Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Identification and Analysis of the Acetylated Status of Poplar Proteins Reveals Analogous N-Terminal Protein Processing Mechanisms with Other Eukaryotes

Background

The N-terminal protein processing mechanism (NPM) including N-terminal Met excision (NME) and N-terminal acetylation (N^α-acetylation) represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown.

Methodology/Principal Findings

To reveal the NPM in poplar, we investigated the N^α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO₂ enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N^α-acetylated) proteins. Most proteins (47, >81%) are subjected to N^α-acetylation following the N-terminal removal of Met, indicating that N^α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N^α-acetylation (NPM) to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs) and N-terminal acetyltransferase (Nat) enzymes in poplar. The N^α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins.

Conclusions/Significance

This study represents the first extensive investigation of N^α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N^α-acetylation of proteins in poplar.     

Preparation and structure of chitosan soluble in wide pH range

Two kinds of chitosans, namely N-acetylated and N-deacetylated chitosan were prepared by the modified processes. They can dissolve in both acid and alkali solution. ¹³C NMR was used to study the basic solution of chitosan, and XRD, FT-IR and SEM were used to study the structure of N-acetylated and N-deacetylated chitosan. The result from X-ray diffraction showed that a transformation of crystal structure occurred during the N-acetylation or N-deacetylation process with the decrease of crystallinity and expansion of crystal lattices. FT-IR spectra revealed that the intermolecular and intramolecular hydrogen bonds were destroyed by both treatments and a looser structure was observed by the SEM. The lower crystallinity, the decreased intermolecular interactions, the more disordered and looser structure were easy for the permeation of LiOH/urea aqueous solution and coordinated with the breakage of intermolecular and intramolecular hydrogen bond by LiOH at low temperature, the prepared chitosans dissolved in LiOH/urea/H₂O mixture.     

Stability of N-acyl groups in methylated α2 → 8-linked oligosialosyl chains toward methanolysis. Analysis by chemical ionization-mass spectrometry

The stability of N-acetyl group of methylated trisaccharide of N-acetylneuraminic acid toward methanolysis under conditions used in methylation analysis was investigated. The analysis of the products obtained after a reaction sequence, methylation-methanolysis-deuterioacetylation, by chemical ionization-mass spectrometry has led to unequivocal conclusion that N-acetyl group of internal 8-O-substituted residue of the methylated oligosialosyl compound is de-N-acetylated under conditions sufficient to cleave glycosidic linkages, whereas the fully methylated nonreducing terminal residue of neuraminic acid is completely resistant to de-N-acetylation. The reaction mechanism to explain these observations is presented.     

通过染色体整合表达古菌乙酰化酶合成N-末端乙酰化胸腺肽β4

胸腺肽β4（Tβ4）是N-末端乙酰化的43肽,具有多种重要生物学功能.其生物合成存在两大难点,即乙酰化修饰和小分子肽的表达.本研究发现来自古菌Sulfolobus solfataricus的乙酰化酶ssArd1可以催化Tβ4的N-末端乙酰化修饰.利用Red同源重组技术将ssArd1基因表达盒整合至E.coli BL21（DE3）染色体的lpxM位点上,构建了可以实现Tβ4N-末端乙酰化修饰的新型宿主E.coli BDA.将Tβ4编码基因融合在改造的微型Spl DnaX Intein的N端,并在Intein的C端添加His标签,构建了表达载体pET-Tβ4-Intein.在E.coli BDA中表达的融合蛋白,经镍亲和层析纯化后用β-巯基乙醇诱导融合蛋白切割释放小分子多肽,获得了具有N-末端乙酰化的Tβ4.     

Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry

A sensitive method for determination of metallothionein (MT) isoform levels in rat liver by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry was developed. Critical steps in sample preparation, like MT extraction, MT saturation with Cd and protein separation, were optimized. This method is capable of measuring levels of 2.0 μg/g liver for metallothionein-1 (MT-1) and 1.3 μg/g liver for metallothionein-2 (MT-2), respectively, with a high recovery of 103% on average. The method described, thus, proved suitable for analyzing metallothionein isoform concentrations even in untreated animals. The ratio of MT-1 to MT-2 was found to be 1:1 on average. MT decomposition during storage was very high in whole livers, but could be reduced by about 80% when extracted liver samples were used.     

