Phosphodiesterolytic activity of samarium(III) mixed ligand complexes containing crown ethers and α-amino acids

Phosphodiesterolytic activity of samarium complexes containing crown ethers and amino acids was systematically studied. Formation constants of mixed ligand Sm–crown ethers–amino acids complexes (crown ethers = 18-crown-6, 15-crown-5 and 12-crown-4 and amino acids = Gly and Arg) were determined at 37.0 °C and 0.50 M NMe₄Cl. Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide(III)-mixed ligands complexes was studied under the same experimental conditions. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis–Menten-type saturation kinetics. High pH values markedly increase the observed activity. Potentiometric titrations results together with kinetic data of all these systems, under identical conditions, allowed us to identify the active species towards hydrolysis. Complexes with phosphodiesterolytic activity are monomeric hydroxylated cationic species. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones.     

Crystallisation of inorganic salts containing 18-crown-6 from ionic liquids

Reaction of the zwitterionic imidazolium salt [(CH₂COOH)(CH₂COO)im] with K₂CO₃ or BaO in the presence of 18-crown-6 affords the salts [(CH₂COO)₂im][K(18-crown-6)] and [(CH₂COO)₂im]₂[Ba(18-crown-6)], respectively. Recrystallisation of these crown complexes from the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide, [emim][Tf₂N], at a water interface, results in the formation of new salts in which the original anion is replaced by Tf₂N⁻. Single crystal X-ray diffraction has been performed on two of the salts. Notably, the potassium structure containing 18-crown-6 and Tf₂N⁻ forms a linear chain coordination polymer that can be regarded as metal organic frameworks (MOFs). Moreover, this study provides insights into the separation of group I and II metal ions using crown ethers in combination with ionic liquids.     

Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents.

Colyophilization or codrying of subtilisin Carlsberg with the crown ethers 18-crown-6, 15-crown-5, and 12-crown-4 substantially improved enzyme activity in THF, acetonitrile, and 1,4-dioxane in the transesterification reactions of N-acetyl-L-phenylalanine ethylester and 1-propanol and that of (+/-)-1-phenylethanol and vinylbutyrate. The acceleration of the initial rate, V(0), ranged from less than 10-fold to more than 100-fold. All crown ethers activated subtilisin substantially, which excludes a specific macrocyclic effect from being responsible. The secondary structure of subtilisin was studied by Fourier-transform infrared (FTIR) spectroscopy. 18-Crown-6 and 15-crown-5 led to a more nativelike structure of subtilisin in the organic solvents employed when compared with that of the dehydrated enzyme obtained from buffer alone. However, the high level of activation with 12-crown-4 where this effect was not observed excluded overall structural preservation from being the primary cause of the observed enzyme activation. The conformational mobility of subtilisin was investigated by performing thermal denaturation experiments in 1,4-dioxane. Although only a small effect of temperature on subtilisin structure was observed for the samples prepared with or without 12-crown-4, both 18-crown-6 and 15-crown-5 caused the enzyme to denature at quite low temperatures (38 degrees C and 56 degrees C, respectively). No relationship between this property and V(0) was evident, but increased conformational mobility of the protein decreased its storage stability. The possibility of a "molecular imprinting" effect was also tested by removing 18-crown-6 from the subtilisin-18-crown-6 colyophilizate by washing. V(0) was only halved as a result of this procedure, an effect insignificant compared with the ca. 80-fold rate enhancement observed prior to washing in THF. This suggests that molecular imprinting is likely the primary cause of subtilisin activation by crown ethers, as recently suggested.     

Aromatic Crown Ethers as Phase Transfer Catalysts in the Synthesis of N-Acetylglucosamine β-Aryl Glycosides

The crown ether-catalyzed glycosylation of phenol, 4-methoxyphenol, and 4-nitrophenol was studied under phase transfer conditions in solid–liquid system. The asymmetric dibenzocrown esters are superior to [3.3]dibenzo-18-crown-6 and 15-crown-5 in the catalysis of these reactions.     

