A CD exciton chirality method for determination of the absolute configuration of threo-beta-aryl-beta-hydroxy-alpha-amino acid derivatives

The absolute configuration of threo-beta-aryl-beta-hydroxy-alpha-amino acids was studied by CD exciton chirality method using 7-diethylaminocoumarin-3-carboxylate as a red-shifted chromophore. Bischromophoric derivatives for a series of threo-beta-aryl-beta-hydroxy-alpha-amino acids (3a-h) were prepared and their CD spectra measured in CH2Cl2. By combining the data of CD and NMR coupling constants, we are able to correlate their preferred conformer (B) and the positive CD to the corresponding (2S,3R)-absolute configuration. These results are consistent with those obtained from serine and threonine derivatives, which represent the simplest form of beta-hydroxy-alpha-amino acids. This CD method could thus become a general method for determining the absolute configuration of threo-beta-aryl-beta-hydroxy-alpha-amino acids.     

Novel nanoparticles composed of chitosan and β-cyclodextrin derivatives as potential insoluble drug carrier

This research was aim to develop novel cyclodextrin/chitosan(CD/CS) nanocarriers for insoluble drug delivery through the mild ionic gelation method previously developed by our lab. A series of different bcyclodextrin(β-CD) derivatives were incorporated into CS nanoparticles including hydroxypropyl-bcyclodextrin(HP-β-CD), sulphobutylether-β-cyclodextrin(SB-β-CD), and 2,6-di-O-methy-β-cyclodextrin(DM-β-CD). Various process parameters for nanoparticle preparation and their effects on physicochemical properties of CD/CS nanoparticles were investigated, such as the type of CD derivatives,CD and CS concentrations, the mass ratio of CS to TPP(CS/TPP), and p H values. In the optimal condition,CD/CS nanoparticles were obtained in the size range of 215–276 nm and with the zeta potential from30.22 m V to 35.79 m V. Moreover, the stability study showed that the incorporation of CD rendered the CD/CS nanocarriers more stable than CS nanoparticles in PBS buffer at p H 6.8. For their easy preparation and adjustable parameters in nanoparticle formation as well as the diversified hydrophobic core of CD derivatives, the novel CD/CS nanoparticles developed herein might represent an interesting and versatile drug delivery platform for a variety of poorly water-soluble drugs with different physicochemical properties.     

Tuning the cavity of cyclodextrins: altered sugar adaptors in protein pores

Cyclodextrins (CDs) have been widely used in host-guest molecular recognition because of their chiral and hydrophobic cavities. For example, β-cyclodextrin (βCD) lodged as a molecular adaptor in protein pores such as α-hemolysin (αHL) is used for stochastic sensing. Here, we have tuned the cavity and overall size of βCD by replacing a single oxygen atom in its ring skeleton by a disulfide unit in two different configurations to both expand our ability to detect analytes and understand the interactions of βCD with protein pores. The three-dimensional structures of the two stereoisomeric CDs have been determined by the combined application of NMR spectroscopy and molecular simulation and show distorted conformations as compared to natural βCD. The interactions of these synthetic βCD analogues with mutant αHL protein pores and guest molecules were studied by single-channel electrical recording. The dissociation rate constants for both disulfide CDs from the mutant pores show ～1000-fold increase as compared to those of unaltered βCD, but are ～10-fold lower than the dissociation rate constants for βCD from wild-type αHL. Both of the skeleton-modified CDs show altered selectivity toward guest molecules. Our approach expands the breadth in sensitivity and diversity of sensing with protein pores and suggests structural parameters useful for CD design, particularly in the creation of asymmetric cavities.     

Native and polymeric β-cyclodextrins in performance improvement of chitosan films aimed for buccal delivery of poorly soluble drugs

β-Cyclodextrin (βCD) and its soluble polymeric derivative (EPIβCD) were used to improve the effectiveness of chitosan-based bucco-adhesive film formulations containing bupivacaine hydrochloride and triclosan as poorly-soluble model drugs. The film formulations were characterized in terms of swelling, mucoadhesion and in vitro drug release, while possible interactions between the components were investigated by DSC and FTIR analyses. For both drugs EPIβCD showed a higher solubilizing efficiency than βCD; however cyclodextrin effectiveness in improving the release rate from film formulations was influenced by their different interactions with chitosan. Free βCD acted as a channelling agent, favouring the film swelling, while EPIβCD due to interaction with chitosan caused an opposite effect. βCD was the optimal partner for bupivacaine-loaded films in terms of film swelling, mucoadhesion and drug release. Contrariwise, EPIβCD was the best partner for triclosan-loaded films, allowing the highest drug release rate increase, due to its higher solubilizing ability with respect to βCD. Addition of the suitable cyclodextrin enabled formulation of buccal films with suitable drug release properties.     

