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Two different chiral selectors synthesised in our laboratory were used to test the possibility of separation for a sample consisting of ten different enantiomeric pairs of dansyl-derivatives of α-amino acids in electrokinetic chromatography. It was possible to observe all the peaks, though only partly resolved, due to the twenty analytes through an accurate strategy of choice of the experimental conditions. As a part of this strategy, a procedure of identification of the single peaks in the electropherograms called LACI (lastly added component identification) has been developed.

Thirteen enantiomeric pairs of α-amino acids derivatised with fluorescein isothiocyanate (FITC-AAs) were separated in capillary electrophoresis (CE) using as chiral selectors the single isomer derivatives (SIDs) 3-monodeoxy-3-monoamino-β- and γ-cyclodextrins. The chiral separation data obtained by these strictly homologous compounds, show different behaviours, allowing to hypothesise a possible structure of the obtained selector-analyte complexes, as well as highlighting the crucial role of the cavity size and the significant effects on the resolution obtained by small differences in the structural characteristics of these analytes. © 2012 Elsevier B.V.

The copper(II) complex of a modified cyclodextrin, namely 6-mono-deoxy-6-[4-(2-aminoethyl)imidazolyl]-β-CD (CDmh), previously synthesized and characterized in our laboratory and already used as chiral selector for ligand exchange capillary electrophoresis (LECE) with optical detection, is investigated here in LECE using electrospray-mass spectrometry (ESI-MS) as the detection device. The potential of this hyphenated method is compared with the results previously obtained with optical detection by studying the chiral resolution of tryptophan racemate. Chiral separation conditions compatible with LECE-ESI-MS could be achieved based on the figures of merit obtained by LECE-UV. Interestingly, the values of LOD obtained by LECE-ESI-MS were significantly better than those obtained by LECE-UV and thus, ESI-MS detection seems to open new perspectives in chiral separations by LECE.

The diaminotrehalose-capped derivative of β-CD, a new member of hemispherodextrins, was synthesized and characterized by NMR spectroscopy. Its protonation constants were determined by potentiometry, and the inclusion of both the enantiomers of dansyl-phenylalanine was investigated by NMR spectroscopy. Its stereoselective properties were exploited in electrokinetic chromatography by separating four enantiomeric pairs of dansyl derivatives of amino acids.

In this work, three modified CDs (mCDs), namely 6-deoxy-6-[1-(2-amino)ethylamino]-beta-CD, 6-deoxy-6-[N-(2-methylamino)pyridine)]-beta-CD and 3-monodeoxy-3-monoamino-beta-CD, are investigated as chiral selectors for CE with LIF (CE-LIF) and CE-TOF-MS. The potential of these three mCDs as chiral selectors in CE was also compared with the unmodified beta-CD and gamma-CD. A group of ten different D- and L-amino acids derivatized with FITC was selected as case-study for CE-LIF and CE-MS comparison. The analyte group includes negatively (D/L-Glu and D/L-Asp), neutral (D/L-Ala and D/L-Asn) and positively (D/L-Arg) charged amino acids. Chiral separation conditions compatible with CE-TOF-MS could be achieved based on the figures of merit obtained by CE-LIF using the different CDs. Interestingly, LOD values obtained by chiral CE-TOF-MS were in the nM range comparable or only slightly worse to those obtained by CE-LIF. Moreover, the time of analysis using mCDs was reduced more than 30% compared with the non-chiral conditions (19.2 versus 28.4 min, respectively), with resolution and efficiency values as high as 5.15 and 843 000 plates/m, respectively. The usefulness of this chiral CE-TOF-MS method was corroborated through the detection of the main D- and L-amino acids found in different real samples including transgenic versus wild soy and vinegar.

