molecules

Review The Reciprocal Principle of Selectand-Selector-Systems in Supramolecular Chromatography †

Volker Schurig

Institute of Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany; [email protected]; Tel.: +49-7071-62605 † This contribution is dedicated to Professor Stig Allenmark at his 80th anniversary.

Academic Editor: Yoshio Okamoto Received: 5 October 2016; Accepted: 7 November 2016; Published: 15 November 2016

Abstract: In selective chromatography and electromigration methods, supramolecular recognition of selectands and selectors is due to the fast and reversible formation of association complexes governed by thermodynamics. Whereas the selectand molecules to be separated are always present in the mobile phase, the selector employed for the separation of the selectands is either part of the stationary phase or is added to the mobile phase. By the reciprocal principle, the roles of selector and selectand can be reversed. In this contribution in honor of Professor Stig Allenmark, the evolution of the reciprocal principle in chromatography is reviewed and its advantages and limitations are outlined. Various reciprocal scenarios, including library approaches, are discussed in efforts to optimize selectivity in separation science.

Keywords: supramolecular chromatography; electromigration methods; selectand; selector; reciprocal principle; combinatorial libraries; cyclopeptides; single-walled nanotubes (SWCNTs); fullerenes; cyclodextrins; calixarenes; congeners; enantiomers; molecular recognition; chiral recognition; chiral stationary phases

1. Introduction Separation approaches in highly selective supramolecular chromatography [1,2], with their most refined expression of stereochemical resolution [3], rely on the fast and reversible non-covalent association equilibrium between functionally and/or structurally complementary partners. The designations selector and selectand were introduced by Mikeš into chromatography to avoid ambiguities that may arise from the use of the notations solvent-solute, ligand-substrate, or host-guest [4]. The new terms were coined in analogy to Ashby’s cybernetic operator-operand terminology [5]. This terminology extends to all supramolecular interactions such as antibody-antigen, receptor-substrate, metal ion-ligand etc. In chromatographic partitioning systems, selectors are employed to separate mixtures of selectands which are added to the mobile phase. The selector is either present as a stationary phase or as an additive to the liquid mobile phase. In order to find an optimized selector for a pair of selectands, the role of selector/selectand can be reversed by the reciprocal principle. One molecule of the selectand is now used as the stationary phase and an array of potential structural related selectors are now employed as analytes in the mobile phase. The best identified candidate is then used as the optimized lead stationary phase for the target selectands to be separated. Thus the reciprocal recognition principle applies to selective separation system when selectors and selectands change their role and belong to two or more forms of congeners, analogues, isomers etc. In the special case of chiral recognition, both enantiomers of selectand and selector must be available. This excludes proteins [6], carbohydrates [7] and antibodies [8], although they represent versatile chiral stationary phases (CSPs). Pertinent examples will be described in the following Sections.

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2. Origin of the Reciprocal Principle in Chromatography 2. Origin of the Reciprocal Principle in Chromatography Following the enantioseparation of [5]–[14]helicenes on the optically active charge-transfer agent Following the enantioseparation of [5]–[14]helicenes on the optically active charge-transfer agent R-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic acid (TAPA, Figure 1) or its 2-butyric acid R-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic acid (TAPA, Figure1) or its 2-butyric acid homologue (TABA) [9–11], Mikeš speculated that the function of the selector and the selectand could homologue (TABA) [9–11], Mikeš speculated that the function of the selector and the selectand could be reversed, i.e., optically active helicenes could be used as resolving agents for racemic compounds be reversed, i.e., optically active helicenes could be used as resolving agents for racemic compounds [4]. [4]. Thus, in a reciprocal fashion, if the selectands A1 and A2 are chromatographically separated on the Thus, in a reciprocal fashion, if the selectands A1 and A2 are chromatographically separated on the selector B1 (or B2), the selectands B1 and B2 are separated on the selector A1 (or A2) as well, whereby selector B1 (or B2), the selectands B1 and B2 are separated on the selector A1 (or A2) as well, whereby the respective pairs A1/A2 and B1/B2 refer to isomers (e.g., stereoisomers) or belong to members of the respective pairs A1/A2 and B1/B2 refer to isomers (e.g., stereoisomers) or belong to members homologous series of compounds (Figure 2). Indeed, it was subsequently demonstrated that P-(+)- of homologous series of compounds (Figure2). Indeed, it was subsequently demonstrated that hexahelicene-7,7′-dicarboxylic acid disodium salt could be used as the chiral stationary phase (CSP) P-(+)-hexahelicene-7,70-dicarboxylic acid disodium salt could be used as the chiral stationary phase as π-donor selector for the enantioseparation of N-2,4-dinitrophenyl(DNP)-α-amino acid esters as (CSP) as π-donor selector for the enantioseparation of N-2,4-dinitrophenyl(DNP)-α-amino acid esters π-acceptor selectands by HPLC [12]. as π-acceptor selectands by HPLC [12].

FigureFigure 1. EnantiomericEnantiomeric separation separation of of racemic racemic [5]–[14]helicenes on ((RR)-()-(−)-2-(2,4,5,7-tetranitro-9-)-2-(2,4,5,7-tetranitro-9- fluorenylideneamino-oxy)propionicfluorenylideneamino-oxy)propionic acid acid (TAPA) (TAPA) linked linked to to silica silica gel gel [4]. [4]. ( (aa)) odd odd numbers; numbers; ( (bb)) even even numbers.numbers. Insertions: Insertions: [7]helicene [7]helicene and and TAPA. TAPA.

FigureFigure 2 2.. TheThe reciprocalreciprocal supramolecularsupramolecular recognition recognition principle. principle. If If the the selector selector B B separates separates the the selectands selectands

AA11 andand A2 A, than2, than the theselector selector A is expected A is expected to separate to separate the selectands the selectands B1 and B2 B. A1 iand and B2i are.A homologousi and Bi are compounds,homologous congeners compounds, or isomers congeners (including or isomers enantiomers). (including enantiomers).

The principle of reciprocity by inverting the role of selectand and selector had first been demonstrated via the differentiation of enantiomers by chiral solvating agents (CSA) in NMR

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MoleculesThe2016 principle, 21, 1535 of reciprocity by inverting the role of selectand and selector had first3 ofbeen 35 demonstrated via the differentiation of enantiomers by chiral solvating agents (CSA) in NMR spectroscopyspectroscopy [ 13[13].]. Pirkle Pirkle et et al. al. stated: stated:the the enantiomersenantiomers ofof secondarysecondary andand tertiary tertiary alcohols alcohols may may be be caused caused to to havehave nonequivalent nonequivalent nmr nmr spectra spectra through through the the use use of of appropriate appropriate optically optically active active amines amines as as solvents. solvents. The The converse converse ofof this this phenomenon phenomenon also also obtains; obtains; amine amine enantiomers enantiomers may may have have nonequivalent nonequivalent nmr nmr spectra spectra in in optically optically active active alcoholsalcohols[ 14[14]] and and Pirkle Pirkle and and Hoover Hoover concluded concluded that that the the roles roles of of an an enantiomeric enantiomeric solute solute and and a a CSA CSAmay may bebe interchangeable interchangeablefor for a a given given pair pair of of compounds compounds [ 15[15].]. The Thereciprocal reciprocalprinciple principlewas was later later extended extendedby by PirklePirkle et et al. al. to to enantioselective enantioselective liquid liquid chromatography chromatography [ 16[16,17].,17]. The The researchersresearchers pointedpointed outout that that one one cancan take take one one chiral chiral stationary stationary phase phase (CSP) (CSP) to to develop develop others: others:if if a a CSP CSP derived derived from from (+)-A (+)-A retains retains (+)-B, (+)-B, thenthen a a CSP CSP comprised comprised of of immobilized immobilized (+)-B (+)-Bshould, should, using using the the samesame interactions,interactions, selectivelyselectively retain retain (+)-A (+)-A[ [18,19].18,19]. OrOr put put in in other other words words by by Pirkle Pirkle and and Pochapsky: Pochapsky:This This‘ bootstrapping‘bootstrapping’’method method of of designing designing reciprocal reciprocal CSPs CSPs fromfrom compounds compounds that that themselves themselves resolve resolve on on existing existing CSPs CSPs is is based based on on the the premise premise that that if if two two molecules molecules show show mutualmutual chiralchiral recognition,recognition, thenthen itit doesdoes notnot mattermatter whichwhich ofof thethe two two is is bound bound to to a a stationary stationary support support for for that that recognitionrecognition toto occur.occur. This This is is in in practice practice not not strictly strictly co correct,rrect, for for the the nature nature of attachment of attachment to the to CSP the CSPoften oftenaffect affectchiral chiral recognition. recognition. Within Within limits, limits, however, however, reciprocity reciprocity is isa auseful useful guide guide to to CSP CSP design [[20].20]. TheThe latterlatter importantimportant restrictions restrictions was was repeatedly repeatedly stressed stressed by by Pirkle Pirkle et et al.: al.:The The twotwo situationssituations areare notnot ‘‘mirrormirror imagesimages’’ andand the the relationship relationship is is only only approximate. approximate. The The manner manner of immobilization,of immobilization, for example,for example, has somehas some bearing bearing upon upon the efficacythe efficacy of the of chiral the chiral recognition recognition process process[19]. Thus[19]. Thus deviations deviations from thefromreciprocal the reciprocalprinciple principle may arise may from arise thefrom environment the environment of the of selector the selector in the in stationary the stationa phase,ry phase, e.g., ase.g., the as result the result of immobilization of immobilization and theand presencethe presence of a spacerof a spacer linking linking the selector the sele toctor a chromatographicto a chromatographic matrix matrix [21]. [21]. AA vivid vivid example example ofof thethe de de novo novo design design of of a a CSP CSP for for a a particular particular target target chiral chiral selectand selectand by by the the reciprocalreciprocalprinciple principle involved involveda a series series of of CSPs CSPs developed developed for for the the separation separation of of the the enantiomers enantiomers of of the the non-steroidalnon-steroidal anti-inflammatoryanti-inflammatory drugdrug (NSAID)(NSAID) naproxennaproxen[ 22[22,23].,23]. ByBy the the so-called so-calledimmobilized immobilized guest guest methodmethod,, a a single single enantiomer enantiomer of of naproxen naproxen (Figure (Figure3a) was3a) was immobilized immobilized to form to form a naproxen-derived a naproxen-derived CSP (FigureCSP (Figure3b) which 3b) which was then was usedthen toused identify to identify a potential a potential candidate candidate from from an array an array of test of racematestest racemates as aas de a novode novo enantioselective enantioselective naproxen naproxen selector selector [23 [23].]. This Thisreciprocal reciprocalstudy study also also identified identified the the structural structural requirementsrequirements forfor enantioselectiveenantioselective recognitionrecognition forfor the the target target racemate racemate naproxen. naproxen. TheThe tailor-madetailor-made Whelk-O1Whelk-O1 CSP (Figure (Figure 3c)3c) not not only only showed showed the thehighest highest enantioseparation enantioseparation factor factorfor naproxen for naproxen (α = 2.25) (αbut= 2.25also) enantioseparated but also enantioseparated structurally structurally related NSAIDs related as well NSAIDs as a ashost well of different as a host racemates of different by a racematescombination by of a combinationface-to-edge, ofand face-to-edge, face-to-face and π-πface-to-face interaction πas-π wellinteraction as hydrogen as well bonding as hydrogen [22,23]. bondingThe Whelk-O1 [22,23]. CSP The offered Whelk-O1 a broad CSP offeredspectrum a broad for enantioseparations spectrum for enantioseparations of aromatic selectands. of aromatic The enantioseparation of bis-, tris- and hexakis-adducts of C60 has also been achieved by HPLC on the selectands. The enantioseparation of bis-, tris- and hexakis-adducts of C60 has also been achieved by HPLCWhelk-O1-CSP on the Whelk-O1-CSP (Section 4). (Section4).

