NMR investigation of the interaction of (+)- and (−)-flurbiprofen with β-cyclodextrin

The sodium salts of (+)-(S)- and (−)-(R)-2-(2-fluoro-4-biphenylyl)propionic acid (flurbiprofen, FBP) form 1:1 inclusion complexes with β-cyclodextrin (β-CD) having different association constants. Proton selective relaxation rate measurements revealed the existence of superior aggregated forms for both complexes (+)-FBP/β-CD and (−)-FBP/β-CD; information about their stereochemistry has been obtained by 2D ROESY analysis. © 1996 Wiley-Liss, Inc.     

Quantification of trace levels of β-cyclodextrin and substitution patterns in hydroxypropyl-β-cyclodextrin using high-performance liquid chromatography with polarimetric detection

A high-performance liquid chromatographic (HPLC) method has been developed for separation and determination of components in hydroxypropyl-β-cyclodextrin (HP-β-CD). The method involves separation on an amino-bonded HPLC column using water–acetonitrile as a mobile phase with a polarimetric HPLC detector for quantification. It provides good selectivity and sensitivity and can also be used to compare different sources of HP-β-CD and to measure batch to batch variation. The similarity of the values of molar optical rotation for β-cyclodextrin (β-CD) and HP-β-CD suggests that a polarimetric HPLC detector may be used with a straightforward area normalization method, to quantify the proportion of β-CD in any HP-β-CD sample. Trace amounts of β-CD in HP-β-CD have been measured to a precision of 0.01%. © 1993 Wiley-Liss, Inc.     

Synthesis of (+)-(S)-ibuproxam and preparation of some new complexes of racemic and (+)-(S)-ibuproxam with β-cyclodextrin and its derivatives

(+)-(S)-Ibuproxam, a prodrug of (+)-(S)-ibuprofen, the pharmacologically active component of ibuprofen, was synthesized in order to minimize side effects (especially gastric irritation) and reduce effective dose. The low water solubility of (+)-(S)-ibuproxam, which prevents rapid dissolution and absorption from the gastrointestinal tract, was overcome by complexation with β-cyclodextrin and its derivatives. The inclusion complex formation was confirmed by differential scanning calorimetry (DSC), by ¹H-NMR spectroscopy, and X-ray powder diffractometry. The physicochemical characteristics of ibuproxam were significantly improved by the complexation. © 1995 Wiley-Liss, Inc.     

Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Molecular modelling of the inclusion complexes between β-cyclodextrin and (R)/(S)-methylphenobarbitone and its application to HPLC

Molecular modelling of β-cyclodextrin and optimisation of its potential energy suggests that a favoured conformation is that distorted from a symmetrical torus. The inclusion of water molecules into the torus cavity simulates the increased stability in an aqueous solvent. Complexes of β-cyclodextrin with (R)- and (S)-enantiomers of methylphenobarbitone have been modelled and energetically optimised by the application of molecular mechanics. The simulations suggests that the guest molecules adopt an orientation in which the phenyl ring is projected into the torus cavity, with in each case the plane of the ring parallel to a longer axis of the distorted torus and slightly displaced from the axis through the torus cavity. It is suggested that the asymmetry in the macrocyclic ring contributes to chiral recognition as a result of additional discriminatory binding to the barbiturate ring residue of each enantiomer, which occupy different 3D geometries. The enantiomers form complexes of different minimum potential energies. The resulting difference in complex stability can be related to the behaviour of β-cyclodextrin, as a mobile phase additive in reverse-phase HPLC to effect chiral separation of rac-medthylphenobarbitone during chromatography. © 1994 Wiley-Liss, Inc.     

Separation of R- and S-Thalidomide by Reversed-Phase HPLC with β-cyclodextrin in the mobile phase

R- and S-Thalidomide were resolved by reversed-phase HPLC on a C-18 column with β-cyclodextrin in the mobile phase. As the concentration of β-cyclodextrin was increased stepwise from 0 to 20 mM, enantiomeric resolution increased and retention times decreased significantly. The influence of different organic modifiers in the mobile phase were evaluated, and ethanol, among others, proved to be effective. Equilibrium constants for the formation of β-cyclodextrin inclusion complexes of R- and S-thalidomide in EtOH-buffer (5:95) were calculated to be 64 and 76 M⁻¹, respectively. © 1996 Wiley-Liss, Inc.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-β-cyclodextrin

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy was synthesized by C₆-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.     

