Degree Of Acylation Research Articles

Structural features of lipid A of Rickettsia typhi

Lipid A isolated from the Rickettsia typhi lipopolysaccharide (LPS) was investigated for its composition and structure using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and electrospray ionization (ESI) combined with the tandem mass spectrometry (MS/MS). Our studies revealed a noticeable compositional and structural heterogeneity of lipid A with respect to the content of phosphate groups and the degree of acylation. It appeared that at least two molecular species were present in lipid A. The major species represented the hexaacyl lipid A consisting of the β-(1--> 6)-linked D-glucosamine (GlcN) disaccharide backbone carrying two phosphate groups. One of them was linked to the glycosidic hydroxyl group of the reducing GlcN I and the other was ester linked to the O-4´ position of the non-reducing GlcN II. The primary fatty acids consisted of two 3-hydroxytetradecanoic [C14:0(3-OH)] and two 3-hydroxyhexadecanoic [C16:0(3-OH)] acids. The former were ester- and the latter amide-linked to both GlcN. Two secondary fatty acids were represented by the octadecanoic (C18:0) and hexadecanoic (C16:0) acids that were ester-linked at the N-2´ and O-3´ positions, respectively. In the minor lipid A species, ester-linked C18:0 was substituted by C16:0 at the C16:0(3-OH) of GlcN II. The R. typhi lipid A resembles structurally the classical forms of enterobacterial lipids A with high endotoxicity.

Tetraacylated Lipopolysaccharide ofYersinia pestisCan Inhibit Multiple Toll‐Like Receptor–Mediated Signaling Pathways in Human Dendritic Cells

Yersinia pestis, the causative agent of plague, showed a temperature-dependent change in lipid A composition, with a reduced degree of acylation when bacteria were grown at 37 degrees C (tetraacylated) versus ambient temperature (hexaacylated). Human monocytes and monocyte-derived dendritic cells (DCs) were exposed to Y. pestis grown at 26 degrees C or 37 degrees C, to their corresponding lipopolysaccharides (LPS-26 degrees C or LPS-37 degrees C), and to ligands of different Toll-like receptors (TLRs), such as LPS from Escherichia coli (TLR4), lipoprotein (TLR2), polyinosinic-polycytidylic acid (poly-IC) (TLR9), and their combinations. Production of cytokines was measured, along with expression of surface markers of DC maturation. Y. pestis grown at 37 degrees C or LPS-37 degrees C induced much lower production of cytokines (such as tumor necrosis factor alpha and interleukins 1beta, 10, and 12) by DCs than did Y. pestis grown at 26 degrees C or LPS-26 degrees C. Expression of the surface markers HLA-DR, CD86, and CD40 by DCs was also reduced in response to treatment with LPS-37 degrees C compared with LPS-26 degrees C. Pretreatment of DCs with LPS-37 degrees C inhibited subsequent stimulation with LPS-26 degrees C, control LPS from E. coli, lipoprotein, or poly-IC. LPS-37 degrees C can inhibit stimulation of DCs not only via TLR4 signaling but also via TLR2 and TLR3. [corrected]

Immunoreactivity of acetylated and succinylated pea proteins

Proteins of pea variety Kwestor, grown in Poland, were modified with acetic or succinic anhydride (0.01–1.0 g/g anhydride/protein). Degree of acylation, electrophoretic properties (SDS-PAGE) and immunoreactivity (by ELISA) were studied. The study indicates that not only the degree of acylation but also the type of anhydrides affected the extent of changes in the immunoreactivity of individual pea proteins. The greatest reductions in the immunoreactivity of albumins and legumin were observed during acylation with 0.2 g anhydrides (by 91–99% and 79–97% during succinylation and acetylation, respectively). The lowest immunoreactivity of vicilin fraction was found when 1.0 g of anhydride was used (about 6% during succinylation and 14.7% during acetylation as compared to the immunoreactivity of vicilin in native pea proteins).

