Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Fungus β-glycosidases: immobilization and use in alkyl-β-glycoside synthesis

Production of β-glycosidases: β-xylosidase and β-glucosidase by the fungus Sclerotinia sclerotiorum was optimized in the presence of different carbon sources. Immobilization supports with different physico-chemical characteristics were evaluated for use in continuous reactors. Immobilization and activity yields were calculated. Among the adsorption on Duolite, Amberlite, Celite and DEAE-sepharose, and entrapment in polyacrylamide gel or reticulation using glutaraldehyde, highest yields were obtained when β-xylosidase was adsorbed on Duolite A 7 and when β-glucosidase was adsorbed on DEAE-sepharose.

Enzyme preparations from S. sclerotiorum cultures were used in a biphasic (alcohol/aqueous) medium for the synthesis of alkyl-glycosides by trans-glycosylation of sugars and long-chain alcohols. The synthesis was studied under different conditions with primary and secondary alcohols as substrates, in the presence of free or immobilized enzyme. Xylan and cellobiose were used for the synthesis of alkyl-xylosides and alkyl-glucosides, respectively. The majority of the immobilized preparations were unable to catalyze the synthesis of alkyl-glycosides.

Highest yields were obtained when using xylan and C₄–C₆-alcohols. The reaction produced alkyl-β-xyloside and alkyl-β-xylobioside, as confirmed by MS/MS. Up to 22 mM iso-amyl-xyloside and 14 mM iso-amyl-xylobioside were produced from iso-amyl alcohol and xylan.     

Conformationally restricted PACAP27 analogues incorporating type II/II′ IBTM β-Turn Mimetics. Synthesis, NMR Structure Determination, and Binding Affinity

To probe the importance of a proposed β-turn within residues S9-R12 of PACAP for recognition by VIP/PACAP receptors, compounds 1 and 2, two conformationally restricted analogues of PACAP27 incorporating respectively (S)- or (R)-IBTM as type II or II′ β-turn dipeptide mimetic at the Y10-S11 position, were synthesized. According to ¹H NMR conformational analyses in aqueous solution and 30% TFE, both PACAP27 and the [S-IBTM^10,11]PACAP27 analogue 1 adopt similar ordered structures. PACAP27 shows an N-terminal disordered region (residues H1-F6) and an -helical conformation within segment T7–L27. For residues S9–R12, our data seem more compatible with a segment of the -helix than with the β-turn previously proposed for this fragment. In compound 1 the -helix, also spanning T7–L27 residues, appears slightly distorted at the N-terminus relative to the native peptide. Although this distortion could lead to the marked decrease in binding affinity of this compound at the VIP/PACAP receptors, the lack of the Y10 side chain in analogues 1 and 2 could also significantly affect the binding of these compounds.     

Solution structure of ω-conotoxin MVIIA using 2D NMR spectroscopy

The solution structure of ω-conotoxin MVIIA (SNX-111), a peptide toxin from the fish hunting cone snail Conus magus and a high-affinity blocker of N-type calcium channels, was determined by 2D NMR spectroscopy. The backbones of the best 44 structures match with an average pairwise RMSD of 0.59 angstroms. The structures contain a short segment of triple-stranded β-sheet involving residues 6–8, 20–21, and 24–25. The structure of this toxin is very similar to that of ω-conotoxin GVIA with which is has only 40% sequence homology, but very similar calcium channel binding affinity and selectivity.     

β-carrageenan: Isolation and characterization

β-Carrageenan, essentially devoid of ester sulfate, was isolated from the hot aqueous extracts of alkali-modified Eucheuma gelatinae, Eucheuma speciosa, and Endocladia muricatum by precipitating the more anionic moieties with a quaternary ammonium salt, isolating the fractions that did not precipitate, then treating these with an anion-exchange cellulose. The β-carrageenan was characterized by chemical analysis, optical rotation, and NMR. Gelling was found to be ion-independent, with T_g = 31–33°C and T_m = 63–70°C. Specific optical rotations of the isolated β-carrageenan samples were more positive than the κ-, λ-, and ι-carrageenans with which they were compared, while agarose, its stereoisomer, exhibited a negative specific rotation. Electrophoresis gels made from β-carrageenan were used to separate DNA fragments which exhibited faster migration than on an agarose gel of comparable concentration, indicating that β-carrageenan has a less restrictive pore structure.     

Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-d-glucan type produced by B. rhodina) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.     