Detailed experimental and theoretical analysis of chiral discrimination: Enantioselective binding of R/S methyl mandelate by β-cyclodextrin

Enantiodifferentiation of methyl mandelate by β-Cyclodextrin in the liquid phase is explored in detail. Temperature dependent studies using a β-cyclodextrin chiral stationary phase provided differential binding enthalpies and entropies. NMR studies revealed where around the host molecule the guest tends to reside. Molecular dynamics simulations correctly predict the retention order and provide an atomistic account of how chiral discrimination takes place. It is found that short range dispersion forces rather than long range coulombic forces are responsible for both complexation and for enantiodiscrimination. The intermolecular hydrogen bonds are not discriminating and the idea that a tight fit of included species within a cyclodextrin cavity be a requirement for chiral discrimination is questioned. © 1996 Wiley-Liss, Inc.     

Enantiomeric separation of tetrahydroisoquinoline alkaloids by high-performance liquid chromatography with β-cyclodextrin as chiral selector

Tetrahydroisoquinoline alkaloids, which are known to be present not only in plants but also in animals, including mammals, can be considered as condensation products of 2-phenylethylamines (e.g., catecholamines) with aldehydes (e.g., acetaldehyde) or 2-oxo acids (e.g., pyruvic acid). In this study the possibility of separating the optical isomers of several tetrahydroisoquinolines by high-performance liquid chromatography was investigated. For isosalsoline, tetrahydropapaveroline and laudanosoline a good enantiomeric separation could be achieved by applying β-cyclodextrin-bonded silica as stationary phase in connection with various mobile phases. With respect to laudanosoline, the addition of β-cyclodextrin as chiral selector to the mobile phase using a C₁₈ reversed-phase column as stationary phase revealed an even higher resolution when compared with the chiral columns. All tested tetrahydroisoquinolines which could be well separated into enantiomers bear a hydroxyl group at carbon atom 7 as a common structural feature. Those alkaloids substituted with a methoxy group on position 7 instead of a hydroxyl group (e.g., salsolidine) failed to be resolved into their optical isomers. Therefore, the presence of a hydroxyl group on C7 of the aromatic ring seems to be conducive to steric discrimination. However, the separation results for 1-carboxysalsolinol were unsatisfactory although this molecule possesses a 7-hydroxyl group. In this case the existence of a carboxyl group on C1 reduced the chiral recognition and thus the enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Separating enantiomers by HPLC on silica dynamically coated with a chiral stationary phase of permethylated β-cyclodextrin

The paper demonstrates that the technique of solvent generated liquid--solid chromatography can be used to create normal phase systems for chiral separations. The chiral adsorption layer was generated by pumping a binary hexane:ethanol eluent containing a small fraction of permethylated β-cyclodextrin through a column packed with microparticulate silica. This technique leads to columns with good time stability and reproducibility. The possibility of generating normal phase systems with permethylated β-cyclodextrin as chiral component via the mobile phase broadens the range of phase system which can be used to separate enantiomers by HPLC.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Stereoisomeric flavour compounds LXVIII. 2-, 3-, and 4-Alkyl-branched acids,part 2: Chirospecific analysis and sensory evaluation

Enantioselective GC analysis of 4-ethyloctanoic and 4-methylheptanoic acid, using heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin as the chiral stationary phase, is described and the sensory properties of several 4-alkyl-branched acids, using gas chromatography-olfactometry (GC-O) equipment and octakis(2,3-di-O-methyl-6-tert-butyldimethylsilyl)-γ-cyclodextrin as the stationary phase, are evaluated. The chirospecific analysis of various 2-, 3-, and 4-alkyl-branched acids from commercially available Roman chamomile (Chamaemelum nobile (L.) Allioni), Parmesan cheese, and subcutaneous mutton adipose tissue, using either GC-GC (MDGC) or GC-MS analytical methods, is described. © 1994 Wiley-Liss, Inc.     

Comparison of capillary electrophoresis and liquid chromatography for the enantiomeric separation of α-phosphonosulfonic acids

The enantiomers of α-phosphonosulfonic acids were completely resolved by capillary electrophoresis using β-cyclodextrin as a chiral selector in a borate electrolyte and HPLC using a chiral AGP column. The methods were used to quantitate the R-enantiomer present as an impurity in the S-enantiomer, a potential cardiovascular drug candiate. Chirality 9:104–108, 1997. © 1997 Wiley-Liss, Inc.     

