Chemo-enzymatic synthesis of amino acid-based surfactants

The application of lipases to the synthesis of amino acid-based surfactants was investigated. Low yields (2–9%) were obtained in the acylation of free amino acids, such as l-serine and l-lysine, as well as their ethyl esters and amides with fatty acids, owing in part to low miscibility of the reactants. When the N-carbobenzyloxy (Cbz)-l-amino acids were used in an effort to improve miscibility of the amino acid derivatives with the acyl donor, a dramatic improvement was observed for N-Cbz-l-serine (92% yield) but not for N _α-Cbz- or N _ζ-Cbz-l-lysine (7 and 2% yield, respectively). As an alternative, and efficient synthesis of N _ζ-acyl-l-lysines was developed, based on the regiospecific chemical acylation of copper(II) lysinate. In pursuit of a general route to amino acid-fatty acid surfactants, the utility of a polyol linker was investigated. Thus, the glycerol ester of N _α′ N _ζ-di-Cbz-l-lysine was prepared and evaluated as a substrate for acylation. As expected, this and other glycer-1-yl esters of N-protected amino acids were excellent substrates for lipase-catalyzed acylation. Their reaction with myristic acid in the presence of Novozyme resulted in the regioselective acylation of the primary hydroxyl group of the glycerol moiety to afford the corresponding 1-O-(N-Cbz-l-aminoacyl)-3-O-myris-toylglycerols with conversions of 50–90%. These were readily deprotected to give a range of 1-O-(aminoacyl)-3-O-myristoyl-glycerols with overall yields of 27–71%.     

Preparation of schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2∶1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15–57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products. Based on presentation at the AOCS Annual Meeting & Expo in Indianapolis, Indiana, April 28–May 1, 1996.     

Drug delivery from nonpeptidic α-amino acid containing polyamides

New nonpeptidic α-amino acid-based polyamides were used for drug delivery. Benzocaine was covalently linked via a spacer to N-protected poly(l-cystyl-l-cystine) and to poly(adipoyl-l-lysine). Its release by α-chymotrypsin attack was followed by UV. It was nearly quantitative within two days with l-cystine-based polyamide. Protected forms of these polyamides were used as matrices. Release of benzocaine entrapped in polyamide from (l-cystine) was controlled by diffusion. When entrapped in polyamide from (l-lysine) it was controlled by diffusion and swelling. Received: 10 January 1997/Accepted: 23 January 1997     

Synthesis and degradation of nonpeptidic α-amino acid-containing polyamides

This paper describes the synthesis of new water-soluble nonpeptidic α-amino acid-containing polyamides. Preliminary results of hydrolytic and enzymatic degradations of l-lysine and l-cystine-based polyamides are also presented. Most of these polymers revealed to be stable towards hydrolytic degradation. Only poly(l-cystyl-l-cystine) PCC IV was rapidly degraded in Tris buffer pH 7.4. Insoluble poly(adipoyl-l-lysine benzyl ester) could be degraded by papain and pepsin. Polyamides from l-cystine were shown to be more susceptible to enzymatic degradation. Trypsin, papain and glutathione reductase degraded PCC IV much more rapidly than Tris buffer 7.4 alone. Received: 6 January 1997/Accepted: 23 January 1997     

Synthesis and polymerization of nucleic acid base substituted L-lysine

Summary Poly-l-lysines containing nucleic acid bases, that is, adenine, thymine, uracil and theophylline were synthesized. The nucleic acid base substituted l-lysine derivatives were prepared from ,N-Cbz-l-lysine and the p-nitrophenyl esters of the carboxyethyl derivatives of the nucleic acid bases. The polymers were obtained from these l-lysine derivatives by the NCA method.     

