Acetylation of cellulose in LiCl-N,N-dimethylacetamide: first report on the correlation between the reaction efficiency and the aggregation number of dissolved cellulose

The acylation of three cellulose samples by acetic anhydride, Ac(2)O, in the solvent system LiCl/N,N-dimethylacetamide, DMAc (4 h, 110 A degrees C), has been revisited in order to investigate the dependence of the reaction efficiency on the structural characteristics of cellulose, and its aggregation in solution. The cellulose samples employed included microcrystalline, MCC; mercerized cotton linters, M-cotton, and mercerized sisal, M-sisal. The reaction efficiency expresses the relationship between the degree of substitution, DS, of the ester obtained, and the molar ratio Ac(2)O/AGU (anhydroglucose unit of the biopolymer); 100% efficiency means obtaining DS = 3 at Ac(2)O/AGU = 3. For all celluloses, the dependence of DS on Ac(2)O/AGU is described by an exponential decay equation: DS = DS(o) - Ae(-[(Ac2O/AGU)/B]); (A) and (B) are regression coefficients, and DS(o) is the calculated maximum degree of substitution, achieved under the conditions of each experiment. Values of (B) are clearly dependent on the cellulose employed: B((M-cotton)) > B((M-sisal)) > B((MCC)); they correlate qualitatively with the degree of polymerization of cellulose, and linearly with the aggregation number, N(agg), of the dissolved biopolymer, as calculated from static light scattering measurements: (B) = 1.709 + 0.034 N(agg). To our knowledge, this is the first report on the latter correlation; it shows the importance of the physical state of dissolved cellulose, and serves to explain, in part, the need to use distinct reaction conditions for MCC and fibrous celluloses, in particular Ac(2)O/AGU, time, temperature.

Some aspects of acetylation of untreated and mercerized sisal cellulose

We report here on some aspects of the acetylation in LiCl/N,N-dimethylacetamide, DMAc, of untreated and mercerized sisal cellulose, hereafter designated as sisal and M-sisal, respectively. Fiber mercerization by NaOH solution has resulted in the following changes: 29.9% decrease in the index of crystallinity; 16.2% decrease in the degree of polymerization and 9.3% increase in α-cellulose content. A light scattering study of solutions of sisal, M-sisal, microcrystalline and cotton celluloses in LiCl/DMAc has shown that they are present as aggregates, with (an apparent) average aggregation numbers of 5.2, 3.2, 9.8, and 35.3, respectively. The presence of these aggregates affects the accessibility of cellulose during its functionalization. A study of the evolution of the degree of substitution, DS, of cellulose acetate as a function of reaction time showed an increase up to 5 h, followed by a decrease at 7 h. Possible reasons for this decrease are discussed. As expected, M-sisal gave a higher DS that its untreated counterpart.

Thermal behavior of cellulose acetate produced from homogeneous acetylation of bacterial cellulose

Cellulose acetate (CA) is one of the most important cellulose derivatives and its main applications are its use in membranes, films, fibers, plastics and filters. CAs are produced from cellulose sources such as: cotton, sugar cane bagasse, wood and others. One promissory source of cellulose is bacterial cellulose (BC). In this work, CA was produced from the homogeneous acetylation reaction of bacterial cellulose. Degree of substitution (DS) values can be controlled by the acetylation time. The characterization of CA samples showed the formation of a heterogeneous structure for CA samples submitted to a short acetylation time. A more homogeneous structure was produced for samples prepared with a long acetylation time. This fact changes the thermal behavior of the CA samples. Thermal characterization revealed that samples submitted to longer acetylation times display higher crystallinity and thermal stability than samples submitted to a short acetylation time. The observation of these characteristics is important for the production of cellulose acetate from this alternative source. (C) 2008 Elsevier B.V. All rights reserved.

Sisal, sugarcane bagasse and microcrystalline celluloses: Influence of the composition of the solvent system N,N-dimethylacetamide/lithium chloride on the solubility and acetylation of these polysaccharides

The present work describes an investigation concerning the acetylation of celluloses extracted from short-life-cycle plant sources (i.e. sugarcane bagasse and sisal fiber) as well as microcrystalline cellulose. The acetylation was carried out under homogeneous conditions using the solvent system N,N-dimethylacetamide/lithium chloride. The celluloses were characterized, and the characterizations included an evaluation of the amount of hemicellulose present in the materials obtained from lignocellulosics sources (sugarcane and sisal). The amount of LiCl was varied and its influence on the degree of acetate substitution was analyzed. It was found that the solvent system composition and the nature of the cellulose influenced both the state of chain dissolution and the product characteristics. The obtained results demonstrated the importance of developing specific studies on the dissolution process as well as on the derivatization of celluloses from various sources.

