic glycopolypeptides have attracted much interest for application in biomedical field as they are structural mimics to the natural glycopeptides or glycoproteins. However, the synthesis methods toward glycopolypeptides are still few or less efficient. Herein, we present a facile route to preparation of glycopolypeptides with highly effective “glycosylation” by click postpolymerization modification. First, an alkyne‐substituted N‐carboxyanhydride (NCA) monomer was synthesized and subsequently polymerized to afford the polypeptide with “clickable” alkyne pendants. The alkyne‐functionalized polypeptide was then “glycosylated” by click reaction of different sugar azides to the alkyne pendants with high efficiency. All the obtained glycopolypeptides were soluble and preferred α‐helix conformation in water. Primary studies on the obtained glycopolypeptides binding with Con A lectin were assessed by turbidimetric assay. The more quantitive studies of the interactions between lectin proteins and the synthetic glycopolypeptides, and the application of these materials as the multivalent ligands are in progress.

     

Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N‐GlcNAcylation

As a unique and unappreciated protein posttranslational modification, arginine N‐glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N‐glycosylation, we report the first general strategy for a rapid and cost‐effective synthesis of glycopeptides carrying single or multiple arginine N‐GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N‐GlcNAcylated peptides or proteins in a sequence‐independent manner.     

Optimization of an α‐(Amino acid)‐N‐carboxyanhydride polymerization using the high vacuum technique: Examining the effects of monomer concentration,polymerization kinetics,polymer molecular weight,and monomer purity

l ‐Ornithine‐based poly(peptides) have been widely utilized in the field of drug delivery, however few studies have been conducted examining the details of polymerization. In this article, the effects of monomer concentration, polymerization kinetics, polymer molecular weight and monomer purity were investigated using l ‐carboxybenzyl (Cbz)‐ornithine as a model monomer. The mechanism of polymerization herein follows the normal amine mechanism to produce poly(peptides) having controlled molecular weights, known chain ends and a narrow polydispersity index (PDI). A preferred monomer concentration range was determined, which required minimal polymerization times and allowed for predictable and reproducible molecular weights with narrow PDIs. The impact of monomer purity on the polymerization was established and monomer purification conditions are reported, which produce high‐purity monomer after a single recrystallization. Additionally, the optimized polymerization conditions and monomer purification protocol were combined with a sequential monomer addition technique to produce high molecular weight poly(ornithine) with a narrow PDI and known chain ends. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1385–1391     

Preparation of pseudo glycoamino acid and its application to glycopeptide synthesis

A pseudo glycoamino acid composed of a 1,3-diol structure and an amino acid was synthesized. This amino acid analog can act as an alternative acceptor to an amino acid containing GlcNAc for transglycosylation by Endo-M. A pseudo glycopeptide was synthesized using the pseudo glycoamino acid by a solid phase procedure. We attempted transglycosylation of N-glycan to the peptide using Endo-M.     

Deprotonation reaction of α‐amino acid N‐carboxyanhydride at 4‐CH position by yttrium tris[bis(trimethylsilyl)amide]

Reaction of yttrium tris[bis(trimethylsilyl)amide] [(TMSN)₃Y] with equivalent L ‐alanine N‐carboxyanhydride (ALA NCA) yields yttrium α‐isocyanato carboxylate ( II ), yttrium ketenyl carbamate ( III ), and hexamethyldisilazane ( V ). The products indicate that 4‐CH group of ALA NCA monomer is deprotonated in addition to 3‐NH group, which has been neglected in NCA chemistry for decades. This result proves the acidity of 4‐CH in NCA and provides the first direct evidence for racemization phenomenon of NCA in strong base in microscopic aspect. Rare earth tris[bis(trimethylsilyl)amide] (TMSN)₃Ln (Ln = Sc, Y, La, Dy, and Lu) compounds are high efficient catalysts for ring‐opening polymerizations of NCAs. Polypeptides can be produced in quantitative yields with narrow molecular weight distributions below 1.3, and block copolypeptides can be facilely prepared by sequential addition method. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Controlled-potential electrosynthesis of glucosaminic acid from glucosamine at a gold electrode

The electrocatalytic oxidation of d-glucosamine (2-amino-2-deoxy-d-glucose) in alkaline and neutral solutions was examined using a carbon felt electrode modified with 2 nm core sized gold nanoparticles (Au_2 nm nanoparticles) and a gold plate electrode. The electrocatalytic voltammetric oxidation curves of d-glucosamine were obtained in both solutions. The voltammetric responses for the electrocatalytic oxidation at a Au_2 nm nanoparticle-modified electrode in both alkaline and neutral solutions were almost the same to those at a gold plate electrode. The oxidized product was identified to be d-glucosaminic acid (2-amino-2-deoxy- d-gluconic acid) generated by the 2-electron oxidation product of d-glucosamine by electrospray ionization time-of-flight mass spectra (ESI TOF-MS). The HPLC results also indicated that the oxidation product was d-glucosaminic acid.The controlled-potential electrolysis of d-glucosamine was performed at the Au_2 nm nanoparticle-modified carbon felt electrodes in both alkaline and neutral solutions. In the alkaline solution, at a potential of −0.2 V, d-glucosaminic acid was formed with a current efficiency of 100%. In the neutral solution, electrolysis at 0.4 V on d-glucosaminic acid was obtained with current efficiencies of 70%.     

Selective enrichment of glycopeptides based on copper tetra(N‐carbonylacrylic) aminephthalocyanine and iminodiacetic acid functionalized polymer monolith

Efficient separation and enrichment of low‐abundance glycopeptides from complex biological samples is the key to the discovery of disease biomarkers. In this work, a new material was prepared by coating copper tetra(N‐carbonylacrylic) aminephthalocyanine and iminodiacetic acid onto poly(glycidyl methacrylate‐pentaerythritol triacrylate) monolith. The monolith was applied to polymer monolithic microextraction for specific capture of glycopeptides coupled with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. The developed monolith exhibited satisfactory efficiency for glycopeptide enrichment with high selectivity and detection sensitivity. When the tryptic digest of immunoglobulin G was used as the sample, total 24 glycopeptides were identified and the detection limit was determined as 5 fmol. When the approach was applied to the analysis of glycopeptides in the mixture of bovine serum albumin and immunoglobulin G (100:1, m/m) digests, 16 glycopeptides could still be observed. Moreover, the monolith was successfully applied to the selective enrichment of glycopeptides from human serum digests, exhibiting great practicability in identifying low‐abundance glycopeptides in complex biological samples.