Reactions of chitosan: 4. Preparation of organosoluble derivatives of chitosan

Fully subtituted di-O-acetyl-N-acetylchitosan (chitin diacetate) has been prepared by a route in which the hydroxyl groups are acetylated prior to N-acetylation. This overcomes the previously reported intramolecular steric hindrance to esterification caused by the N-acetamido group. The resultant products were of high viscosity but had a limited solubility range. Di-O-acrylcarbamate derivatives of N-acetylchitosan (chitin) have been produced by a similar route, whilst di-O-arylcarbamate-N-arylureidochitosans have been prepared directly from chitosan. These products also have limited solubility ranges and have inherent viscosities similar to that of di-O-acetylchitosan prepared from the same batch of chitosan.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Synthesis of 3-O-acyl esters of serine and threonine

The 3-O-acyl derivatives of serine and threonine have been prepared by reacting oleoyl chloride and palmitoyl chloride with N-t-butoxycarbonyl (N-T-BOC) serine and N-t-BOC threonine. The t-BOC group was removed by treatment with 4 N HCl in dioxane. The products were identified by proton magnetic resonance spectroscopy, infrared spectroscopy, elemental analysis and chromatographic properties. The O-acyl serines and O-acyl threonines were converted to their methyl esters by treatment with boron trifluoride in methanol and were converted to their dinitrophyl derivatives by treatment with dinitrofluorobenzen (DNFB). The yield of the dinitrophenyl derivatives was very high but the yield of methyl esters was low due mainly to methanolysis and loss of the fatty acyl group. The O-acyl serines and O-acyld threonines prepared will provide standards for researchers who are interested in identifying fatty acids esterified to serine and threonine hydroxyl groups in membrane proteins.     

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.     

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.     

A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides.

A rapid and quantitative procedure is described for the re-N-acetylation of amino sugar methyl glycosides prior to their analysis by gas-liquid chromatography. Two equivalents of pyridine are added to acidic methanolysates containing amino sugars, serving both to neutralize the acid and to act as a catalyst for the subsequent N-acetylation reaction with acetic anhydride. The N-acetylation is quantitative and complete within 10 min at ambient temperature. Excess acetic anhydride is destroyed by solvolysis with the methanolic solvent. The procedure has been used effectively for methanolysates containing 0.01–2.0 mg/ml glucosamine. The procedures utilizing ion-exchange columns and insoluble salts are thus circumvented and all reaction byproducts are volatile. The procedure is therefore ideally suited for the simultaneous workup of numerous samples for analytical procedures such as gas-liquid chromatography.     

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.     

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.     

Isolation of isoaccepting tRNAs

The N-hydroxysuccinimide ester of succinated polyethylene oxide (polyethylene glycol 6000) has been prepared. The ester has been used to make the N-acyl derivatives of valyl-tRNA and phenylalanyl-tRNA from E. coli K-12. Because of the large molecular weight, high solubility in phenol, and the binding to Corning porous glass of polyethylene oxide, the acyl derivative, N-(succinated polyethylene oxide)-aminoacyl-tRNA, has been separated from unreacted tRNA. Since the reaction is reasonably specific for the amino group of the amino acid, large purifications have been obtained for tRNA_val and tRNA_phe. Evidence is presented to show that the ester can react with tRNA at a slow rate. The limitations on the purification due to this reaction are quantitatively evaluated. The highest ratios, pmoles aminoacyl-tRNA/ OD₂₆₀, obtained for valyl-tRNA and phenylalanyl-tRNA were 800 and 360.     

2-epi-fortimicin B. Participation of 1-N-benzyloxycarbonylamino and 1-acetamido groups in solvolysis of 2-O-(methylsulfonyl)fortimicin B derivatives

Synthesis of 2-epi-fortimicin B has been accomplished by processes involving solvolyses of both 1-N-benzyloxycarbonyl- and 1-N-acetyl-2-O-(methylsulfonyl)fortimicins B, which occur with participation of the carbonyl oxygen atoms of the 1-N-acyl groups. The results illustrate both the greater effectiveness of acetamido groups in neighboring-group participation relative to benzyloxycarbonylamino groups, and the sensitivity of the nature of the products to the reaction conditions.     

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.     