The Structure- and Metal-dependent Activity of Escherichia coli PgaB Provides Insight into the Partial De-N-acetylation of Poly-β-1,6-N-acetyl-D-glucosamine

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In Escherichia coli, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the periplasmic protein PgaB is required for polysaccharide intercellular adhesin-dependent biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of PgaB in complex with Ni²⁺ and Fe³⁺ have been determined to 1.9 and 2.1 Å resolution, respectively, and its activity on β-1,6-GlcNAc oligomers has been characterized. The structure of PgaB reveals two (β/α)_x barrel domains: a metal-binding de-N-acetylase that is a member of the family 4 carbohydrate esterases (CE4s) and a domain structurally similar to glycoside hydrolases. PgaB displays de-N-acetylase activity on β-1,6-GlcNAc oligomers but not on the β-1,4-(GlcNAc)₄ oligomer chitotetraose and is the first CE4 member to exhibit this substrate specificity. De-N-acetylation occurs in a length-dependent manor, and specificity is observed for the position of de-N-acetylation. A key aspartic acid involved in de-N-acetylation, normally seen in other CE4s, is missing in PgaB, suggesting that the activity of PgaB is attenuated to maintain the low levels of de-N-acetylation of PNAG observed in vivo. The metal dependence of PgaB is different from most CE4s, because PgaB shows increased rates of de-N-acetylation with Co²⁺ and Ni²⁺ under aerobic conditions, and Co²⁺, Ni²⁺ and Fe²⁺ under anaerobic conditions, but decreased activity with Zn²⁺. The work presented herein will guide inhibitor design to combat biofilm formation by E. coli and potentially a wide range of medically relevant bacteria producing polysaccharide intercellular adhesin-dependent biofilms.     

Specific Synthesis of Neurostatin and Gangliosides O-Acetylated in the Outer Sialic Acids Using a Sialate Transferase

Gangliosides are sialic acid containing glycosphingolipids, commonly found on the outer leaflet of the plasma membrane. O-acetylation of sialic acid hydroxyl groups is one of the most common modifications in gangliosides. Studies on the biological activity of O-acetylated gangliosides have been limited by their scarcity in nature. This comparatively small change in ganglioside structure causes major changes in their physiological properties. When the ganglioside GD1b was O-acetylated in the outer sialic acid, it became the potent inhibitor of astroblast and astrocytoma proliferation called Neurostatin. Although various chemical and enzymatic methods to O-acetylate commercial gangliosides have been described, O-acetylation was nonspecific and produced many side-products that reduced the yield. An enzyme with O-acetyltransferase activity (SOAT) has been previously cloned from the bacteria Campylobacter jejuni. This enzyme catalyzed the acetylation of oligosaccharide-bound sialic acid, with high specificity for terminal alpha-2,8-linked residues. Using this enzyme and commercial gangliosides as starting material, we have specifically O-acetylated the gangliosides’ outer sialic acids, to produce the corresponding gangliosides specifically O-acetylated in the sialic acid bound in alpha-2,3 and alpha-2,8 residues. We demonstrate here that O-acetylation occurred specifically in the C-9 position of the sialic acid. In summary, we present a new method of specific O-acetylation of ganglioside sialic acids that permits the large scale preparation of these modified glycosphingolipids, facilitating both, the study of their mechanism of antitumoral action and their use as therapeutic drugs for treating glioblastoma multiform (GBM) patients.     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

Detection of N-acetyl derivative of 2-amino-6-methyldipyrido[1,2-α: 3′,2′-d]imidazole (Glu-P-1) in Glu-P-1-injected rats

In order to confirm in vivo N-acetylation of 2-amino-6-methyl-dipyrido[1,2-a:3′,2′-d]imidazole (GluP-1) in rats, we developed a new high-performance liquid chromatography (HPLC) method for detecting the N-acetyl derivative of Glu-P-1 in animal organs. Using this method, N-acetyl-Glu-P-1 was detected in rat liver, kidney and intestinal contents 6 h after intraperitoneal injection of Glu-P-1. This fact provides evidence to support in vivo N-acetylation of Glu-P-1 in rats.     

