Lipase catalysed acylation of hydroxylamine and hydrazine derivatives

The lipase catalysed acylation of hydroxylamine-and hydrazine as well as their derivatives by octanoic acid is very efficient. Cross-linked crystals of Candida rugosa lipase (ChiroCLEC-CR) mediated the conversion of racemic ibuprofen into (S)-ibuproxam. A number of lipases also catalysed the condensation of hydrazine with an excess of octanoic acid giving N,N′-dioctanoylhydrazine. The hydrazide of 2-(4-isobutylphenyl)propanoic acid (ibuprofen), prepared by non-enzymatic reaction of ibuprofen methyl ester with hydrazine, acted as nucleophile towards several lipases that do not accept ibuprofen derivatives as acyl donor.     

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

Enantiomerically pure alkylphosphonate compounds RR′P(O)PNP (R=C_nH_2n+1, R′=OY with Y=C_n′H_2n′+1 with n=n′ or n≠n′; PNP=p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (₅₀) leading to half inhibition, and the chain length, R or Y was observed. (R₁₁Y₁₆)s were the best inhibitor of HPL and (R₁₀Y₁₁)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.     

Synthetic routes to mono-N-functionalized P2N2-ligands

Three different synthetic routes have been explored for the synthesis of the mono-N-substituted phosphinoamine N-ethyl,N′bis[2(diphenylphosphino)phenyl]propane-1,3-diamine: (a) selective alkylation of N,N′bis[2(diphenylphosphino)phenyl]propane-1,3-diamine; (b) linkage of the different fragments of N-ethyl,N′bis[2(diphenylphosphino)phenyl]propane-1,3-diamine; (c) selective acylation of N,N′bis[2(diphenylphosphino)phenyl]propane-1,3-diamine followed by acetyl reduction. While approaches (a) and (b) were unsuccessful, N-ethyl,N′bis[2(diphenylphosphino)phenyl]propane-1,3-diamine was obtained by route (c) via separation of the mono- and di-alkylated P₂N₂-species obtained from reduction, through complexation of Ni(NO₃)₂6H₂O followed by demetallation reaction with KCN. Additional related phosphinoamine chelates and phosphonium adducts were synthesized and characterized by conventional physico–chemical techniques.     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

A facile enzymatic process for the preparation of ibuprofen ester prodrug in organic media

A novel glucopyranoside derivative of ibuprofen, ibuprofen methyl -d-glucopyranoside ester was synthesized via immobilized lipase-catalyzed esterification between racemic ibuprofen and methyl -d-glucopyranoside in non-aqueous medium. An appropriate product concentration (4.6 mg ml⁻¹) was achieved by optimization of reaction conditions, such as solvent type, reaction temperature, enzyme concentration and initial concentration of substrates. Comparing with the parent drug ibuprofen in physicochemical properties, the glucopyranoside derivative of ibuprofen has better hydrophilicity. The chemical structure of the ibuprofen ester was confirmed to be methyl 6-O-(2′-(4′-isobutylphenyl) propionyl) -d-glucopyranoside.     

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R′ series of compounds produced no significant affect on the high δ-opioid receptor affinity (K_i=0.035–0.454 nM), but dramatically decreased that for the μ-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R′): phenyl (Ph) (5′–8′) elicited a greater reduction in μ-affinity (40–70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i) a loss of δ-agonism coupled with the appearance of potent δ antagonism (4′–7′) (pA₂=8.14–9.47), while 1 exhibited only a 160-fold decreased δ agonism (1′) and the δ antagonism of 8 enhanced >10-fold (pA₂=10.62, 8′); and (ii) a consistent loss of μ-affinity resulted in enhanced δ-opioid receptor selectivity. With the exception of compound 1′, the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent δ-selective antagonists.     