Substituent control of selective alkali metal ion extraction by amidocrown η-butadienyl complexes of molybdenum(II)

Substituted η³-butadienyl complexes containing amide-armed crowns (X) of general formula [MoCl(CO)₂(η³-CH₂C(COX)CCH₂)(phen)]_n (phen=1,10-phenanthroline) were prepared and investigated for their ability to extract alkali metal ions from a mixed phase system. Reaction of the chlorocarbonyl precursor (1) with 1-aza-15-crown-5, 4^′-aminobenzo-15-crown-5, 2-aminomethyl-15-crown-5, 4^′-aminobenzo-18-crown-6 or 2-aminomethyl-18-crown-6 gave monomeric complexes (n=1), and addition of sodium tetraphenylboron to the 15-crown-5-substituted complexes gave the corresponding sodium salts. Dinuclear complexes (n=2) were formed by reaction of 1 and 1,7-diaza-15-crown-5 or 4,4^′(5^′)-diaminobenzo-15-crown-5. Comparison of amidobenzo- and 2-amidomethyl-15-crown-5-substituted complexes showed enhanced sodium transport properties for the latter, and spectroscopic and molecular modeling studies suggested complexation occurred by concerted action of the amide and crown.     

Variability of crown ether toxicity

Crown ethers are toxic to Escherichia coli. At a sublethal dosage, the crown ether affects the three phases in the bacterial growth curve as evidenced by an appearance of a lag period, an occasional decrease in the stationary phase at a lower microbial population. Potassium ion but not sodium ion can reduce the lag induced by the presence of 18-crown-6. On the contrary, the presence of either potassium ion or sodium ion lengthens the lag due to substituted 18-crown-6 ethers. Explanations to this variable toxicity are proposed.     

Large acceleration of alpha-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents

The effects of 18-crown-6 on the synthesis of peptides catalyzed by alpha-chymotrypsin are reported. Lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 results in a 425-fold enhanced activity when the reaction between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide is carried out in acetonitrile. Addition of crown ether renders the dipeptide synthesis in nonaqueous solvents catalyzed by alpha-chymotrypsin possible on a preparative scale. The acceleration is observed in different solvents and for various peptide precursors. Copyright 1998 John Wiley & Sons, Inc.     

Silver and thallium(I) complexation with dibenzo-16-crown-4.

Dibenzo-16-crown-4 (1) indicates high silver and thallium(I) ion selectivity over sodium, potassium, and rubidium ion evaluated from the solvent extraction of metal picrates, while its cation-binding ability is lower than those of dibenzo-18-crown-6 (2) and dibenzo-22-crown-6 (3). Taking account of the highest thallium(I) ion selectivity for 1 obtained from extraction experiments, PVC membrane thallium(I)-selective electrodes based on 1 are prepared. The electrode shows the best potentiometric selectivity coefficients for thallium(I) over potassium and rubidium than those of 2 and 3, and commercially available bis(crown ether)s (4).     

Structural, molecular mechanics, and DFT study of cadmium(II) in its crown ether complexes with axially coordinated ligands, and of the binding of thiocyanate to cadmium(II)