A physical organic chemistry approach to dissolution of cellulose: effects of cellulose mercerization on its properties and on the kinetics of its decrystallization

The effects of alkali treatment on the structural characteristics of cotton linters and sisal cellulose samples have been studied. Mercerization results in a decrease in the indices of crystallinity and the degrees of polymerization, and an increase in the alpha-cellulose contents of the samples. The relevance of the structural properties of cellulose to its dissolution is probed by studying the kinetics of cellulose decrystallization, prior to its solubilization in LiCl/N,N-dimethylacetamide (DMAc). Our data show that the decrystallization rate constants and activation parameters are only slightly dependent on the physico-chemical properties of the starting celluloses. This multi-step reaction is accompanied by a small enthalpy and large, negative, entropy of activation. These results are analyzed in terms of the interactions within the biopolymer chains during decrystallization, as well as those between the two ions of the electrolyte and both DMAc and cellulose.

Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine - A mechanism for the slow acetylator phenotype and substrate-dependent down-regulation

Many drugs and chemicals found in the environment are either detoxified by N-acetyltransferase 1 (NAT1, EC 2.3.1.5) and eliminated from the body or bioactivated to metabolites that have the potential to cause toxicity and/or cancer. NAT1 activity in the body is regulated by genetic polymorphisms as well as environmental factors such as substrate-dependent down-regulation and oxidative stress. Here we report the molecular mechanism for the low protein expression from mutant NAT1 alleles that gives rise to the slow acetylator phenotype and show that a similar process accounts for enzyme down-regulation by NAT1 substrates. NAT1 allozymes NAT1 14, NAT1 15, NAT1 17, and NAT1 22 are devoid of enzyme activity and have short intracellular half-lives (similar to4 h) compared with wild-type NAT1 4 and the active allozyme NAT1 24. The inactive allozymes are unable to be acetylated by cofactor, resulting in ubiquitination and rapid degradation by the 26 S proteasome. This was confirmed by site-directed mutagenesis of the active site cysteine 68. The NAT1 substrate p-aminobenzoic acid induced ubiquitination of the usually stable NAT1 4, leading to its rapid degradation. From this study, we conclude that NAT1 exists in the cell in either a stable acetylated state or an unstable non-acetylated state and that mutations in the NAT1 gene that prevent protein acetylation produce a slow acetylator phenotype.

Activation of HTLV-I transcription in the presence of Tax is independent of the acetylation of CREB-2 (ATF-4).

We have previously demonstrated that the bZIP transcription factor CREB-2, also called ATF-4, trans-activates, in association with the viral protein Tax, the human T-cell leukemia virus type I (HTLV-I) promoter. In this study, we have examined whether CREB-2 acetylation affects transcriptional activation mediated by Tax. We present evidence that CREB-2 is acetylated in vitro and in vivo. CREB-2 is acetylated in two regions: the basic domain of the bZIP (from amino acid residue 270 to 300) and the short basic domain (from 342 to 351) located downstream from the bZIP. We also demonstrate that CREB-2 is acetylated by p300/CBP but not by p/CAF. Moreover, replacement of lysine by arginine in the basic domains decreases the trans-activating capacity of CREB-2. However, in the presence of Tax, the HTLV-I transcription remains fully activated by these CREB-2 mutants. Although we cannot totally exclude that the mutations could also affect CREB-2 structure and activity independent of acetylation, our results suggest that activation of the viral promoter in the presence of Tax is independent of the CREB-2 acetylation.

Acetylation of cell wall is required for structural integrity of the leaf surface and exerts a global impact on plant stress responses.

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Enzyme loading dependence of cellulose hydrolysis of sugarcane bagasse

The enzymatic hydrolysis of steam-pretreated sugarcane bagasse, either delignified or non-delignified, was studied as a function of enzyme loading. Hydrolysis experiments were carried out using five enzyme loadings (2.5 to 20 FPU/g cellulose) and the concentration of solids was 2% for both materials. Alkaline delignification improved cellulose hydrolysis by increasing surface area. For both materials, glucose concentrations increased with enzyme loading. On the other hand, enzyme loadings higher than 15 FPU/g did not result in any increase in the initial rate, since the excess of enzyme adsorbed onto the substrate restricted the diffusion process through the structure.

Radical acetylation of 2`-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k(2) = 1.0 X 10(4) M-1 s(-1)) when compared with monocarbonyl substrates (k(2) < 10(3) M-1 s(-1)). Phosphate ions (100-500 MM) do not affect the rate of spontaneous peroxynitrite decay, but the H2PO4- anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a(N) = a(H) = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2`-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS"". These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.

First stereocontrolled acetylation of a hydroxypropargylpiperidone by lipase CALB

Hydroxypropargylpiperidones rac-1-3 were efficiently obtained by a one-pot three-component coupling reaction; enantioenriched propargylpiperidones were then obtained by a kinetic resolution process using the lipase from Candida antarctica. Lipase CALB has been shown to efficiently catalyse the stereocontrolled acetylation of hydroxypropargylpiperidones rac-3 by promoting stereodiscrimination at the carbinolic centre. The enzymatic catalytic processes allow the separation of the (S,R)- and (S,S)-3 diastereoisomers into the corresponding acetates produced as a (R,S)- and (R,R)-6 diastereoisomeric pair. The CALB was able to discriminate the stereogenic centre of the secondary (R)-enantiomer of rac-3 according to the Kaslauzkas rule. The remote stereogenic centre was not discriminated by the lipase. The functionalised enantioenriched diastereoisomers obtained are important building blocks in organic synthesis. (C) 2010 Elsevier Ltd. All rights reserved.

