Cyclodextrins: Past and Present

Cyclodextrins (CDs) are cyclic oligosaccharides produced by enzymatic degradation of starch. The most common CDs are the main natural ones, α, β and γ, which are constituted of 6, 7 and 8 glucopyranose units, respectively. The CD structure forms a torus or doughnut ring and the molecule actually exists as a truncated cone. The outer side of the toroid is hydrophilic in nature due to the hydroxyl groups of the glucopyranose units while the internal cavity is relatively apolar. Thus, CDs have a high potential to entrap entirely or partially a wide variety of compounds in a process known as complexation. This gives them new physico-chemical properties and characteristics. The main applications of CDs in drug formulation rely on CD complexation and include the protection of easily oxidizable molecules or the improvement of aqueous solubility. The use of CDs in analytical chemistry is based on his host-guest recognition property, known as supramolecular complex formation. Currently, CDs are successfully used in molecular recognition-based methods like chromatographic separations, spectroscopic and electroanalyses. Quiral analytical separations are a CD area of special relevance. In this work, attention is paid to more recent references, especially to selected reviews

NMR study on the stabilization and chiral discrimination of sulforaphane enantiomers and analogues by cyclodextrins

Sulforaphane (SFN), a phytochemical isolated from broccoli, is an important antitumoral compound with additional beneficial effect on other important diseases. However, the chemical instability of SFN has hampered its clinical use. In order to circumvent this problem, we report the first comparative study on the inclusion complexes of SFN and SFN homologues with different cyclodextrins by NMR spectroscopy. From this study it has been shown that α-CD is the most indicated cyclodextrin for the stabilization of SFN and SFN homologues, and that the highest affinity constant is that of the isothiocyanate obtained from the wasabi. Furthermore, the study of the inclusion complexes of α-CD and the non-natural SFN and analogues with S absolute configuration at sulfur shows for the first time that α-CD is able to discriminate between the two enantiomers, with the natural R enantiomers forming the inclusion complexes with higher affinity.Ministerio de Economía yCompetitividad (grants No. CTQ2016-78580-C2-1-R, and CTQ2016-78580-C2-2-R)Junta de Andalucía (P11-FQM-8046)EvgenPharm

Enantioseparation of Alkylaryl Sulfoxides Using Capillary Zone Electrophoresis

Alkylaryl sulfoxides possess a chiral sulfur atom easily identifiable by capillary zone electrophoresis (CZE). Separation of chiral alkylaryl sulfoxides has already been accomplished by modified method of CZE known as micellar electrokinetic chromatography (MEKC). However, no articles have been published on the enantioseparation of alkylaryl sulfoxides using capillary zone electrophoresis. A series of sulfoxides were synthesized, purified and identified via NMR. Enantioseparation was performed using CZE employing a 10 mmol. Phosphate buffer (pH 4.0, 25% acetonitrile, 2% sulfated-β-cyclodextrin). Synthesis of these sulfoxides will be presented along with the results of the procedure’s optimization. Separation of the sulfoxide enanitomers relies on the partitioning between the chiral additive (sulfated-β-cyclodextrin) and buffer solution

