Trehalose Esters Research Articles

The synthesis of certain fatty acid ester derivatives of trehalose and an investigation of their emulsifying properties and bioactivities

Bio-based surfactants exerting both functional and nutritional properties are needed in the food industry. This study reports the one-pot synthesis, using a Novozym 435-catalyzed trans-esterification protocol, of a new family of such compounds, namely a homologous series of 6-O-acylated trehalose and 6,6′-di-O-acylated trehalose derivatives incorporating long-chain fatty acid residues (12–22 carbons). These mono- and di-esters exhibited HLB values within the 8.3 to 14.7 range and their performance as surface-active compounds, penetration enhancers and anti-cancer agents have been evaluated. They were found to be excellent emulsifiers, maintaining oil-in-water emulsions with 566–742 nm mean oil-particle sizes and with the stability of these only gradually deteriorating over a 15-day storage period and during in vitro digestion. Mono-esters 2–4 embodying shorter side-chains (12–16 carbons) proved to be excellent foaming agents while congeners 4–6 incorporating longer side-chains (16–20 carbons) served as penetration enhancers capable of reversibly opening tight junctions in Caco-2 cell mono-layers. Mono-esters 5–7, as well as di-esters 11–13, exerted weak cytotoxic effects on seven human cancer cell lines. This work should serve as a useful guide for the development of the multi-functional properties of such trehalose esters in the food and related industries.

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis

AbstractIn Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram‐scale synthesis of four stereo‐isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn‐methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

Total Synthesis of a Mycolic Acid from Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, mycolic acids and their glycerol, glucose, and trehalose esters (“cord factor”) form the main part of the mycomembrane. Despite their first isolation almost a century ago, full stereochemical evaluation is lacking, as is a scalable synthesis required for accurate immunological, including vaccination, studies. Herein, we report an efficient, convergent, gram‐scale synthesis of four stereo‐isomers of a mycolic acid and its glucose ester. Binding to the antigen presenting protein CD1b and T cell activation studies are used to confirm the antigenicity of the synthetic material. The absolute stereochemistry of the syn‐methoxy methyl moiety in natural material is evaluated by comparing its optical rotation with that of synthetic material.

Trehalose-Rich, Degradable Hydrogels Designed for Trehalose Release under Physiologically Relevant Conditions

Trehalose, a natural disaccharide, is primarily known for its ability to protect proteins from inactivation and denaturation caused by a variety of stress conditions. Furthermore, over the past few years, it has emerged as a promising therapeutic candidate for treatment of neurodegenerative diseases. Herein, we examine the attachment of trehalose to polymers for release under selected physiologically relevant conditions. The proposed strategies are evaluated specifically using hydrogels undergoing simultaneous degradation during trehalose release. These materials are fabricated via copolymerization of the appropriate acrylamide-type monomers with polymerizable trehalose esters or benzylidene acetals. This provides trehalose release in a slightly alkaline (i.e., pH 7.4) or mildly acidic (i.e., pH 5.0) environment, respectively. Using this method materials containing up to 51.7 wt% of trehalose are obtained. The presented results provide a solid basis for future studies on polymeric materials intended for trehalose release in biological systems.

Enzymatic synthesis of fatty acid esters of trehalose: Process optimization, characterization of the esters and evaluation of their bioactivities.

One-pot synthesis of caprylic, lauric and palmitic acid mono- and diesters of trehalose was catalyzed by the lipase Fermase CALB™ 10000. An optimized molar conversion of 35% of trehalose to its palmitate esters was obtained in acetone at 60°C with a trehalose:palmitic acid molar ratio of 1:5 in 4h. Trehalose fatty acid esters (THFAE) were purified by column chromatography and characterized using TLC, HPTLC, HR-MS, ATR-FTIR, and differential scanning calorimetry. THFAE were studied for their antimicrobial potential against four bacterial, and two fungal species. Trehalose monolaurate and trehalose dicaprylate demonstrated MIC of 0.45mM and 16mM against Pseudomonas aeruginosa and Escherichia coli, respectively. Trehalose monocaprylate showed the highest inhibition of biofilm forming property against Staphylococcus aureus (86.25%) at 99.2mM and trehalose dipalmitate had lowest IC50 of 13.23mM. Furthermore, their anti-inflammatory property was studied in vitro using 15-LOX inhibition assay and human red blood cell membrane stabilization assay. In the confirmatory in vivo tests using carrageenan-induced rat paw edema assay, inflammation in disease control group reached up to 63% as against 32% and 20% for trehalose dilaurate and diclofenac treated groups, respectively. THFAE can hence find potential applications in pharmaceuticals, functional foods, and nutraceuticals.