The possibility of using capillary electrophoresis-electrospray ionisation (ESI)/MS to separate and quantify cobalt and iron was explored. It was necessary to tackle and overcome problems from different sources, some of them being rather unpredictable. The results obtained suggest the occurrence of oxidation processes of metal ions during the formation of the electrospray. The quantitative oxidation of cobalt(II) to cobalt(III), a process at our knowledge never described before, appears particularly interesting. The results obtained, though not optimised for sensitivity appear promising, since a limit of detection of the order of tenths of picograms was obtained. Further, from the comparison with the results obtained by optical detection, the use of CE-ESI/MS appears advantageous.

A capped derivative of beta-CD (THALAH) was synthesized and characterized by NMR spectroscopy at different pH values. A trehalose moiety, bonded through beta-alanine bridges to the CD cavity, is included in the capping unit, giving peculiar properties to this molecule. The hemispherodextrin thus obtained was tested as a chiral selector in EKC. At neutral pH, the monocationic species of THALAH behaves as a very efficient selector separating successfully all the 11 tested enantiomeric pairs of dansyl-derivatives of amino acids, some of them even at concentrations as low as 0.15 mM. The differences observed in the migration order among the different systems give suggestions about the mechanism of molecular recognition between the selector and the analytes.

Six different racemates of the profen family were used as analytes in order to test the chiral selector properties of three members of a new class of cyclodextrin derivatives, hemispherodextrins (HMs), in capillary electrophoresis. In addition to experiments carried out to separate each enantiomeric pair one by one, other experiments were carried out on samples containing all six enantiomeric pairs. Electropherograms were obtained either by adding a single HM to the background electrolyte (BGE), or a binary mixture of HMs. The results obtained confirm the excellent chiral selector properties of the HMs, and furthermore show that these compounds can also be used for achiral selection. When mixing different HMs, a complementary effect in chiral selectivity is observed, which, in our opinion, deserves further study.

A histamine derivative of β-cyclodextrin functionalised at the secondary rim was synthesised and characterised by optical and NMR spectroscopy. Its binary systems both with proton and copper(II) were thermodynamically characterised through pH-metric potentiometry. In addition, the ternary systems with the enantiomers of tryptophan, phenylalanine and alanine were investigated. Thermodynamic stereoselectivity was observed for both the tryptophan and phenylalanine enantiomers. The properties of the synthesised cyclodextrin derivative as a chiral selector were verified in chiral ligand-exchange capillary electrophoresis (CLECE) towards the enantiomeric pairs of some amino acids.

This review highlights recent progresses in the chiral recognition and separation of amino acid enantiomers obtained by capillary electromigration techniques, using different chiral selectors and especially cyclodextrins, covering the literature published from January 2010 to March 2014. Sections are dedicated to the use of derivatization reagents and to the possibility to enantioseparate underivatized amino acids by using either ligand exchange capillary electrophoresis (LECE) and capillary electrophoresis (CE) coupled on line with mass spectrometry. A short insight on frontier nanomaterials is also given.

Potentiometric and spectroscopic methods were used to investigate the stability and bonding details of the copper(II) complexes of β-cyclodextrin (β-CD) functionalized by histamine through the imidazole N-1 (CDmh) and a number of enantiomeric pairs of amino acidates (AaO−) where AaO− is alaninate (AlaO−), phenylalaninate (PheO−) or tryptophanate (TrpO−). Stability constant values were determined at 25 °C and I = 0.1 mol dm−3 (KNO3). Enantioselectivity was observed in the copper(II) mixed complex formation of L/D-TrpO− in aqueous solution and [Cu(CDmh)(L-TrpO)]+ was seen as being favoured over [Cu(CDmh)(D-TrpO)]+. In contrast, the AlaO− systems did not show any differences in the stability of copper(II) diastereomeric ternary complexes. The c.d. spectra of the complexes containing aromatic L-amino acids showed much higher intensity (Δe) compared with the spectra for the corresponding D-amino acids, the difference Δ(Δe) increasing in proportion to the size of the side chain. Subtle differences were found to exist between the ESR parameters of the diastereomeric complexes with TrpO−. However, these tended to disappear upon the addition of a less polar solvent. The results obtained are consistent with a model where the complexation of the L enantiomer to copper already bound to the histamine residue is favoured by the inclusion of the aromatic side chain in the cyclodextrin cavity, this in turn being due to the preferential cis arrangement of the amino nitrogens in the two ligands.

