Characterization of highly sulfated cyclodextrins

A class of highly sulfated cyclodextrins (HS-CDs) was developed for enantiomeric separation of chiral compounds by capillary electrophoresis (CE). The HS-CDs were produced by a facile single-step direct sulfation of cyclodextrin using sulfur trioxide-trimethylamine complex in dimethylformamide. Characterization of the HS-CDs by electrospray ionization mass spectrometry and by CE using a well-established indirect detection method indicated the species have very narrow heterogeneity in terms of degree of sulfation. Elemental analysis of the HS-alpha-, beta- and gamma-CDs showed that the average sulfate contents were 11, 12, and 13 per CD molecule, respectively. The 13C NMR of HS-CDs is consistent with the structural assignment of nearly complete sulfation at C-6 primary hydroxyl groups and partial sulfation at the C-2 secondary hydroxyls (>70%), while the C-3 hydroxyls remain unsubstituted. Enantiomeric separation by CE using the HS-CDs as chiral selectors showed that HS-alpha-, beta- and gamma-CDs complement each other by exhibiting different chiral selectivities, resulting in resolution of many chiral neutral, acidic and basic compounds of greatly varying structural features. The part of HS-CD that interacts with the guest molecule during complexation and, therefore, the receiving end of the cyclodextrin hydrophobic bucket was surrounded with largely regiospecifically substituted C-2 sulfates and intact C-3 hydroxyls, both at the equatorial positions. Such global regiospecific structural arrangement in HS-CDs provides differential diasteroisomeric complexation is proposed to be the principal contributing factor in the resolving racemates.     

Carboxymethylated cyclodextrin derivatives as chiral NMR discriminating agents

Procedures to prepare cyclodextrins with carboxymethyl groups incorporated selectively at the primary (6-position) or secondary (2-position) are described. Complexation properties of the primary and secondary carboxymethylated derivatives of α-, β-, and γ-cyclodextrins are compared to native cyclodextrins and indiscriminately substituted carboxymethylated cyclodextrins, using pheniramine, chlorpheniramine, and brompheniramine as substrates. The stoichiometry of association of these substrates with the α-cyclodextrins is 1:1, whereas with the γ-cyclodextrins, a 2:1 substrate:cyclodextrin complex forms. Data for the β-cyclodextrins suggest that there is a mix of 1:1 and 2:1 substrate–cyclodextrin complexes. The position of the carboxymethyl groups on the cyclodextrin does not appear to alter the geometry of substrate–cyclodextrin association. The effectiveness of the carboxymethylated cyclodextrins as chiral NMR discriminating agents is compared with the native cyclodextrins. In all cases, the indiscriminately substituted α-, β-, and γ-cyclodextrins are more effective at enantiodistinction with the cationic substrates than native cyclodextrins or the derivatives with carboxymethyl groups at the primary or secondary positions. Among α-, β-, and γ-indiscriminately substituted cyclodextrins, there was no clearly optimal candidate for chiral NMR discrimination studies. The indiscriminately substituted carboxymethyl cyclodextrins are effective water-soluble chiral NMR discrimination reagents for cationic substrates.     

Synthesis of New Regioselectively Sulfated Hyaluronans for Biomedical Application

Sulfated glycosaminoglycans (GAGs) display various biological effects which are strongly influenced by the degree of sulfation and the position of sulfate groups within the polymer. Hyaluronan, a non-sulfated GAG, represents a readily accessible educt to synthesize structural analogues of sulfated GAGs mimicking their biological activity. Different strategies were developed and evaluated to synthesize hyaluronan sulfates with a free primary hydroxyl group at C-6' and sulfated secondary hydroxyl groups. Applying selective desulfation methods of high-sulfated hyaluronan by means of silylating agents, products regioselectively desulfated at the primary C-6' but also partly the C-4' position were obtained. A pathway using benzoyl ester protecting groups to block the primary hydroxyl function of Hya during the sulfation resulted in a high-sulfated product, functionalized only at the secondary hydroxyl groups.     