(a) (b)(c)

FigureFigure 3. 3.( (aa)) Structure Structure of of naproxen; naproxen; ( b(b)) A A single single enantiomer enantiomer of of naproxen naproxen attached attached covalently covalently or or ionically ionically onon silica; silica; ( c()c The) The optimized optimized CSP CSP Whelk-O1 Whelk-O1 (commercialized (commercialized by by Regis Regis Chemical Chemical Co., Co., Morton Morton Grove, Grove, IL, USA).IL, USA). Reprinted Reprinted (adapted) (adapted) with with permission permission from from [23]. [23].

Some preliminary work on the use of the reciprocal principle has been reported for the design of Some preliminary work on the use of the reciprocal principle has been reported for the design pyrethroid specific CSPs [24]. In a preliminary attempt to design novel CSPs for the resolution of the of pyrethroid specific CSPs [24]. In a preliminary attempt to design novel CSPs for the resolution stereoisomers of the insecticide cypermethrin, the principle of reciprocal interaction has been considered of the stereoisomers of the insecticide cypermethrin, the principle of reciprocal interaction has been via the following rationale: ‘If a CSP based on a pure enantiomer (+)-A resolves a racemate (+/−)-B, then a considered via the following rationale: ‘If a CSP based on a pure enantiomer (+)-A resolves a racemate single pure enantiomer (+)- or (−)-B should resolve (+/−)-A. This is true if no major point of interaction is (+/−)-B, then a single pure enantiomer (+)- or (−)-B should resolve (+/−)-A. This is true if no major point changed when attaching B to silica’. It should be mentioned that by employing the CSP (−)-A instead of of interaction is changed when attaching B to silica’. It should be mentioned that by employing the CSP

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(−)-A instead of (+)-A, a reversal of the elution order of (−/+)-B would obtain. Likewise by employing the CSP (−)-B instead of (+)-B, again a reversal of the elution order of (−/+)-A would obtain. The biologically most active (1R cis S) stereoisomer of cypermethrin has been structurally modified by an allylic group, followed by free radical addition or hydrosilylation reaction on two different silanes and linking (i) a short-chain monofunctional unit and (ii) a long-chain trifunctional unit, respectively, to silica. As cypermethrin can be resolved on N-(3,5-dinitrobenzoyl)-phenylglycine CSP, the reciprocal interaction of the cypermethrin CSP with racemic N-3,5-dinitrobenzoyl-phenylglycinepropylamide was claimed [24]. The actual objective to use the cypermethrin-derived CSPs to identify and optimize the interaction with racemic test compounds from which a single enantiomer will then be bound to silica to afford a cypermethrin-specific CSP still awaits its realization [24].

2.1. Capillary Electromigration Methods (CE) In enantioselective capillary electromigration methods, the non-racemic chiral selector is added to the background electrolyte as part of the mobile phase. The method of enantioseparation of charged selectands with neutral selectors is referred to capillary zone electrophoresis (CZE), whereas the method of enantioseparation of neutral selectands with charged selectors, entailed with self-mobility, is referred to electrokinetic chromatography (EKC) [25]. Chankvetadze argued that from a mechanistic point of view there is no principle difference whether the selectand or the selector is charged in electromigration methods [26]. The philosophy behind this concept has been stated as follows: if a neutral chiral selector can resolve the enantiomers of a charged chiral analyte, then the enantioseparation of the neutral chiral analyte should also be possible with the charged chiral selector [26]. Thus another type of the reciprocal principle applies to electromigration methods. Whereas the enantioseparation of neutral chiral selectands on charged chiral selectors were at first considered not possible in earlier studies of EKC, it was later successfully realized based on the reciprocal concept. Thus positively charged cyclodextrin derivatives [25,27] and negatively charged cyclodextrin derivatives [27–30] were used for the enantioseparation of a multitude of neutral chiral compounds [31,32]. Another reciprocal principle in the achiral electromigration domain has not yet been realized, but may be anticipated. Native α-, β-, γ-cyclodextrin [33] and, more recently, δ-cyclodextrin [34] have been used as an added mobile phase selector for molecular association with various positively or negatively charged selectands in CZE. By inverting their role, a charged selector may be able to separate various cyclodextrin congeners preceding δ-CD (α-, β-, γ-) and, more importantly, congeners following up δ-CD (>δ). In enantioselective capillary electrochromatography (enantio-CEC), a non-racemic chiral selector is used as CSP coated on the inner capillary wall. As the first examples, racemic 1-phenylethanol [35,36] and some non-steroidal anti-inflammatory drugs (NSAIDs, Figure4) were enantioseparated on Chirasil-Dex (permethylated β-cyclodextrin chemically linked to polydimethylsiloxane and thermally immobilized on the inner wall of a 80 cm (effective) × 50 µm I.D. capillary column). In order to find the best cyclodextrin selector for a given racemic analyte by the reciprocal approach, the target molecule may now be used as CSP, whereas the modified cyclodextrin is added to the mobile phase. However, as the cyclodextrins are only available in one enantiomeric form (comprised of D-glucose building blocks) two columns coated with the L- and D-target molecule, respectively, are required. As shown in Figure5 the highest enantioselectivity for phenylalanilol was observed with hydroxypropyl-β-cyclodextrin due to a large retention time difference on the two mirror image CSPs [37]. Although not related to the reciprocal principle, the following approach is of interest in its own right. A dual chiral recognition system can be obtained when one cyclodextrin selector is used as the CSP whereas another one is added to the mobile phase (combination of CEC and EKC). As the enantioselectivities of the bonded and free CDs were opposite, the incremental addition of the sulfonated β-cyclodextrin to a Chirasil-Dex-coated capillary led to an inversion of the elution order of racemic 1,1’-bi-2,2’-naphthol-hydrogenphosphate (20 mM borate/phosphate (pH 7) buffer, at 30 kV) [29]. Molecules 2016, 21, 1535 5 of 35 Molecules 2016, 21, 1535 5 of 35 Molecules 2016, 21, 1535 5 of 35

Figure 4. Enantioseparation of profens by electrochromatography on Chirasil-β-Dex coated and FigureFigure 4.4. EnantioseparationEnantioseparation of profens by electrochromatography on on Chirasil- Chirasil-ββ-Dex-Dex coated coated and and immobilizedimmobilized (df == 0.20 0.20 μµm)m) on on an an 80 80cm cm (effective) (effective) × 50× μm50 I.D.µm fused-silica I.D. fused-silica capillary capillary at 20 °C at and 20 ◦30C kV. and immobilized (dff = 0.20 μm) on an 80 cm (effective) × 50 μm I.D. fused-silica capillary at 20 °C and 30 kV. Buffer: 20 mM Tris/HCl at pH 7. Reproduced from S. Mayer, Doctoral Thesis, University of Tübingen, 30Buffer: kV. Buffer: 20 mM 20 Tris/HCl mM Tris/HCl at pH 7. atReproduced pH 7. Reproduced from S. Mayer, from Doctoral S. Mayer, Thesis, Doctoral University Thesis, of University Tübingen, of Tübingen, Germany, 1993. Tübingen,Tübingen, Tübingen,Germany, Germany,1993. 1993.

Figure 5. Retention time differences of three modified β-cyclodextrins (β-CD as mono-2.6- FigureFigure 5. Retention time differences of three modified β-cyclodextrinsβ (β-CD as mono-2.6-β dimethylphenylcarbamate)Retention on time silica-bound differences D- and of L-phenylalaninol three modified by the-cyclodextrins CEC-experiment ( (50-CD mM as dimethylphenylcarbamate) on silica-bound D- and L-phenylalaninol by the CEC-experiment (50 mM mono-2.6-dimethylphenylcarbamate)ammonium acetate, pH 6.5, 15 kV, column: on silica-bound 68 cm eff. D×- 50 and μmL-phenylalaninol I.D.) [37]. by the CEC-experiment (50ammonium mM ammonium acetate, acetate,pH 6.5, 15 pH kV, 6.5, column: 15 kV, column: 68 cm eff. 68 × cm 50 eff.μm ×I.D.)50 [37].µm I.D.) [37]. 2.2. Combinatorial Library Approaches in Achiral Supramolecular Chromatographic Systems 2.2. Combinatorial Library Approaches in Achiral Supramolecular Chromatographic Systems 2.2. Combinatorial Library Approaches in Achiral Supramolecular Chromatographic Systems The reciprocal principle has been used for the rational design of an optimized selector for The reciprocal principle has been used for the rational design of an optimized selector for molecularThe reciprocal recognition.principle Another has been optimization used for the principle rational is design based ofon an the optimized use of combinatorial selector for molecular selector molecular recognition. Another optimization principle is based on the use of combinatorial selector recognition.libraries aimed Another at identifying optimization the prime principle selector is basedfor a given on the enantioselective use of combinatorial separation selector system. libraries This libraries aimed at identifying the prime selector for a given enantioselective separation system. This approach can also be combined with the reciprocal principle [38,39]. It has generally been accepted approach can also be combined with the reciprocal principle [38,39]. It has generally been accepted

Molecules 2016, 21, 1535 6 of 35 aimed at identifying the prime selector for a given enantioselective separation system. This approach can also be combined with the reciprocal principle [38,39]. It has generally been accepted that the screening of libraries of selectors anchored to solid supports allows the identification of key structural elements responsible for host/guest interaction and molecular recognition through binding assays with soluble selectands. Combinatorial methods have been classified into two major categories. One is based on the synthesis of mixtures and another is based on the parallel synthesis of pure library components (also called the high-throughput approach). In the former approach, a mixture of library components is synthesized and screened simultaneously for the desired property. In the parallel library approach, each library component is synthesized and screened individually. The advantage of the parallel approach is that the library is generally better characterized, whereas the random library technique is advantageous in that a large number of components can be synthesized and screened more efficiently. A classification of the various combinatorial approaches for the preparation and use of CSPs for enantioseparations has been summarized [40]. Peptide libraries have been used to optimize selectors that bind specifically to a desired target protein in the area of protein purification. Solid phase ‘one-bead-one-peptide’ (OBOP) parallel combinatorial libraries have been successfully employed to search for affinity ligands for large proteins. Thus the immobilized linear hexapeptide Try-Asn-Phe-Glu-Val-Leu was identified as a potential host for the purification of S-protein by affinity chromatography [41]. Also an affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu has been developed for the purification of fibrinogen. The selector was identified by screening the solid-phase combinatorial peptide library using an immunostaining technique [42]. In a reversed reciprocal fashion, affinity chromatography employing the immobilized S-protein was used for the screening of affinity peptide selectands from a soluble peptide library consisting of octamers with glycine (Gly) at both termini of each peptide, i.e., Gly-X-X-X-X-X-X-Gly. The six variable centre positions were constructed using random sequences of the six L-amino acids Tyr, Asn, Phe, Glu, Val and Leu. Peptides that were retained specifically on the immobilized S-protein column were eluted by 2% acetic acid. The peptides in the acid eluate were further separated using reversed-phase HPLC. Each separated peptide fraction was collected and the peptide sequences deconvoluted by mass spectrometry (MS/MS). The screenings of the peptide library resulted in twelve affinity peptides and eight out of them contained the essential sequence Asn-Phe-Glu-Val [43].