II. N-acetyl human β-endorphin-(1–31) alleviates the morphine withdrawal syndrome in rodents: A comparative study with clonidine

The potential effect of intracerebroventricular (icv) N-acetyl human β-endorphin-(1–31) on morphine dependence was examined in mice and rats. Animals were rendered tolerant-dependent by subcutaneous (sc) implantation of an oily suspension (10 ml/Kg mouse and 3 ml/Kg rat) containing 0.1 g/ml of morphine. After 72 h of chronic morphine, 1 mg/Kg sc naloxone precipitated in both species a withdrawal syndrome that was moderate in animals pretreated with the acetylated derivative of β-endorphin. Doses of 28 fmols/rat or 80 fmols/mouse N-acetyl human β-endorphin-(1–31) reduced the number of animals presenting the jumping behaviour, as well as the number of jumps recorded. Moreover, less than half of the rats presented the other withdrawal signs evaluated: squeak on touch, diarrhoea, chattering, chewing, ptosis and body shakes. This activity could be observed when N-acetyl human β-endorphin was injected 1 h to 24 h before naloxone; longer intervals resulted in a significant loss of this activity. The ₂ agonist clonidine given icv at pmol-nmol doses decreased the incidence of morphine withdrawal syndrome. Combinations of these two substances generally did not produce any further enhancement of the effects of clonidine and N-acetyl β-endorphin when used alone. Icv injections of the antagonist of ₂-adrenoceptors yohimbine prevented both clonidine and N-acetyl β-endorphin-(1–31) from reducing the jumping behaviour displayed by morphine-abstinent mice. It is suggested that N-acetyl β-endorphin produces this alleviation of the morphine withdrawal syndrome by improving the efficiency of ₂-mediated agonist effects after acting on a neural substrate that is distinct from the μ opioid receptor binding site.     

Binding of the benzodiazepine ligand [H]-Ro 15–1788 to brain membrane of the saltwater fish Mullus surmuletus

The distribution and the pharmacological properties of the binding of the benzodiazepine receptor antagonist [³H]-Ro 15–1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H imidazol [1,5-a] 1,4 benzodiazepine) were compared in some brain membranes of the saltwater teleost fish, Mullus surmuletus: only a single population of [³H]-Ro 15–1788 binding sites was detected. The binding was saturable and reversible with a high affinity, revealing a significant population of binding sites (K_d value of 2.1 ± 0.2 nM and B_max value of 1400-900 fmol mg⁻¹ of protein, depending on fish length). The highest concentration of benzodiazepine recognition sites labelled with [³H]-Ro 15–1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. In order to explore behavioural selectivity as a consequence of multiple receptor subtypes, six benzodiazepine receptor ligands, flunitrazepam (5-(2-fluoro-phenyl)-1,3,dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepine-2-one), alpidem, (N,N-dipropyl-6-chloro-2-(4-chlorophenyl) imidazo [1,2-a] pyridine-3-acetamide) zolpidem {N,N,6, trimethyl-2-(4-methyl-phenyl) imidazo [1,2-a] pyridine-3-acetamide hemitartrate}, methyl β carboline-3-carboxylate (βCCM), Ro 15–1788 and Ro 5–4864 (4′-chlorodiazepam), were tested in vitro by binding of [³H]-Ro 15–1788 to membrane preparations from various brain areas of Mullus surmuletus. Displacement studies showed a similar rank order of efficacy of various unlabelled ligands. In all regions of the brain and in the spinal cord, GABA potentiate [³H]-flunitrazepam binding in a similar order, suggesting that the BDZ recognition sites are part of the GABA_A receptor structure. These results suggest that central-type benzodiazepine receptors are present in one class of benzodiazepine binding sites in the saltwater teleost fish brain of Mullus surmuletus (type I-like). Here we report initial evidence of homogeneity of subtypes of central benzodiazepine receptors in the spinal cord of the saltwater teleost fish, Mullus surmuletus.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

The contribution of beta1 integrins to neuronal migration and differentiation depends on extracellular matrix molecules

The interaction of β1 integrin receptors and different extracellular matrix molecules during neuronal development was investigated by comparing both migration and morphological differentiation of D3 wild-type embryonic stem (ES) cell line-derived neural precursor cells with those of the β1 integrin knockout ES cell line G201. Analysing neurosphere explants on laminin and fibronectin as major β1 integrin ligands, the maximal spreading of outward migrating neuronal cells was determined. Compared with gelatine as a standard substrate, migration was found to be significantly increased for D3-derived neurospheres on fibronectin and laminin-1. These matrix effects were found to be even enhanced for G201 preparations. In addition, also the differentiation of wild-type and β1 integrin −/− neurones – as determined by MAP-2- and HNK-1-immunoreactive processes – was found to be increased on fibronectin and laminin when compared to gelatine standards. In the respective knockout preparations on these matrices, again perturbation effects were less pronounced than on gelatine. Our observations indicate that laminin and fibronectin are involved both in β1 integrin-dependent and -independent signalling mechanisms during neurogenesis. Upregulation of compensatory mechanisms such as β1 integrin-independent receptors for laminin and fibronectin might be responsible for the much less pronounced perturbations of G201 neural precursor migration and differentiation on these two substrates than on gelatine.     