Synthesis of the chiral selector heptakis(6‐O‐methyl)‐β‐cyclodextrin by phase‐transfer catalysis and hydrazine‐mediated transfer‐hydrogenation

The exhaustive primary‐side alkylation of cyclodextrins has never been achieved directly. The undesired and simultaneous derivatization of the secondary hydroxyl moieties generates intricate isomeric mixtures that are challenging to purify, analyse and characterize. The aim of this study was to develop a chromatography‐free and up‐scalable strategy towards the preparation of per‐6‐O‐methylated cyclodextrin and to test the compound as potential chiral selector. The target molecule was prepared according to a five‐step synthesis by using methyltriphenylphosphonium bromide as catalyst under heterogeneous conditions. The removal of benzyl moieties, used as temporary secondary‐side protecting groups, was attained by applying hydrazine‐carbonate in the presence of Pd/C. All the intermediates were obtained in high yields, thoroughly characterized and their purity was assessed by ad‐hoc developed HPLC methods. The per‐6‐O‐methylated β‐cyclodextrin showed promising chiral recognition ability as background electrolyte additive in cyclodextrin‐modified capillary electrophoresis using the recreational drug methylene‐dioxypyrovalerone as model compound. Additionally, a model for the inclusion geometry between the single isomer host and the selected drug was developed based on the extensive 2D NMR analysis. The versatility of the proposed synthetic strategy opens the way to the industrial production of homogeneously primary‐alkylated cyclodextrins and to their wide application in chiral separation of various drugs.     

Ruthenium and Osmium Podate Cyclodextrins with Dual-function Recognition Sites for Luminescent Sensing

A multifunctionalised podand cyclodextrin ligand, β-CD-(urebpy)⁷, with urea--bipyridine binding sites leads to ruthenium and osmium, {Ru[β-CD-(urebpy)⁷]}[PF⁶]² {Os[β-CD-(urebpy)⁷]}[PF⁶]², cyclodextrins. The bipyridine ligands are preorganised by the cyclodextrin cavity encapsulating the ruthenium and osmium core to give photoactive metallocyclodextrins. The podate cyclodextrin complexes show characteristic ruthenium and osmium tri-bipyridine luminescence. It is demonstrated that the ruthenium cyclodextrins participate in sensing schemes through both the cyclodextrin cavity and the urea cage at the bottom of the cyclodextrin rim. Luminescence quenching of the ruthenium emission is observed by addition of anthraquinone guests in the cyclodextrin cavity or addition of dihydrogen phosphate anion.     

Thermolysin catalyses the synthesis of cyclodextrin esters in DMSO

Fatty acid esters of cyclodextrins (CDs) were synthesised in a one-step reaction with native CDs as acyl acceptors and vinyl-activated fatty acid esters as acyl donors. Immobilised preparations of thermolysin, subtilisin, the alkaline protease AL-89 and Candida antarctica lipase B were investigated for their catalytic properties regarding transesterification in solvents of increasing hydrophilicity. The synthesis of cyclodextrin fatty acid esters was proved to be catalysed enzymatically by thermolysin in DMSO. The obtained products were analysed by TLC and their structures characterised by NMR, MS and FTIR spectroscopy. With vinyl decanoate as acyl donor β-CD was esterified at all seven glucose C-2 positions resulting in heptakis(2-O-decanoyl)-β-cyclodextrin as the major product. With vinyl butyrate, substitution occurred at all the C-2 and partially at the C-3 or C-6 positions resulting in an average degree of substitution of nine. Between 20% and 25% (w/w) of the acyl donor was converted to esters in 20 h corresponding to an estimated total conversion of the acyl acceptor in the case of maltosyl-β-CD. In the subtilisin and AL-89 catalysed reactions, product formation was simultaneously catalysed non-enzymatically by inorganic buffer salts in aprotic, hydrophilic solvents and with the lipase no products were formed in any of the solvents investigated.     

甲基丙烯酸N,N-二甲氨基乙酯与丙烯酰β-环糊精共聚水凝胶的合成及其性能研究

用不同的方法合成了两种结构不同的丙烯酰 β 环糊精酯 (β CD 3 A和 β CD 6 A) ,以此为单体与甲基丙烯酸N ,N 二甲氨基乙酯 (DMAEMA)通过氧化还原自由基引发聚合 ,合成出两类含 β 环糊精结构单元的新型水凝胶 .用核磁共振 ,红外光谱及元素分析对两种单体及共聚物的结构和组成进行了表征 .溶胀实验结果表明 ,两类水凝胶均具有较好的pH、温度及离子强度敏感性 ,且因其交联网络结构不同 ,其溶胀性能有所差异     

Cal-B-catalyzed alkoxycarbonylation of a-ring stereoisomeric synthons of 1alpha,25-dihydroxyvitamin D3 and 1alpha,25-dihydroxy-19-nor-previtamin D3: a comparative study. first regioselective chemoenzymatic synthesis of 19-nor-A-ring carbonates