Monolignol acylation and lignin structure in some nonwoody plants: A 2D NMR study

Lignins from three nonwoody angiosperms were analyzed by 2D NMR revealing important differences in their molecular structures. The Musa textilis milled-wood-lignin (MWL), with a syringyl-to-guaiacyl (S/G) ratio of 9, was strongly acylated (near 85% of side-chains) at the γ-carbon by both acetates and p-coumarates, as estimated from 1H– 13C correlations in C γ-esterified and C γ–OH units. The p-coumarate H 3,5–C 3,5 correlation signal was completely displaced by acetylation, and disappeared after alkali treatment, indicating that p-coumaric acid was esterified maintaining its free phenolic group. By contrast, the Cannabis sativa MWL (S/G ∼0.8) was free of acylating groups, and the Agave sisalana MWL (S/G ∼4) showed high acylation degree (near 80%) but exclusively with acetates. Extensive C γ-acylation results in the absence (in M. textilis lignin) or low abundance (4% in A. sisalana lignin) of β–β′ resinol linkages, which require free C γ-OH to form the double tetrahydrofuran ring. However, minor signals revealed unusual acylated β–β′ structures confirming that acylation is produced at the monolignol level, in agreement with chromatographic identification of γ-acetylated sinapyl alcohol among the plant extractives. In contrast, resinol substructures involved 22% side-chains in the C. sativa MWL. The ratio between β–β′ and β -O-4′ side-chains in these and other MWL varied from 0.32 in C. sativa MWL to 0.02 in M. textilis MWL, and was inversely correlated with the degree of acylation. The opposite was observed for the S/G ratio that was directly correlated with the acylation degree. Monolignol acylation is discussed as a mechanism potentially involved in the control of lignin structure.

The Acylation and Phosphorylation Pattern of Lipid A from Xanthomonas Campestris Strongly Influence its Ability to Trigger the Innate Immune Response in Arabidopsis

Lipopolysaccharides (LPSs) are major components of the cell surface of Gram-negative bacteria. LPSs comprise a hydrophilic heteropolysaccharide (formed by the core oligosaccharide and the O-specific polysaccharide) that is covalently linked to the glycolipid moiety lipid A, which anchors these macromolecules to the external membrane. LPSs are one of a group of molecules called pathogen-associated molecular patterns (PAMPs) that are indispensable for bacterial growth and viability, and act to trigger innate defense responses in eukaryotes. We have previously shown that LPS from the plant pathogen Xanthomonas campestris pv. campestris (Xcc) can elicit defense responses in the model plant Arabidopsis thaliana. Here we have extended these studies by analysis of the structure and biological activity of LPS from a nonpathogenic Xcc mutant, strain 8530. We show that this Xcc strain is defective in core completion and introduces significant modification in the lipid A region, which involves the degree of acylation and nonstoichiometric substitution of the phosphate groups with phosphoethanolamine. Lipid A that was isolated from Xcc strain 8530 did not have the ability to induce the defense-related gene PR1 in Arabidopsis, or to prevent the hypersensitive response (HR) that is caused by avirulent bacteria as the lipid A from the wild-type could. This suggests that Xcc has the capacity to modify the structure of the lipid A to reduce its activity as a PAMP. We speculate that such effects might occur in wild-type bacteria that are exposed to stresses such as those that might be encountered during plant colonization and disease.

Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma

BackgroundThe genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood.ResultsWe report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species.ConclusionOur results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.