Kinetics of formation of fluorescent products from hexanal andl-lysine in a two-phase system

Kinetics of formation of fluorescent condensation products from hexanal andl-lysine (or itsN-acetylated forms) including mass-transfer has been studied in a two-phase system consisting of lysine (or lysine derivative) in a aqueous phosphate buffer and a 1-octanol solution of hexanal as model for formation of fluorophores between protein and carbonyl compounds in peroxidizing biological systems. The initial rate of formation of fluorescent products in the aqueous phase was found to be proportional to the concentration of hexanal and lysine and to increase in both phases with increasing pH in the aqueous phase, in contrast to a higher-order dependence on hexanal in the octanol phase. At pH=6.8, the temperature dependence of the appearance of fluorescent products corresponds to apparent energies of activation of 63 kJ·mol⁻¹ and 87 kj·mol⁻¹ in the aqueous phase and the octanol phase, respectively. Fluorescent condensation products appeared faster in the octanol phase. However, by a kinetic analysis, the fluorescent products were shown to be formed in the aqueous phase, corresponding to the lower energy of activation and to the simple second-order kinetics, and subsequently distributed between the aqueous phase and the octanol phase.l-Lysine reacted faster thanN _α-acetyl-l-lysine which reacted faster thanN _ε-acetyl-l-lysine. Using fluorescence quantum yields, determined to be 1.4·10⁻² in octanol and 8·10⁻³ in water at pH 6.8, an apparent partition coefficient of 17 (octanol/water) was determined for the condensation product ofl-lysine. The steady-state fluorescence in the octanol phase was attributed to two components with fluorescence life-time at 25°C of 0.7±0.05 ns and 5.1±0.2 ns, assigned to hexanal and the condensation product, respectively. The emission spectra, were resolved in the two components using phase-sensitive detection, and the condensation product had emission maximum at 405 nm.     

Poly(<Emphasis Type="SmallCaps">l</Emphasis>-lysine) as a polychelatogen to remove toxic metals using ultrafiltration and bactericide properties of poly(<Emphasis Type="SmallCaps">l</Emphasis>-lysine)–Cu<Superscript>2+</Superscript> complexes

Poly(l-lysine) is a water-soluble, synthetic polypeptide containing functional amine groups that help remove di- and trivalent metal ions from aqueous solutions. This polymer’s removal properties were studied under different experimental conditions: (1) competitive and non-competitive conditions; (2) different pHs; and (3) filtration factors. Under the conditions of the Liquid-Phase Polymer-Based Retention (LPR) technique, the cooper (II) ion interaction was found to be selective and efficient when compared to other divalent metal ions studied. However, interestingly, this selectivity disappeared when trivalent metal ions were present. The polymer–metal ion interactions are based on the amino groups of the side-chains as well as the polypeptide backbone chain. The removal of metal ions was strongly dependent on the pH. By structural characterization with FT-IR and EPR spectroscopy, participation of the amide and mainly amine groups was found to take place for the coordination. For the Cu²⁺, coordination through four amine nitrogen donor atoms in the primary coordination sphere was detected. Antibacterial activity tests were conducted with the poly(l-lysine)–Cu²⁺ complex and showed a higher activity in comparison with the precursors Cu²⁺ and poly(l-lysine) at the same concentrations for E. coli (6538P), a Gram-negative bacterium, and S. aureus (ATCC), a Gram-positive bacterium.     

Synthesis, characterization and surface properties of 1-N-l-tryptophan-glycerol-ether surfactants

A novel homologous series of 1-N-l-tryptophanglycerol-ether surfactants was synthesized and characterized. The precursor compounds, 3-alkyloxy-1-chloropropan-2-ols, were prepared from epichlorohydrin and aliphatic alcohols with alkyl chain lengths of 9–16 carbon atoms. Tryptophan was then attached to the monosubstituted glycerol backbone from its α-amino group through an α-NH-C bond. Structural assignment of the new compounds was made on the basis of elemental analysis and spectroscopic data. Critical micelle concentrations of the new surfactants, as well as the negative logs of the surfactant concentrations required to reduce the surface tension of the solvent by 20 mN/m (pC ₂₀) and the interfacial areas occupied by the surfactant molecules, were calculated from aqueous surface tension measurements using the Wilhelmy plate technique.     