A comparative study of alkoxycarbonylation processes of both 19-nor-A-ring and A-ring stereoisomers of 1alpha,25-dihydroxyvitamin D3 analogues catalyzed by Candida antarctica lipase B (CAL-B) has been described. The presence of the methyl group in the A-ring at C-2, as in 3-6, has a determining role in the regioselectivity of the biocatalysis, mainly allowing the hydroxyl group at C-5 position to react. For the 19-nor-A-ring stereoisomers 7-10, which lack the C-2 methyl group, the configurations at C-3 and C-5 have a high influence in the selectivity exhibited by CAL-B. Thus, each couple of enantiomers showed opposing regioselectivities depending on the C-3 configuration. When C-3 possesses an (S)-configuration, enzymatic alkoxycarbonylations took place at the C-5-(R) or C-5-(S) hydroxyl groups. However, if the chiral centers at C-3 are (R), CAL-B alkoxycarbonylated the C-3-(R) hydroxyl group independently of the configuration at C-5. The corresponding carbonates are useful A-ring precursors of 1alpha,25-dihydroxyvitamin D3 analogues, selectively modified at the C-1 or C-3 positions. In addition, an improved synthesis of cis A-ring synthons 5 and 6 is described using a Mitsunobu methodology.     

Mono‐6A‐(4‐methoxybutylamino)‐6A‐β‐cyclodextrin as a chiral selector for enantiomeric separation

A new member of the family of methoxylalkylamino monosubstituted β‐cyclodextrins, mono‐6^A‐(4‐methoxybutylamino)‐6^A‐β‐cyclodextrin, has been developed as a chiral selector for enantioseparation in capillary electrophoresis. This amino cyclodextrin exhibited good enantioselectivities for 16 model acidic racemates including three dansyl amino acids at an optimum pH of 6.0. Excellent chiral resolutions over six were obtained for α‐hydroxy acids and 2‐phenoxypropionic acids with 3.0 mM chiral selector. The good chiral recognition for α‐hydroxyl acids was attributed to inclusion complexation, electrostatic interactions, and hydrogen bonding. The hydrogen‐bonding‐enhanced chiral recognition was revealed by NMR spectroscopy. The chiral separation of acidic racemates was further improved with the addition of methanol (≤10 vol%) as an organic additive.     

Functionalization of cyclodextrins via reactions of 2,3-anhydrocyclodextrins

Three types of reactions of 2,3-anhydro-beta-cyclodextrins, namely nucleophilic ring-opening, reduction to 2-enopyranose, and reduction to 3-deoxypyranose, have been investigated to regio- and stereoselectively functionalize the secondary face of beta-cyclodextrin. Upon treatment with various nucleophiles, both 2,3-mannoepoxy and 2,3-alloepoxy-beta-cyclodextrins are found to undergo nucleophilic ring-opening reaction generating 3- and 2-modified cyclodextrin derivatives. In each case, the 3-position is more easily accessible than the 2-position. By using these ring-opening reactions, imidazolyl, iodo, azido, and benzylmercapto groups are selectively introduced to the secondary face of beta-cyclodextrin in place of the 2- or 3-hydroxyl groups. The functionalized cyclodextrins have either modified glucosidic subunits or modified altrosidic subunits that make the hydrophobic cavity slightly distorted from that of native beta-cyclodextrin. Thiourea also reacts with the cyclodextrin epoxides. In this case, thiirane and olefin species are generated instead of any ring-opening products. By ameliorating the reaction condition, cyclodextrin olefin, diene, and triene derivatives are prepared in moderate to good yields. Reduction of per[6-(tert-butyldimethyl)silyl]-beta-cyclodextrin permannoepoxide with lithium aluminum hydride produces the per(3-deoxy)-beta-cyclomannin. All these chemically modified cyclodextrins are structurally well characterized and most of them are expected to serve as versatile scaffolds for diverse purposes such as the construction of catalysts and development of synthetic receptors and molecular containers.     