Controllable selective enzymatic synthesis of N-acyl and O-acylpropranolol vinyl esters and preparation of polymeric prodrug of propranolol

Controllable selective synthesis strategy of polymerizable N-acyl and O-acylpropranolol vinyl derivatives was developed by enzyme-catalyzed acylation of propranolol using divinyl dicarboxylates with different carbon chain length as acyl donor. The influence of parameters including enzyme, solvents and chain length of acyl donor on the reaction was investigated in detail. Lipase AY30 in diisopropyl ether demonstrated high selectivity towards the amino group of propranolol, while lipase M from Mucor javanicus in dioxane acylated selectively the hydroxyl group of propranolol. N-Acylpropranolol (3a–3c) and O-acylpropranolol vinyl (4a–4c) derivatives were obtained successfully, and can be used for preparing functional macromolecular prodrugs of beta-blockers drugs. N-(Vinyladipoyl)propranolol (NVAP) was copolymerized with methyl methacrylate (MMA) using AIBN as initiator. The obtained polymeric prodrug was characterized with IR, NMR and GPC. The poly(NVAP-co-MMA) has M_n of 3.23 × 10⁴, and M_w/M_n of 1.66.     

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.     

Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors

O-Peracetylated 1-(β-d-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated β-d-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-β-d-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(β-d-glucopyranosyl)-3,5-diphenyl- as well as 3-(β-d-glucopyranosyl)-1,5-diphenyl biurets besides 1-(β-d-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(β-d-glucopyranosyl)-5-(β-d-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated β-d-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of β-d-gluco, β-d-galacto and β-d-xylo configurations. O-Acyl protected 1-(β-d-glucopyranosyl)-3-(β-d-glycopyranosylcarbonyl)ureas were obtained from the reaction of β-d-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of β-d-gluco and β-d-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary α-amylase.     

Structural Basis for the De-N-acetylation of Poly-β-1,6-N-acetyl-d-glucosamine in Gram-positive Bacteria

Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaB_Ad) has been determined to 1.7 Å resolution. The structure of IcaB_Ad reveals a (β/α)₇ barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaB_Ad is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni²⁺, Co²⁺, and Zn²⁺. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Development of carbamate-tethered coumarins as phototriggers for caged nicotinamide

The syntheses of 7-diethylaminocoumarin- or modified DEACM-nicotinamide and 6-bromo-7-methoxycoumarin- or BMCM-nicotinamide have been accomplished by reaction of nicotinoyl isocyanate with the corresponding coumarin allylic alcohol derivatives. The resulting compounds contain an N-acyl O-alkyl carbamate as a new type of linkage for the caging of nicotinamide with a coumarin phototrigger, which undergoes cleavage upon photolysis. Our design of specific caged-nicotinamides was based upon NBO and TD-FT calculations to predict absorption wavelengths and photocleavage potential. This work provides a potentially general method for the caging of amides with coumarin photolabile protecting groups.     

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.     

Microbial models of mammalian metabolism. Aromatic hydroxylation

The potential for selected microorganisms to hydroxylate aromatic substrates in a manner analogous to mammalian systems has been studied. Based on literature precedence and prior experience, 11 microorganisms were chosen from among a variety of genera of fungi and bacterial species and were incubated with 13 model compounds including acetanilide, acronycine, aniline, anisole, benzene, benzoic acid, biphenyl, chlorobenzene, coumarin, naphthalene, nitrobenzene, trans-stilbene, and toluene. In most instances, the microbial model system yielded patterns of phenolic metabolites similar to those reported with cytochrome P₄₅₀ monooxygenases of hepatic microsomes and/or in vivo mammalian systems. Furthermore, N-acetylation of aniline, N-deacetylation of acetanilide, and O-demethylation of anisole were found with certain organisms. The potential usefulness of microbial systems for the synthesis of preparative quantities of mammalian metabolites of foreign organic compounds is discussed.     

The linkage of 4-O-methyl-L-galactose in the sulphated polysaccharide of Aeodes ulvoidea

Methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-β-d-glucofuranoside (11) was obtained in six steps from the known methyl 3-O-allyl-2-benzamido-2-deoxy-5,6-O-isopropylidene-β-d-glucofuranoside. Mild acid hydrolysis, followed by benzylation gave the 5,6-dibenzyl ether. The benzamido group was exchanged for an acetamido group by strong alkaline hydrolysis, followed by N-acetylation, and the allyl group was isomerized into a 1-propenyl group that was hydrolyzed with mercuric chloride. Treatment of 11 with l-α-chloropropionic acid and with diazomethabe gave methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucofuranoside which formed on mercaptolysis the internal ester 16, further converted into 2-acetamido-4-O-acetyl-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-d-glucose diethyl dithioacetal (18) by alkaline treatment followed by esterification with diazomethane and acetylation. Attempts to remove the O-acetyl group of the corresponding dimethyl acetal 20 with sodium methoxide in mild conditions were not successful.