Characterization of depolymerized residues from extremely low acid hydrolysis (ELA) of sugarcane bagasse cellulose: Effects of degree of polymerization, crystallinity and crystallite size on thermal decomposition

Sugarcane bagasse cellulose was subjected to the extremely low acid (ELA) hydrolysis in 0.07% H2SO4 at 190, 210 and 225 degrees C for various times. The cellulose residues from this process were characterized by TGA, XRD, GPC, FIR and SEM. A kinetic study of thermal decomposition of the residues was also carried out, using the ASTM and Kissinger methods. The thermal studies revealed that residues of cellulose hydrolyzed at 190, 210 and 225 degrees C for 80,40 and 8 min have initial decomposition temperature and activation energy for the main decomposition step similar to those of Avicel PH-101. XRD studies confirmed this finding by showing that these cellulose residues are similar to Avicel in crystallinity index and crystallite size in relation to the 110 and 200 planes. FTIR spectra revealed no significant changes in the cellulose chemical structure and analysis of SEM micrographs demonstrated that the particle size of the cellulose residues hydrolyzed at 190 and 210 degrees C were similar to that of Avicel. (C) 2011 Elsevier B.V. All rights reserved.

Post-translational lysine-acetylation of Ran and its regulation by Sirtuin deacetylases

Through recent advances in high-throughput mass spectrometry it has become evident that post-translational N-(epsilon)-lysine-acetylation is a modification found on thousands of proteins of all cellular compartments and all essential physiological processes. Many aspects in the biology of lysine-acetylation are poorly understood, including its regulation by lysine-acetyltransferases and lysine-deacetylases (KDACs). Here, the role of this modification was investigated for the small GTP-binding protein Ran, which, inter alia, is essential for the regulation of nucleocytoplasmic transport. To this end, site-specifically acetylated Ran was produced in E. coli by genetic code expansion. For five previously identified sites, Ran acetylation was tested regarding its impact on the intrinsic GTP hydrolysis rate, the assembly of export complexes (modeled in vitro with the export receptor CRM1 and the export substrate Spn1) and the interaction of Ran with its GTPase activation protein RanGAP and RanBP1. Overall, mild effects of Ran acetylation were observed for intrinsic and RanGAP-stimulated GTP hydrolysis rates. The interaction of active Ran with RanBP1 was negatively influenced by Ran acetylation at K159. Moreover, CRM1 bound to Ran acetylated at K37, K99 or K159 interacted more strongly with Spn1. Thus, lysine-acetylation interferes with essential aspects of Ran function. An in vitro screen was performed to identify potential Ran KDACs. The NAD+-dependent KDACs of the Sirtuin class showed activity towards two acetylation sites of Ran, K37 and K71. The specificity of Sirtuins was further analyzed based on an additional Ran acetylation site, K38. Since deacetylation of RanAcK38 was much slower compared to RanAcK37, di-acetylated RanAcK37/38 was tested next. The deacetylation rate of di-acetylated Ran was comparable to that of RanAcK37. Deacetylation experiments under single turnover conditions revealed that deacetylation occurs first at the K38 site in the di-acetylated RanAcK37/38 background. The ability of Sirtuins to deacetylate two adjacent AcKs was further investigated based on two proteins, which had previously been found to be di-acetylated and targeted by Sirtuins, namely the tumor suppressor protein p53 and phosphoenolpyruvate carboxykinase 1 (PEPCK1). p53 was readily deacetylated at two di-acetylation sites (K372/372 and K381/382), whereas PEPCK1 was not deacetylated in vitro. Taken together, these results have important implications for the substrate specificity of Sirtuins.

Hydrogen peroxide bleaching of cellulose pulps obtained from brewer`s spent grain

Brewer`s spent grain (BSG) was evaluated for bleached pulp production. Two cellulose pulps with different chemical compositions were produced by soda pulping: one from the original raw material and the other from material pretreated by dilute acid. Both of them were bleached by a totally chlorine-free sequence performed in three stages, using 5% hydrogen peroxide in the two initial, and a 0.25 N NaOH solution in the last one. Chemical composition, kappa number, viscosity, brightness and yield of bleached and unbleached pulps were evaluated. The high hemicellulose (28.4% w/w) and extractives (5.8% w/w) contents in original BSG affected the pulping and bleaching processes. However, soda pulping of acid pretreated BSG gave a cellulose-rich pulp (90.4% w/w) with low hemicellulose and extractives contents (7.9% w/w and < 3.4% w/w, respectively), which was easily bleached achieving a kappa number of 11.21, viscosity of 3.12 cp, brightness of 71.3%, cellulose content of 95.7% w/w, and residual lignin of 3.4% w/w. Alkaline and oxidative delignification of acid pretreated BSG was found as an attractive approach for producing high-purity, chlorine-free cellulose pulp.

Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium

This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of Retinal Pigment Epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.52.0 swelling/mm3), high mechanical strength (2048 MPa) and non-pyrogenicity (2.12 EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.