Diastereoselective synthesis of [alpha]-tocopherol

Vitamin E exists in eight different forms, four tocopherols and four tocotrienols. All possess a hydrophobic side chain, which allows them to penetrate into biological membranes. Their second common feature is a chromanol moiety with a hydroxyl group that can donate a hydrogen atom. These properties make vitamin E a very important radical chain-breaking antioxidant in living organisms, and therefore also an industrial product. α-Tocopherol is the member of the vitamin E family that is preferentially absorbed and accumulated in humans. There are three stereocenters in α-tocopherol, whereas RRR-α-tocopherol (1) is the natural and biologically most active form. The R-configuration at C(2) is essential in order to be recognised by the α-tocopherol transport protein and thus maintained in the plasma. The biological activity rendered RRR-α-tocopherol (1) a synthetic target. In this work two novel syntheses of RRR-α-tocopherol (1) are presented. Both syntheses involve a highly diastereoselective epoxidation of the bis-protected phytyl hydroquinone 132 as the key step followed by a cyclisation to form the chromanol ring (figure 70). In order to find suitable stereoselective epoxidation catalysts, cyclodextrin-based catalysts were prepared and tested (figure 71). However, none of these catalysts were reactive or selective enough to be applicable to the epoxidation of bis-protected phytyl hydroquinones. However, the asymmetric Shi epoxidation proved to be a suitable epoxidation method for this purpose. A number of bis-protected phytyl hydroquinones were synthesised and subsequently epoxidised under Shi epoxidation conditions. The highest diastereoslectivity could be obtained for substrate 132. By applying Shi ketone 114, which is derived from D-fructose, epoxide 148 could be obtained with 96% de, whereas if the enantiomer ent-114 (synthesised in 5 steps from L-sorbose) was used, 147 was formed with 97% de (figure 71). The epoxide function in 148 could be selectively opened with hydride. Further transformations led to the hydroquinone 155, which could be cyclised under acidic conditions to form the chromanol ring of 1 with the desired R-configuration at C(2) (figure 72). α-Tocopherol could be synthesised in 8 steps with 93% de via this route. To the best of my knowledge, this highly diasteroselective synthesis of α-tocopherol (1) is one of the shortest in terms of numbers of steps, employing a commercially available organocatalyst. In a different approach an acid supported, “anti-Baldwin” epoxide ring opening of desilylated 147 under inversion of configuration led to the 6-membered chromanol ring. α-Tocopherol could be synthesised in 10 steps and with 93% de. This synthesis was carried out in collaboration with Julien Chapelat. To the best of our knowledge this is the second application of an organocatalyst to the construction of chromanols, having a tetrasubstituted chiral carbon centre, in high diastereoselectivity.7

Chiral stationary phases and applications in gas chromatography

Chiral compounds are ubiquitous in nature and play a pivotal role in biochemical processes, in chiroptical materials and applications, and as chiral drugs. The analysis and determination of the enantiomeric ratio (er) of chiral compounds is of enormous scientific, industrial, and economic importance. Chiral separation techniques and methods have become indispensable tools to separate chiral compounds into their enantiomers on an analytical as well on a preparative level to obtain enantiopure compounds. Chiral gas chromatography and high-performance liquid chromatography have paved the way and fostered several research areas, that is, asymmetric synthesis and catalysis in organic, medicinal, pharmaceutical, and supramolecular chemistry. The development of highly enantioselective chiral stationary phases was essential. In particular, the elucidation and understanding of the underlying enantioselective supramolecular separation mechanisms led to the design of new chiral stationary phases. This review article focuses on the development of chiral stationary phases for gas chromatography. The fundamental mechanisms of the recognition and separation of enantiomers and the selectors and chiral stationary phases used in chiral gas chromatography are presented. An overview over syntheses and applications of these chiral stationary phases is presented as a practical guidance for enantioselective separation of chiral compound classes and substances by gas chromatography

Comparison of various chiral stationary phases for the chromatographic separation of chiral pharmaceuticals

Many pharmaceuticals contain active ingredients that have more than one stereoisomer. An important concern is the recognition that these different stereoisomers do not necessarily have identical, or even desirable biological activity. Consequently, analytical methods for the analysis and separation of enantiomers are important in the proper development of a marketed pharmaceutical product. In this research, direct HPLC methods for the chromatographic separation of oxyphene optical isomers have been developed and optimized using three types of chiral stationary phases. The research carried out a systematic study of the conditions for the separation of oxyphene optical isomers using synthetic polymer chiral stationary phase of cellulose tris (3, 5-dimethylphenylcarbamate) Chiralcel OD, ß-cyclodextrin chiral stationary phase, and a1-acid glycoprotein chiral stationary phase. The methods using the ß-cyclodextrin and Chiralcel OD columns provide for the accurate determination of the optical purity (as low as 0.1%) of each enantiomer, in the presence of the other major enantiomer. The performance of these chiral stationary phases is also compared