2.3. Combinatorial Cyclopeptide Library Approaches in Chiral Capillary Electrophoresis Combinatorial cyclohexapeptide libraries were used as novel multicomponent chiral additives for enantioseparation in capillary electrophoresis (CE) by Jung and Schurig et al. in 1996 [44]. It was inferred that the time-consuming screening of a multitude of individual potential chiral selectors can be avoided by employing a whole selector library. The random library approach is then followed by deconvolution steps. The sub-libraries with reduced heterogeneity can then be employed to identify the most enantioselective selectors. In general, cyclopeptide libraries of high molecular diversity can be obtained by including structural variations. By using the nearly 200 commercially available amino acids of defined chirality, 2006 = 64 × 1012 individual cyclohexapeptides are in principle accessible. The number of cyclopeptides can further be increased by varying the ring size and by including non-peptidic components, derivatives with side chain and backbone modifications, and selected building blocks with inverted chirality [44]. Three libraries consisting of 183 = 5832 individual cyclohexapeptides were synthesized as a random mixture and were characterized by electrospray mass spectrometry [44]. Each library had the composition cyclo(O-O-X-X-X-O) and consisted of three defined positions (O) and three randomized positions (X) which represented a mixture of 18 of the common L-α-amino acids except cysteine and tryptophan. To aid ring closure, one position (O) consisted of unnatural D-alanine (ala). When added to the mobile phase in CE, the libraries cyclo(Asp-Phe-X-X-X-ala), cyclo(Arg-Lys-X-X-X-ala) and cyclo(Arg-Met-X-X-X-ala), respectively, enantioseparated Tröger’s base and N-2,4-dinitrophenyl (DNP)- or Fmoc-D,L-glutamic acid (α = 1.04–1.13) (Figure6)[44]. Molecules 2016, 21, 1535 7 of 35 Molecules 2016, 21, 1535 7 of 35

Figure 6. Enantioseparation of racemic Tröger’s base and DNP-D,L-glutamic acid with combinatorial Figure 6. Enantioseparation of racemic Tröger’s base and DNP-D,L-glutamic acid with combinatorial cyclohexapeptides by CE. Conditions: 10 mmol cyclopeptide library cyclo(OOXXXO) in phosphate buffer cyclohexapeptidespH 7.4 (20 mmol). by CE. Capillary: Conditions: 50 cm (effective) 10 mmol × cyclopeptide50 μm I.D., 20 librarykV (left, cyclomiddle)(OOXXXO) and 10 kV in(right), phosphate buffer pHdetection 7.4 (20 at mmol).260 nm (left) Capillary: and 340 nm 50 (middle cm (effective) and right)× [44].50 µm I.D., 20 kV (left, middle) and 10 kV (right), detection at 260 nm (left) and 340 nm (middle and right) [44]. The result raised the question whether all 183 individual components (which are present in the library at high dilution and probably in a non-equimolar ratio) possess the same or, more likely, different 3 Theenantioselectivities result raised the with question individual whether ‘losers and all winners’. 18 individual It may even components be conceived (which that some are members present in the library atexert high an opposite dilution elution and order probably of the selectand in a non-equimolar thus reducing the ratio) overall possess enantioselectivity. the same Cooperative or, more likely, differenteffects enantioselectivities between the individual with individualselectors were ‘losers believed and to winners’.be absent [44]. It may To identify evenbe the conceived prime selector that some membersin exertthe library, an opposite Chiari et elutional. used an order interesting of the deconvolution selectand thus strategy reducing [45]. the overall enantioselectivity. CooperativeThis effects approach between was performed the individual in two steps: selectors (i) the were synthesis believed of sublibraries to be absent with a [ 44progressively]. To identify the increased number of defined positions and (ii) the evaluation of their ability to act as chiral selectors prime selector in the library, Chiari et al. used an interesting deconvolution strategy [45]. in CE for a set of DNP-D,L-amino acids. First of all, Chiari et al. deconvoluted the library cyclo(Arg- ThisLys-X-X-X- approachβ-Ala) was by performed substituting in D two-Ala steps:(ala) of (i) the the library synthesis cyclo(Arg-Lys-X-X-X-ala) of sublibraries with of Jung a progressively and increasedSchurig number et al. [44] of by defined achiral β positions-alanine (Table and 1, (ii) S.1.1) the [45]. evaluation Then a number of theirthe sublibraries ability toS.2.1, act S.2.2 as chiral selectorsand in S.2.3 CE forwere a setprepared, of DNP- wherebyD,L-amino proline acids.was delibera Firsttely of all, selected Chiari for etone al. of deconvoluted the positions X as the it library cyclo(Arg-Lys-X-X-X-facilitates hexapeptideβ-Ala) cyclization. by substituting However,D-Ala only (ala) proline of the in position library fourcyclo (Table(Arg-Lys-X-X-X-ala) 1, S.2.2) exerted a of Jung and Schurighigh enantioselectivity et al. [44] by achiral of the βsublibrary.-alanine (Table1, S.1.1) [ 45]. Then a number the sublibraries S.2.1,

S.2.2 and S.2.3Table were 1. List prepared, of sublibraries whereby of cyclohexapeptides proline was used deliberately in the deconvolution selected process. forone X stands of the for one positions X as it facilitatesof hexapeptide the 18 of the common cyclization. L-α-amino However, acids except only cysteine proline and tryptophan. in position Reprinted four with (Table permission1, S.2.2) exerted a high enantioselectivityfrom [45]. Copyright of the (1998) sublibrary. American Chemical Society.

Numbering Cyclohexapeptide Library Table 1. List of sublibraries of cyclohexapeptides used in the deconvolution process. X stands for one S.1.1: cyclo(Arg-Lys-X-X-X-β-Ala) α of the 18 of the common L- -aminoS.2.1: acids exceptcyclo cysteine(Arg-Lys-Pro-X-X- and tryptophan.β-Ala) Reprinted with permission from [45]. Copyright (1998) AmericanS.2.2: Chemicalcyclo Society.(Arg-Lys-X-Pro-X-β-Ala) S.2.3: cyclo(Arg-Lys-X-X-Pro-β-Ala) NumberingS.3.1: cyclo Cyclohexapeptide(Arg-Lys-Val-Pro-X- Libraryβ-Ala) S.3.2: cyclo(Arg-Lys-Met-Pro-X-β-Ala) S.1.1:S.3.3: cyclo(Arg-Lys-Ile-Pro-X-(Arg-Lys-X-X-X-ββ-Ala)-Ala) S.2.1:S.3.4: cyclocyclo(Arg-Lys-Leu-Pro-X-(Arg-Lys-Pro-X-X-ββ-Ala) S.2.2:S.3.5: cyclocyclo(Arg-Lys-X-Pro-X-(Arg-Lys-Tyr-Pro-X-β-Ala) S.2.3:S.3.6: cyclocyclo(Arg-Lys-X-X-Pro-(Arg-Lys-Phe-Pro-X-ββ-Ala) S.4.1: cyclo(Arg-Lys-Tyr-Pro-Val-β-Ala) S.3.1: cyclo(Arg-Lys-Val-Pro-X-β-Ala) S.4.2: cyclo(Arg-Lys-Tyr-Pro-Met-β-Ala) cyclo β S.3.2:S.4.3: cyclo(Arg-Lys-Met-Pro-X-(Arg-Lys-Tyr-Pro-Ile-β-Ala)-Ala) β S.3.3:S.4.4: cyclo(Arg-Lys-Tyr-Pro-Leu-(Arg-Lys-Ile-Pro-X- β-Ala)-Ala) β S.3.4:S.4.5: cyclocyclo(Arg-Lys-Leu-Pro-X-(Arg-Lys-Tyr-Pro-Tyr-β-Ala)-Ala) β S.3.5:S.4.6: cyclocyclo(Arg-Lys-Tyr-Pro-X-(Arg-Lys-Tyr-Pro-Phe-β-Ala) β S.3.6: cyclo(Arg-Lys-Phe-Pro-X- -Ala) S.4.1: cyclo(Arg-Lys-Tyr-Pro-Val-β-Ala) S.4.2: cyclo(Arg-Lys-Tyr-Pro-Met-β-Ala) S.4.3: cyclo(Arg-Lys-Tyr-Pro-Ile-β-Ala) S.4.4: cyclo(Arg-Lys-Tyr-Pro-Leu-β-Ala) S.4.5: cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) S.4.6: cyclo(Arg-Lys-Tyr-Pro-Phe-β-Ala) Molecules 2016, 21, 1535 8 of 35

The optimized library cyclo(Arg-Lys-X-Pro-X-β-Ala) (S.2.2) was then semipreparatively resolved into three fractionsMolecules 2016 by, 21, reversed-phase 1535 HPLC. The second fraction exerted the highest enantioselectivity8 of 35 Molecules 2016, 21, 1535 8 of 35 and amino acidThe analysisoptimized library revealed cyclo(Arg-Lys-X-Pro-X- that this fractionβ-Ala) (S.2.2) contained was then the semipreparatively hydrophobic resolved amino into acids Val, Met, Ile, LeuthreeThe and fractions optimized Phe. by This reversed-phaselibrary gave cyclo(Arg-Lys-X-Pro-X- rise HPLC. to theThe synthesissecondβ-Ala) fraction (S.2.2) of exertedwas the then six the semipreparatively sublibraries highest enantioselectivity S.3.1–S.3.6. resolved into and (Table1). The sublibrarythreeamino fractionscyclo acid (Arg-Lys-Tyr-Pro-X-analysis by reversed-phase revealed that HPLC. thisβ-Ala) fractionThe second (S.3.5) contained fraction was the selectedexerted hydrophobic the for highest the amino furtherenantioselectivity acids deconvolution,Val, Met, and Ile, since aminoLeu and acid Phe. analysis This gave revealed rise to that the thissynthesis fraction of thecontained six sublibraries the hydrophobic S.3.1–S.3.6. amino (Table acids 1). TheVal, sublibrary Met, Ile, Tyr contains an aromatic system useful for detection purposes. Now the six sublibraries S.4.1–S.4.6 Leucyclo and(Arg-Lys-Tyr-Pro-X- Phe. This gave riseβ -Ala)to the (S.3.5) synthesis was ofselected the six for sublibraries the further S.3.1–S.3.6. deconvolution, (Table since 1). The Tyr sublibrarycontains an were synthesizedcycloaromatic(Arg-Lys-Tyr-Pro-X- system (Table useful1). Inβfor-Ala) adetection similar (S.3.5) waspurposes. fashion selected Now positionfor thethe furthersix sublibraries 5 wasdeconvolution, optimized S.4.1–S.4.6 since were by Tyr a containssynthesized preceding an HPLC separationaromatic(Table into 1). threesystem In a similar fractionsuseful fashionfor detection and position again purposes. 5 identifyingwas optimizedNow the sixhydrophobic by sublibraries a preceding S.4.1–S.4.6 HPLC amino separation were acids synthesizedas into being three essential for enantioselectivity.(Tablefractions 1). Inand a similaragain Phe identifying andfashion Tyr position inhydrophobic position 5 was optimized 5amino provided acids by a as preceding the being highest essential HPLC enantioselectivity separationfor enantioselectivity. into three and hence fractionsPhe and and Tyr again in position identifying 5 provided hydrophobic the highest amino enantioselectivityacids as being essential and hence for enantioselectivity. the final defined the final defined cyclohexapeptide selector (Figure7) has been selected for the enantioseparation of Phecyclohexapeptide and Tyr in position selector 5 (Figure provided 7) has the been highest selected enantioselectivity for the enantioseparation and hence of theDNP- finalD,L -glutamicdefined DNP-D,L-glutamiccyclohexapeptideacid (α = 1.24) acid (Figure selector (α = 8) 1.24) [45].(Figure (Figure 7) has8 been)[45 selected]. for the enantioseparation of DNP-D,L-glutamic acid (α = 1.24) (Figure 8) [45].

cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) Figure 7. The optimized cyclohexapeptide library component, deconvoluted by Chiari et al. [45]. Figure 7.FigureThe optimized 7. The optimized cyclohexapeptide cyclohexapeptide librarylibrary compon component,ent, deconvoluted deconvoluted by Chiari by et Chiarial. [45]. et al. [45].