Regioselective enzymatic aminoacylation of lobucavir to give an intermediate for lobucavir prodrug

Synthesis of lobucavir prodrug, L-valine, [(1S,2R,3R)-3-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (BMS 233866), requires regioselective coupling of one of the two hydroxyl groups of lobucavir (BMS 180194) with valine. Either hydroxyl group of lobucavir could be selectively aminoacylated with valine by using enzymatic reactions. N-[(Phenylmethoxy)carbonyl]-L-valine, [(1R,2R,4S)-2-(2-amino-6-oxo-1H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester (3, 82.5% yield), was obtained by selective hydrolysis of N,N′-bis[(phenylmethoxy)carbonyl]bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester (1) with lipase M, and L-valine, [(1R,2R,4S)-2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-4-(hydroxymethyl)cyclobutyl]methyl ester monohydrochloride (4, 87% yield) was obtained by hydrolysis of bis[L-valine], O,O′-[(1S,2R,3R)-3-(2-amino-6-oxo-1H-purin-9-yl)cyclobuta-1,2-diyl]methyl ester, dihydrochloride (2), with lipase from Candida cylindracea. The final intermediate for lobucavir prodrug, N-[(phenylmethoxy)carbonyl]-L-valine, [(1S,2R,4R)-3-(2-amino-6-oxo-1H-purin-9-yl)-2-(hydroxymethyl)cyclobutyl]methyl ester (5), could be obtained by transesterification of lobucavir using ChiroCLEC™ BL (61% yield), or more selectively by using immobilized lipase from Pseudomonas cepacia (84% yield).     

Synthesis of oligonucleotides containing a new azobenzene fragment with efficient photoisomerizability

The azobenzene derivatives possessing substituents of ROCH₂CH₂O- and-CH₂CH₂OR′ or -CONHCH₂CH₂OR′ at p,p′-positions, where R and R′ are 4,4′-dimethoxytrityl and 2-cyanoethyl-N,N′-diisopropylphophoramidite, have been synthesized for linking two oligonucelotide segments. It has been found that the azobenzene linkers efficiently undergo trans–cis isomerization by exposing to UV light. The conversion efficiency showed slight dependence on structure or conformation of oligonucleotides attached to the azobenzene chromophore. The cis-form of the azobenzene in oligonucleotides was sufficiently stable at low temperature under dark. The present findings would open the way for light switch of nucleic acid structures.     

Biocatalyst engineering exerts a dramatic effect on selectivity of hydrolysis catalyzed by immobilized lipases in aqueous medium

It has been found that enantioselectivity of lipases is strongly modified when their immobilization is performed by involving different areas of the enzyme surface, by promoting a different degree of multipoint covalent immobilization or by creating different environments surrounding different enzyme areas. Moreover, selectivity of some immobilized enzyme molecules was much more modulated by the experimental conditions than other derivatives. Thus, some immobilized derivatives of Candida rugosa (CRL) and C. antarctica-B (CABL) lipases are hardly enantioselective in the hydrolysis of chiral esters of (R,S)-mandelic acid under standard conditions (pH 7.0 and 25°C) (E<2). However, other derivatives of the same enzymes exhibited a very good enantioselectivity under nonstandard conditions. For example, CRL adsorbed on PEI-coated supports showed a very high enantio-preference towards S-isomer (E=200) at pH 5. On the other hand, CABL adsorbed on octyl-agarose showed an interesting enantio-preference towards the R-isomer (E=25) at pH 5 and 4°C. These biotransformations are catalyzed by isolated lipase molecules acting on fully soluble substrates and in the absence of interfacial activation against external hydrophobic interfaces. Under these conditions, lipase catalysis may be associated to important conformational changes that can be strongly modulated via biocatalyst and biotransformation engineering. In this way, selective biotransformations catalyzed by immobilized lipases in macro-aqueous systems can be easily modulated by designing different immobilized derivatives and reaction conditions.     