Flavonoids inhibiting glycation of bovine serum albumin: affinity-activity relationship

Protein glycation leads to the formation of advanced glycation end-products (AGEs), which contribute to the pathogenesis of diabetic complications. The structure-activity relationship of dietary flavonoids for inhibiting the glycation of bovine serum albumin (BSA) in vitro was subjected to a detailed investigation. The structure-activity relationship revealed that: 1) the hydroxylation on ring B of the flavones enhanced the inhibition and the hydroxyl groups at the C-5 and C-7 positions of flavones favoured the inhibition; 2) the optimal number of hydroxyl groups on ring B of the flavonols was one (at the C-3 position) and the methylation of flavonols weakened the inhibition; 3) the methoxylation at the C-6 position and methylation at C-4′ position of genistein clearly enhanced the inhibition; 4) the hydroxyl groups at the C-5 and C-7 positions of flavanones were in favour of the inhibition; 5) the glycosylation of flavonoids significantly weakened the inhibition. Obvious linear affinity-activity relationships exist between the BSA-flavonoid interaction and flavonoids as BSA glycation inhibitors (R² = 0.76585). The flavonoids with a higher affinity to BSA exhibited a stronger inhibition of the glycation of BSA.     

Bilayer vesicles of amphiphilic cyclodextrins: host membranes that recognize guest molecules

A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of alpha-, beta-, and gamma-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicles in aqueous solution. The bilayer vesicles were characterized by transmission electron microscopy, dynamic light scattering, dye encapsulation, and capillary electrophoresis. The molecular packing of the amphiphilic cyclodextrins was investigated by using small-angle X-ray diffraction of bilayers deposited on glass and pressure-area isotherms obtained from Langmuir monolayers on the air-water interface. The bilayer thickness is dependent on the chain length, whereas the average molecular surface area scales with the cyclodextrin ring size. The alkyl chains of the cyclodextrins in the bilayer are deeply interdigitated. Molecular recognition of a hydrophobic anion (adamantane carboxylate) by the cyclodextrin vesicles was investigated by using capillary electrophoresis, thereby exploiting the increase in electrophoretic mobility that occurs when the hydrophobic anions bind to the nonionic cyclodextrin vesicles. It was found that in spite of the presence of oligo(ethylene glycol) substituents, the beta-cyclodextrin vesicles retain their characteristic affinity for adamantane carboxylate (association constant K(a)=7.1 x 10(3) M(-1)), whereas gamma-cyclodextrin vesicles have less affinity (K(a)=3.2 x 10(3) M(-1)), and alpha-cyclodextrin or non-cyclodextrin, nonionic vesicles have very little affinity (K(a) approximately 100 M(-1)). Specific binding of the adamantane carboxylate to beta-cyclodextrin vesicles was also evident in competition experiments with beta-cyclodextrin in solution. Hence, the cyclodextrin vesicles can function as host bilayer membranes that recognize small guest molecules by specific noncovalent interaction.     

Synthesis of cyclic methacrylic acid oligomers by atom transfer radical polymerization

Poly(methacrylic acid) (PMAA) oligomers were synthesized by combining template polymerization and copper‐mediated atom transfer polymerization with multivinyl monomer of β‐cyclodextrin (CD) having 20.4 methacryloyl groups on both primary and secondary hydroxyl group sides of CD scaffold, with 1,3‐dibromobutane as an initiator. The initiation and propagation sites of polymerized sequence of β‐CD were connected by postpolymerization of polymerized products with CuBr and tris[(2‐dimethylamino)ethyl]amine (Me₆TREN) in a methanol/water mixture of 10 wt % of water. Polymerized and cyclized sequences, PMAA oligomers formed on the primary and the secondary hydroxyl group sides, were detached from β‐CD scaffold by hydrolysis. Molecular weights of PMAA oligomers were measured by GPC and matrix assisted laser desorption ionization time‐of‐flight mass measurement. By ¹H NMR measurements, it was found that three types of cyclic PMAA were obtained by postpolymerization. The cyclization preferentially occurred on the secondary hydroxyl group side than on the primary hydroxyl group side. From the structures of cyclic PMAA, two reaction positions were proposed. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 6262–6271, 2005     

Addressing the Structural Complexity of Fluorinated Glucose Analogues: Insight into Lipophilicities and Solvation Effects

In this work, we synthesized all mono-, di-, and trifluorinated glucopyranose analogues at positions C-2, C-3, C-4, and C-6. This systematic investigation allowed us to perform direct comparison of ¹⁹F resonances of fluorinated glucose analogues and also to determine their lipophilicities. Compounds with a fluorine atom at C-6 are usually the most hydrophilic, whereas those with vicinal polyfluorinated motifs are the most lipophilic. Finally, the solvation energies of fluorinated glucose analogues were assessed for the first time by using density functional theory. This method allowed the log P prediction of fluoroglucose analogues, which was comparable to the C log P values obtained from various web-based programs.     