The role of relativistic effects (RE) in the structures of Cd(II) complexes with crown ethers, and the reason the ‘soft’ Cd(II) strongly prefers to bind to SCN⁻ through N, are considered. The synthesis and structures of [Cd(18-crown-6)(thiourea)₂] (ClO₄)₂.18-crown-6 (1) and [Cd(Cy₂-18-crown-6)(NCS)₂] (2) are reported. (18-crown-6 = 1,4,7,10,13,16-hexaoxacyclooctadecane; Cy₂-18-crown-6 = cis-anti-cis-2,5,8,15,18,21-hexaoxatricylo[20.4.0.0(9,14)]hexacosane). In 1 Cd is coordinated in the plane of the crown which has close to D_3d symmetry, with long Cd-O bonds averaging 2.688 Å. The two thiourea molecules form relatively short Cd-S bonds that average 2.468 Å, with an S-Cd-S angle of 164.30°. This structure conforms with the idea that Cd(II) can adopt a near-linear structure involving two covalently-bound donor atoms (the S-donors) with short Cd-S bonds, which resembles gas-phase structures for species such as CdCl₂. The structure of 2 is similar, with the two SCN⁻ ligands N-bonded to Cd, with short Cd-N bonds of 2.106 Å, and N-Cd-N angle of 180°. The crown in 2 forms long Cd-O bonds that average 2.698 Å. Molecular mechanics calculations suggest that a main reason Cd(II) prefers to bind to SCN⁻ through N is that when bound through S, the small Cd-S-C angle, which is typically close to 100°, brings the ligand into close contact with other ligands present, and causes steric destabilization. In contrast, the Cd-N-C angles for SCN⁻ coordinated through N are much larger, being 171.4° in 2, which keeps the SCN⁻ groups well clear of the crown ether. DFT (density functional theory) calculations are used to generate the structures of [Cd(18-crown-6)(H₂O)₂]²⁺ (3) and [Cd(18-crown-6)Cl₂] (4). In 3, the Cd(II) is bound to only three O-donors of the macrocycle, with Cd-O bonds averaging 2.465 Å. The coordinated waters form an O-Cd-O angle of 139.47°, with Cd-O bonds of 2.295 Å. In contrast, for 4, the Cd is placed centrally in the cavity of the D_3d symmetry crown, with long Cd-O bonds averaging 2.906 Å. The Cl groups form a Cl-Cd-Cl angle of 180°, with short Cd-Cl bonds of 2.412 Å. With ionically bound groups on the axial sites of[Cd(18-crown-6)X₂] complexes, such as with X = H₂O in 3, the Cd(II) does not adopt linear geometry involving the two X groups, with long Cd-O bonds to the O-donors of the macrocycle. With covalently-bound X = Cl in 4, short Cd-Cl bonds and a linear [Cl-Cd-Cl] unit results, with long Cd-O bonds to the crown ether.     

Coordination compounds of divalent lanthanides with crown ethers

New complexes LnI₂·18-crown-6 (Ln-Sm, Tm, Dy, Nd) and LnJ₂·dibenzo-18-crown-6 (Ln-Sm, Tm) were synthesized using the solutions of LnI₂ in THF. The compounds obtained oxidize quickly in air, but are relatively stable in an inert atmosphere. The Tm²⁺ complex is decomposed by light. The compounds obtained are poorly soluble in THF, the Sm²⁺ and Tm²⁺ compounds are soluble in CH₃CN, forming solutions with a period of half oxidation of 170 h and 6 min, respectively. Iodide ions of the complexes can be substituted for Cl^? during treatment of the compounds by solution of LiCl in THF. The reflection spectra of the compounds synthesized are similar to the absorption spectra of Ln²⁺ in THF, although a shift of bands towards the short wave region is observed.The study of the Ln²⁺ oxidation kinetics in H₂O, CH₃CN, THF in the presence of crown ethers has shown that their stability is influenced not only by the type of solvent, relative solubility and stability of complexes Ln²⁺ and Ln³⁺, but also by phenyl groups, and by decreasing stability of Dy²⁺ and Nd²⁺.     

Development of functional imidazole derivatives: the influence of alkaline metal cations on the rates of CL reactions of 2-(phenyl and 4-dimethylaminophenyl)-4-hydroperoxy-4-3',4'-(15-crown-5)phenyl-5-3'-4'-(15-crown-5)phenyl-4H-isoimidazoles.

Although several investigations have focused on luminescence modulation by chelation with metal cations using bidentate ligands or crown ether systems, a bis(crown ether) system has not yet been used for modulation of chemiluminescence (CL) reactions. In the CL reaction of 2-(phenyl and 4-dimethylaminophenyl)-4-hydroperoxy-4-3',4'-(15-crown-5)phenyl-5-3',4'(15-crown-5)phenyl-4H-isoimidazoles 2a and b possessing a bis(15-crown-5 ether) moiety, the rate acceleration was observed in the presence of K(+), Rb(+) and Cs(+) due to the holding effect of the bis-crown moiety, but no rate acceleration was observed by Li(+) and Na(+) due to the template effect of the crown moiety. The acceleration of the CL reaction rates is ascribable to the conformational change induced by the scissor-like motion of the bis-crown moiety assisted by the holding effect.     

Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids

(+)-18-crown-6 tetracarboxylic acid (18C6H(4)) has been used as a chiral selector for various amines and amino acids. To further clarify the structural scaffold of 18C6H(4) for chiral separation, single crystal X-ray analysis of its glycine(+) (1), H3O+ (2), H5O2+ (3), NH4+ (4), and 2CH3NH3+ (5) complexes was performed and the guest-dependent conformation of 18C6H(4) was investigated. The crown ether ring of 18C6H4 in 3, 4, and 5 took a symmetrical C2 or C2-like conformation, whereas that in 1 and 2 took an asymmetric C1 conformation, which is commonly observed in complexes with various optically active amino acids. The overall survey of the present and related complexes suggests that the molecular conformation of 18C6H4 is freely changeable within an allowable range, depending on the molecular shape and interaction mode with the cationic guest. On the basis of the present results, we propose the allowable conformational variation of 18C6H4 and a possible transition pathway from its primary conformation to the conformation suitable for chiral separation of racemic amines and amino acids.     

Experimental attempt to simulate receptor site environment. A 500-MHz 1H nuclear magnetic resonance study of enkephalin amides

The amides of Leu5-enkephalin, Met5-enkephalin, and three analogues, D-Ala2,Leu5-enkephalin, (AcO)Tyr1,Met5-enkephalin, and (AcO)Tyr1,D-Ala2,Met5-enkephalin, have been studied by means of 1H NMR spectroscopy in two different solvent systems: Me2SO-d6 and CDCl3. In the latter solvent the peptides were dissolved as complexes with 18-crown-6-ether, a coronand that binds strongly to the NH3+ groups. The crown ether complexation and the apolar solvent were used to simulate the anionic subsite of the receptor and the hydrophobic environment of the receptor cavity, respectively. The very unusual amide proton chemical shifts and their temperature coefficients suggest the presence of folded conformations in CDCl3 for all peptides, consistent with several models of opioid receptors and with the crystal structure of Leu5-enkephalin. The differences among the proposed cyclic conformations of the five peptides may be correlated, in part, with their different biological activity. All peptides in Me2SO-d6 are characterized by complex mixtures of extended fully solvated conformations.     

Why do crown ethers activate enzymes in organic solvents?

One of the major drawbacks of enzymes in nonaqueous solvents is that their activity is often dramatically low compared to that in water. This limitation can be largely overcome by crown ether treatment of enzymes. In this paper, we describe a number of carefully designed new experiments that have improved the insights into the mechanisms that are operative in the crown ether activation of enzymes in organic solvents. The enhancement of enzyme activity upon addition of 18-crown-6 to the organic solvent can be reconciled with a mechanism in which macrocyclic interactions of 18-crown-6 with the enzyme play an important role. Macrocyclic interactions (e.g., complexation with lysine ammonium groups of the enzyme) can lead to a reduced formation of inter- and intramolecular salt bridges and, consequently, to lowering of the kinetic conformational barriers, enabling the enzyme to refold into thermodynamically stable, catalytically (more) active conformations. This assumption is supported by the observation that the crown-ether-enhanced enzyme activity is retained after removal of the crown by washing with a dry organic solvent. A much stronger crown ether activation is observed when 18-crown-6 is added prior to lyophilization, and this can be explained by a combination of two effects: the before-mentioned macrocyclic complexation effect, and a less specific, nonmacrocyclic, lyoprotecting effect. The magnitude of the total crown ether effect depends on the polarity and thermodynamic water activity of the solvent, the activation being highest in dry and apolar media, where kinetic conformational barriers are highest. By determination of the specific activity of crown-ether-lyophilized enzyme as a function of the enzyme concentration, the macrocyclic crown ether (linearly dependent on the enzyme concentration) and the nonmacrocyclic lyoprotection effect (not dependent on the enzyme concentration) could be separated. These measurements reveal that the contribution of the nonmacrocyclic effect is significantly larger than the macrocyclic refolding effect.     

On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.     

Studies on the mechanism of crown-ether-induced activation of enzymes in non-aqueous media.

Studies on the mechanism of crown-ether-induced activation are described in this paper. Michaelis Menten kinetics of -chymotrypsin in toluene in the presence and absence of 18-crown-6 showed that only V_max is increased upon crown ether treatment. Parallel Lineweaver–Burk plots indicate that crown ethers do not activate the enzyme by specific interactions in the active site, such as transition state stabilization or facilitated transport of water molecules. Increased V_max values of crown-ether-treated enzyme most probably originate from conformational changes, which alter k_cat as well as the amount of catalytically active enzyme.     