Identification of acridinyl hydrazides as potent aspartic protease inhibitors

We have identified acridinyl derivatives as potent aspartic protease inhibitors by virtual screening of in-house library of synthetic compounds. Enzyme inhibition experiments showed that both compounds inhibit human cathepsin D and Plasmodium falciparum plasmepsin-II in nanomolar ranges. The IC₅₀ values against cathepsin D and plasmepsin-II of compound-Nar103 were found to be 9.0 ± 2.0 and 4.0 ± 1.0 nM and of compound-Nar110 were 0.5 ± 0.05 and 0.13 ± 0.03 nM, respectively. Ligand docking predicted the binding of acridinyl derivatives at the substrate-binding cleft, where hydrazide part of the inhibitors interact with the S1–S1′ subsite residues including catalytic aspartates. The phenyl ring and acridinyl moiety of the inhibitors were predicted to interact with S2/S3 and S2′/S3′ subsite residues.     

Carbon–carbon-linked (pyrazolylphenyl)oxazolidinones with antibacterial activity against multiple drug resistant gram-positive and fastidious gram-negative bacteria

In an effort to expand the spectrum of activity of the oxazolidinone class of antibacterial agents to include Gram-negative bacteria, a series of new carbon–carbon linked pyrazolylphenyl analogues has been prepared. The -N-substituted methyl pyrazole (10) in the C3-linked series exhibited very good Gram-positive activity with MICs ≤0.5–1 μg/mL and moderate Gram-negative activity with MICs=2–8 μg/mL against Haemophilus influenzae and Moraxella catarrhalis. This analogue was also found to have potent in vivo activity with an ED₅₀=1.9 mg/kg. β-Substitution at the C3-linked pyrazole generally results in a loss of activity. The C4-linked pyrazoles are slightly more potent than their counterparts in the C3-linked series. Most of the analogues in the C4-linked series exhibited similar levels of activity in vitro, but lower levels of activity in vivo than 10. In addition, incorporation of a thioamide moiety in selected C4-linked pyrazole analogues results in an enhancement of in vitro activity leading to compounds several times more potent than eperezolid, linezolid and vancomycin. The thioamide of the N-cyanomethyl pyrazole analogue (34) exhibited an exceptional in vitro activity with MICs of ≤ 0.06–0.25 μg/mL against Gram-positive pathogens and with MICs of 1 μg/mL against fastidious Gram-negative pathogens.     

Whole-cell bioconversion of β-sitosterol in aqueous–organic two-phase systems

The selective cleavage of the β-sitosterol side-chain by free Mycobacterium sp. NRRL B-3805 cells was used as a model system for the study of solvent effects in a whole-cell bioconversion in two phase aqueous–organic media. This multi-step degradation pathway leads to the production of 4-androstene-4,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) as a minor product. In an attempt to correlate the substrate and cell partition effects and solvent hydrophobicity (log P) with biocatalytic activity, 15 carboxylic acid esters with log P values between 3 and 10 were screened. The results indicated that the toxicity of the tested solvents in this system could not be correlated to their log P, but seemed to depend on their ability to accumulate in the cells, as these showed a strong affinity towards the organic phase. Different solvent/aqueous ratios and hydrodynamic conditions were further tested in the solvent systems (phthalates) showing significant biodegradation activity. The bioconversion rate was generally not much affected by the stirring speed in the employed range (150–300 rpm) but was strongly influenced by the aqueous/organic phase ratio. Results suggest that the bioconversion takes place at the interphase, its rate being possibly limited by mass transport inside the organic phase.     