Conformational changes due to vicinal glycosylation: The branched α-l-Rhap(1–2)[β-d-Galp(1–3)]-β-d-Glc1-OMe trisaccharide compared with its parent disaccharides*

Conformations of the α-l -Rhap(1-2)-β-d -Glc1-OMe and β-d -Galp(1-3)-β-d -Glc1-OMe disaccharides and the branched title trisaccharide were examined in DMSO-d₆ solution by ¹H-nmr. The distance mapping procedure was based on rotating frame nuclear Overhauser effect (NOE) constraints involving C- and O-linked protons, and hydrogen-bond constraints manifested by the splitting of the OH nmr signals for partially deuteriated samples. An “isotopomer-selected NOE” method for the unequivocal identification of mutually hydrogen-bonded hydroxyl groups was suggested. The length of hydrogen bonds thus detected is considered the only one motionally nonaveraged nmr-derived constraint. Molecular mechanics and molecular dynamics methods were used to model the conformational properties of the studied oligosaccharides. Complex conformational search, relying on a regular Φ,Ψ-grid based scanning of the conformational space of the selected glycosidic linkage, combined with simultaneous modeling of different allowed orientations of the pendant groups and the third, neighboring sugar residue, has been carried out. Energy minimizations were performed for each member of the Φ,Ψ grid generated set of conformations. Conformational clustering has been done to group the minimized conformations into families with similar values of glycosidic torsion angles. Several stable syn and anti conformations were found for the 1→2 and 1→3 bonds in the studied disaccharides. Vicinal glycosylation affected strongly the occupancy of conformational states in both branches of the title trisaccharide. The preferred conformational family of the trisaccharide (with average Φ,Ψ values of 38°, 17° for the 1→2 and 48°, 1° for the 1→3 bond, respectively) was shown by nmr to be stabilized by intramolecular hydrogen bonding between the nonbonded Rha and Gal residues. © 1998 John Wiley & Sons, Inc. Biopoly 46: 417–432, 1998     

Cytotoxic (9βH)‐Pimarane and (9βH)‐17‐Norpimarane Diterpenes from the Tuber of Icacina trichantha

Three (9βH)‐pimaranes, 1, 2 , and 3 , and two (9βH)‐17‐norpimaranes, 4 and 5 , belonging to a rare compound class in nature, were obtained from the tubers of Icacina trichantha for the first time. Compound 1 is a new natural product, and 2 – 5 have been previously reported. The structures were elucidated based on NMR and MS data, and optical rotation values. The absolute configurations of (9βH)‐pimaranes were unambiguously established based on X‐ray crystallographic analysis. Full NMR signal assignments for the known compounds 2, 4 , and 5 , which were not available in previous publications, are also reported. All five isolates displayed cytotoxic activities on MDA‐MB‐435 cells (IC₅₀ 0.66–6.44 μM ), while 2, 3 , and 4 also exhibited cytotoxicities on HT‐29 cells (IC₅₀ 3.00–4.94 μM ).     

Ligand‐regulated oligomerization of β2‐adrenoceptors in a model lipid bilayer

The β₂‐adrenoceptor (β₂AR) was one of the first Family A G protein‐coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified β₂AR site‐specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the β₂AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into β₂AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.     

Strategy combining chiral chromatography with β-cyclodextrin and stereospecific enzyme reaction detection with hydroxysteroid dehydrogenases for resolving steroid epimers

Steroids are chiral molecules with multiple stereogenic centers. Studies of their intermediary metabolism often require analytical techniques to separate the isomers and determine their stereochemistry. Methods for resolving steroid stereoisomers by HPLC using β-cyclodextrin in the mobile phase are reported. Even with the improved selectivity of cyclodextrin chromatography, not all isomers within a steroid series can be resolved. Additional specificity is achieved by reaction detection using postcolumn reactors containing hydroxysteroid dehydrogenases stereospecific for the configuration of the hydroxy functions of steroids. The enzymes catalyze the oxidation of hydroxysteroids and reduction of the coenzyme NAD to NADH. NADH, which is highly fluorescent, is detected at the nanogram levels. Isomers not separated by chromatography were effectively resolved by reaction detection with stereospecific enzymes. © 1992 Wiley-Liss, Inc.