Factors determining the reaction temperature of the solvent-free enzymatic synthesis of trehalose esters

Solvent-free synthesis encourages the design of processes and products that reduce the use and generation of hazardous chemicals. Given the importance of developing greener methodologies, we sought to determine the factors influencing the reaction temperature required for solvent-free, enzymatic synthesis of sugar esters such as trehalose (TRE) esters, using Novozyme 435 as the enzyme catalyst. The use of lauric acid (La) and ethyl laurate (LaEt) as acyl donors did not affect the activation temperature for the generation of trehalose diesters (TDEs), despite the differences in corresponding by-products (water and ethanol). However, when glucose (GLU) and La were employed as reaction substrates as a comparison, glucose monoester (GME) generation readily occurred at much lower temperatures than with the TRE esters, even without a water collection device. Moreover, when the glass transition temperature (Tg) of the sugar substrates increased, a higher reaction temperature was required. These results suggest that while the activation temperature of the reaction did not correlate with the boiling point of the by-product, it did correlate with the glass transition temperature (Tg) of the trehalose substrates. Thus, our work demonstrates the importance of the physical state of amorphous matrices in determining the optimal reaction temperature of a solvent-free sugar synthesis.

New synthetic lipid antigens for rapid serological diagnosis of tuberculosis.

BackgroundDuring pulmonary tuberculosis (PTB) antibodies are generated to trehalose esters of mycolic acids which are cell wall lipids of Mycobacterium tuberculosis (Mtb). Attempts have been made to use these complex natural mixtures in serological tests for PTB diagnosis.AimThe aim of this work was to determine whether a serological test based on a panel of defined individual trehalose esters of characteristic synthetic mycolic acids has improved diagnostic accuracy in distinguishing patients with culture positive PTB from individuals who were Mtb culture negative.MethodOne hundred serum samples from well-characterized patients with presumptive tuberculosis, and diagnosed as having pulmonary smear and culture positive TB, or being culture and smear negative were evaluated by ELISA using different combinations of synthetic antigens and secondary antibodies. Using cut-off values determined from these samples, we validated this study blind in samples from a further 249 presumptive TB patients.ResultsWith the first 100 samples, detailed responses depended both on the precise structure of the antigen and on the secondary antibody. Using a single antigen, a sensitivity/specificity combination for smear and culture positive PTB detection of 85 and 88% respectively was achieved; this increased to 96% and 95% respectively by a statistical combination of the results with seven antigens. In the blind study a sensitivity/specificity of 87% and 83% was reached with a single antigen. With some synthetic antigens, the responses from all 349 samples were significantly better than those with the natural mixture. Combining the results for seven antigens allowed a distinction between culture positive and negative with a ROC AUC of 0.95.ConclusionWe have identified promising antigen candidates for serological assays that could be used to diagnose PTB and which could be the basis of a much-needed, simple, rapid diagnostic test that would bring care closer to communities.

Down-regulation of PE11, a cell wall associated esterase, enhances the biofilm growth of Mycobacterium tuberculosis and reduces cell wall virulence lipid levels.

PE11 (Rv1169c or LipX) is a cell wall associated esterase/lipase of Mycobacterium tuberculosis (Mtb). Evidences suggest that PE11 is expressed by Mtb both in vitro and in vivo. Previous studies have shown that PE11 leads to modification in cell wall lipid content and enhanced virulence when expressed in the non-pathogenic surrogate Mycobacterium smegmatis. Since cell wall lipids often play different roles in pathogenic and non-pathogenic mycobacteria, we investigated the role of PE11 in its host, Mtb. Mtb with lowered expression of PE11 (PE11 knock-down) displayed significant changes in colony morphology and cell wall lipid profile, confirming the role of PE11 in cell wall architecture. In addition, the levels of phthiocerol dimycocerosates, a cell wall virulence factor, were decreased. Levels of trehalose esters and free mycolic acids were increased. In contrast to M. smegmatis expressing Mtb PE11, a role reversal was observed in Mtb with respect to pellicle/biofilm formation. The PE11 knock-down Mtb strain showed significantly enhanced aggregation and early biofilm growth in detergent-free medium, compared to the wild-type. Knock-down strain also showed nearly 27-fold up-regulation of a fibronectin attachment protein (Rv1759c), linking biofilm growth with over-expression of bacterial proteins that help in aggregation and/or binding to host extracellular matrix. The knock-down also resulted in poor virulence of Mtb in PMA (phorbol 12-myristate 13-acetate) treated and PMA+IFN-γ treated THP-1 macrophages. Therefore, the study not only links PE11 to cell wall virulence lipids but also reveals the involvement of this cell wall associated esterase in down-regulation of biofilm in Mtb.