Microhabitat selection and daily movements of two rodents (Necromys lasiurus and Oryzomys scotti) in Brazilian Cerrado, as revealed by a spool-and-line device

Studies on patterns of habitat use by mammals are necessary for understanding the mechanisms involved in their distribution and abundance. In this study, we used the spool-and-line method to investigate habitat utilization by two sigmodontine rodents from Brazilian Cerrado, Necromys lasiurus and Oryzomys scotti. We conducted the study in a Cerrado area in central Brazil (15°56′S and e 47°56′W) where the animals were caught in an area of 7.68 ha of Cerrado sensu stricto. Captured individuals were marked, equipped with a spool-and-line device, and released at the same capture point. The next day we followed the thread to record their daily movements and find their nests. To investigate microhabitat selection we compared habitat characteristics along trails of each studied species with general habitat characteristics of the study area. Although the mean 24-h distance was greater for N. lasiurus (mean ± SE: 41.9 ± 42.2 m, N=3) than for O. scotti (28.7 ± 14.2 m, N=6) this difference was not significant (Mann–Whitney test, U=26, P>0.6). We detected significant differences among observed microhabitats variables of both species and available microhabitat characteristics as determined by discriminant analysis (Wilks′s lambda F=3.001; df=14, 116; P<0.001). Both species were associated to microhabitat characteristics whose values differed markedly from the overall available habitat. Along the first canonical discriminant function of the DFA both them were associated with greater grass height than the mean height available and along the second axis N. lasiurus selected areas with higher fruit availability and more shelters than those selected by O. scotti. For stronger inferences regarding differential patterns of habitat utilization by Cerrado rodents we suggest the simultaneous use of both spool-and-line and standard trapping methods.     

1,10-Phenanthroline-5,6-dione as a building block for the synthesis of homo- and heterometallic complexes

1,10-Phenanthroline-5,6-dione (C₁₂H₆N₂O₂ (1)) reacts with V(η⁶-mesitylene)₂ and Ti(η⁶-toluene)₂ affording coordination compounds of general formula M(O,O′---C₁₂H₆N₂O₂)₃ (M=Ti (2); M=V (3)) which further react with TiCl₄ or TiCp₂(CO)₂ yielding the tetrametallic species M(O,O′---C₁₂H₆N₂O₂---N,N′)₃(M′L_n)₃ (M=V, M′L_n=TiCl₄ (4); M=Ti, M′L_n=TiCp₂ (5); M=V, M′L_n=TiCp₂ (6)). The complex salt [Fe(N,N′---C₁₂H₆N₂O₂)₃][PF₆]₂ (7) has been obtained from iron(II) chloride tetrahydrate and 1 in the presence of NH₄PF₆. The reaction of 7 with TiCp₂(CO)₂ affords the tetrametallic derivative [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)₃(TiCp₂)₃][PF₆]₂ (8). TiCl₂(THF)₂ reacts with MCp₂(O,O′---C₁₂H₆N₂O₂) to give MCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (M=Ti (9); M=V (10)). By reaction of TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (9) with C₁₂H₆N₂O₂, the bimetallic derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂) (11) has been prepared, which readily adds to TiCl₄, to give the trimetallic titanium derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₄ (12). VCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (10) reacts with the tris-chelate iron(II) cation 7 affording the heptametallic cationic complex [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)TiCl₂(N,N′---C₁₂H₆N₂O₂---O,O′)VCp₂]₃ ⁺² isolated as the hexafluorophosphate 13.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

Antibacterial and antiproliferative activity of cationic fullerene derivatives

We examined the antibacterial and antiproliferative activities of alkylated C₆₀-bis(N,N-dimethylpyrrolidinium iodide) derivatives. The fullerene derivatives inhibited bacteria and cancer cell growth effectively. However, the fullerene derivatives with a long alkyl chain did not show antibacterial activity.     

Chemoenzymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP-sialic acid synthetases