Reactivity of {(Ph3P)Pt[μ-η-H---SiH(Ar)]}2 (Ar=2-isopropyl-6-methylphenyl) with phosphines. X-ray crystal structure of trans-{(dppe)Pt[μ-SiH(Ar)]}2

The dinuclear Pt---Si complex {(Ph₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-1a–cis-1b=3:1; IMP=2-isopropyl-6-methylphenyl) reacted with basic phosphines such as 1,2-bis(diphenylphosphino)ethane (dppe) and dimethylphenylphosphine (PMe₂Ph) to afford different dinuclear Pt---Si complexes with loss of H₂, {(P)₂Pt[μ-SiH(IMP)]}₂ [P=dppe, trans-2a (major), cis-2b (trace); PMe₂Ph, 3 (trans only)]. Complexes 2 and 3 were characterized by multinuclear NMR spectroscopy and X-ray crystallography (2a). In contrast, the reaction of 1a,b with the sterically demanding tricyclohexylphosphine (PCy₃) afforded {(Cy₃P)Pt{μ-η²-H---SiH(IMP)]}₂ (trans-4a–cis-4b 2:1) analogous to 1a,b where the central Pt₂Si₂(μ-H)₂ core remains intact but the PPh₃ ligands have been replaced by PCy₃. Complexes 4a and 4b was characterized by multinuclear NMR and IR spectroscopies.     

Microbiological transformations 44. Optimisation of a new Baeyer–Villigerase activity: application to the stereospecific oxidation of 3-phenylcyclobutanone

A screening was achieved out of 80 microbial strains in order to detect new “Baeyer–Villigerase” activities, using bicyclo[3.2.0]hept-2-en-6-one 1 as a test substrate. Such a new and interesting activity was discovered in the fungus Cunninghamella echinulata. Using this strain, oxidation of prochiral 3-phenyl-cyclobutanone 5 was examined. After an optimisation of the experimental conditions, the corresponding γ-butyrolactone 6 was obtained in 71% yield and 98% ee.     

Organization of 3β-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358–1364] of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) and cytochrome P450_scc (cyt. P450_scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3β-HSD and cyt. P450_scc is observed during the purification of 3β-HSD from mitochondria. The behaviour of 3β-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450_scc. Immunoprecipitations from mitochondria with either anti-cyt. P450_scc or anti 3β-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 μM between 3β-HSD and P450_scc was obtained. These observations suggest that P450_scc and 3β-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Solubilization of active GLP-1 (7–36)amide receptors from RINm5F plasma membranes

Glucagon-like peptide-1 (7–36)amide (GLP-1 (7–36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7–36)amide have been described in RINm5F cells. We have solubilized active GLP-1 (7–36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-β-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7–36)amide to the solubilized receptor was inhibited conentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K_a= 0.26 ± 0.03 nM and B_max= 65.4 ± 21.24 fmol/mg of protein for the membrane-bound receptor and K_a= 22.54 ± 4.42 μM and B_max= 3.9 ± 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-γ-S nor GDP-β-S affected binding or labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7–36)amide receptor in a functional state and opens up the possibility for further molecular characterization of the receptor protein.     

The crystal and molecular structures of lithium trimethylenediaminetetraacetatoferrate(III) trihydrate Li[Fe(trdta)]·3H2O and sodium ethylenediamine-N,N′-diacetato-N,N′-di-3-propionatoferrate(II) pentahydrate Na[Fe(eddda)]·5H2O

The crystal structures of Li[Fe(trtda)]·3H₂O and Na[Fe(eddda)]·5H₂O (trtda = trimethylenediaminetetraacetate and eddda = ethylenediamine-N,N′-diacetate-N,N′-di-3-propionate) have been determined by single crystal X-ray diffraction techniques. The former crystal was monoclinic with the space group P2₁/n,a = 17.775(3),b = 10.261(1),c = 8.883(2)Å, β = 95.86(4)° and Z = 4. The latter was also monoclinic with the space group P2₁/n,a = 6.894(2),b = 20.710(6),c = 13.966(3)Å, β = 101.44(2)° and Z = 4. Both complex anions were found to adopt an octahedral six-coordinated structure with all of six ligand atoms of trdta⁴⁻ or eddda⁴⁻ coordinated to the Fe(III) ion, unlike the corresponding edta⁴⁻ complex which is usually seven-coordinate with the seventh coordination site occupied by H₂O. Of the three geometrical isomers possible for the eddda complex, the trans(O₅) isomer was actually found in the latter crystal. Factors determining the structural types of metal–edta complexes are discussed in detail.     

C NMR study on peracetylated derivatives of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative

Peracetylated samples of cyclomaltoheptaose (β-cyclodextrin, β-CD) and its methylated derivative were studied by ¹³C NMR. The acetyl carbonyl carbon signal in peracetylated β-CD was resolved into a triplet, and the three peaks were assigned by long-range C---H COSY and INAPT techniques. The individual carbonyl peak was found to be indicative of the location of the acetyl group on the 2, 3, and 6 position in the glucose residues. An acetylated derivative of a partly methylated β-CD was also subjected to ¹³C NMR analysis to determine the distribution of acetyl and, subsequently, methyl groups on the glucose residues.