Can an inactivating agent increase enzyme activity in organic solvent? Effects of 18‐crown‐6 on lipase activity,enantioselectivity, and conformation

Lipase from Burkholderia cepacia (lipase BC) and lipase B from Candida antarctica (CALB) show an increase of the transesterification activity in toluene (up to 2.4- and 1.7-fold, respectively), when lyophilized with 18-crown-6. Nevertheless, the increase was observed only for low (less than 100) 18-crown-6/lipase molar ratio, while at higher ratios, the activity decreased for both enzymes to values lower than those obtained in the absence of the additive. In 1,4-dioxane, the activation is lower for lipase BC (1.7-fold) and for CALB (1.5-fold). Concerning enantioselectivity, tested in the kinetic resolution of 6-methyl-5-hepten-2-ol, only in the case of CALB, an effect of the additive (the E value varied from about 120 to 280) was observed. In water, 4% (w/w) of 18-crown-6 caused a loss of activity in the hydrolysis of p-nitrophenyl laurate of about 88 and 99.75%, compared to that observed in the absence of the crown ether for CALB and lipase BC, respectively. These data and the conformational analysis of both lipases, carried out by FT/IR spectroscopy indicate that the enzyme inactivation in water and in organic solvents at 18-crown-6/lipase molar ratios, higher than 100 might be due to conformational changes caused by the additive. Instead, at molar ratios lower than 100, 18-crown-6 might increase the activity - particularly, in toluene - thanks to the fact that in its presence, the enzyme has an hydrogen bonds pattern, more similar to that in water. This suggests that the additive would be able to provide the enzyme with more water.     

Synthesis and structural characterization of two monomeric potassium phenolates

Reaction of 2,6-dibenzylphenol and 2,6-bis-(2-methoxybenzyl)phenol with potassium hexamethyldisilazide in the presence of 18-crown-6 yielded [K(18-crown-6)(Odbp)] and [K(18-crown-6)(OdbpOMe)], respectively. Both compounds were mononuclear with seven-coordinate potassium and displayed C-H?O and C-H?π intermolecular hydrogen bonding.     

Nonaqueous synthesis of a selectively modified, highly anionic sulfopropyl ether derivative of cyclomaltoheptaose (beta-cyclodextrin) in the presence of 18-crown-6

A highly anionic cyclomaltooligosaccharide (cyclodextrin, CD) derivative containing sulfopropyl functional groups on the primary face of the CD was synthesized. Heptakis(2,3-di-O-methyl)cyclomaltoheptaose [heptakis(2,3-di-O-methyl)-beta-cyclodextrin] was reacted with 1,3-propane sultone and potassium hydride (KH) in anhydrous tetrahydrofuran in the presence of 18-crown-6 to yield highly substituted potassium heptakis(2,3-di-O-methyl-6-O-sulfopropyl)cyclomaltoheptaose [heptakis(KSPDM)-beta-CD] with an average degree of substitution (DSCE) of 6.9 as determined by inverse detection capillary electrophoresis (CE). The principal species in the product is the fully substituted heptakis(KSPDM)-beta-CD. Complete NMR assignments of the hydrogen and carbon atoms are made using a combination of gCOSY and gHSQC. In the absence of 18-crown-6, the reaction generates a mixture of multiply charged derivatives with average DSCE of 4.1. The possible roles of the crown ether in the reaction are discussed. The ROESY NMR spectrum of the inclusion complex that forms between heptakis(KSPDM)-beta-CD and 2-naphthoic acid in D2O reveals that 2-naphthoic acid inserts with the carboxyl group toward the derivatized primary rim of the cyclodextrin.     

Supramolecular photochemical synthesis of an unsymmetrical cyclobutane.

2-styrylbenzothiazole (1) and cinnamic acid (2) derivatives containing 15-crown-5 ether moieties form a supramolecular assembly in the presence of Ba(2+) cations in acetonitrile. The assembly is stabilized by hydrogen bonding between the heterocyclic N atom of 1 and the proton of the carboxylic group of 2, by sandwich Ba(2+) complex formation between the crown ether moieties of 1 and 2, and by pi-pi stacking interactions. Irradiation of solutions containing these supramolecular complexes leads to highly specific formation of an unsymmetrical cycloadduct. This investigation provides an interesting example of supramolecular control of [2 + 2]-photocyclization in solution.