Figure 8. Enantioseparation of D,L-DNP-amino acids in a running electrolyte containing cyclo(Arg- FigureLys-Tyr-Pro-Tyr- 8. Enantioseparationβ-Ala) (10 ofmM D, Lin-DNP-amino 20 mM sodium acids phosphate in a runni bungffer, electrolyte pH 7.0) containingas chiral mobile cyclo(Arg- phase Figure 8. Enantioseparation of D,L-DNP-amino acids in a running electrolyte containing Lys-Tyr-Pro-Tyr-additive (CMPA)β-Ala) in CE. (10 Column:mM in 20 57 mM cm sodium (effective) phosphate × 50 μ mbu I.D.;ffer, pH−20 7.0)kV, as15 chiral °C. Reprinted mobile phase with cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) (10 mM in 20 mM sodium phosphate buffer, pH 7.0) as chiral mobile additivepermission (CMPA) from [45].in CE. Copyright Column: ( 1998)57 cm American (effective) Chemical × 50 μm Society. I.D.; − 20 kV, 15 °C. Reprinted with ◦ phase additivepermission (CMPA) from [45]. in CE. Copyright Column: (1998) 57 American cm (effective) Chemical× Society.50 µm I.D.; −20 kV, 15 C. Reprinted with permissionThe from overall [45]. deconvolution Copyright (1998) process American required Chemicalthe synthesis Society. and the evaluation of 15 sublibraries insteadThe ofoverall the 54 deconvolution syntheses considered process byrequired a classical the syntprocedurehesis and of seri theal evaluation deconvolution of 15 [46].sublibraries insteadIn of a followthe 54 synthesesup study, severalconsidered cyclo byhexa a claspeptidesical andprocedure cylcohepta of seripeptideal deconvolution analogues of [46]. the identified The overalllibraryIn a membersfollow deconvolution up were study, studied several process as cyclo chiral requiredhexa selectorspeptide thein and countercurrent synthesiscylcoheptapeptide and capillary the analogues evaluationelectrophoresis of the identified of [47]. 15 sublibrariesAt instead oflibrary the operating 54 members syntheses pH were of 7.0, considered studied all of asthe chiral selectors by selectors a classical bear ain positive countercurrent procedure charge,of whilecapillary serial the electrophoresisanalyte deconvolution bears a [47].negative [ 46At]. In a followthecharge. operating upA poly(acrylamide-co-allyl study, pH of 7.0, several all of the cyclo selectors amidehexa peptideof bear D-gluconic a positive and acid-co-allylglycidylcylco charge,hepta whilepeptide the ether)analyte analogues was bears coated a negative onto of the the identified charge. A poly(acrylamide-co-allyl amide of D-gluconic acid-co-allylglycidyl ether) was coated onto the library members were studied as chiral selectors in countercurrent capillary electrophoresis [47].

At the operating pH of 7.0, all of the selectors bear a positive charge, while the analyte bears a negative charge. A poly(acrylamide-co-allyl amide of D-gluconic acid-co-allylglycidyl ether) was coated onto the capillary to suppress the EOF to ensure that the analyte and selector would migrate in opposite directions by counter-current CE. The following changes were made in the deconvoluted cyclohexapeptide cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala). (i) Tyr in position five was Molecules 2016, 21, 1535 9 of 35

substituted by Tyr(3-NO2), Phe, Phe(4-NO2) and Trp; (ii) Tyr in position three was substituted by positively charged Lys, negatively charged Glu and neutral Leu; (iii) Ala was inserted into cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) in the subsequent positions 1–6 to yield conformationally flexible cycloheptapeptides with positional variety. All positively charged cyclohexapeptides are effective Molecules 2016, 21, 1535 9 of 35 chiral selectors for the enantioseparation of DNP-α-amino acids with resolutions factors Rs = 1–5. The chargecapillary in position to suppress 3 did the notEOF haveto ensure any that influence the analyte on and enantioselectivity selector would migrate of DNP-AAs. in opposite directions However, all of the cyclobyhepta counter-currentpeptides failed CE. toThe resolve following DNP-amino changes were acids made [47 ].in This the findingdeconvoluted suggested cyclohexapeptide that the size and rigiditycyclo of the(Arg-Lys-Tyr-Pro-Tyr- cyclopeptide systemβ-Ala). was(i) Tyr important in position fi forve was ensuring substituted chiral by Tyr(3-NO discrimination.2), Phe, Phe(4-NO In subsequent2) NMR studiesand Trp; it (ii) was Tyr recognized in position thr thatee thewas presencesubstituted of by aromatic positively moieties charged Lys, carrying negatively electron-withdrawing charged Glu and neutral Leu; (iii) Ala was inserted into cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) in the subsequent substituents, i.e., nitro groups, were essential for enantiorecognition via π-π stacking interactions and positions 1–6 to yield conformationally flexible cycloheptapeptides with positional variety. All amide/aromaticpositively chargedn-π interactions cyclohexapeptides [47,48 ].are effective chiral selectors for the enantioseparation of DNP-α- Basedamino on acids the optimized with resolutionscyclo (Arg-Lys-Tyr-Pro-Tyr-factors Rs = 1–5. The chargeβ-Ala) in position cyclohexapeptide 3 did not have any selector influence deconvoluted on by Chiarienantioselectivity et al. [45], Jungof DNP-AAs and Schurig. However, et all al. of the also cyclo screenedheptapeptides related failed to cyclopeptides resolve DNP-amino with small structuralacids variations [47]. This finding [49]. Thus suggested aromatic that the tyrosine size and wasrigidity replaced of the cyclopeptide by phenylalanine system was and important tryptophane for ensuring chiral discrimination. In subsequent NMR studies it was recognized that the presence of in position 3 and 5. In both cases the enantioselectivity was reduced for several DNP-amino aromatic moieties carrying electron-withdrawing substituents, i.e., nitro groups, were essential for acids (Tableenantiorecognition2, Figure9) via and π- theπ stacking choice interactions of the aromatic and amide/aromatic amino acid n-π had interactions a pronounced [47,48]. effect on enantioselectivity.Based on the When optimized the five-membered cyclo(Arg-Lys-Tyr-Pro-Tyr- ring ofβ-Ala) proline cyclohexapeptide was opened, selector the deconvoluted rigid structure of the cyclohexapeptideby Chiari et al. [45], was Jung released and Schurig and et accompanied al. also screened by related ring cyclopepti extension.des with Thus small when structural proline was substitutedvariations by 6-aminohexanoic [49]. Thus aromatic acid tyrosine in cyclo was (Arg-Lys-Tyr-replaced by phenylalaninePro-Tyr-β -Ala),and tryptophane no enantioseparation in position 3 at all was observedand 5. In for both various cases the DNP-amino enantioselectivity acids was [49 redu]. Enantioselectivityced for several DNP-amino also broke acids down(Table 2, when Figure the 9) amino and the choice of the aromatic amino acid had a pronounced effect on enantioselectivity. When the acid sequence in cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) was changed to cyclo(Arg-Pro-Lys-Tyr-Tyr-β-Ala) five-membered ring of proline was opened, the rigid structure of the cyclohexapeptide was released and cycloand(Arg- accompaniedPro-Tyr-Lys-Tyr- by ring extension.β-Ala). Thus Thus when the sequenceproline was ‘ aromaticsubstituted amino by 6-aminohexanoic acid-proline-aromatic acid in amino acid’ (Tyr-cycloPro(Arg-Lys-Tyr--Tyr) wasPro essential-Tyr-β-Ala), for no chiral enantioseparation recognition. at all Finally was observed the ring for sizevarious of DNP-amino the cyclopeptide was probedacids [49]. (Table Enantioselectivity3)[ 49]. The also highest broke down enantioselectivity when the amino was acid observedsequence in with cyclo(Arg-Lys-Tyr- cyclohexapeptides. In agreementPro-Tyr- withβ-Ala) results was changed of De to Lorenzi cyclo(Arg- etPro al.-Lys-Tyr-Tyr- [47], enantioselectivityβ-Ala) and cyclo was(Arg- lostPro for-Tyr-Lys-Tyr- conformationallyβ- flexible cycloAla). Thushepta thepeptides. sequence Surprisingly, ‘aromatic amino this acid-pro dropline-aromatic was even more aminopronounced acid’ (Tyr-Pro-Tyr) with was cyclo essentialpentapeptides for chiral recognition. Finally the ring size of the cyclopeptide was probed (Table 3) [49]. The highest β (Table3).enantioselectivity When the cyclohexapeptide was observed withcyclo cyclo(Arg-Lys-Tyr-Pro-Tyr-hexapeptides. In agreement-Ala) with was results opened of De up Lorenzi to the linear peptideet Arg-Lys-Tyr-Pro-Tyr-Lys al. [47], enantioselectivity was by lost exchanging for conformationallyβ-Ala for flexible Lys no cyclo enantioselectivityheptapeptides. Surprisingly, for DNP-amino acids wasthis observed drop was even [49]. more Thus pronounced the cyclo withhexa cyclopeptidepentapeptidescyclo(Arg-Lys-Tyr-Pro-Tyr- (Table 3). When the cyclohexapeptideβ-Ala) identified by Chiari etcyclo al.(Arg-Lys-Tyr-Pro-Tyr- (Figure7)[ 45] indeedβ-Ala) was represents opened up the to the most linear favourable peptide Arg-Lys-Tyr-Pro-Tyr-Lys selector which could by not be optimizedexchanging further. β-Ala for Lys no enantioselectivity for DNP-amino acids was observed [49]. Thus the cyclohexapeptide cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) identified by Chiari et al. (Figure 7) [45] indeed represents the most favourable selector which could not be optimized further. Table 2. Resolution factors Rs of DNP-amino acids on cyclohexapeptides by CE (conditions as in Figure6)[49]. Table 2. Resolution factors Rs of DNP-amino acids on cyclohexapeptides by CE (conditions as in Figure 6) [49]. Amino Acid/Cyclopeptide DNP-Glu DNP-Norleu DNP-Meth DNP-Ala DNP-Norval DNP-Threo Amino Acid/Cyclopeptide DNP-Glu DNP-Norleu DNP-Meth DNP-Ala DNP-Norval DNP-Threo β cyclo(Arg-Lys-Tyr-Pro-Tyr-cyclo(Arg-Lys-Tyr-Pro-Tyr--Ala)β-Ala) 4.974.97 3.80 3.80 5.46 2.27 2.27 3.64 3.64 2.10 2.10 β cyclo(Arg-Lys-Phe-Pro-Phe-cyclo(Arg-Lys-Phe-Pro-Phe--Ala)β-Ala) 2.332.33 1.63 1.63 1.96 1.03 1.03 1.66 1.66 0.59 0.59 β cyclo(Arg-Lys-Trp-Pro-Trp-cyclo(Arg-Lys-Trp-Pro-Trp--Ala)β-Ala) 1.691.69 1.31 1.31 1.21 1.47 1.47 1.93 1.93 0.90 0.90