Quantitation of Human Metallothionein Isoforms: A Family of Small,Highly Conserved,Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.The metallothioneins (MTs)¹ are a family of small, highly conserved proteins with the specific capacity to bind metal ions (1–3). Mammalian MTs, typically 61 to 68 amino acid residues in length, contain 20 invariant cysteine residues that form two distinct metal-binding domains. Up to seven or eight metal ions may be coordinated per MT. Many functions have been attributed to this redox-active protein, including zinc homeostasis; heavy metal detoxification; metal exchange; metal transfer; and protection against oxidative damage, inflammatory responses, and other cellular stresses (4–6). Changes in MT expression have been associated with human pathologies including cadmium-induced renal toxicity (7), neurodegeneration (8), and many forms of cancer (9, 10). The understanding of these changes is complicated by the 11 functional MT genes, seven pseudogenes, and four MT-like genes encoded in the genome, most of which contain only small differences in amino acid sequence (11). Seventeen of the 18 genes and pseudogenes are clustered together on chromosome 16, which is known to be enriched for intrachromosomal duplications (12). The various MT gene products differ in their patterns of mRNA and protein expression in human tissues and cell lines. Immunohistochemical detection using antibodies that do not discriminate between MT-1 and MT-2 isoforms indicates wide tissue and cell type distribution of MTs, as illustrated with the MT-1A entry of the Human Protein Atlas (13, 14). Measurements of individual MT mRNA levels, however, clearly demonstrate differential expression of specific MT-1 isoforms in human tissues and cell lines (15–17). The MT-3 (18, 19) and MT-4 (20) mRNAs are expressed in even narrower ranges of cell types.An abundance of immunohistochemical and mRNA measurements show that alteration of MT isoform expression is correlated with a variety of cancers (9, 10). For example, several studies show that the expression of specific MT isoforms is altered in invasive ductal breast carcinomas. Elevated MT-2A (21) or MT-1F (22) is correlated with increased proliferation or tumor grade, respectively. Expression of MT-3 is associated with poor prognosis (23, 24). The MT-1E isoform is found in estrogen-receptor-negative (ER⁻), but not estrogen-receptor-positive (ER⁺), tumors (25) and cell lines (26). Parallel assessment of changes in MT protein expression via immunohistochemistry supports the mRNA data up to a point. Except for antibodies specific for the MT-3 isoform (27), all commercially available MT antibodies are pan-specific for the MT-1, MT-2, and MT-4 protein isoforms (28). This is because epitopes recognized by antibodies raised against MT-1 or MT-2 are limited to the first five residues of the acetylated N terminus, which are invariant among all MT-1, MT-2, and MT-4 isoforms (29–31). This includes the commercially available E9 antibody that has been used to demonstrate the overexpression of MT in a wide variety of human cancers (28, 32, 33). In general, the overexpression of MT in various cancers has been associated with resistance to anticancer therapies and linked to a poor prognosis.The mounting evidence that specific MT isoforms may be useful prognostic and diagnostic markers for cancers highlights the need for alternative approaches to the assessment of MT isoform expression at the protein level. A few mass-spectrometry-based studies have succeeded in identifying the complement of MT isoforms in human cells (34, 35). Though top-down approaches hold promise for the quantitation of MTs based on the unique masses of intact isoforms (34, 36), this has yet to be exploited. Inductively coupled plasma MS has been used to quantify total metal-bound MTs in cells and tissues, but it cannot assign relative abundance values of MT isoforms because the proteins are reduced to their elemental composition with this technique. Thus far, MALDI-MS has been used in parallel with inductively coupled plasma MS for the qualitative identification of isoforms (35). Bottom-up quantitative approaches specifically targeting MTs have not yet been reported.The use of mass spectrometry to quantify MT isoforms is not straightforward. The N-terminal tryptic peptide of each human MT isoform encompasses the only sequence that distinguishes all 12 and therefore may be used for their identification and quantitation in complex biological samples from cells and tissues (34). Any attempt at quantitation of this family of small, highly conserved, cysteine-rich proteins therefore requires reproducible detection of these signature peptides.An optimized bottom-up proteomic method is presented here that is capable of identifying and quantifying all isoforms that constitute the human MT gene family in a single experiment. The approach is comparable in sensitivity and dynamic range to quantitative PCR methods used to measure mRNA levels. Quantitative and qualitative differences between mRNA and protein expression indicate that isoform-specific measurements of protein levels complement and extend our understanding of MT isoform expression in complex biological samples. The method was applied to the characterization of MT isoforms in ER⁺ and ER⁻ breast cancer cell lines. Protein and mRNA measurements showed the same complement of isoform expression, confirming differential MT expression between ER⁺ and ER⁻ cell lines. The mass spectrometry assay further showed dramatic differences in the abundance of protein and mRNA in specific isoforms, an observation that has not been previously reported.     