Circular Dichroism of Amino Acids: Following the Structural Formation of Phenylalanine

Circular dichroism (CD) is frequently used to assess the secondary structure of peptides and proteins, whereas less attention has been given to their building blocks, that is, single amino acids, as they do not possess a secondary structure. Here, we follow the CD signal of amino acids and reveal that several acids exhibit a unique CD pattern as a function of their concentration. Accordingly, we propose an eight‐level classification of the CD signal of the various amino acids. Special focus is given to the CD pattern of phenylalanine (Phe), for which we observe the formation of an ultra‐narrow CD peak (full width at high maximum of only 5 nm). This CD peak can be attributed to the formation of Phe‐based chiral structural features. Further support for the formation of an ordered structure is given by using NMR, and the additional self‐assembly process of Phe to tubular structures.     

Circular dichroism spectra of DNA hairpins studied by the green function method

The circular dichroism (CD) spectrum of a large biological molecule represented as an array of interacting chromophores is investigated. The configuration-averaged Green function formalism is developed to describe the CD and absorption spectra. The perturbation theory expansion is derived for absorption and CD spectra in the case of strong interaction of chromophores with their environment (solvent and/or internal dynamics) compared to their interaction with each other. We apply this formalism to study CD spectra of DNA hairpins.     

Effect of β- and γ-cyclodextrins and their methylated derivatives on the degradation rate of benzylpenicillin

Benzylpenicillin is characterized by instability in aqueous media, low oral bioavailability, and short biological half-life. Since their degradation products can cause allergic reactions, β-lactam antibiotics are supplied as a powder for injection and require special precaution when stored and re-constituted in water. An efficient method for β-lactam stabilization in the aqueous environment could reduce allergic side effects and facilitate their handling. Previously we proposed that complexation with cyclodextrins (CDs) is a way to achieve this goal. The purpose of the present study was to investigate the effect of methylation of CD on the chemical stability of benzylpenicillin. Kinetic studies revealed that degree of methylation of the CD molecule determines whether the CD has destabilizing or stabilizing effect on the β-lactam. The fully methylated βCD derivative Trimeb stabilizes benzylpenicillin while partial methylation of βCD only decreases to some extent the catalytic effect of native βCD. The complexes of all investigated CDs were also studied by DSC, FT-IR and NMR spectroscopy.     

Large‐Ring Cyclodextrins as Chiral Selectors for Enantiomeric Pharmaceuticals

Large‐ring cyclodextrins (CD) are cyclic glucans composed of 9 or more α‐1,4‐linked glucose units. They are minor side products of bacterial glucanotransferases (CGTases, EC 2.4.1.19) and have previously been available only in very small amounts for studies of their properties in supramolecular complex formation reactions. We engineered a CGTase to synthesize mainly large‐ring CD facilitating their preparation in larger amounts. By reversed phase chromatography, we obtained single CD samples composed of 10 to 12 glucose units (CD10, CD11, and CD12) with a purity of >90 %. Their identity was confirmed by high resolution mass spectrometry and fragmentation analysis. We demonstrated the non‐toxicity of CD10–CD12 for human cell lines by a cell proliferation assay and impedimetric monitoring. We then showed that CD10 and CD11 are efficient chiral selectors for the capillary electrophoretic separation of the enantiomeric pharmaceuticals fluvastatin, mefloquine, carvedilol, and primaquine.     

Solubilized, spaced polyalanines: a context-free system for determining amino acid alpha-helix propensities

The logical design principles behind a system of properly water-solubilized, spaced polyalanines are presented. Peptides conforming to these design principles are shown to be unaggregated, and their helical properties as measured by the circular dichroism (CD) residue ellipticity at 222 nm, [theta](222), are shown to be dependent upon the lengths of their alanine regions. It is further demonstrated that CD contributions of the alanine cores are independent of the CD contributions attributable to other features of the peptides. The CD response of these polyalanines to variations in temperature and salt or denaturant concentration is described. CD data for a series of peptides with Ala(n) cores varying in length from 12 to 45 residues are presented that allow calculation of the helical propensity, w(Ala), in a purely alanine context. Mathematical modeling of these unprecedented data reveals the insufficiency of currently accepted literature helicity modeling parameters. A modification to the standard Lifson-Roig algorithm is introduced based on hydrogen-bonding cooperativity.     