Regioselective Silylations of C-2 Hydroxyl Groups of Cyclodextrins Dependent on Reaction Temperature

Silylations of the C-2 hydroxyl group of cyclodextrins were carried out using t-butyldimethylsilyl imidazole in the presence of 4A molecular sieves in N,N-dimethylformamide. A unique aspect of this silylation method is the temperature dependence of the regioselectivity; silylation at 0 °C regioselectively favored the C-6 position to afford mono-6-O-t-butyldimehylsilyl-cyclodextrins, whereas silylation at 140 °C exhibited high regioselectivity on the C-2 hydroxyl group.     

Molecular Recognition Studies on Modified Cyclodextrins

This account describes our research progress in recent years in the areas of the molecular recognition studies on modified cyclodextrins, including positively charged cyclodextrins, cyclodextrin derivatives with hydrophobic substituent, and dimeric cyclodextrins. Calorimetric titration and various spectrometric techniques were employed to determine the complex stability constants, as well as the thermodynamic parameters, for their inclusion complexation with diverse guest molecules. The results obtained have been discussed from the viewpoint of size/shape‐matching, induced‐fit, geometric compensation, and multiple recognition. Thermodynamically, the compensatory relationship between ΔH and TΔS was found to be exhibited in the inclusion complexation of modified cyclodextrin.     

Synthesis of proline-modified analogues of the neuroprotective agent glycyl-l-prolyl-glutamic acid (GPE)

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH₂ group with sulfur and/or incorporation of two methyl groups at C-5.     

13C-NMR spectral studies on the distribution of substituents in some cellulose derivatives

¹³C-NMR spectra of trityl cellulose (Tr-Cell), tosyl cellulose (Ts-Cell), cellulose S-methyl xanthate (Cell-M-Xan), and cellulose formate (CF) in dimethylsulfoxide-d₆ were analyzed at 50.4 MHz. It was found that the distribution of substituents in the anhydroglucose units of these cellulose derivatives can be estimated from their ring carbon spectra. The results showed that (i) in Tr-Cell having degree of substitution (DS) lower than 1, the hydroxyl groups at C-6 carbon position are selectively tritylated, (ii) in the case of Ts-Cell, the difference in the relative DS value among three different types of hydroxyl groups is not large, although the relative reactivities of hydroxyl groups toward tosylation decrease in the order C-6 > C-2 > C-3, (iii) in Cell-M-Xan, the hydroxyl groups at C-3 carbon position are mainly substituted, and (iv) the ease of formylation is C-6 > C-2 > C-3. The 100.8 MHz ¹³C-NMR spectra of O-methyl cellulose (MC) revealed that the reactivity order in commercial MC prepared from alkali cellulose is C-6 ? C-2 > C-3. Concerning MC, its water solubility was also discussed in terms of the distribution of substituents along the cellulose chain.     

Rapid transacylations of activated ester substrates bound to the primary side β-cyclodextrin-cyclen conjugate and its M2+ complexes

The ability of cyclodextrins to effect rapid transacylations of bound substrates has been well studied. One important difference between cyclodextrin and enzyme-mediated transacylation is the pH required. Because the pK _a of a cyclodextrin secondary-side hydroxyl group is about 12, transacylations are accelerated in the presence of cyclodextrin under basic conditions (pH > 10.5). In 1988, our group reported the synthesis of cyclodextrin with attached cyclen-Co(III) complexes; significant acceleration in the reaction withp-nitrophenyl acetate was observed only with the primary side derivative. Of course, metalloenzymes utilize M²⁺ and not M³⁺ catalytic centers; in addition, large rate accelerations in the transacylations of both activated and unactivated substrates have been observed previously in systems utilizing M²⁺ ions (e.g., Zn, Cu, Ni) as well as M³⁺ ions (e.g. Co, Ir, Cr). In this paper, we describe the ability of CD-cyclen-M²⁺ conjugates to transacylate activated esters, amides, and phosphates. In addition, the ability of the apoenzyme mimic to effect transacylations was examined.     

Selectively monomodified cyclodextrins. Synthetic strategies

Monomodifications of cyclodextrins to give selectively 2-, 3-, or 6-substituted product is a challenging task because of the number of hydroxyl groups that can potentially react with the incoming reagent. The principles and the methods involved in manipulations of the differences in the chemistry of these hydroxyl groups to control the outcome of an electrophilic reaction with them to produce monoalkylated (ether-linkaged) cyclodextrin derivatives are discussed and illustrated.