Chemo-enzymatic synthesis of arginine-based gemini surfactants

A novel chemo-enzymatic synthesis of arginine-based gemini cationic surfactants bis(Args) is reported. These compounds consist of two single N(alpha)-acyl-arginine structures connected through the alfa-carboxylic groups of the arginine residues by a alpha, omega-diaminoalkane spacer chain. N(alpha)-Acyl-L-arginine alkyl ester derivatives were the starting building blocks for the synthesis. The best strategy found consisted of two steps. First, the quantitative acylation of one amino group of the spacer by the carboxylic ester of the N(alpha)-acyl-arginine took place spontaneously, at the melting point of the alpha,omega-diaminoalkane, in a solvent-free system. The second step was the papain-catalyzed reaction between another N(alpha)-acyl-arginine alkyl ester and the free aliphatic amino group of the derivative formed in the first step. Reactions were carried out in solid-to-solid and solution systems using low-toxic potential solvents. Changes in reaction performance and product yield were studied for the following variables: organic solvent, support for enzyme deposition and substrate concentration. The best yields (70%) were achieved in solid-to-solid systems and in ethanol at a(w) = 0.07. Bis(Args) analogs of 8, 10 and 12 carbon atoms using 1,3-diaminopropane and 1, 3-diamino-2-hydroxy-propane as hydrocarbon spacers were prepared at the 6-7 gram level employing the methodology developed. The overall yields which include reaction and purification varied from 51% to 65% of pure (97-98% by HPLC) product.     

Screening of plant peptidases for the synthesis of arginine-based surfactants

Partially purified preparations with proteolytic activity, obtained from South American native plants, were used as biocatalysts in condensation reactions of N-protected arginine alkyl ester derivatives with decylamine and dodecylamine in low-water content systems. The final products are cationic surfactants with potential application as emulsifiers and preservatives. Most of the proteolytic extracts were obtained from latex of species belonging to the Asclepiadaceae family (araujiain from Araujia hortorum, asclepain c from Asclepias curassavica and funastrain from Funastrum clausum). Hieronymain was obtained from unripe fruits of Bromelia hieronymi (Bromeliaceae). Plant proteases from commercial sources (papain and bromelain) were also tested as catalysts in the same reactions. Araujiain and funastrain furnished good reaction conversions (60–84%, with a ratio synthesis/hydrolysis of 2–5) similar to those obtained with commercial papain. Moreover, araujiain was the biocatalyst which rendered the best conversions (60%) for the synthesis of the two novel Bz-Arg-NH-dodecylamide (Bz-Arg-NHC₁₂) and Bz-Arg-NH-decylamide (Bz-Arg-NHC₁₀) derivatives. Moderate to poor conversions (10–50%, showing a ratio synthesis/hydrolysis of 0.5–1) were achieved with asclepain c, hieronymain and bromelain. The screening presented in this work revealed that, although these are structurally similar, their behavior for the synthesis of this kind of products differ among them.     

Enzymatic synthesis of amide surfactants from ethanolamine

The condensation of a primary amine with fatty acids has been studied to determine optimum conditions for selective formation of amide surfactants via enzymatic amidification. Monoacylated ethanolamide and the diacylated amide-ester can be isolated from the reaction mixture, but the monoacylated ester cannot be isolated. The selectivity of the reaction depends on the solubility of the intermediate amide. Continuous precipitation of this product decreases the amount of amide-ester produced. Solubility values of the desired product (amide) are reported for different conditions.In acetonitrile, the ethyl ester of the corresponding fatty acid has been used successfully to avoid formation/precipitation of the ion-pair of the precursor reagents. In this medium, use of the transacylation reaction permits one to accelerate the reaction without producing a significant change in the selectivity toward the intermediate amide. This strategy is not successful in n-hexane where the solubilities of both ethanolamine and its ion-pair with lauric acid are similar.Results obtained for high loadings of substrates have been analyzed. In n-hexane and acetonitrile, the kinetics of the direct acylation reactions are controlled by the limited solubility of the ion pair formed by the two precursor reagents For the transacylation reaction in acetonitrile, at a sustrate loading of 2 mol l(-1,) selective production of as much as 92 mole percent N-acyl ethanolamine was observed in only 1.5 h.     