Comparing cyclodextrin derivatives as chiral selectors for enantiomeric separation in capillary electrophoresis

A total of 26 different cyclodextrin (CD) derivatives with different functional groups and degrees of substitution were tested against 35 basic pharmaceutical compounds in an effort to investigate their effectiveness as chiral selectors for enantiomeric separation in capillary electrophoresis (CE). Testing was performed under the same conditions using a low pH buffer (25 mM phosphate buffer at pH approximately 2.5). Five CD derivatives, namely, highly sulfated-beta-CD, highly sulfated-beta-CD, hydroxypropyl-beta-CD (degree of substitution approximately 1), heptakis-(2,6-O-dimethyl)-beta-CD, and heptakis(2,3,6-O-trimethyl)-beta-CD were identified to be most effective for enantiomeric separations and have a wide range of enantiomeric selectivity towards the model compounds. Over 90% of the model compounds were enantiomerically resolved with the five identified CD derivatives, at a minimum resolution of 0.5. An additional 20 compounds were also tested to demonstrate the validity of the identified CD derivatives. The five CD derivatives were recommended as the starting chiral selectors in developing enantiomeric separation methods by CE.     

Inclusion complexes of <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoic acid esters and γ-cyclodextrin

The alkyl esters of p-hydroxybenzoic acid (parabens) are commonly used as preservatives in cosmetics, food products and pharmaceutical formulations because of their wide range antimicrobial activity. However, the usage of parabens in aqueous media has been hampered, especially parabens with long alkyl chains, due to their low aqueous solubility. One approach to increase their solubility is cyclodextrins (CDs) complexation to form water-soluble inclusion complexes. γCD has the widest hydrophobic central cavity and the highest water solubility among natural CDs. Hence, inclusion complexes between γCD and parabens of various alkyl chain lengths were investigated. Results from phase-solubility studies show that methyl- and ethylparaben form various complexes of paraben/γCD (i.e. 1:1, 2:1, etc.) while the 1:1 complex was dominant in propyl- and butylparaben/γCD complex solution. Moreover, the effect of the paraben complexation on the critical aggregation concentration (cac) of γCD in aqueous solutions was determined. It was found that the longer the paraben alkyl chain was the more influence it had on the γCD cac. In pharmaceutical formulations the mixture of parabens (i.e. binary, ternary and quaternary) has been used to maximize antimicrobial effect. It is important to determine how mixtures of parabens affect the solubility of γCD and its cac values upon formation of inclusion complexes. Competition of the different parabens for a space in the γCD central cavity was evaluated by comparing the γCD cac values obtained in presence of the individual parabens and their mixtures.     

Determination of stability constants of tauro- and glyco-conjugated bile salts with the negatively charged sulfobutylether-β-cyclodextrin: comparison of affinity capillary electrophoresis and isothermal titration calorimetry and thermodynamic analysis of the interaction

The aim of the present work was to investigate the interaction between bile salts present in the intestine of man, dog and rat with the negatively charged cyclodextrin (CD), sulfobutylether-β-cyclodextrin (SBEβCD). The interactions between bile salts and CDs are of importance for the release of CD-complexed drugs upon oral administration. This makes a good understanding of this particular interaction important for rational drug formulation. SBEβCD is a modified CD, which has attracted particular interest in formulation science. It is unique in the sense that it carries approximately seven negatively charged side chains, which can potentially interact electrostatically with the guest molecule. Bile salts are negatively charged at physiological pH, and the concomitant repulsion from SBEβCD could potentially reduce their affinity for this CD and hence their ability to expel drugs delivered as SBEβCD complexes. However, this study has demonstrated that the interaction, between a bile salts and SBEβCD is only slightly weaker than the corresponding interactions with natural βCD. Significant differences between the thermodynamics of bile salt complexes with respectively HPβCD and SBEβCD were found, when comparing the same degree of substitution. This underscores the importance of the substituents on the interactions of modified CDs with bile salts.     

Can one derive the conformational preference of a beta-peptide from its CD spectrum?