Figure 9.FigureInfluence 9. Influence of the of changethe change of of Tyr Tyr for for Phe Phe inin the cyclohexapeptide cyclohexapeptide for enantioseparation for enantioseparation of of norvalinenorvaline (conditions (conditions as in as Figure in Figure8)[ 498) ].[49].

Molecules 2016, 21, 1535 10 of 35 Molecules 2016, 21, 1535 10 of 35

TableTable 3. ResolutionResolution factors factors Rs ofR sDNP-aminoof DNP-amino acids acidson cyclopeptides on cyclopeptides of varying of ring varying size by ring CE size (conditions by CE (conditionsas in Figure as 6) in[49]. Figure 6)[49].

Amino Acid/Cyclopeptide DNP-Glu DNP-Norleu DNP-Meth DNP-Ala DNP-Norval DNP-Threo Amino Acid/Cyclopeptide DNP-Glu DNP-Norleu DNP-Meth DNP-Ala DNP-Norval DNP-Threo cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) 4.97 3.80 5.46 2.27 3.64 2.10 cyclocyclo(Arg-Lys-Tyr-Pro-Tyr-(Lys-Lys-Tyr-Tyr-Tyr-Tyr-Lys)β-Ala) 4.970.36 3.80 1.41 5.46 1.53 2.27 1.05 1.46 3.64 0 2.10 cyclo (Lys-Lys-Tyr-Tyr-Tyr-Tyr-Lys)cyclo(Lys-Tyr-Arg-Tyr-β-Ala) 0.360 1.410.62 1.530 1.050.35 0.37 1.46 0 0 cyclo(Lys-Tyr-Arg-Tyr-β-Ala) 0 0.62 0 0.35 0.37 0 cyclo(Arg-Lys-Tyr-Tyr-cyclo(Arg-Lys-Tyr-Tyr-β-Ala)β-Ala) 0.230.23 0.730.73 0.430.43 0.340.34 0.55 0.55 0 0

2.4. Reciprocal Principle in Combinatorial Approaches Utilizing Cyclopeptides—A Failure and a Success 2.4. Reciprocal Principle in Combinatorial Approaches Utilizing Cyclopeptides—A Failure and a Success Instead of a deconvolution strategy, a reciprocal LC approach has been considered by Jung, Schurig Instead of a deconvolution strategy, a reciprocal LC approach has been considered by Jung, et al. to identify an enantioselective hit component in the deconvoluted library (Figure 10) [49]. Schurig et al. to identify an enantioselective hit component in the deconvoluted library (Figure 10)[49].

FigureFigure 10.10. IllustrationIllustration of thethe identificationidentification ofof anan optimaloptimal selectandselectand in aa smallsmall librarylibrary exhibitingexhibiting thethe highesthighest enantioselectivityenantioselectivity towardtoward aa chiralchiral selectorselector usedused eithereither inin the S-- oror R-configuration-configuration asas revealedrevealed byby thethe largestlargest differencedifference ofof itsits retentionretention (earmarked(earmarked byby thethe palepale blueblue colourcolour atat thetheright). right).

Since the cyclopeptide library exists only in one enantiomeric form (obtained from L-amino acids) Since the cyclopeptide library exists only in one enantiomeric form (obtained from L-amino acids) two different columns containing the target racemate separately in the L- and D-form, respectively, are two different columns containing the target racemate separately in the L- and D-form, respectively, are required. The sub-library component present in the mobile phase which exhibits the largest difference required. The sub-library component present in the mobile phase which exhibits the largest difference in its retention on the two mirror-image selectors, should also show the highest enantioselectivity in in its retention on the two mirror-image selectors, should also show the highest enantioselectivity the reciprocal system when used as chiral selector for the racemic non-bonded candidate used as in the reciprocal system when used as chiral selector for the racemic non-bonded candidate used as selectand (Figure 10). selectand (Figure 10). In initial experiments L- or D-DNP-alanine silica (Figures 11 and 12) was filled into In initial experiments L- or D-DNP-alanine silica (Figures 11 and 12) was filled into 100 µm 100 μm I.D. capillaries. Cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) was selected as the analyte as it shows high I.D. capillaries. Cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) was selected as the analyte as it shows high enantioselectivity toward racemic DNP-alanine (Figure 8) [45]. Micro-HPLC measurements were enantioselectivity toward racemic DNP-alanine (Figure8)[ 45]. Micro-HPLC measurements were performed in the reversed phase mode (H2O), in the organic polar mode (methanol/acetonitrile) and performed in the reversed phase mode (H O), in the organic polar mode (methanol/acetonitrile) and in the normal phase mode (toluene, n-hexane/2-propanol).2 However, no measurable retention time in the normal phase mode (toluene, n-hexane/2-propanol). However, no measurable retention time differences for the two mirror image stationary phases were observed. Also the coating of 50 μm I.D. differences for the two mirror image stationary phases were observed. Also the coating of 50 µm I.D. capillaries with L- or D-5-fluoro-2,4-DNP-alanine in the presence of aminopropyltrimethoxysilane in capillaries with L- or D-5-fluoro-2,4-DNP-alanine in the presence of aminopropyltrimethoxysilane in toluene and the investigation of cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) by pressure-supported open-tubular toluene and the investigation of cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala) by pressure-supported open-tubular capillary electrochromatography (OT-CEC) did not reveal differences in retention times as expected capillary electrochromatography (OT-CEC) did not reveal differences in retention times as expected from the reciprocal principle [49]. This unexpected result reinforces the limitation of the reciprocal from the reciprocal principle [49]. This unexpected result reinforces the limitation of the reciprocal principle as enantioselectivity may be reduced by non-specific interactions on silica. This might be principle as enantioselectivity may be reduced by non-specific interactions on silica. This might be circumvented by end-capping of silanol groups or elongation of the linker categories [38,39]. circumvented by end-capping of silanol groups or elongation of the linker categories [38,39].

Molecules 2016, 21, 1535 11 of 35 Molecules 2016, 21, 1535 11 of 35 Molecules 2016, 21, 1535 11 of 35

Figure 11. Linking of L- or D-DNP-alanine to silica (5 μm) via the amine route [49]. FigureFigure 11. 11.Linking Linking of ofL L-- or orD D-DNP-alanine-DNP-alanine to to silica silica (5 (5 μµm)m) via via the the amine amine route route [49]. [49 ].

Figure 12. Linking of L- or D-DNP-alanine to silica (5 μm) via the epoxide route [49]. FigureFigure 12. 12.Linking Linking of ofL L-- or orD D-DNP-alanine-DNP-alanine to silica silica (5 (5 μµm)m) via via the the epoxide epoxide route route [49]. [49 ]. Since the cyclohexapeptide cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala), used as chiral mobile phase selector Since the cyclohexapeptide cyclo(Arg-Lys-Tyr-Pro-Tyr-β-Ala), used as chiral mobile phase selector in CE,Since enantioseparates the cyclohexapeptide DNP-glutamiccyclo(Arg-Lys-Tyr-Pro-Tyr- acid (Figure 8), the reciprocalβ-Ala), principle used as chiral was also mobile tested phase by adding selector in CE, enantioseparates DNP-glutamic acid (Figure 8), the reciprocal principle was also tested by adding in CE,L-DNP-Glu enantioseparates in a first run, DNP-glutamic and D-DNP-Glu acid (Figurein a second8), the run,reciprocal to the principlebackground was electrolyte also tested in bythe adding CE L-DNP-Glu in a first run, and D-DNP-Glu in a second run, to the background electrolyte in the CE experiment. The corresponding elution time of the added cyclohexapeptide was determined by indirect L-DNP-Gluexperiment. in The a first corresponding run, and D elution-DNP-Glu time inof athe second added run,cyclohexapeptide to the background was determined electrolyte by inindirect the CE UV-detection. Unexpectedly also no striking difference of the elution time of the cyclohexapeptide was experiment.UV-detection. The Unexpectedly corresponding also elution no striking time ofdifferen the addedce of cyclohexapeptidethe elution time of wasthe cyclohexapeptide determined by indirect was observed for the mirror-image situation of the two experiments between 15–20 °C and at voltages of UV-detection.observed for Unexpectedlythe mirror-image also situation no striking of the difference two experiments of the elutionbetween time 15–20 of °C the and cyclohexapeptide at voltages of 10–25 kV, again reinforcing the limitation of the reciprocal concept [49]. ◦ was10–25 observed kV, again for the reinforcing mirror-image the limitation situation of of the the reciprocal two experiments concept [49]. between 15–20 C and at voltages For screening a mixture of a combinatorial library for its best selector, also a reciprocal approach of 10–25For kV, screening again reinforcing a mixture theof a limitationcombinatorial of the libraryreciprocal for itsconcept best selector, [49]. also a reciprocal approach was developed by Li et al. in line with Figure 10 [50]. The model study concerned the optimized wasFor developed screening by a mixtureLi et al. ofin aline combinatorial with Figure library10 [50].for The its model best selector, study concerned also a reciprocal the optimizedapproach enantioseparation of (1-naphthyl)leucine ester with a small peptide library (4 × 4 = 16) containing Leu, wasenantioseparation developed by Li of et(1-naphthyl)leucine al. in line with Figure ester with 10[ a50 small]. The peptide model library study (4 concerned× 4 = 16) containing the optimized Leu, Ala, Gly and Pro and four different N-acyl components. In a reciprocal fashion, L-(1-naphthyl)leucine enantioseparationAla, Gly and Pro of and (1-naphthyl)leucine four different N-acyl ester components. with a small In peptidea reciprocal library fashion, (4 × L4-(1-naphthyl)leucine = 16) containing Leu, ester was first immobilized and used as CSP for the enantiomeric peptide libraries obtained from L- Ala,ester Gly was and first Pro immobilized and four different and usedN-acyl as CSP components. for the enantiomeric In a reciprocal peptidefashion, librariesL-(1-naphthyl)leucine obtained from L- and D-amino acids, respectively. L- and D-sublibraries, exhibiting the largest retention differences on esterand was D-amino first acids, immobilized respectively. and L- used and D as-sublibraries, CSP for theexhibiting enantiomeric the largest peptide retention libraries differences obtained on