The 3-hydroxy group of one glucopyranosinic ring of γ-cyclodextrin was selectively substituted with an amino moiety to obtain a new compound able to complex copper(II). Indeed, the new ligand, an altro-γ-CD, forms stable complexes with Cu(II), as the analogous 3-amino derivative β-CD previously exploited for the chiral separation of some amino acids by ligand exchange mechanism in capillary electrophoresis. Furthermore, the ligand forms a stable inclusion complex with anthraquinone-2-sulfonate.

A self-complementary decadeoxynucleotide (5′d (CTGGATCCAG)2) was synthesized for the purposes of this study. The systems formed by this decadeoxynucleotide and the dipeptides of the diastereoisomeric pairs Ala–Ala, Ala–Trp and Leu–Phe both with and without copper(II) were investigated by CD spectroscopy. The ternary systems were also submitted to an e.s.r. investigation. The formation of ternary copper(II) complexes was ascertained. In these complexes, the influence exerted on the B-DNA type conformation of the double strand as observed by CD spectroscopy strongly suggests intramolecular ligand–ligand interactions which have not been previously described for this type of systems. The discussion centers on how the type and disposition of the dipeptide side-chains influences the type of interaction in the different complexes, as inferred by the experimental results obtained.

A new capped derivative of cyclomaltoheptaose (CDTHCM)was synthesized by the reaction of6,6′-dideoxy-6,6′-di(S-cysteamine)-α,α′-trehalose with6A,6D-dideoxy-6A,6D-diiodo-cyclomaltoheptaose. TheCDTHCM obtained was characterized, together with itsbehaviour towards protonation. The CDTHCM/ACS(anthraquinone-2-sulfonic acid sodium salt) system wasinvestigated by 1H NMR spectroscopy and by i.c.d.(induced circular dichroism) at two different pHs. Thedeep inclusion of ACS within the CDTHCM cavity, withan association constant about six times largercompared to the β-CD/ACS system was found at pH 6.

DSC measurements of binary, decanucleotide/L,L or L,D Ala-Trp, and ternary decanucleotide/L,L or L,D Ala-Trp/Copper(II), systems have been carried out. The results obtained show the different behaviour of the two diastereoisomers both in the binary and in the ternary systems, ascribable to the different disposition of the side-chains in the dipeptides.

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

In chiral separation, capillary electrophoresis (CE) is a leading technique and, due to the absence of any stationary phase, it is an all-in-solution process, thus allowing accurate study of separation mechanisms. This review focuses on the leading role played by knowledge of solution equilibria for a deep understanding of the phenomena involved in the chiral-discrimination mechanisms at a molecular level in chiral ligand-exchange CE. Speciation diagrams have been accurately calculated on the basis of the stability-constant values reported in the literature for the evaluation of the species present in solution and their concentrations. This analytical approach turned out to be very useful in understanding electrophoretic behavior and should be used systematically to predict and to optimize chiral separations.

A thermodynamic and proton NMR study has been carried out in order to characterize the behaviour of 2′-deoxyadenylyl-(3′→5′)-2′-deoxyadenosine towards the protonation. Conformational changes occurring following two protonation steps are discussed.

The new platinum(II) complexes of β-cyclodextrin functionalized at the primary position with ethylenediamine or with propylenediamine were synthesized and characterized by mass spectrometry, electronic spectrophotometry, and multinuclear NMR spectroscopy. Platinum(II) complexation makes the cavity more asymmetric. These complexes were tested in vitro for their cytotoxic activity. The relevance of the low activity observed regarding the interaction between the cell and the cyclodextrin cavity is discussed.