Synthesis of sphingosine analogues by diastereospecific amination of enantiopure trans-gamma, delta-unsaturated-beta-hydroxyesters

Sphingolipids play critical roles in signal transduction, intercellular membrane trafficking and cell growth. As bioactive sphingolipids, ceramide and sphingosine have been implicated in activating anti-proliferative and apoptotic responses in various cancer cells. Conversely, metabolic conversion of ceramide into sphingosine 1-phosphate, ceramide 1-phosphate and glucosylceramide regulates cell proliferation and suppresses ceramide programmed cell death. Many anticancer drugs and stress-induced agonists have been developed to increase endogenous ceramide levels. Sphingosine/ceramide analogues reportedly enhance antitumor activity and have been proposed as a potential new class of chemotherapeutic agents. Among these, sphingosines with aromatic substituents in the side chain often exhibit stronger biological activity compared to natural sphingosines. While a large number of synthetic pathways to sphingosine analogues have been described in the literature, very few pathways provide analogues with high stereospecificity. This dissertation describes a novel synthetic pathway to aromatic sphingosines which is highly stereospecific, provides good yields, uses commonly available reagents, and is versatile in terms of its potential to provide a large family of sphingosine analogues for future research. The entry into the synthetic pathway toward sphingosine analogues involves first the enantiospecific synthesis of trans-γ, δ-unsaturated β-hydroxyesters using biocatalytic reduction of trans-γ, δ-unsaturated-β-ketoesters with commercially available ketoreductases. With the advantages of compatibility with carbon-carbon double bonds, high enantioselectivity, broader substrate acceptance, mild environmentally-friendly reaction conditions and easy separation, the biocatalytic reduction was found to be superior compared to other more traditional chemical methods. Indeed, both (R) and (S)- enantiomers of trans-γ, δ-unsaturated β-hydroxyesters are synthesized by one or more ketoreductases in excellent stereochemical purity and high yield. The enantiopure trans-y, δ-unsaturated β-hydroxyesters were used to prepare erthyro-sphingosine analogues with aromatic substituents in the side chain. The strategy is based on the diastereospecific amination of trans-γ, δ-unsaturated β-hydroxyesters to introduce the amino group and establish anti N-Boc-α-hydrazine-β-liydroxyesters. Proper E1cB non-reductive elimination is essential for the successful cleavage of N-N bond of the hydrazine group. No racemization is detected during the entire course of the synthetic pathway. A total novel synthetic route of sphingosine analogues was thus accomplished with stereochemically pure intermediates and products

Stereospecific capillary electrophoresis assays for methionine sulfoxide reductases