Molecules 2016, 21, 1535 12 of 35

from L- and D-amino acids, respectively. L- and D-sublibraries, exhibiting the largest retention Molecules 2016, 21, 1535 12 of 35 differences on the L-configurated CSP, were deconvoluted and the best selector for the racemate was identifiedthe L-configurated [50]. The optimizedCSP, were deconvoluted peptide Leu-Gly and the was best then selector attached for the to silica.racemate It enantioseparated was identified [50]. racemic The (1-naphthyl)leucineoptimized peptide Leu-Gly ester with was an then enantioseparation attached to silica. factorIt enantioseparatedα = 3. Thus racemic it was indeed (1-naphthyl)leucine demonstrated thatester CSPs with can an be enantioseparation developed by a reciprocal factor α chromatographic= 3. Thus it was indeed approach demonstrated using enantiomeric that CSPs libraries. can be developed by a reciprocal chromatographic approach using enantiomeric libraries. 2.5. On-Bead Library Combinatorial Approaches 2.5.For On-Bead the enantioselective Library Combinatorial screening Approaches of a target racemate, Weingarten et al. used a resin library containingFor the 60 membersenantioselective of a multimodal screening of receptor a target which racemate, were Weingarten bonded to polystyreneet al. used a beadsresin library (100 µ m) (Figurecontaining 13)[ 6051 members]. The chemicallyof a multimodal encoded, receptor solid-phase which were supported bonded to polystyrene library was beads generated (100 μm) by a mix-and-split(Figure 13) [51]. protocol The chemically employing encoded, three classessolid-phase of chiral supported building library blocks was (Figuregenerated 13 by). Thea mix-and- rationale behindsplit protocol this non-chromatographic employing three classes screening of chiral method building was blocks stated (Figure as follows: 13). IfThe a tightly rationale binding behind and this highly enantioselectivenon-chromatographic(e.g., Rs >screening 10) resin ormethod other solid was material stated wereas follows: readily available,If a tightly then binding it would and be possiblehighly to resolveenantioselective compounds (e.g., by simplyRs > 10) stirring resin orthe other racemate solid material with such were a readily resin andavailable, filtering. then Theit would screening be possible method to employedresolve compounds differently by simply coloured stirring enantiomeric the racemate target with such molecules a resin and that filtering. were labelledThe screening with differentlymethod employed differently coloured enantiomeric target molecules that were labelled with differently coloured dyes. Thus a quasi-racemate included the blue L-α-amino acid derivative and the red coloured dyes. Thus a quasi-racemate included the blue L-α-amino acid derivative and the red D-α- D-α-amino acid derivative. Proline was selected as the test compound. The idea was to treat an amino acid derivative. Proline was selected as the test compound. The idea was to treat an equimolar equimolar mixture of these coloured probe molecules with a library of chiral selectors on synthetic mixture of these coloured probe molecules with a library of chiral selectors on synthetic beads in beads in which each bead carried a different chiral selector (one-bead-one-selector, OBOS). It was which each bead carried a different chiral selector (one-bead-one-selector, OBOS). It was reasoned reasoned that a highly enantioselective binding would yield beads that were either red or blue, whereas that a highly enantioselective binding would yield beads that were either red or blue, whereas non- non-enantioselectiveenantioselective binding binding would would yield yield beads beads that that were were brown. brown. Thus Thus the thebest best selector selector was was directly directly visualizedvisualized through through a a two-colour two-colour differentialdifferential binding screening. screening. The The reddest reddest and and bluest bluest beads beads found found withwith each each probe probe were were then then picked picked andand decodeddecoded to de determinetermine the the structures structures of of their their associated associated chiral chiral selectors.selectors. Finally Finally the the leading leading resolvingresolving resins were were individually individually resynthesized resynthesized on on a gram a gram scale scale and and againagain treated treated with with excess excess red red andand blueblue proline en enantiomers.antiomers. Subsequent Subsequent treatment treatment with with NaOMe NaOMe and and HPLCHPLC quantification quantification of of the the released released dyesdyes providedprovided a a determination determination of of the the enantiomeric enantiomeric excess excess (ee) ( eeof) of thethe proline proline derivatives derivatives bound bound toto thethe beads.beads. The iden identifiedtified enantioselective enantioselective resolving resolving resin resin was was then then testedtested via via its its ability ability to to kinetically kinetically resolveresolve racemicracemic proline ester ester in in solution solution [51]. [51 ].

FigureFigure 13. 13.Structure Structure of of the the multimodalmultimodal re receptorceptor array array and and kinetic kinetic resolution resolution of ofdifferently differently dye-tagged dye-tagged solid-supportedsolid-supported amino amino acids acids from from a 60-member a 60-member library. library. Module Module A contains A contains 15 different 15 differentD- and D-L -aminoand acids,L-amino module acids, B module contains BRR containsor SS RRdiamine or SS diamine enantiomers, enantiomers, and the and receptor the receptor module module C contains C containsRRRR or SSSSRRRRenantiomers or SSSS enantiomers resulting in resulting 15 × 2 × in2 15 = 60× 2 different × 2 = 60 library different members library [members51]. Figure [51]. 13 Figurereproduced 13 withreproduced permission with from permission [52]. from [52].

MoleculesMolecules2016 2016, 21, 21, 1535, 1535 1313 of of 35 35

2.6. On-Bead Combinatorial Approach of Individual Library Members 2.6. On-Bead Combinatorial Approach of Individual Library Members Fréchet et al. used an on-bead combinatorial approach to the rational design of optimized CSPs for HPLC.Fréchet At et first al. used a library an on-bead of 3 × 12 combinatorial = 36 L-α-amino approach acid anilides to the rational(consisting design of 3 of α-amino optimized acids CSPs and for12 HPLC.aromatic At amines) first a library was synthesized of 3 × 12 = 36in Lsolution,-α-amino then acid attached anilides (consistingto functionalized of 3 α -aminomacroporous acids andpolymeric 12 aromatic methacrylate/dimethacrylate amines) was synthesized beads in solution, which were then attachedpacked into to functionalized a ‘library column’ macroporous and tested polymericfor the enantioseparation methacrylate/dimethacrylate of the target beads analyt whiche. The were lead packed selector into aof ‘library the library, column’ i.e., and L-proline-1- tested for theindananilide, enantioseparation employed of the for target the HPLC analyte. enan Thetioseparation lead selector of ofthe the target library, racemate i.e., L-proline-1-indananilide, N-(3,5-dinitrobenzoyl)- employedleucine diallylamide, for the HPLC was enantioseparationidentified by a deconvolut of theion target process racemate using N11-(3,5-dinitrobenzoyl)-leucine ‘sublibrary columns’ of lower diallylamide,diversity, each was of identifiedwhich containing by a deconvolution a CSP with a process reduced using number 11 ‘sublibrary of library columns’components of [53,54]. lower diversity,Fréchet et each al. also of whichcombined containing the library a CSP approach with a and reduced the reciprocal number principle of library for components the development [53,54 ].of Fréchetenantioselective et al. also CSPs combined for HPLC the library[55,56]. approachThus accord anding the to reciprocalFigure 14,principle a single enantiomer for the development (+)-T of the oftarget enantioselective racemate was CSPs at first for HPLCimmobilized [55,56 ].onto Thus a polymeric according support to Figure and 14 ,the a single resulting enantiomer CSP was (+)-T used offor the the target HPLC racemate screening was of atsublibraries first immobilized of racemic onto compounds a polymeric that support have been and prepared the resulting by parallel CSP wascombinatorial used for the synthesis. HPLC screening The best enantioseparated of sublibraries of me racemicmber compoundsof this library that (±)-S have is then been prepared prepared as bythe parallel single enantiomer combinatorial (−)-S, synthesis. coupled to The a solid best support enantioseparated and used in member the second ofthis step library for the (±required)-S is thenenantioseparation prepared as the of the single target enantiomer racemate (±)-T. (−)-S, This coupled reciprocal to amethodology solid support has and been used exemplified in the second for the steptarget for racemate the required N-(3,5-dinitrobenzoyl)-leucine enantioseparation of the and target the parallel racemate library (±)-T. components This reciprocal of racemicmethodology 4-aryl-1,4- hasdihydropyrimidines been exemplified forobtained the target in a racemateone-pot Nthree--(3,5-dinitrobenzoyl)-leucinecomponent reaction (Biginelli and thedihydropyrimidine parallel library componentsthree-component of racemic condensation 4-aryl-1,4-dihydropyrimidines reaction) [56]. Hydrogen-bonding obtained in a one-pot and π three-component-π interactions played reaction a (Biginellidominant dihydropyrimidine role in chiral recognition. three-component condensation reaction) [56]. Hydrogen-bonding and π-π interactions played a dominant role in chiral recognition.

FigureFigure 14. 14.Reciprocal Reciprocalcombinatorial combinatorial approach approach to the to optimization the optimization of a CSP. of Reproduced a CSP. Reproduced with permission with ofpermission the Royal Societyof the Royal of Chemistry Society of from Chemistry [55]. from [55].

LibrariesLibraries of of potential potential chiral chiral selectors selectors have have also also been been prepared prepared by by the the Ugi Ugi three-component three-component reaction reaction ofofβ β-lactams-lactams andand screened for for their their enantioselectivity enantioselectivity by by using using the the reciprocalreciprocal approachapproach involving involving a CSP a CSPcomprised comprised of the of theimmobilize immobilizedd model model target target compound compound N-(3,5-dinitrobenzoyl)-N-(3,5-dinitrobenzoyl)-L-leucineL-leucine [57]. [ 57The]. Thelead lead candidate candidate identified identified from from the the library library of of race racemicmic phenylamides phenylamides of 2-oxo-azetidine2-oxo-azetidine aceticacetic acid acid derivativesderivatives was was subsequently subsequently synthesized synthesized in in bulk. bulk. The The up-scaled up-scaled racemate racemate was was then then resolved resolved by by chiral chiral preparativepreparative HPLC HPLC and and one one enantiomer enantiomer was was immobilized immobilized on on a a solid solid support. support. The The obtained obtained CSP CSP showed showed enantioselectivities of α = 3 for various amino acid derivatives. Interestingly, the enantioselectivities

Molecules 2016, 21, 1535 14 of 35 enantioselectivities of α = 3 for various amino acid derivatives. Interestingly, the enantioselectivities observed were even higher than those obtained during the reciprocal screening on the immobilized target compound N-(3,5-dinitrobenzoyl)-L-leucine [57].