The functionalized cyclodextrin 6-deoxy-6-[1-(2-amino)ethylamino]-β-cyclodextrin was synthesized, and an NMR, EPR, pH-metric, and calorimetric investigation was carried out in aqueous solution in order to ascertain its behaviour towards protonation and copper(II) complex formation. The thermodynamic parameters of the ternary complex formation with alanine, phenylalanine and tryptophan enantiomeric pairs were also determined (25° C andI=0.1 mol dm−3). No thermodynamic enantioselectivity was observed in these systems, while a chiral, though poor, discrimination was observed in LEC: c.d. spectra also show enantiomeric stereoselectivity. The results of the present investigation, compared with previously reported results, suggest the occurence of acis-complex ⇆trans-complex equilibrium in such systems.

The newly synthesized 6-derivative of β-cyclodextrin with histamine, bound through the N-1 of imidazole was used as a ligand for copper(II) ion, the ligand being characterized by NMR spectroscopy and the copper(II) complex by EPR spectroscopy; the complex was used in chiral ligand exchange chromatography and was able to separate L- and D-tryptophan.

The diaminotrehalose-capped derivative of β-CD, a new member of hemispherodextrins, was synthesized and characterized by NMR spectroscopy. Its protonation constants were determined by potentiometry, and the inclusion of both the enantiomers of dansyl-phenylalanine was investigated by NMR spectroscopy. Its stereoselective properties were exploited in electrokinetic chromatography by separating four enantiomeric pairs of dansyl derivatives of amino acids.

The review focuses on the role of ionic or ionisable single isomer derivatives (SIDs) of cyclodextrins on the separation of chiral analytes in capillary electrophoresis (CE), covering the period since the year 2000. The advantages of using pure compounds are discussed, as well as the ways to optimise the separations in the context of a rational approach to these techniques. Specific attention is paid to the modulation of the selector-analyte interaction. The advantage due to a detailed knowledge of equilibria occurring in solution during the CE run is underlined, particularly in the case of the presence of metal complexes, as occurs in chiral ligand exchange capillary electrophoresis (CLECE).

The three isomeric dihistamine derivatives of β-cyclodextrin (cyclomaltoheptaose) have been prepared and investigated by 1H- and 13C-n.m.r. spectroscopy. For each isomer, neither of the imidazole rings is included in the cavity, but a reciprocal influence of the histamine chains is observed, suggesting that they have an organised structure.

Following a previous thermodynamic and spectroscopic investigation, 13 copper(II)–dipeptide complexes have been characterized by electrospray ionization mass spectrometry (ESI-MS) and visible optical spectroscopy. The data obtained confirm the presence of the species singled out by the thermodynamic approach, showing the ability of electrospray mass spectrometry to give evidence of the presence of a water molecule in the copper(II) in-plane coordination. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Autolysis of Saccharomyces cerevisiae yeast is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work, a genetically modified yeast (LS11) is compared to its isogenic wild type strain (BY4741) after autolysis. Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC–LIF) is used to identify and quantify the main d- and l-amino acids from both strains after accelerated autolysis. The procedure includes amino acids extraction, derivatization with FITC and chiral-MEKC–LIF separation in a background electrolyte composed of 100 mM sodium tetraborate, 30 mM SDS, 20 mM β-CD at pH 10.0. The d- and l-forms of Arg, Asn, Ala, Glu and Asp, corresponding to the major amino acids found in these samples, are separated in less than 30 min with efficiencies up to 800,000 plates/m and high sensitivity (i.e., LODs as low as 40 nM were obtained for d-Arg for a signal to noise ratio of three). From these results it is corroborated that the genetic modification brings a faster autolysis of the yeast, releasing a higher amount of l-amino acids to the medium in a short time. Interestingly, the pattern of release of d-amino acids was also different between the transgenic and the conventional yeast strains., This work was supported by Projects AGL2005-05320-C02-01 and Consolider Ingenio 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Educación y Ciencia), S-505/AGR-0153 (ALIBIRD, Comunidad de Madrid) and HA2006-0057 (Ministerio de Educación y Ciencia).