Thermal degradation of anthocyanins and its impact on color andin vitroantioxidant capacity

The aim of the current study was to thoroughly investigate the structural changes of anthocyanins at pH 3.5 in purified fractions from black carrot, elderberry and strawberry heated over 6 h at 95 degrees C. Degradation products were monitored by HPLC-DAD-MS(3 )to elucidate the prevailing degradation pathways. In addition, alterations of color and antioxidant properties observed upon heating were scrutinized. Most interestingly, the degradation pathways at pH 3.5 were found to differ from those at pH 1. Among others, chalcone glycosides were detected at 320 nm in heat-treated elderberry and strawberry pigment isolates, and opening of the pyrylium ring initiated anthocyanin degradation. In the case of acylated anthocyanins, acyl-glycoside moieties were split off from the flavylium backbone, first. Finally, for all pigment isolates, phenolic acids and phloroglucinaldehyde were the terminal degradation products as remainders of the B- and A-ring, respectively. Maximum and minimum antioxidant stabilizing capacities were found in black carrot and strawberry, respectively, which was explained by the high degree of acylation in the former. After heating, decline of trolox equivalent antioxidant capacity (TEAC) was observed in all samples, which was attributed to both anthocyanins and their colorless degradation products following thermal exposure. As deduced from the ratio of TEAC value and anthocyanin content, the loss of anthocyanin bioactivity could not be compensated by the antioxidant capacity of newly formed colorless phenolics upon heating.

Acylation Determines the Toll-like receptor (TLR)-dependent Positive Versus TLR2-, Mannose Receptor-, and SIGNR1-independent Negative Regulation of Pro-inflammatory Cytokines by Mycobacterial Lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.

Fine Discrimination in the Recognition of Individual Species of Phosphatidyl-myo-Inositol Mannosides fromMycobacterium tuberculosisby C-Type Lectin Pattern Recognition Receptors

The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb.

Influence of Acylation of a Peptide Corresponding to the Amino-Terminal Region of Endothelial Nitric Oxide Synthase on the Interaction with Model Membranes

Covalent attachment of fatty acids to proteins is a common form of protein modification which has been shown to influence both structure and interaction with membranes. Endothelial nitric oxide synthase (eNOS) is dually acylated by the fatty acids myristate and palmitate. We have synthesized four peptides corresponding to the first 28 amino acids of the N-terminal region of eNOS. Besides the nonacylated eNOS sequence, three additional peptides with different degrees of acylation have been obtained: myristoylated, doubly palmitoylated, and dually myristoylated and doubly palmitoylated. Acylation itself, myristic and/or palmitic, confers the peptide the ability to adopt extended conformations, indicated by the fact that the CD spectrum of all acylated peptides has a minimum at approximately 215 nm characteristic of beta-sheet structure. The nonacylated sequence interacts with model membranes composed of acidic phospholipids probably through ionic interactions with the polar headgroup of the phospholipids. However, the acylated peptides are able to insert deeply into the hydrophobic core of both neutral and acidic phospholipids, maintaining the spectral features of extended conformations. When DMPC vesicles containing cholesterol and sphingomyelin at 10% were used, the insertion of the triacylated peptide almost completely canceled the thermal transition, although the interaction of the other acylated peptides also reduced the transition amplitude but to a much lower extent and affected only the acyl chains in the fluid state.

The Acylation State of Mycobacterial Lipomannans Modulates Innate Immunity Response through Toll-like Receptor 2

Detection of Mycobacterium tuberculosis antigens by professional phagocytes via toll-like receptors (TLR) contributes to controlling chronic M. tuberculosis infection. Lipomannans (LM), which are major lipoglycans of the mycobacterial envelope, were recently described as agonists of TLR2 with potent activity on proinflammatory cytokine regulation. LM correspond to a heterogeneous population of acyl- and glyco-forms. We report here the purification and the complete structural characterization of four LM acyl-forms from Mycobacterium bovis BCG using MALDI MS and 2D (1)H-(31)P NMR analyses. All this biochemical work provided the tools to investigate the implication of LM acylation degree on its proinflammatory activity. The latter was ascribed to the triacylated LM form, essentially an agonist of TLR2, using TLR2/TLR1 heterodimers for signaling. Altogether, these findings shed more light on the molecular basis of LM recognition by TLR.