Synthèse du méthyl-2-acétamido-2-désoxy-β-D-glucofuranoside et de dérivés

Methyl 2-acetamido-2-deoxy-5,6-O-isopropylidene-β-D-glucofuranoside was prepared in excellent yield from methyl 2-benzamido-2-deoxy-5,6-O-isopropylidene-β-D-glucofuranoside by alkaline hydrolysis, followed by selective N-acetylation. Treatment with 60% acetic acid at room temperature gave syrupy methyl 2-acetamido-2-deoxy-β-D-glucofuranoside, characterized by a crystalline tri-O-p-nitrobenzoyl derivative. The same treatment, at 100° gave methyl 2-acetamido-2-deoxy-β-D-glucopyranoside. In an alternative procedure, the selective N-acetylation was performed after acetic acid hydrolysis of methyl 2-amino-2-deoxy-5,6-O-isopropylidene-β-D-glucofuranoside. Several derivatives of methyl 2-acetamido-2-deoxy-β-D-glucofuranoside were prepared and compared with the corresponding pyranosides. The furanoside structure was clearly demonstrated by mass spectrometry and periodate oxidation.     

Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques.

The potential of hyphenated techniques based on a combination of microbore reversed-phase (RP) HPLC or capillary zone electrophoresis (CZE) with inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS) was demonstrated for the characterization of metal complexes with metallothionein in rat liver and kidney. The mixture of MT complexes was isolated from the tissues by size-exclusion LC and further characterized in neutral pH conditions (pH 6.8-7.2) by RP-HPLC or CZE. The metal stoichiometry and the molar mass of the eluted complexes was measured by ICP-MS and ES-MS, respectively. An additional dimension to the analysis was achieved by post-column acidification of the chromatographic eluent that allowed the determination of the molecular weight of the demetallated complexes with 10-fold higher sensitivity. The approach allowed the detection of two major metallothionein (MT) isoforms (MT-1 and MT-2) in liver and one MT isoform in kidney. The actual number of peaks in chromatograms and electropherograms was bigger because of the formation of mixed Cd-Cu complexes of the same MT isoform that showed different hydrophobicities.     

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.     

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Crystallinity of Partially N-Acetylated Chitosans

In order to search for a chitosan with low crystallinity, partially N-deacetylated chitins (PDC) and partially N-acetylated chitosans (PAC) with a low degree of N-acetylation (DAc) were examined by X-ray powder diffraction measurements. Three PDC samples, having less than 30% DAc and prepared by solid-state deacetylation, gave X-ray powder patterns showing the presence of α-chitin, a hydrated crystal of chitosan, or their mixture, respectively. When these PDC samples were treated with an acid-alkali, however, reduced crystallinity was observed. By annealing in water at 160 or 200°C, the latter PDC having DAc less than approx. 22% gave powder patterns indicating the presence of an anhydrous crystal which may spoil the chitosan’s functionality. In contrast, PAC prepared by N-acetylating pure chitosan (DAc=0%) in a swollen state, which can be expected to have random copolymers in the chain, was always less crystallized than PDC, this crystallinity depending on the molecular weight. In the case of high-molecular-weight PAC samples, whose DAc was in the range of 5–21%, the effect of high molecular weight on reducing crystallinity was larger than that of the degree of N-acetylation.     

Sialic acid O-acetylation: From biosynthesis to roles in health and disease

Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.