The thermodynamic stereoselectivity of a wide series of dipeptide complexes with proton and copper (II) ions is examined. In particular, it is shown that non-covalent interactions are the driving forces of the chirality discrimination. Furthermore, the diagnostic character of ΔH° and ΔS° values is brought to light and compared with the spectroscopy approach for correlating the stereoselectivity with the structural properties of complexes.

Enantiomeric pairs of 11 dansyl derivatives of alpha-amino acids were used as analytes in electrokinetic chromatography to test the ability as chiral selectors of two pure derivatives of beta-cyclodextrin: the ethylendiamine derivative in primary position (CDen) and a member of a new class of receptors, the cysteamine-bridged hemispherodextrin THCMH. The selectivity obtained by the presence of the hemispherodextrin, appears particularly promising as shown by the large values of resolution obtained. The importance of a detailed analysis of these data is discussed in terms of suggestions for a rational approach to separation science.

Five pure CD derivatives synthesized in our laboratory were used as chiral selectors in the presence of copper(II) ion. Three enantiomeric pairs of amino acids were submitted to separation experiments in CE, by exploiting the ligand exchange mechanism. The results obtained in the investigated systems, together with those of the analogous systems previously studied, clearly show the usefulness of this technique in chiral separations. By comparing the ligand exchange CE results with potentiometric results, either reported elsewhere or studied here for the first time (system Cu/CDampy/tyrosine), it has been possible to rationalise the separation results. The importance of the availability of pure selectors, and to characterise them both spectroscopically and thermodynamically is discussed.

An aqueous solution spectroscopic (Vis and EPR) study of the copper(II) complexes with the Ac-HGGG-NH2 and Ac-PHGGGWGQ-NH2 polypeptides (generically designated as L) suggests square base pyramids ascribable to [Cu(L)H(-2)] complex species, which contain three nitrogen donor atoms, arising from imidazole and peptide groups, in the equatorial plane and for a pseudo-octahedral geometry in the case of [CuLH-3]- and [Cu(L)H-4]2- which have four nitrogen donor atoms in their equatorial plane. The coordination sphere of the copper complex in the [Cu(L)H(-2)] species, which is present at neutral pH values, is completed by two oxygen donor atoms. ESI-MS spectra ascertained that water molecules are not present in the coordination equatorial plane of this latter species, in comparison with other copper(II) complexes with ligands bearing nitrogen and oxygen donor atoms and surely having equatorial water molecules. This indicates the coordination of a carbonyl oxygen atom in the equatorial plane has to be invoked. However, no direct proof about the involvement of a carbonyl group oxygen donor atom apically linked to copper was obtained, due to the flexibility of these structures at room temperature. Additionally, the low A(ll) value leads one to consider another oxygen atom of a carbonyl group being involved in the apical bond to copper in a fast exchange fashion. This apical interaction, which may also involve a water molecule, is more pronounced in the Cu-Ac-HGGG-NH2 than in the analogous Cu-Ac-PHGGGWGQ-NH2 system, probably because of the presence of tryptophan and proline in the polypeptide sequence.

The bis-amino AB derivative of beta-cyclodextrin on the secondary rim was synthesised and spectroscopically characterised by different techniques. Its binary systems both with protons and copper(II) were thermodynamically characterised by pH-metric potentiometry. In addition the ternary systems with each of the enantiomers of tryptophan and alanine were investigated. A thermodynamic stereoselectivity was observed for the tryptophan enantiomers and this was exploited to separate them by capillary electrophoresis through a ligand exchange mechanism (LECE). LECE separation of racemates of phenylalanine and tyrosine was also obtained.

6-O-Succinil-beta-cyclodextrin (CDsuc6) was synthesized with very good yield by one pot synthesis and characterized by NMR spectroscopy and ESI-MS. It was used as a chiral selector in capillary electrophoresis to resolve catecholamine racemates, namely norepinephrine, epinephrine, terbutaline and norphenilephrine. The CE experiments at pH 5.6 show very promising selector ability by 6-O-succinil-beta-cyclodextrin for the chiral recognition of all the catecholamines tested, while at pH 9.2, only racemic terbutaline was successfully separated.

