Srđanka Tomić

Quinuclidinium-imidazolium compounds: synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice

Abstract Four compounds were prepared: 3-oxo-1- methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH2)3- (III) and -CH2-O-CH2- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD50) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD50 dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD50 doses of Soman.

PARTIAL PURIFICATION OF ESTERASES FROM RABBIT SERUM AND THEIR USE IN REGIOSELECTIVE DEACYLATIONS OF SUGARS

14C-Labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside was used as a substrate for the detection of esterase activity in the isolation of esterase II from rabbit serum. On treatment of methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside with rabbit serum and esterase II, the 6-O-pivaloyl group was removed selectively. Likewise, the 6-O-pivaloyl group was removed selectively from methyl 2-acetamido-2-deoxy-3,4,6-tri-O-pivaloyl-alpha-D-glucopyranoside.

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.

Syntheses of partially pivaloylated d-glucopyranoses: new substrates for the esterase from rabbit serum

Abstract The selective pivaloylation (pivaloyl chloride-pyridine) of d -glucopyranose proceeds by two pathways after initial 6-pivaloylation, namely, successive esterification of ( a ) positions 1, 3, 4, and 2 (major pathway) and ( b ) positions 2, 1, 4, and 3 (minor pathway). Numerous di- ( 3 and 4 ), tri ( 5–9 ), and tetra-pivalates ( 10–12 ) have been prepared and characterised. On treatment of the 2,6-dipivalate ( 4 ) with rabbit serum or partially purified esterase II, PivO-6 was hydrolysed selectively, whereas the 1,6-dipivalate ( 3 ) underwent partial 1 → 2 acyl migration to give 4 and enzymic de-esterification of 3 and 4 occurred simultaneously.

Synthesis, intramolecular migrations and enzymic hydrolysis of partially pivaloylated methyl α-d-mannopyranosides

A series of methyl O-pivaloyl-alpha-D-mannopyranosides was synthesized using pivaloyl chloride in pyridine. The 3,6-di-O-pivaloyl derivative 6 undergoes intramolecular transesterification in neutral conditions (buffer, pH 7.2) to give its 2,6-di-O-pivaloyl analogue 5. The course of this migration was followed using 14C-labelled 6. As opposed to 6 compound 5 was shown to be a good substrate for esterases present in rabbit serum. Thus, regioselective enzymic hydrolysis led to the preferential cleavage of the 2-OPiv group to yield a mixture of 2- and 6-O-monopivalates in a ratio of 1:2.6.

Synthesis and intramolecular transesterifications of pivaloylated methyl α-d-galactopyranosides

Abstract Selective pivaloylations of methyl α- d -galactopyranoside have been studied under various reaction conditions. Partially pivaloylated products were submitted to additional acetylations. The structures were established by 1 H and 13 C NMR spectroscopies. Both, 2,6- and 3,6-dipivalates underwent intramolecular cyclization in neutral conditions (phosphate buffered saline, pH 7.2) to give a stable 2,3-orthoacid with a parallel 6→4 migration of the pivaloyl group.

Esterase catalyzed regioselective hydrolyses of acetylated monosaccharides

Abstract Deacylations of fully acylated D -glucopyranoses, methyl D -glucopyranosides, 2-acetamido-2-deoxy- D -glucopyranoses glucopyranoses and methyl 2-acetamido-2-deoxy- D -glucopyranosides catalyzed by rabbit serum or the esterase isolated from rabbit serum were investigated. Depending on the structure of the acyl protecting group a high degree of regioselectivity is observed. Products of enzymic hydrolysis followed by intramolecular migrations of acetyl groups are also described.

A comprehensive evaluation of novel oximes in creation of butyrylcholinesterase-based nerve agent bioscavengers

A well-considered treatment of acute nerve agents poisoning involves the exogenous administration of butyrylcholinesterase (BChE, EC 3.1.1.8) as a stoichiometric bioscavenger efficient in preventing cholinergic crises caused by acetylcholinesterase (AChE, EC 3.1.1.7) inhibition. An additional improvement in medical countermeasures would be to use oximes that could reactivate BChE as well to upgrade bioscavenging from stoichiometric to oxime-assisted catalytic. Therefore, in this paper we investigated the potency of 39 imidazolium and benzimidazolium oximes (36 compounds synthesized for the first time) to be considered as the reactivators specifically designed for reactivation of phosphylated human BChE. Their efficiency in the reactivation of paraoxon-, VX-, and tabun-inhibited human BChE, as well as human AChE was tested and compared with the efficiencies of HI-6 and obidoxime, used in medical practice today. A comprehensive analysis was performed for the most promising oximes defining kinetic parameters of reactivation as well as interactions with uninhibited BChE. Furthermore, experimental data were compared with computational studies (docking, QSAR analysis) as a starting point in future oxime structure refinement. Considering the strict criteria set for in vivo applications, we determined the cytotoxicity of lead oximes on two cell lines. Among the tested oxime library, one imidazolium compound was selected for preliminary in vivo antidotal study in mice. The obtained protection in VX poisoning outlines its potential in development oxime-assisted OP-bioscavenging with BChE.

Synthesis and immunostimulating properties of novel adamant-1-yl tripeptides.

The aim of this work was to prepare L‐ and D‐(adamant‐1‐yl)‐Gly‐L‐Ala‐D‐isoGln peptides in order to study their adjuvant (immunostimulating) activities. Adjuvant activity of adamant‐1‐yl tripeptides was tested in the mouse model using ovalbumin as an antigen and in comparison to the peptidoglycan monomer (PGM; β‐D‐GlcNAc‐(1→4)‐D‐MurNAc‐L‐Ala‐D‐isoGln‐mesoDAP(εNH2)‐D‐Ala‐D‐Ala) and structurally related adamant‐2‐yl tripeptides.

Mannosylated N-Aryl Substituted 3-Hydroxypyridine-4-Ones: Synthesis, Hemagglutination Inhibitory Properties, and Molecular Modeling

Structural alterations of the aglycon portions of α‐mannosides influence their inhibitory potency toward type 1‐fimbriated Escherichia coli. The aim of our work was to prepare and explore inhibitory properties of novel mannosylated N‐aryl‐substituted 3‐hydroxypyridine‐4‐ones because they possess needed structural characteristics as possible FimH antagonists. Hemagglutination inhibitory tests showed that the examined 3‐hydroxypyridine‐4‐one α‐mannosides exhibited better inhibitory activity than methyl α‐d‐mannopyranoside used as a reference compound. Molecular modeling studies revealed the specific interactions responsible for the observed binding activities toward the mannose‐specific FimH lectin. The activity depends on the substituent in p‐position on the aglycon aromatic ring.