The Structure of Uncommon Lipid A from the Marine Bacterium Marinomonas communis ATCC 27118T

Lipid A was obtained in a high yield (27%) by the hydrolysis of lipopolysaccharide from the marine gamma proteobacterium Marinomonas communis ATCC 27118T with 1% AcOH. Using chemical analysis and ID and 2D NMR spectroscopic and fast atom bombardment mass spectrometric methods, it was shown to be beta-(1',6)-linked D-glucosaminobiose 1-phosphate acylated with (R)-3-dodecanoyl- or (R)-3-decanoyloxydecanoic acid, (R)-3-[(R)-3-hydroxydecanoyloxy)]decanoic acid, and (R)-3-hydroxydecanoic acid at the C2, C2' and C3 positions, respectively. Uncommon structural peculiarities (a low acylation and phosphorylation degree) of the M. communis lipid A in comparison with those of terrestrial bacteria may be of pharmacological interest. The potential physiological meaning of this lipid A and compounds of similar structure are discussed.

Temperature-Dependent Variations and Intraspecies Diversity of the Structure of the Lipopolysaccharide of Yersinia pestis,

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.

Synthesis and biodegradation of starch‐graft‐poly(hexylene adipate)

AbstractPoly(hexylene adipate) (PHA) was synthesized by the direct condensation of hexandiol and adipic acid with p‐toluene sulfonic acid as a catalyst, dimethyl benzene as a solvent. The terminal carboxyl group of the polyester was then converted to acyl chloride by a solution of SOCl2 in toluene. The degree of acylation (DA) was very much dependent on the molecular weight and the type of the other terminal group of the PHA (hydroxyl or capped with an ester group). A high concentration of SOCl2 increased DA, but the scission of the polyester chain was observed. The graft reaction of the acylated PHA with starch was carried out in dried dimethyl sulfoxide. The results show that the percentage grafting (G) and graft efficiency were determined by several factors, including the weight ratio of the reactants, reaction temperature, and molecular weight of PHA. Biodegradation tests in both the laboratory and the natural environment indicated that the degradation rate of starch‐g‐PHA was much faster than that of PHA. For example, the weight loss of PHA was 1.3% after 36 days of natural soil burial, whereas the weight loss of starch‐g‐PHA (G = 39.1%) reached 28% after the same time period. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 848–854, 2003

Efficient scavenger receptor-mediated hepatic targeting of proteins by introduction of negative charges on the proteins by aconitylation: the influence of charge density and size of the proteins molecules.

The in vivo disposition characteristics of some aconitylated proteins in mice were studied after intravenous injection in relation to their molecular properties such as overall negative charge and size of the molecules. Superoxide dismutase (SOD, M(w)=32000) and bovine serum albumin (BSA, M(w)=67000) were used to produce aconitylated derivatives with a different extent of modification. Aconitylated SOD (Aco-SOD) was only moderately taken up by the liver in spite of its negative charge density, whereas aconitylated BSA (Aco-BSA) with a smaller charge density was taken up by the liver very efficiently. Aco-BSA was more rapidly cleared by the liver than succinylated BSA due to the introduction of more anionic groups, especially when the degree of modification was low. Interestingly, highly aconitylated BSAs exhibited significant accumulation in the kidney at higher doses, especially when the hepatic uptake was saturated. Further analysis that was based on a physiological pharmacokinetic model including a saturable hepatic uptake process revealed that the higher the number of negative charges on the proteins, the higher was the apparent affinity of aconitylated proteins for the hepatic SRs. In general, the affinity of aconitylated proteins was higher than that of succinylated proteins when the degree of acylation was the same. Thus, the present study indicates that apart from charge density on the proteins the molecular size of the proteins is important for SR-mediated uptake in the liver. Aconitylation of proteins seems more suitable than succinylation for targeting of proteins to the liver nonparenchymal cells, in particular, at a low degree of acylation of the proteins at which the enzymatic activity is better retained for sufficient negative charges introduced.