2.7. Batch-Screening of Peptide Libraries and the Reciprocal Principle Previous studies by Pirkle, Hyun et al. had shown that the enantiomers of racemic N-(3,5-dinitrobenzoyl)-peptide selectands are enantioseparated on a number of CSPs [58]. It was therefore reasoned that in a reciprocal fashion N-(3,5-dinitrobenzoyl)-peptide CSPs should be capable of enantioseparating various racemic compounds [59]. In an off-column batch-screening approach, Welch et al. probed the selector-selectand interaction directly, rather than resorting to a bonded analogue of the selectand used as CSP and packed into a column [60,61]. Thus the individual components of a parallel library of 50 silica-supported N-(3,5-dinitrobenzoyl)-dipeptide CSPs were prepared by solid phase synthesis on aminopropylsilica (APS) particles [59]. Whereas the ‘aa1’ position (Figure 15, top) consisted of the five D-α-amino acids Phg, Val, Pro, Gln and Phe, the ‘aa2’-position (Figure 15, top) was comprised of the same five α-amino acids in the L- and D-form, respectively, giving rise to 50 different dipeptides. The members of the library were subsequently screened for the enantioselective recognition of the test racemate N-(2-naphthyl)alanine diethylamide by an efficient batch-screening process. Thus individual vials containing approximately 50 mg of the candidate CSPs were placed into an autosampler and a dilute solution of the target racemate was added to each vial. The concentration (1 mL; 10−5 M in 2-propanol/n-hexane (20/80, v/v)) of the analyte solution was low enough to avoid saturation of the available interaction sites on the CSP. During equilibration using a rotary platform shaker, the individual enantiomers were partitioned between the solid and liquid phases of the vial. After equilibration, enantioselective HPLC of 50 µL injections of the supernatant solution in each vial afforded two peaks for the individual enantiomers. A 1:1 ratio revealed no enantioselectivity of the adsorbent, whereas ratios up to 1:2.72 indicated the presence of an enantioselective adsorbent. It was found that the homochiral combinations (DD) of the library components exhibited a higher enantioselectivity than the heterochiral combinations (LD) [60]. The presence of steric bulk in the ‘aa 2’ position and of hydrogen-bonding groups in the ‘aa1’ position proved to be important for enantiomeric discrimination of the test racemate. APS-bonded D-Gln-D-Val-DNB, D-Pro-D-Val-DNP and D-Gln-D-Phe-DNP were identified as the leading enantioselective selectors for racemic N-(2-naphthyl)alanine diethylamide. Based on these findings, an improved library of individual dipeptides was designed. It included amino acids with side chain functional groups such as Glu, His, and Arg, Whereas the ‘aa1’ position (Figure 15, top) consisted of the seven L-α-amino acids Gln, Asn, Ser, His, Asp, Arg and Glu, the ‘aa2’-position (Figure 15, top) was comprised of the L-α-amino acids Val, Leu, Ileu, Phe, Tyr, tert-Leu and Trp and the D-α-amino acids Val, Leu, Ileu, Phe, Tyr and Trp, respectively, giving rise to 91 different dipeptides. N-(2-naphthyl)alanine diethylamide was enantioseparated on APS-bonded L-Glu-L-Leu-DNP with the high enantioselectivity of α = 20.74 (Figure 15, bottom) [61]. Up to 100 mg of the compound could be enantioseparated using an analytical column (4.6 mm × 250 mm I.D.) in a single injection. The versatile approach of Welch et al. permitted the identification of the necessary structural requirements for chiral recognition by comparing the relative performance of various CSPs in the library. The enantioselective library approach, commercialized jointly by Regis Technology and MediChem Research [reported in: Chem. Eng. News 1999, 11, p. 101], may also prove useful for the discovery of improved selectors for other types of supramolecular chromatographic interactions. Molecules 2016, 21, 1535 15 of 35

Molecules 2016, 21, 1535 15 of 35

Figure 15.Figure Top 15.: DNB-dipeptide Top: DNB-dipeptide libraries libraries used used as as CSP CSP forfor the the enantioseparation enantioseparation of N of-(2-naphthyl)alanineN-(2-naphthyl)alanine diethylamide. Bottom: The optimized aminopropylsilica-bonded L-Glu-L-Leu-DNB selector enantioseparates diethylamide. Bottom: The optimized aminopropylsilica-bonded L-Glu-L-Leu-DNB selector the test racemate with high enantioselectivity. Column: 4.6 mm × 250 mm I.D., eluent: 2-propanol/n- enantioseparates the test racemate with high enantioselectivity. Column: 4.6 mm × 250 mm I.D., hexane (20/80 v/v). Reprinted with permission from [61]. Copyright (1999) American Chemical Society. eluent: 2-propanol/n-hexane (20/80 v/v). Reprinted with permission from [61]. Copyright (1999) AmericanAn Chemical improved Society. strategy for the evaluation of common CSPs by the straightforward single-vial- equilibration-approach of Welch et al. was based on rapid LC/MS screening of isotopically labelled pseudoenantiomers present in the supernatant instead of the HPLC analysis of the enantiomers [62]. An improved strategy for the evaluation of common CSPs by the straightforward single-vial- Concurrently, another batch-wise screening method was described by Wang and Li [63]. The equilibration-approachapproach was almost of Welchidentical et to al. that was of based Welch onet al. rapid with LC/MS the exception screening that the of incubation isotopically was labelled pseudoenantiomersperformed withpresent the selector in the attached supernatant to a polymeri insteadc resin, of the instead HPLC of analysisa chromatographic of the enantiomers support. The [62]. Concurrently,reliability of the another established batch-wise stepwise solid-phase screening Merrifield-type method was peptide described synthesis by was Wang considered andLi [63]. The approachas an advantage. was almost However, identical it was to also that recogniz of Welched that et the al. screening with the resu exceptionlt would not that necessarily the incubation was performedcorrelate with the the chromatographic selector attached experiment to a polymeric as no chromatographic resin, instead support of a was chromatographic used for the batch support. screening step [38,39]. Crucial elements of the approach were the reciprocity of chromatographic The reliability of the established stepwise solid-phase Merrifield-type peptide synthesis was considered separation and the use of enantiomeric libraries. Thus, a potential chiral selector from the parallel library as an advantage.was linked onto However, a solid-phase it was resin. also The recognized racemic an thatalyte thein the screening proper solv resultent was would then allowed not necessarily to correlateequilibrate with the with chromatographic this potential selector experiment on the re assin. no The chromatographic enantiomeric ratio supportof the analyte was usedin the for the batch screeningsupernatant step was [38 analysed,39]. Crucial after the elements equilibration of the period approach by circular were dichroism. the reciprocity A selectiveofchromatographic adsorption separationof one and of thethe two use enantiomers of enantiomeric to the resin libraries. was indicative Thus, a of potential an efficient chiral chiral selector selector. fromIt was thethen parallel linked onto a chromatographic support, and its enantioselectivity toward the target analyte was library was linked onto a solid-phase resin. The racemic analyte in the proper solvent was then allowed evaluated. The feasibility of the parallel library screening procedure was compared with the model to equilibratesystem withstudied this earlier potential of the ch selectoriral HPLC on enantioseparation the resin. The enantiomericof N-(1-naphthyl)leucine ratio of ester the analyteusing a in the supernatant16-member was analysed small library after [50]. the equilibration period by circular dichroism. A selective adsorption of one of theBluhm two et enantiomers al. simplified the to thereciprocal resin system was by indicative using onlyof one an library efficient (all-L)chiral which was selector. equilibrated It was then linked ontoon two a different chromatographic columns containing support, the immobilized and its enantioselectivity target racemate (1-naphthyl)leucine toward the targetester either analyte in was evaluated.the L The- or feasibilityenantiomeric of D-form, the parallel followed library by incubation screening of each procedure CSP with was a mixture compared library with [64]. The the model adsorbed members of the library on the two columns were washed off and analysed by a reversed system studied earlier of the chiral HPLC enantioseparation of N-(1-naphthyl)leucine ester using a phase HPLC assay. Components with the same retention time but different intensities in both 16-memberchromatograms small library represented [50]. an optimized selector. Its chemical structure was then determined by BluhmLC-MS et or al. LC-MS-MS. simplified In the the reciprocalreciprocal fashion,system the optimized by using candidate, only one i.e., library immobilized (all-L) N which-3,5- was equilibrateddinitrobenzoyl- on two differentL-leucine columnsester, was containing used as CSP the to immobilizedenantioseparate target racemic racemate N-(1-naphthyl)leucine (1-naphthyl)leucine ester eitherester inwith the theL high- or enantioseparation enantiomeric D factor-form, α = followed 12 [64]. In an by optimized incubation study, of a each 200-member CSP with parallel a mixture library [64]. The adsorbed members of the library on the two columns were washed off and analysed by a reversed phase HPLC assay. Components with the same retention time but different intensities in both chromatograms represented an optimized selector. Its chemical structure was then determined by LC-MS or LC-MS-MS. In the reciprocal fashion, the optimized candidate, i.e., immobilized N-3,5-dinitrobenzoyl-L-leucine ester, was used as CSP to enantioseparate racemic N-(1-naphthyl)leucine ester with the high enantioseparation factor α = 12 [64]. In an optimized Molecules 2016, 21, 1535 16 of 35 study, a 200-member parallel dipeptide library was created and screened with 10-undecenyl-N- (1-naphthyl)leucinate [65]. The library was prepared using Fmoc-protected α-amino acids with amino-methylated polystyrene (AMPS) resin as solid support. The library contained three modules. Modules 2 and 3 consisted of ten different L-α-amino acids, whereas the N-terminating module 1 was an acetyl group or a 3,5-dinitrobenzoyl (DNB) group. Each single library member was incubated with a solution of racemic 10-undecenyl-N-(1-naphthyl)leucinate. After equilibration, the supernatant was collected and assessed for enantiomeric excess ee by circular dichroism (CD). In another study, an 81-member dipeptide library was evaluated for the target compound containing a stereogenic phosphorus atom, i.e., tert-butyl-1-(2-methylnaphthyl)phosphane oxide [66]. The member with the highest enantioselectivity was DNB-D-Asn-L-Thr-Abu-AMPS (Abu = 4-amino- butanoic acid). It was attached to aminopropylsilica and used as CSP for the target racemate in HPLC. However, the enantioselcetivities between the batch-experiment and the chromatographic experiment did not match due to the different solid supports. Therefore a longer linker had to be employed to immobilize the chiral selector onto silica to observe an enantioseparation factor of α = 3.2 for the racemic phosphine oxide. A 121-member peptide library was also tested for the enantioseparation of atropisomeric 1,10-bi-2,20-naphthol. A trityl-protected di-asparagine selector (Fmoc-Asn(Trt)-Asn(Trt)) yielded a separation factor of α = 7.2 [67]. By the reciprocal principle, it can be assumed that the target racemate 1,10-bi-2,20-naphthol, for which a highly enantioselective peptide has been identified, could also be used as a CSP for the enantioseparation of the racemic peptides. Another high throughput screening protocol was proposed for chiral selector discovery. It was modeled after the protocol for biological screening of candidate drugs from chemical libraries. The procedure was based on target distribution between an aqueous phase and an organic phase [68].