CD spectroscopy is often used to elucidate the secondary structure of peptides built from non-natural amino acids such as beta-amino acids. The interpretation of such CD spectra is not always unambiguous. Here, we present a case where two beta-hexapeptides, a dimethyl-beta-hexapeptide indicated as DM-BHP (A) and its nonmethylated analogue indicated as BHP (B), exhibit similar CD spectra, whereas they are expected to differ in secondary structure. The structural properties of both peptides were studied by molecular dynamics simulation, and from the resulting trajectories, the corresponding CD spectra were calculated. Starting from a fully extended conformation, BHP is observed to form a 3(14)-helix, while DM-BHP remains unfolded. However, even though these two peptides hardly share any conformations, their calculated CD spectra are alike and show the same features as the experimentally measured ones. Our results imply that a particular CD pattern can be induced by spatially different structures, which makes it difficult to derive the conformational preference of a peptide from its CD spectrum alone. To gain more insight into the relationship between the preferred conformation of a peptide and its CD spectrum, more accurate methods to calculate the CD spectrum for a given conformation are required.     

Solid-phase synthesis of CD40L mimetics

The C3-symmetric molecule has been previously shown to mimic CD40 ligand (CD40L) homotrimers and to display effector functions. This molecule consists of a cyclic hexapeptide core containing the repetition of the D-Ala-L-Lys motif. The side chains of the lysine residues have been modified by appending the CD40L-derived sequence 143Lys-Gly-Tyr-Tyr146 via a 6-aminohexanoic acid residue as a spacer. The present report describes a general solid-phase synthesis approach to and related trimeric architectures. In addition, their CD40 binding properties as well as their effector functions have been evaluated.     

Simultaneous chiral discrimination of multiple profens by cyclodextrin-modified capillary electrophoresis in normal and reversed polarity modes

Simultaneous enantioseparations of nine profens for their accurate chiral discrimination were achieved by capillary electrophoresis (CE) in the normal polarity (NP) mode with a single cyclodextrin (CD) system and in the reversed polarity (RP) mode with a dual CD system. The single CD system in the NP mode employed heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) added at 75 mM-100 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) as the optimum run buffer. The dual CD system operated in the RP mode used 30 mM TMbetaCD and 1.0% anionic carboxymethyl-beta-cyclodextrin dissolved in pH 3.0, 100 mM phosphoric acid-triethanolamine buffer containing 0.01% hexadimethrine bromide added to reverse the electroosmotic flow. Fairly good enantiomeric resolutions and the opposite enantiomer migration orders were achieved in the two modes. Relative migration times to internal standard under respective optimum conditions were characteristic of each enantiomer with good precision (< 2% relative standard deviation, RSD), thereby enabling to crosscheck the chemical identification of profens and also their accurate chiralities. The method linearity in the two modes was found to be adequate (r > or = 0.9991) for the chiral assay of the profens investigated. Simultaneous enantiomeric purity test of ibuprofen, ketoprofen and flurbiprofen in a mixture was feasible in a single analysis by the present method.     

UVA and UVB decrease the expression of CD44 and hyaluronate in mouse epidermis, which is counteracted by topical retinoids

The transmembrane glycoprotein CD44 is currently thought to be the main cell surface receptor for the glycosaminoglycan hyaluronate. We previously showed that (1) CD44 regulate keratinocyte proliferation; (2) topical retinoids dramatically increase the expression of CD44, hyaluronate and hyaluronate synthase (HAS)s in mouse epidermis; (3) topical retinaldehyde restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus lesions; and (4) retinaldehyde-induced proliferative response of keratinocytes is a CD44-dependent phenomenon and requires the presence of HB-EGF, erbB1 and matrix metalloproteinases. In this study, we analyzed the effect of UV irradiation on the levels of epidermal hyaluronate and CD44 in mice, as well as its potential prevention by topical retinoids. UVA (10 J/cm(2)) or UVB (1 J/cm(2)) irradiation significantly decreased the expression of CD44 and hyaluronate in the epidermis of hairless mice after 2 h. Expression of both epidermal CD44 and hyaluronate was reconstituted within 24 h. Topical application of retinaldehyde for 3 days prior to UVA or UVB irradiation prevented the decrease of CD44 and hyaluronate expression. Topical retinol and retinoic acid also increased the basal levels of epidermal CD44 and hyaluronate, although their preventive effect on UV-induced decrease of these molecules was less pronounced as compared to topical retinaldehyde. These data confirm the relationships between retinoid and CD44 pathways, although the primary target(s) of UV leading to CD44 and hyaluronate degradation remain to be elucidated.     