The synthesis of single enantiomers of trans-alkene containing mycolic acids and related sugar esters

Routes to single enantiomers of hydroxy, methoxy and ketomycolic acids containing an α-methyl-trans-alkene unit as well as related trehalose, glucose, arabinose and glycerol esters are described. In serodiagnostic assays to detect antibodies to mycobacterial lipids, the trehalose esters give higher responses to the serum of patients with culture positive tuberculosis than to that of culture negative patients.

Synthesis of wax esters and related trehalose esters from Mycobacterium avium and other mycobacteria

The synthesis of mycobacterial mycolic acid related wax esters and of their trehalose di- and mono-esters is described. The trehalose dimycolates (TDMs) synthesised activated bone marrow derived dendritic cells (BMDCs) in vitro more strongly than trehalose dibehenate or the trehalose monomycolates (TMMs). The inflammatory effects were similar to those of TDM from either a synthetic keto- or methoxy-mycolic acid, but somewhat stronger than those of a TDM from an α-mycolic acid. In vivo, the effects of one wax ester TDM were similar to those of the methoxy-MA and α-mycolic acid TDMs and trehalose dibehenate.

Enzymatic synthesis, purification and identification of bioactive trehalose ester derivatives for health applications

In the present work, we attempted to produce novel trehalose ester derivatives catalyzed by lipase using trehalose and lipoic acid as substrates. Our aim was to select the lipase with the highest catalytic efficiency as a biocatalyst to efficiently produce trehalose ester derivatives with the lowest production cost for further industrial applications. The highest conversion yields of trehalose lipoate (75.9±1.9%) were obtained at the following optimal conditions: reaction temperature 40°C, reaction time 4 days, substrate molar ratio 1:4 (trehalose:lipoic acid), total enzyme activity 3000 PLU (propyl laurate units), four stepwise additions of lipoic acid (0.03mmol/day) and a cosolvent ratio of 4:1 [DMSO:2-methyl-2-butanol(2M2B)]. After isolation and purification, the carboxyl group of lipoic acid was determined to be connected to the C6 hydroxyl group of trehalose using nuclear magnetic resonance. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity (%) of purified 6-o-trehalose lipoate was at least 2.5-fold higher than that of lipoic acid. This bioactive trehalose ester exhibits improved antioxidant activity, which may have higher health benefits than those of lipoic acid, in various functional product applications.

Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis

A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

A trehalose ester from Lancea tibetica

A phytochemical study of the 95% ethanolic extract of the whole plant of Lancea tibetica Hook. f. et Thoms. led to the isolation of a new trehalose ester, 6-O-undecanoyl-α,β-trehalose (1), along with 23 known compounds (2–24), of which compounds 2–17 were isolated from this plant for the first time. The structures of these compounds were established on the basis of spectroscopic methods. Compound 1 was evaluated for their in vitro anti-proliferative activities against MCF-7, NCI-H460 and Hep-G2 tumour cell lines. Compound 1 exhibited potent inhibitory activity against NCI-H460 cell growth, in contrast to moderate cytotoxic activity against MCF-7 and Hep-G2 cells.

Characteristics of Sugar Surfactants in Stabilizing Proteins During Freeze–Thawing and Freeze–Drying

Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze-thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze-thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze-thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze-thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10-C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze-thawing and freeze-drying increased at the sugar ester concentrations of 1-10 and 10-100 μM, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced.

Synthesis and photocuring of cinnamoyl trehalose esters

AbstractA trehalose cinnamoyl ester (TC) was synthesized from trehalose and cinnamoyl chloride in dimethylformamide (DMF) in the presence of triethylamine and 4‐(N,N‐dimethylamino)pyridine. The product was characterized by 1H NMR spectroscopy to reveal that the reaction proceeded. Two different types of TCs were synthesized by changing the feed ratio of cinnamoyl chloride to trehalose. When the feed ratio of cinnamoyl chloride to trehalose was 8 (TC8), the degree of substitution (DS) was 8.0, while it was 4.2 when the feed ratio was 4 (TC4). Photocuring was confirmed by observing changes in UV absorption spectrum and FT Infrared (IR) spectrum. After 5 min of UV irradiation, solubility in chloroform significantly decreased. A transparent thin coating film of TC was easily prepared by casting from a chloroform solution on a Petri dish and UV irradiation was carried out over a simple photomask. After TC within the non‐irradiated region was removed by flash soaking with chloroform, the shape of the photomask appeared. A scanning electron microscope (SEM) measurement revealed that the surface of the photocured coating film was smooth and that the edge of the photocured TC had a characteristic feature. Biodegradation of the photocured TC and non‐irradiated TC was examined by the biochemical oxygen demand (BOD) method using activated sludge. Copyright © 2007 John Wiley & Sons, Ltd.