Diacyl glycerol arginine-based surfactants: biological and physicochemical properties of catanionic formulations

In this paper, we report on a catanionic vesicles-based strategy to reduce the cytotoxicity of the diacyl glycerol arginine-based synthetic surfactants 1,2-dimyristoyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1414RAc) and 1,2-dilauroyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1212RAc). The behavior of these surfactants was studied either as pure components or after their formulation as pseudo-tetra-chain catanionic mixtures with phosphatidylglycerol (PG) and as cationic mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) used as control. The antimicrobial activity of the negatively charged formulations against Acinetobacter baumannii was maintained with respect to the surfactant alone, while a significant improvement of the antimicrobial activity against Staphylococcus aureus was observed, together with a strong decrease of hemolytic activity. The influence of the net charge of the catanionic vesicles on membrane selectivity was studied using model membranes. The dynamics of surface tension changes induced by the addition of 1414RAc/PG aqueous dispersions into phospholipid monolayers composed of zwitterionic DPPC as model system for mammalian membranes and of negatively charged PG mimicking cytoplasmic membrane of Gram-positive bacteria was followed by tensiometry. Our results constitute a proof of principle that tuning formulation can reduce the cytotoxicity of many surfactants, opening their possible biological applications.     

Design, synthesis, and biocompatibility of an arginine-based polyester

Polycations are very useful in biotechnology. However, most existing polycations have high toxicity that significantly limits their clinical translation. We designed poly(ethylene argininylaspartate diglyceride) (PEAD) that is based on arginine, aspartic acid, glycerol, and ethylene glycol. A set of in vitro assays demonstrated that PEAD exhibited no cytotoxicity at 1 mg/mL, which is at least 100 times higher than the widely used polycation-polyethylenimine. Subcutaneous injection of 1 mg PEAD in rats did not cause an adverse response acutely or after 4 weeks. Zeta potential measurements revealed that PEAD has high affinity to biological polyanions such as DNA and hyaluronic acid. This polycation represents a new platform of biocompatible polycations that may lead to clinical innovations in gene therapy, controlled release, tissue engineering, biosensors, and medical devices.     

The binding of cationic surfactants by DNA

Isotherms for the binding of dodecyltrimethylammonium (DTA⁺) and tetradecyltrimethylammonium (TTA⁺) ions by DNA in aqueous solution at 30°C are reported. The binding isotherms were determined using a potentiometric technique with cationic surfactant-selective electrodes. The DNA concentrations used are 5 × 10^?4 and 10^?3 equiv./kg, Surfactant concentrations varying from 3 × 10^?6M to the critical micelle concentration. The influence of added NaCl (0.01 M) on the binding process is studied. The binding process is shown to be highly cooperative. Applying the binding theory of Schwarz and of Satake and Yang, binding constants and cooperativity parameters can be calculated. The binding constant K is found to be 1.2kT larger for TTA⁺ than for DTA⁺ in salt-free solution, and 1.4kT larger for TTA⁺ than for DTA⁺ in 0.01 M NaCl. The cooperativity parameter u is about 1.4kT larger for TTA⁺ in salt-free solution and 1.2kT larger in 0.01 M NaCl. It is concluded that the hydrophobic part of the bound surfactant is not completely immersed in the hydrophobic DNA core, but also interacts with other surfactant molecules. This situation is compared to the case of micelle formation.     

Purification of non-toxic, biodegradable arginine-based gemini surfactants, bis(Args), by ion exchange chromatography

This paper presents an environmentally improved procedure for the preparative purification of a series of arginine-based gemini surfactants. The technique used was cation-exchange chromatography. Mixtures of boric-borate buffer, co-solvent (ethanol), and sodium chloride were tested as eluents. The influence of the buffer pH and the amount of co-solvent on the chromatographic process was studied for the model compound bis(Nalpha-lauroyl-L-arginine) 1,3-propanediamide dihydrochloride, C3(LA)2, and purification conditions were established. The method was scaled-up to the multigram level for C3(LA)2 and the rest of the series. The proposed preparative procedure involves simple equipment, low cost materials, and minimal amounts of solvent (water/ethanol), with low toxicity.     

Enzymatic synthesis of oligosaccharides

Abstract: The biological interest of oligosaccharides is growing very rapidly, and necessitates the development of efficient synthesis reactions. The stereo- and regio-selectivity of enzyme catalysis is a key advantage in this field, as a complementary tool to the chemical approach. Two types of enzymes can be applied to the obtention of oligosaccharides: Hydrolytic enzymes, which can catalyze either reverse hydrolysis (thermodynamic control) or transglycosylation (kinetic control) synthesis reactions; and transferase enzymes, which can use simple carbohydrates from agricultural origin as glycosyl donors.     