The systems that the 3-amino derivative of beta-cyclodextrin (CD3NH2) forms with the proton, the copper(II) ion and each of the enantionters of certain amino acids (alanine, phenylalanine, tyrosine and tryptophan) were investigated. The enantioselectivity shown by the potentiometric measurements carried out on the phenylalanine ternary systems was exploited in capillary electrophoresis by ligand exchange capillary electrophoresis (LECE) to obtain the separation of phenylalanine racemate. The tyrosine racemate was also separated by LECE. The comparison between thermodynamic and capillary electrophoresis (CE) results is discussed, in order to get a better insight into the separation mechanism.

A beta-cyclodextrin substituted by an imidazole-bound histamine (CDmh) was successfully used to separate underivatised tryptophan racemate in capillary electrophoresis in the presence of copper(II) ion by the ligand exchange mechanism. To the best of our knowledge, this is the first example of a cyclodextrin derivative behaving as the first coordination sphere ligand in the complex added to the background electrolyte (BGE).

The newly synthesized 6-derivative of β-cyclodextrin with histamine, bound through the N-1 of imidazole was used as a ligand for copper(II) ion, the ligand being characterized by NMR spectroscopy and the copper(II) complex by EPR spectroscopy; the complex was used in chiral ligand exchange chromatography and was able to separate L- and D-tryptophan.

A capped cyclodextrin derivative (THCMH), called hemispherodextrin, was observed to behave as a very efficient chiral selector for a variety of phenoxyacid enantiomeric pairs, both at pH 6 and pH 9. The very low concentration necessary to obtain separation was particularly impressive. The behaviour of THCMH was compared with that of other hemispherodextrins and cyclodextrin derivatives and the conclusions are reported. Some interesting conclusions are drawn by comparing the behaviour of THCMH with that of other hemispherodextrins reported elsewhere.

In order to obtain better abiotic receptors for analytical and kinetic applications, the new capped derivative of cyclomaltoheptaose (CDTH) was synthesized by reaction of 6, 6’-dideoxy-6, 6’-di(S-cysteamine)-α, α’-trehalose with 6A, 6D-dideoxy-6A, 6D-diiodo-cyclo-maltoheptaose. The CDTH-ACS (anthraquinone-2-sulfonic acid sodium salt) system was investigated, by 1H NMR spectroscopy and by i.c.d. (induced circular dicroism), and a deep inclusion of ACS inside the CDTH cavity, with an association constant about six times larger with respect to ACS — β-CD system was found.

The origins of thermodynamic stereoselectivity in proton and copper(II) complexes of diastereoisomeric dipeptides with aromatic side chains were investigated. The use of a combined approach (potentiometric and calorimetric measurements together with spectroscopic studies) made it possible to examine the different forces that drive stereoselectivity. The aromatic side chains seem to assist proton complex formation (in different ways) in comparison with copper(II) complexation. This different role of side-chain residues is ascribed to a possible d-π interaction existing in both the copper(II) complexes with the two diastereoisomeric dipeptides.

The complex formation of [SnMe2]2+ ion with iminodiacetate, oxydiacetate and thiodiacetate ligands has been investigated by potentiometric, calorimetric and multinuclear magnetic resonance measurements in aqueous solution at 25 °C and I= 0.1 mol dm–3(KNO3). Both the thermodynamic values and NMR parameters revealed the flexibility of the organometallic ion, as regards both the Me–Sn–Me angle and its ability to adopt unusual asymmetric stereochemistries. A new stereochemistry was found for the five-co-ordinated iminodiacetate complex, the formation of which exhibits three-site kinetics.

The two different recognition sites of the cyclodextrin (CD) and of the cyclopeptide are present together in the cyclo-(L-histidyl-L-histidyl) capped β-CD 1, the synthesis of which is now reported; compound 1 is able to coordinate metal ions.