Release mechanism of insulin encapsulated in trehalose ester derivative microparticles delivered via inhalation

The aim of this study was to evaluate properties of amorphous oligosaccharide ester derivative (OED) microparticles in order to determine drug release mechanisms in the lung. Trehalose OEDs with a wide range of properties were synthesised using conventional methods. The interaction of spray dried amorphous microparticles (2–3 μm) with water was investigated using attenuated total reflectance Fourier transform infra-red spectroscopy (ATR-FTIR) and dynamic vapour sorption (DVS). The in vivo performance of insulin/OED microparticles was assessed using a modified Higuchi kinetic model. A modified Hansen solvent parameter approach was used to analyse the interactions with water and in vivo trends. In water or high humidity, OED powders absorb water, lose relaxation energy and crystallise. The delay of the onset of crystallisation depends on the OED and the amount of water present. Crystallisation follows first order Arrhenius kinetics and release of insulin from OED microparticles closely matches the degree of crystallisation. The induction period depends on dispersive interactions between the OED and water while crystallisation is governed by polarity and hydrogen bonding. Drug release from OED microparticles is, therefore, controlled by crystallisation of the matrix on contact with water. The pulmonary environment was found to resemble one of high humidity rather than a liquid medium.

Enzymatic synthesis of trehalose esters having lipophilicity

To improve trehalose lipophilicity, trehalose was regioselectively esterified with vinyl fatty acid esters in dimethyl formamide by protease from Bacillus subtilis to give 6- O-lauroyltrehalose, 6- O-myristoyltrehalose, 6- O-palmitoyltrehalose, 6- O-stearoyltrehalose, 6- O-oleoyltrehalose and 6- O-linoleoyltrehalose. The influence of structural variation by changing fatty acid substitute was examined by measurement of the surface tension and biodegradability.

Trehalose esters from the aspen fungus Hyphozyma lignicola.

Chemical investigation of the metabolites produced when a new fungus, Hyphozyma lignicola, isolated from blackgalls on aspen, was grown in malt extract liquid medium resulted in the isolation and identification of five trehalose esters 1-5, which are unusual fungal metabolites. The major compound is 3,6-di-O-acetyl-2-O-octadecanoyl-alpha,alpha-D-trehalose [2]. The structures were established by the analysis of nmr and fabms data in combination with chemical degradation.

An improved synthesis of trehalose 6-mono and 6,6′-dicorynomycolates and related esters

A simplified synthesis of 6-mono and 6,6′-di-corynomycolate esters of α,α-trehalose, and related compounds, was achieved by coupling the (hydroxyl-protected) acids to the partially trimethylsilylated sugar in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine. As acid reactants, (2- RS,3- RS)-3-hydroxy-2-tetradecyloctadecanoic acid ( dl-corynomycolic acid) and its 2 RS,3 SR diastereomer were prepared from methyl palmitate by sequential Claisen condensation, reduction, chromatographic separation, and saponification. Reaction with tert-butylchlorodimethylsilane (imidazole) gave the disubstituted ether-ethers, which were converted into the required 3- tert-butyldimethylsilyl ethers by partial hydrolysis. 6-Linked monocorynomycolate was obtained in excellent yield (78%) from the reaction of the RS, SR acid with the known heptakis- O-(trimethylsilyl)trehalose, and in good yield from equimolar portions of RS, RS acid and hexakis- O-(trimethylsilyl)trehalose. An excess (2.5-molar portions) of the RS, RS acid gave the 6,6′-diester (69%). The mono- and di-palmitate were similarly obtained from (Me 3Si) 6-trehalose. The mono ( RS, RS)-(Me 3Si) 6-trehalose coupling product was partially resolved on a silica gel column into its RR and SS diastereomers, the former corresponding to the naturally occurring trehalose monocorynomycolate. All coupling products were deprotected to free trehalose esters by treatment first with K 2CO 3 in methanol, then tetrabutylammonium fluoride-trifluoroacetic acid in oxolane.

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei.

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals. Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations. By observing the labelling of the different mycolate pools in the cell, i.e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell-wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.