Enzymatic synthesis of Tinuvin

Coupling of 3-(3-tert-butyl-4-hydroxyphenyl) propionic acid methylester to 1H-benzotriazole using a laccase from Trametes hirsuta was studied. The potentially resulting coupling product Tinuvin 1130 is an important UV-absorber used in polymer based materials. Oxidation of the phenol by the laccase led to homomolecular coupling reactions while the laccase did not attack 1H-benzotriazole. Due to the homomolecular reaction of the phenol in the presence of laccase coupling of phenol and 1H-benzotriazole was only observed when 1H-benzotriazole was applied in four-fold molar excess. The reaction was monitored by UV/vis spectroscopy, TLC and MS (ion trap) analysis. Coupling of 1H-benzotriazole took place in ortho position according to the postulated mechanism.     

Enzymatic synthesis of alkyds

Lipases were used as catalysts in the synthesis of "all-trans" polyester oligomers in organic solvents. Esters of fumaric acid and 1,4-butane diol served as the substrates in the enzyme-catalyzed polytransesterification. No isomerization of the double bond was found under the mild conditions of enzymatic catalysis used by us, as opposed to the extensive isomerization found during chemical polycondensation. The alkoxy leaving group of the ester fumarate was found to be responsible for the rate of transesterification. Low (M(w) approximately 600-800) and high (M(w) = 1250) molecular weight alkyds were synthesized depending on whether tetrahedrofuran or acetonitrile, respectively, was used as the solvent.     

Enzymatic synthesis of aza-l-tyrosines

Tyrosine phenol-lyase from Citrobacter freundii synthesizes 2-aza-L-tyrosine and 3-aza-L-tyrosine from 3-hydroxypyridine and 2-hydroxypyridine, respectively, and ammonium pyruvate.     

Enzymatic synthesis of L-cysteine

O-Acetylserine sulfhydrase in the form of a crude extract from Salmonella typhimurium LT2 was used for the production of L-cysteine from L-O-acetylserine and sodium hydrosulfide at pH 7.0 and 25 degrees C. The two substrates have quite different pH stability relationships. O-Acetylserine readily rearranges to N-acetylserine and the rate of this O --> N acyl transfer reaction increases at higher pH, temperature, and concentration of O-acetylserine. On the other hand, sodium hydrosulfide is more soluble at a higher pH. A stirred-tank bioreactor with a continuous substrate feed was employed to overcome this problem. The O-acetylserine feed was stored at its saturation level (2.05M) at pH 5.0, and the sodium hydrosulfide feed was dissolved at 2.05-2.3M without pH adjustment (pH >/= 11.5). Both substrates were simultaneously introduced into the bioreactor. The performance of the bioreactor was optimized by employing an automatic feedback control system to regulate the concentration of O-acetylserine in the bioreactor. This feedback control system was based on the fact that as the bioconversion proceeds, protons are produced along with cysteine. A pH controller thus detected the decrease in pH and activated the substrate pumps. After mixing in the bioreactor, these two substrate solutions behaved as a base due to the high alkalinity of sodium hydrosulfide. Thus, substrate infusion started when the pH was lower than the set point, i.e., the reaction pH, and stopped when the pH was raised higher than the set point. The amount of substrate introduced was determined by the alkalinity of the mixture of the two substrates, which in turn was controlled by the concentration of sodium hydrosulfide. After optimizing the sodium hydrosulfide concentration and the substrate feed rate, the bioconversion gave a productivity of 3.6 g L-cysteine/h/g dry cell weight S. typhimurium, an L-cysteine titer of 83 g/L and a molar yield based on O-acetylserine of 94%.     

Enzymatic synthesis of amylose

Amylose is a linear polymer of α-1,4-linked glucose and is expected to be used in various industries as a functional biomaterial. However, pure amylose is currently not available for industrial purposes, since the separation of natural amylose from amylopectin is difficult. It is known that amylose has been synthesized using various enzymes. Glucan phosphorylase, together with its substrate, glucose-1-phosphate, is the most suitable system for the production of amylose since the molecular size of amylose can be controlled precisely. However, the problem with this system is that glucose-1-phosphate is too expensive for industrial purposes. This review summarizes our work on the enzymatic synthesis of essentially linear amylose, together with recent progress in the production of synthetic amylose using sucrose or cellobiose through the combined actions of phosphorylases.