Enzymatic synthesis of N-acyl-l-amino acids in a glycerol-water system using acylase I from pig kidney

N-Medium- and long-chain acyl-l-amino acids were enzymatically synthesized from the corresponding l-amino acids and fatty acids using a reverse hydrolysis. Enzymes that are suitable for the synthetic reaction of N-acyl-l-amino acids were screened on the basis of hydrolytic activity toward N-lauroyl-l-glutamic acid as an indicator. Acylase I from pig kidney (EC 3.5.1.14) showed the highest N-acyl-l-amino acid hydrolytic activity among 57 commercially available enzymes tested. Acylase I effectively catalyzed the synthesis of N-lauroyl-l-amino acids except for N-lauroyl-l-proline and N-lauroyl-l-tyrosine in a glycerol-water system. Under the optimized reaction conditions, N-lauroyl-l-arginine and N-lauroyl-l-glutamic acid were obtained in conversions of 82 and 44%, respectively. The equilibrium constants calculated from the conversion obtained were 5.6, 15.4, 18.0, and 39.4 for the syntheses of N-lauroyl-l-glutamic acid, N_α-lauroyl-l-lysine, N-lauroyl-l-glutamine, and N-lauroyl-l-methionine, respectively. N-Acyl-l-arginines with myristic acid and palmitic acid as the fatty acid were also synthesized using acylase I.     

An Improved Method for Analysis of Glucosylceramide Species Having cis-8 and trans-8 Isomers of Sphingoid Bases by LC–MS/MS

High-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) approaches have enabled high selectivity and sensitivity for the identification and quantification of glucosylceramide molecular species. Here we demonstrate that HPLC–ESI–MS/MS is an efficient method for characterizing plant glucosylceramide species having the cis-8 and trans-8 isomers of sphingoid bases. Complete baseline separation was achieved using a high-carbon-content octadecylsilyl column and a simple binary gradient comprising methanol and water. The result of 2-hydroxy fatty acid composition achieved by HPLC–ESI–MS/MS was compared with that achieved by gas chromatography with flame ionization detection (GC–FID), indicating that the two methods yield similar molar compositions. The current method should be applicable to seeking the active components of glucosylceramide species from plant materials in response to biological challenges.     

Synthesis and characterization of polyamides based on natural monomers: l-lysine and l-aspartic acid

Nonpeptidic diamine-diacid type polyamides were prepared from natural α-amino acids, l-lysine and l-aspartic acid, under mild conditions. l-lysine carboxylic group was protected as a benzyl ester; l-aspartic acid amino group was protected as benzyloxycarbonyl (Z) or t-butyloxycarbonyl (BOC) derivatives. The activated ester method provided polyamides with protected amino and carboxyl side groups. The deprotection of these side groups revealed to be perfectly selective when the amino groups were protected as t-butyloxycarbonyl derivatives. Received: 20 February 1997/Revised: 8 April 1997/Accepted: 14 April 1997     

Insight into Isomeric Diversity of Glycated Amino Acids in Maillard Reaction Mixtures

Maillard reactions generate a wide array of amino acid- and sugar-derived intermediates; the isomeric mixtures of glycated amino acids are of particular interest. Excluding stereoisomers, regioisomers, and various anomers, most amino acids can form two monoglycated and three N,N-diglycated isomers when reacted with sugars during the Maillard reaction. Using synthetic Schiff bases and Amadori compounds as standards, we have demonstrated that diagnostic ions obtained from MS/MS fragmentations in negative ionization mode can be used effectively for the discrimination between glucose-derived Schiff bases and their corresponding Amadori compounds in both mono- and diglycated forms. The utilization of these diagnostic ions and isotopic labeling in the glycine/glucose model system revealed that milling glucose/glycine mixtures for 30 min/30 Hz at ambient temperature produced monoglycated glycine in equal proportions of Amadori and Schiff base forms, whereas diglycated glycine was a mixture of the three isomers: Schiff-Schiff, Schiff-Amadori, or Amadori-Amadori in approximately equal molar proportions. The above results were further corroborated using a synthetic histidine Amadori product, N,N-difructosyl-β-alanine, dipeptides, and ribose. Using mechanochemistry as a convenient synthetic tool in combination with MS/MS diagnostic ions, the isomeric diversity of the early stages of the Maillard reaction can be revealed.     