Three C terminal His₆-tagged recombinant microbial CMP–sialic acid synthetases [EC 2.7.7.43] cloned from Neisseria meningitidis group B, Streptococcus agalactiae serotype V, and Escherichia coli K1, respectively, were evaluated for their ability in the synthesis of CMP–sialic acid derivatives in a one-pot two-enzyme system. In this system, N-acetylmannosamine or mannose analogs were condensed with pyruvate, catalyzed by a recombinant sialic acid aldolase [EC 4.1.3.3] cloned from E. coli K12 to provide sialic acid analogs as substrates for the CMP–sialic acid synthetases. The substrate flexibility and the reaction efficiency of the three recombinant CMP–sialic acid synthetases were compared, first by qualitative screening using thin layer chromatography, and then by quantitative analysis using high performance liquid chromatography. The N. meningitidis synthetase was shown to have the highest expression level, the most flexible substrate specificity, and the highest catalytic efficiency among the three synthetases. Finally, eight sugar nucleotides, including cytidine 5′-monophosphate N-acetylneuraminic acid (CMP–Neu5Ac) and its derivatives with substitutions at carbon-5, carbon-8, or carbon-9 of Neu5Ac, were synthesized in a preparative (100–200 mg) scale from their 5- or 6-carbon sugar precursors using the N. meningitidis synthetase and the aldolase.     

Influence of bulky substituents on the regioselective group-transfer reactions of diorganozinc compounds with N, N′-bis(2,6-di-isopropylphenyl)-1,4-diaza-1,3-butadiene

Diorganozinc compounds R₂Zn (R=alkyl or aryl) react with N,N′-bis(2,6-di-isopropylphenyl)-1,4-diaza-1,3-butadiene, (i-Pr₂Ph)N=CHCHp=N(i-Pr₂Ph) (i-Pr₂Ph-DAB) to give thermally unstable 1:1 coordination complexes R₂Zn(i-Pr2Ph-DAB), which subsequently undergo a slow regioselective alkyl or aryl group-transfer reaction from the zinc atom to an imine-nitrogen or a carbon atom of the NCCN system of the i-Pr₂Ph-DAB ligand. In the case of R=methyl, n-propyl, n-butyl, s-butyl, neopentyl and benzyl, C-alkylation occurs with a subsequent 1,2- hydrogen shift in the amino-imino skeleton affording RZn[(i-Pr₂Ph)N-CH₂-CR=N(i-Pr₂Ph)], whereas for R=t- butyl the C-alkylated product t-BuZn[(i-Pr₂Ph)N---CH(t-Bu)---CH=N(i-Pr₂Ph)] is stable. Surprisingly, diphenylzinc reacts with i-Pr₂Ph-DAB exclusively to give the N-arylated product PhZn[(i-Pr₂Ph)N=CHCH=N(Ph)(i-Pr₂Ph)].     

H NMR study of the interaction of N,N′,N″-triacetyl chitotriose with Ac-AMP2, a sugar binding antimicrobial protein isolated from Amaranthus caudatus

The interaction between Ac-AMP2, a lectin-like small protein with antimicrobial and antifungal activity isolated from Amaranthus caudatus, and N,N′,N″-triacetyl chitotriose was studied using ¹H NMR spectroscopy. Changes in chemical shift and line width upon increasing concentration of N,N′,N″-triacetyl chitotriose to Ac-AMP2 solutions at pH 6.9 and 2.4 were used to determine the interaction site and the association constant K_a. The most pronounced shifts occur mainly in the C-terminal half of the sequence. They involve the aromatic residues Phe¹⁸, Tyr²⁰ and Tyr²⁷ together with their surrounding residues, as well as the N-terminal Val-Gly-Glu segment. Several NOEs between Ac-AMP2 and the N,N′,N″-triacetyl chitotriose resonances are reported.     

Kinetic and modelling studies on the lipase catalysed enantioselective esterification of (±)-perillyl alcohol

Several lipases were kinetically studied with the aim to exploit their enantioselectivity in the esterification of (S)-(−) and (R)-(+)-perillyl alcohol with decanoic acid. Most of the lipases studied exhibited stereopreference towards the R-enantiomer with apparent E-values from 3.8 to 0.6, calculated as the initial esterification rates ratio for the individual enantiomers. In an attempt to interpret the structural basis of enantioselectivity, modelling studies were performed with two of these lipases, Candida cylindracea lipase (CcL) and Pseudomonas cepacia lipase (PcL) based on their previously determined X-ray crystal structures. The results derived from modelling studies confirm their stereopreferences towards the R-enantiomer, since increased conformational energy of the S-ester was found compared to the R-ester.