Met, either bound in proteins or in its free form is easily oxidized to Met(O) by reactive oxygen species. Met(O)-containing proteins accumulate during ageing and may play a role in degenerative diseases. Because of the chirality of the sulfoxide moiety, Met(O) exists as a pair of diastereomers Met-S-(O) and Met-R-(O). For the reduction of the diastereomers, stereospecific methionine sulfoxide reductase (Msr) enzymes exist. MsrA reduces free and protein-bound Met-S-(O), while MsrB reduces protein-bound Met-R-(O) with little affinity for free Met-R-(O). The latter is reduced by fRMsr. The aim of the present study was the development of stereospecific CE assays for Msr enzymes in order to study the stereospecificity of the enzymes. The substrates included Met(O), Met(O) derivatives as well as Met(O)-containing peptides. Upon establishment of the separation conditions, the methods were validated and subsequently applied for assaying recombinant human and fungal MsrA and MsrB enzymes. An off-line assay was developed using free Met(O) as substrate. The separation of the Met(O) diastereomers, the product Met as well as β-alanine as internal standard was achieved upon derivatization of the analytes with dabsyl chloride by a MEKC method in a SMIL-coated capillary. The capillary coating consisted of a first layer of polybrene and a second layer of dextran sulfate providing a stable strong cathodic EOF and, consequently, highly repeatable analyte migration times. The assay was subsequently applied to screen the stereospecific activity of recombinant human MsrA and fungal fRMsr enzymes as well as to determine the Michaelis-Menten kinetics. An advantage of the present assay over existing assays is the fact that free Met(O) can be used as the natural substrate. In the assay, DTT was used as reducing agent for Msr recycling instead of using the coupled reaction involving Trx and Trx reductase so that no additional enzymes were required. An in-capillary electrophoretically mediated microanalysis (EMMA) assay was established using Fmoc-Met(O) as substrate. The separation of diastereomers of Fmoc-Met(O), the product Fmoc-Met and the internal standard Fmoc-ß-alanine was also achieved in a SMIl-coated capillary by a MEKC method. The partial filling mode was applied because the BGE contained SDS, which would lead to the denaturation of the enzyme. An injection sequence of incubation buffer-enzyme-substrate-enzyme-incubation buffer was selected. The assay was optimized with regard to the mixing time with a mixing voltage and subsequently applied for the analysis of stereospecificity of human MsrA and MsrB2. The Michaelis-Menten constant, Km, and the maximum velocity, Vmax, were determined. Essentially identical data were determined by the EMMA mode compared to off-line incubations. Compared to the off-line assay, the EMMA assay was fully automated and required smaller amounts of enzyme and substrates, thus, reducing the overall cost of the assay. For the development of an assay using peptide substrates, the C-terminally dinitrophenyl-labeled, N-acetylated pentapeptide KIFM(O)K was chosen. The separation of the KIFM(O)K diastereomers and the reduced peptide KIFMK was achieved in a BGE containing sulfated β-CD and 15-crown-5 as buffer additives. The system was optimized using experimental design with regard to the buffer pH, ionic strength, sulfated β-CD and 15-crown-5, as well as capillary temperature and separation voltage. A fractional factorial response IV design was employed for the identification of the significant experimental factors and a five-level circumscribed central composite design for the final method optimization. The assay was successfully applied for the characterization of the stereospecificity of recombinant human MsrA and MsrB2 including the determination of the Michaelis-Menten kinetic data. Using this peptide substrate, lower Km values but significantly higher kcat constants were observed as compared to literature data reported for the substrates dabsyl-Met(O) and Fmoc-Met(O). Thus, the present pentapeptide-based assay may represent Msr activities towards protein-bound Met(O) in a better way as compared to simple amino acid derivative-based assays. In order to better understand previous results that MsrA enzyme had a preference for peptide substrates with positively charged residues flanking Met(O), a group of peptide substrates was employed in combination with wild-type fungal MsrA and three mutants. The present data were in general agreement with the previous non-stereospecific assay except that the DE mutant (Glu99, Glu134) was not found to be more active than the wild-type enzyme for the reduction of KDM(O)DK. Interestingly, a reversed substrate preference was observed between the mutants DN (Glu99, Asn134) and EQ (Gln99, Asp134) reducing KDM(O)NK and KNM(O)DK. The asymmetric negative charges in the active center may explain this behavior. Molecular modelling was performed to rationalize the specificity and indicated that the conserved residue Glu99 in the active site of MsrA was buried in the Met-S-(O) binding groove, which might contribute to the right placing of Met-S-(O) and, consequently, to the catalytic activity of MsrA. Finally, the dual selector system composing of a CD and a crown ether for the separation of the Met(O) peptide diastereomers was studied systematically using a series of N-acetylated Met(O) pentapeptides with a Dnp label at the C-terminus. Depending on the amino acid sequence and the applied CD, the addition of crown ethers, especially of the Krpytofix® diaza-crown ethers, resulted in a significant improvement of the resolution of the diastereomers of peptides containing basic amino acids. Charged CD derivatives such as carboxymethyl-ß-CD and sulfated ß-CD were superior compared to native ß-CD. In contrast, the diastereomer separation of Met(O) peptides containing uncharged amino acids was found superior in a MEKC system in the presence of CDs