A novel ∈-lysine acylase from <Emphasis Type="Italic">Streptomyces mobaraensis</Emphasis> for synthesis of <Emphasis Type="Italic">N∈</Emphasis>-acyl-<Emphasis Type="SmallCaps">l</Emphasis>-lysines

A novel ∈-lysine acylase (N ₆-acyl-l-lysine amidohydrolase; EC 3.5.1.17) was isolated from Streptomyces mobaraensis and purified to homogeneity by SDS-PAGE from the culture broth. The purified enzyme was monomeric, with a molecular mass of approximately 60 kDa. The enzyme was inactivated by the presence of 1,10-phenanthroline and activated in the presence of Co²⁺ and Zn²⁺. The enzyme showed a pH optimum of 8.0 and was stable at temperatures of up to 50°C for 1 h at pH 8.0. The enzyme specifically catalyzed the hydrolysis of the amide bond of various N∈-acyl-l-lysines. Furthermore, the enzyme efficiently catalyzed the synthesis of N∈-acyl-l-lysines with fatty and aromatic acyl groups in an aqueous buffer. In the syntheses of N∈-decanoyl-l-lysine, N∈-lauroyl-l-lysine, and N∈-myristoyl-l-lysine, the product precipitated and the yield was 90% or higher using 10 mM FA and 0.5 M l-lysine as the substrate.     

Sensitivity of positive ion mode electrospray ionization mass spectrometry (ESI MS) in the analysis of purine bases in ESI MS and on-line electrochemistry ESI MS (EC/ESI MS)

A cone-shaped MS inlet and on-line electrochemistry (EC) were used to enhance the ionization efficiency in electrospray ionization mass spectrometry (ESI MS) of purine bases. A pathway of positive ion mode ESI may involve oxidation of purine bases, guanine, adenine, xanthine and hypoxanthine, by 1e⁻, 1H⁺ processes. The electrospray process generates dimers of purine bases that are detected in ESI MS as protonated ions, except for xanthine, for which a protonated radical dimer is detected. Thus electrochemical oxidation of purine bases during ESI may generate reactive radicals that can subsequently dimerize. Dimer formation is facilitated in ESI MS when the carrier solution pH is high. The positive ion mode ESI MS ionization is consistent with the reactivity of the bases toward oxidation. Furthermore, the formation of the protonated ions, and Na⁺ and K⁺ adducts of the bases, expected in positive ion ESI MS, are observed. In addition, unusual H-bonding of purine bases guanine and xanthine is confirmed by ESI MS. Application of low EC voltage to the on-line EC cell in EC/ESI MS improves the sensitivity and correlates with the decrease of the intensity of the dimers, possibly as a result of their further oxidation.     

Natural antioxidants produced in oxidized lipid/amino acid browning reactions

1-Substituted pyrroles (1 and2) and1-substituted 2-(1′-hydroxypropyl)pyrroles (3–5) were produced in reactions between a lipid peroxidation product, 4,5(E)-epoxy-2(E)-heptenal, and the amino acid lysine. The antioxidative activity of compounds1–5 was studied. Oxidative stability was evaluated in refined soybean oil containing compounds1–5, butylated hydroxytoluene (BHT),n-propyl gallate orl-lysine, at concentrations of 50–200 ppm. Oils were either oxidized at 60°C and oxidation products determined by the thiobarbituric acid-reactive substances assay, or they were oxidized at 110°C by the Rancimat method. Although both methods gave similar results, greater differences were observed at 60°C than at 110°C. Addition of compounds1–5,l-lysine, BHT, and propyl gallate significantly (P<0.01) protected the oil against oxidation. The effectiveness order found was:l-lysine << compounds3–4 < compounds1–2 < compound5 ≈ BHT << propyl gallate.     

Development of an LC–MS/MS Method for the Analysis of Free Sphingoid Bases Using 4-Fluoro-7-nitrobenzofurazan (NBD-F)

The molecular species of sphingoid bases were tagged with the fluorescent amino group reagent, 4-fluoro-7-nitrobenzofurazan (NBD-F). The NBD-sphingoid bases were analyzed by a highly selective and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) technique capable of reliable detection of several fmol of the derivatives. Lipid extracts from plant samples were derivatized with NBD-F, and all nine species of free sphingoid bases present in plant sphingolipids were separated and quantified for the first time; a complete baseline resolution was achieved for cis-8 and trans-8 isomers of sphingoid bases by reversed phase HPLC on a C₁₈ column. The extraction and derivatization procedures and LC–MS/MS method can facilitate the progress of the studies for seeking the active components of sphingoid bases species in response to biological challenges.     

Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides and their chemical properties

A facile and efficient synthesis of the carboxyl-linked glucosides of bile acids is described. Direct esterification of unprotected bile acids with 2,3,4,6-tetra-O-benzyl-d-glucopyranose in pyridine in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent afforded a mixture of the α- and β-anomers (ca. 1∶3) of the 1-O-acyl-d-glucoside benzyl ethers of bile acids, which was separated effectively on a C₁₈ reversedphase chromatography column (isolated yields of α- and β-anomers are 4–9% and 12–19%, respectively). Subsequent hydrogenolysis of the α- and β-acyl glucoside benzyl ethers on a 10% Pd−C catalyst in acetic acid/methanol/EtOAc (1∶2∶2, by vol) at 35°C under atmospheric pressure gave the corresponding free esters in good yields (79–89%). Chemical specificities such as facile hydrolysis and transesterification of the acyl glucosides in various solvents were also discussed.     

A new ceramide from the basidiomycete Russula cyanoxantha

A new phytosphingosine-type ceramide (1) was isolated along with nine other compounds—5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol, 5α,8α-epidioxy-(24S)-ergosta-6-en-3β-ol, (24S)-ergosta-7-ene-3β,5α,6β-triol,(22E,24R)-ergosta-7, 22-dien-3β,5α,6β-triol, inosine, adenine, l-pyroglutamic acid, fumaric acid, and d-allitol from the ethanol and chloroform/methanol extract of the fruiting bodies of the basidiomycete Russula cyanoxanotha (Schaeff.) Fr. The structure of (1) was established as (2S,3S,4R,2′R)-2-(2′-hydroxytetracosanoylamino) octadecane-1,3,4-triol by means of spectroscopic and chemical methods.     

Synthesis of (S)-α-amino oleic acid

An efficient synthesis of (S)-α-amino oleic acid was developed. The fully protected FA derivative was obtained in four steps starting from methyl (2S)-2-[bis(tert-butoxycar-bonyl)amino]-5-oxopentanoate. These steps are (i) olefination of the starting aldehyde with the appropriate phosphonate anion, (ii) hydrogenation of the double bonds, (iii) controlled reduction of ω-ethyl ester to an aldehyde in the presence of α-methyl ester, and (iv) a Wittig reaction of the latter aldehyde with the suitable ylide. Free α-amino oleic acid was prepared after deprotection of the amino group followed by saponification in a total yield of 24%. N-tert-Butoxycarbonyl-protected amino oleic acid and the corresponding amino alcohol were prepared in high yield. The structures of the products have been established by various spectroscopic techniques.     

Biocatalytic synthesis of some chiral drug intermediates by oxidoreductases

Chiral intermediates were prepared by biocatalytic processes with oxidoreductases for the chemical synthesis of some pharmaceutical drug candidates. These include: (i) the microbial reduction of 1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanone (1) to R-(+)-1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1-piperazinyl]-1-butanol (2) [R-(+)-BMY 14802], an antipsychotic agent; (ii) the reduction of N-4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (3) to the corresponding chiral alcohol (4), an intermediate for d-(+)-N-4-{1-hydroxy-2-[(-methylethyl)amino]ethyl}phenyl methanesulfonamide [d-(+) sotalol], a β-blocker with class III antiarrhythmic properties; (iii) biotransformation of Nɛ-carbobenzoxy (CBZ)-l-lysine (7) to Nɛ-CBZ-l-oxylysine (5), an intermediate needed for synthesis of (S)-1-[6-amino-2-{[hydroxy(4-phenylbutyl)phosphinyl]oxy}1-oxohexyl]-l-proline (ceronapril), a new angiotensin converting enzyme inhibitor (6) and (iv) enzymatic synthesis of l-β-hydroxyvaline (9) from α-keto-β-hydroxyisovalerate (16). l-β-Hydroxyvaline (9) is a key chiral intermediate needed for the synthesis of S-(Z)-{[1-(2-amino-4-thiazolyl)-2-{[2,2-dimethyl-4-oxo-1-(sulfooxy)-3-azetidinyl] amino}-2-oxoethylidene]amino}oxyacetic acid (tigemonam) (10), an orally active monobactam.