Capillary electrophoretic separation and computational modeling of inclusion complexes of β‐cyclodextrin and 18‐crown‐6 ether with primaquine and quinocide

The antimalarial drug primaquine (PQ) and its contaminant, the positional isomer quinocide (QC) have been successfully separated using capillary electrophoresis with either β‐cyclodextrin (β‐CD) or 18‐crown‐6 ether (18C6) as chiral mobile phase additive. The interactions of the drugs with cyclodextrins and 18C6 were studied by the semiempirical method (Parametric Model 3) PM3. Theoretical calculations for the inclusion complexes of PQ and QC with α‐CD, β‐CD and 18C6 were performed. Data from the theoretical calculations are correlated and discussed with respect to the electrophoretic migration behavior. More stable complexes are predicted for the PQ–β‐CD and PQ–18C6 complexes. The coelution of PQ and QC when α‐CD was used as buffer additive can be explained by their comparable stabilities of the inclusion complex formed, while significant differences in the complexation stabilities of the drugs with β‐CD is responsible for their separation. The stronger hydrogen bonding in PQ–18C6 system is responsible for the separation between PQ and QC when 18C6 was used as chiral mobile phase additive. Copyright © 2009 John Wiley & Sons, Ltd.     

Aptamer-containing surfaces for selective capture of CD4 expressing cells

Aptamers have recently emerged as an excellent alternative to antibodies because of their inherent stability and ease of modification. In this paper, we describe the development of an aptamer-based surface for capture of cells expressing CD4 antigen. The glass or silicon surfaces were modified with amine-terminated silanes and then modified with thiolated RNA aptamer against CD4. Modification of the surface was first characterized by ellipsometry to demonstrate assembly of biointerface components and to show specific capture of recombinant CD4 protein. Subsequently, surfaces were challenged with model lymphocytes (cell lines) that were either positive or negative for CD4 antigen. Our experiments show that aptamer-functionalized surfaces have similar capture efficiency to substrates containing anti-CD4 antibody. To mimick capture of specific T-cells from a complex cell mixture, aptamer-modified surfaces were exposed to binary mixtures containing Molt-3 cells (CD4+) spiked into Daudi B cells (CD4-). 94% purity of CD4 cells was observed on aptamer-containing surfaces from an initial fraction of 15% of CD4. Given the importance of CD4 cell enumeration in HIV/AIDS diagnosis and monitoring, aptamer-based devices may offer an opportunity for novel cell detection strategies and may yield more robust and less expensive blood analysis devices in the future.     

In Silico Design and Selection of New Tetrahydroisoquinoline-Based CD44 Antagonist Candidates

CD44 promotes metastasis, chemoresistance, and stemness in different types of cancer and is a target for the development of new anti-cancer therapies. All CD44 isoforms share a common N-terminal domain that binds to hyaluronic acid (HA). Herein, we used a computational approach to design new potential CD44 antagonists and evaluate their target-binding ability. By analyzing 30 crystal structures of the HA-binding domain (CD44HAbd), we characterized a subdomain that binds to 1,2,3,4-tetrahydroisoquinoline (THQ)-containing compounds and is adjacent to residues essential for HA interaction. By computational combinatorial chemistry (CCC), we designed 168,190 molecules and compared their conformers to a pharmacophore containing the key features of the crystallographic THQ binding mode. Approximately 0.01% of the compounds matched the pharmacophore and were analyzed by computational docking and molecular dynamics (MD). We identified two compounds, Can125 and Can159, that bound to human CD44HAbd (hCD44HAbd) in explicit-solvent MD simulations and therefore may elicit CD44 blockage. These compounds can be easily synthesized by multicomponent reactions for activity testing and their binding mode, reported here, could be helpful in the design of more potent CD44 antagonists.     

THE ROLE OF TRANSFERRIN RECEPTOR (CD71) IN PHOTODYNAMIC THERAPY OF ACTIVATED AND MALIGNANT LYMPHOCYTES USING THE HEME PRECURSOR δ-AMINOLEVULINIC ACID (ALA)

Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71 +) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA-induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71 + T lymphocytes from a patient with cutaneous T-cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to norma] lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA-PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.