Interaction of porin from Yersinia pseudotuberculosis with different structural forms of endogenous lipopolysaccharide.

The interaction of Yersinia pseudotuberculosis porin solubilized in deoxycholate with the S- and R-forms of endogenous lipopolysaccharide (LPS) was studied by the quenching of intrinsic protein fluorescence. The samples of S-LPS differed both in the length of O-specific polysaccharide (n = 1 and 4) and in the acylation degree of the 3-hydroxytetradecanoic acid residues of the lipid A moiety (12-66%). R-LPS (12%) binding to porin was found to occur with positive cooperativity on two integrated structural regions of the R-LPS macromolecule, namely, core oligosaccharide and lipid A. The mode of porin interaction with low-acylated S-LPSs (15 or 20%) coincided with a model involving three types of binding sites. The shape of Scatchard curves of binding indicates that a complex formation between porin and low-acylated S-LPS is cooperative at low and moderate ligand concentration, whereas at near-saturating LPS concentrations porin binds to LPS independently on two types of binding sites. The O-specific polysaccharide chain in the S-LPS macromolecule increases the affinity of its interaction with porin in comparison with R-LPS-porin binding. A significant increase (to 66%) in the degree of S-LPS acylation substantially changed its porin-binding character: the process became anti-cooperative with lowered affinity. Thus, the features of LPS-porin interaction significantly depend on the conformational changes in the LPS molecule due to expanding of its hydrophobic region.

Control of lysine reactivity in four-helix bundle proteins by site-selective pKa depression: expanding the versatility of proteins by postsynthetic functionalization.

Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.

Quadrupole ion-trap mass spectrometry to locate fatty acids on lipid A from Gram-negative bacteria

The structure of lipid A released by mild acid hydrolysis from lipopolysaccharide from two strains of Shigella flexneri with different degrees of acylation was characterized using electrospray ionization (ESI) and ion-trap mass spectrometry. The lipid A was analyzed underivatized with ESI in negative-ion mode. With multiple stages of fragmentation (MS n ), both the degree of acylation and the positions of the fatty acids on the disaccharide backbone could be determined. It was possible to determine the degree of acylation by the MS n technique, where in each MS stage the parent ion was an ion where one fatty acid had been eliminated. One way to determine the location of the fatty acids was by identifying cross-ring fragments of the reducing sugar from parent ions containing different numbers of fatty acids. Another was by identifying a possible charge-driven release of fatty acids situated close to a phosphate group. The fatty acids were otherwise eliminated by a charge-remote fragmentation mechanism. The combined data show the usefulness of ion-trap mass spectrometers for this type of analysis.

Modification of cassava starch by using propionic anhydride and properties of the starch-blended polyester polyurethane

Chemical modification of the cassava starch was conducted through acylation by using propionic anhydride as an esterifying reagent. The reaction was carried out in the presence of a pyridine catalyst and the effects of reaction variables such as the anhydride content, reaction time and reaction temperature on the degree of acylation were investigated. Results from Fourier transform infrared spectroscopy suggested that the hydroxyl groups in the starch molecules were converted into ester groups, accompanied by an increase in water stability of the starch. The propionyl content was found to be non-linearly dependent with the reaction variables. Scanning electron micrographs of the modified starch-blended polyurethane showed a better interfacial adhesion than the normal starch-blended polyurethane. However, results from the soil burial test showed that the modified starch experienced a slower biodegradation than the normal starch.

Acylation of 6-O-Functionalized Cellulose Ethers with Diphenylketene

Two 6-O-tritylcelluloses and one 6-O-thexyl- dimethylsilylcellulose were acylated with diphenylketene in the presence of different bases under mild conditions. The resulting novel, doubly functionalized cellulose derivatives exhibit a total degree of substitution of 2, irrespective of the excess of ketene used in the reaction. The degree of acylation proved to be base-dependent. The use of bulky pyridine bases resulted in a lower acylation than the use of pyridine itself.