3. Single-Walled Carbon Nanotubes, SWCNTs In single-walled carbon nanotubes (SWCNTs), a single layer graphene sheet is rolled-up into a tubular structure. The use of SWCNTs as selective stationary phases for the chromatographic separation of various classes of achiral compounds has been reviewed [1]. SWCNTs exist in three forms, i.e., two achiral structures (armchair and zig-zag) and a chiral structure. Chiral single-walled carbon nanotubes (n,m)-SWCNTs are characterized by n,m- indices, where n and m are coordination numbers of the carbon atoms in the hexagonal network. By convention, when n is greater than m, the species is right-handed or P (for positive), and when m is greater than n, the species is left-handed or M (for negative). (n,m)-SWCNTs represent interesting supramolecular entities for the reciprocal chromatographic selector/selectand relationship. The enantiomers of nine pre-separated single chirality (n,m)-SWCNTs of varying enantiomeric purity were separated on Sephacryl gel containing allyl-dextran as the chiral selector by gel permeation chromatography (Figure 16)[69]. The successful enantioseparation was proved by their opposite CD-spectra. The enantiomeric purities are not known and they varied for different (n,m)-single chirality SWCNTs with the second eluted enantiomer being more enriched [69]. The fractionation of (n,m)-SWCNTs by countercurrent chromatography also led to chiral recognition [70]. The method was based on the partition of sodium deoxycholate (SDC) dispersed (n,m)-SWCNTs in a polyethylene glycol/dextran aqueous two-phase system. The (n,m)-dependent bimodal elution peak width of the chiral nanotubes were interpreted as enantiomeric recognition in the presence of chiral SDC and polymeric dextran [70]. Dextran represents a complex branched glucan (a polysaccharide made of D-glucose building blocks). By the reciprocal approach, nonracemic (n,m)-SWCNTs may be considered in the future as novel CSPs for the enantiomeric separation of lower linear dextrins (‘acyclodextrins’, maltooligosaccharides) which are readily available in both enantiomeric forms obtained from D- and L-glucose, respectively. Incidentally, acetylated/silylated dextrins with a different degree of oligomerization and devoid of a molecular cavity have been used as CSPs for the enantioseparation of derivatized amino acids and various underivatized racemic compounds by GC, thus highlighting the role of the polar external surface of the selector for chiral recognition in contrast to the well-established inclusion mechanism exerted by cyclodextrins [71]. Molecules 2016, 21, 1535 17 of 35 Molecules 2016, 21, 1535 17 of 35

Figure 16. Illustration of the enantioseparation of P- and M-SWCNTs by gel permeation liquid Figure 16. Illustration of the enantioseparation of P- and M-SWCNTs by gel permeation liquid chromatography on an allyl-dextran stationary phase. Reprinted with permission from [69]. Copyright chromatography on an allyl-dextran stationary phase. Reprinted with permission from [69]. Copyright (2014) American Chemical Society. (2014) American Chemical Society. Using molecular dynamics simulations, Raffaini and Ganazzoli investigated in a theoretical studyUsing the molecularadsorption dynamicsand denaturation simulations, of an oligopeptide Raffaini and formed Ganazzoli by 16investigated chiral L-α-amino in a acids theoretical and studyhaving the a adsorption helical structure and denaturation in the native of state an oligopeptideon both the inner formed and by the 16 outer chiral surfaceL-α-amino of the acids chiral and having(10,20)- a helicaland (20,10)-SWCNTs structure in the with native an opposite state on handedness, both the inner and and of the armchair outer surface (16,16)-SWCNT of the chiral (10,20)-nanotube and with (20,10)-SWCNTs a similar diameter with for an comparison opposite handedness, [72]. The molecular and of simulations the armchair indicated (16,16)-SWCNT that the nanotubeenantioseparation with a similar of the diameter chiral carbon for comparison nanotubes [ 72by]. chromatographic The molecular simulations methods are indicated feasible thatusing the enantioseparationoligopeptides of a of sufficient the chiral length. carbon It was nanotubes also suggested by chromatographic that natural oligopeptides methods are could feasible not only using oligopeptidesbe used to separate of a sufficient enantiomeric length. Itnanotubes, was also suggestedbut in the thatreciprocal natural fashion oligopeptides also chiral could nanotubes not only of be usedsingle to separate handedness enantiomeric could be nanotubes,considered as but chiral in the selectorsreciprocal forfashion racemic also oligopeptides chiral nanotubes (Giuseppina of single handednessRaffiani, personal could be communication, considered as 2016). chiral selectors for racemic oligopeptides (Giuseppina Raffiani, personalUnspecified communication, chiral (n,m 2016).)-SWCNTs were tested as CSPs for the enantioseparation of twelve classes of racemic pharmaceuticals by nano-LC [73,74]. The finding would require that the commercially Unspecified chiral (n,m)-SWCNTs were tested as CSPs for the enantioseparation of twelve available chiral (6,7)-SWCNT (carbon >90%, 0.7–0.9 nm diameter by fluorescence) obtained from Sigma- classes of racemic pharmaceuticals by nano-LC [73,74]. The finding would require that the Aldrich was nonracemic or even enantiomerically pure. Neither the mode of the required enantiomeric commercially available chiral (6,7)-SWCNT (carbon >90%, 0.7–0.9 nm diameter by fluorescence) enrichment of the synthetic racemic (6,7)-SWCNT nor its chiroptical data was reported. Some racemic obtained from Sigma-Aldrich was nonracemic or even enantiomerically pure. Neither the mode compounds showed a deviation of the expected 1:1 peak ratio and no enantioseparation was noticed for ofthe chiral required (7,8)-SWCNTs. enantiomeric Also enrichment in another report of the of synthetic the continuous-flow racemic (6,7)-SWCNT enantioseparation nor itsof carvedilol chiroptical datawith was fluorescent reported. detection Some racemic on chiral compounds carbon nanotu showedbes, chiroptic a deviation data and of enantiomeric the expected compositions 1:1 peak ratio andof nothe enantioseparationchiral selector were wasnot mentioned noticed for [75]. the As chiral chiral (7,8)-SWCNTs. SWCNTs contain Also equal in amounts another reportof left- and of the continuous-flowright-handed helical enantioseparation structures, their of carvediloluse as CSP withrequires fluorescent the separation detection of these on non-superimposable chiral carbon nanotubes, mirror chiropticimage forms data into and single enantiomeric enantiomers compositions of defined ofenantiomer the chiralic selectorpurity as were outlined not mentionedby Peng et al. [75 [76].]. As chiral SWCNTsIn containorder to equal investigate amounts whether of left- andthe right-handeduse of SWCNTs helical can structures,improve enantioseparations their use as CSP requires on an the separationexisting of CSP, these comprised non-superimposable of the chiral mirror nonracemic image forms ionicinto liquid single (R enantiomers)-N,N,N-trimethyl-2-aminobutanol- of defined enantiomeric puritybis(trifluoromethane-sulfon)imidate, as outlined by Peng et al. [76]. two capillary columns, i.e., one containing the chiral ionic liquid aloneIn orderand the to other investigate containing whether the chiral the ionic use ofliquid SWCNTs together can with improve the SWCNTs enantioseparations were tested by onGC an existing[77]. It CSP,was shown comprised that the of capillary the chiral column nonracemic containing ionic SWCNTs liquid (improvedR)-N,N,N the-trimethyl-2-aminobutanol- enantioselectivity of the bis(trifluoromethane-sulfon)imidate,chiral ionic liquid. The ‘nonchiral’ role two of capillarythe SWCNT columns, was due i.e., to onean in containingcrease of the the surface chiral area ionic of liquidthe aloneinner and capillary the other wall containing by formation the chiral of a layer ionic with liquid a skeletal together network, with the leading SWCNTs the were enhanced tested retention by GC [77 ]. It wastimes, shown which that improved the capillary the resolution column factor containing Rs for the SWCNTs enantiomers improved [77]. the enantioselectivity of the chiral ionic liquid. The ‘nonchiral’ role of the SWCNT was due to an increase of the surface area of the 4. Chromatography of Fullerenes and the Reciprocal Principle inner capillary wall by formation of a layer with a skeletal network, leading the enhanced retention times, whichThe molecular improved recognition the resolution of fullerene factor congenersRs for the on enantiomers various selectors [77]. by liquid chromatography has been reviewed [1,78,79], including enantioseparation of chiral native fullerenes or its derivatives 4.[1]. Chromatography For more selective of Fullerenesmolecular recognition and the Reciprocal leading toPrinciple large separation factors between C60 and C70 (α > 2), a supramolecular approach was first employed by Hawkins et al. [80]. As a strong π-acceptor The molecular recognition of fullerene congeners on various selectors by liquid chromatography has been reviewed [1,78,79], including enantioseparation of chiral native fullerenes or its derivatives [1].

Molecules 2016, 21, 1535 18 of 35

MoleculesMolecules 2016 2016, ,21 21, ,1535 1535 1818 of of 35 35 For more selective molecular recognition leading to large separation factors between C60 and C70 selectorselector(α > 2), for afor supramolecular thethe ππ-donor-donor fullerenefullerene approach selectands,selectands, was first a employeda CSPCSP containingcontaining by Hawkins NN-(3,5-dinitrobenzoyl)-phenylglycine-(3,5-dinitrobenzoyl)-phenylglycine et al. [80]. As a strong π-acceptor (DNBPG),(DNBPG),selector for originally originally the π-donor developed developed fullerene by by selectands,Pirkle Pirkle et et al. al. afor for CSP enantioseparations enantioseparations containing N-(3,5-dinitrobenzoyl)-phenylglycine [23], [23], was was employed employed (Figure (Figure 17). 17). (DNBPG), originally developed by Pirkle et al. for enantioseparations [23], was employed (Figure 17).

FigureFigureFigure 17.17. 17. HPLCHPLCHPLC separationseparation separation ofof of CC 606060 (12.2(12.2(12.2 min)min) min) andand and CC C707070 (23.5(23.5(23.5 min)min) min) onon on aa a Pirkle-typePirkle-type Pirkle-type ionicallyionically ionically DNBPGDNBPG DNBPG containingcontainingcontaining columncolumn column (250(250 (250 ×× × 101010 mmmm mm I.D.)I.D.) I.D.) elutedeluted eluted withwith with nn-hexane-hexanen-hexane atat at5.05.0 5.0 mL/minmL/min mL/min andand and detecteddetected detected atat 280280 at 280 nm,nm, nm, αα ==α 2.252.25= 2.25 (r.t.).(r.t.). (r.t.). ReprintedReprinted Reprinted withwith with permissionpermission permission fromfrom from [[80].80]. [80 CopyrightCopyright]. Copyright (1990)(1990) (1990) AmericAmeric Americananan ChemicalChemical Chemical Society.Society. Society.

An unprecedented increase in retention of C6060 and C7070 with increasing column temperature was AnAn unprecedented unprecedented increase increase in in retention retention of of C C60 andand C C70withwith increasing increasing column column temperature temperature was was observedobservedobserved byby by PirklePirkle Pirkle andand and WelchWelch Welch onon on thethe the covalentlycovalently covalently bondedbonded bonded πππ-acidic-acidic-acidic NNN-(3,5-dinitrobenzoyl)-phenylglycine-(3,5-dinitrobenzoyl)-phenylglycine-(3,5-dinitrobenzoyl)-phenylglycine (DNBPG)(DNBPG)(DNBPG) containing containing containing column column column (250 (250 (250 × × 4.6 4.6× mm mm4.6 I.D.) mmI.D.) (Figur I.D.)(Figur (Figureee 18) 18) [81]. [81]. 18 Van’t )[Van’t81]. Hoff Hoff Van’t plots plots Hoff revealed revealed plots the revealedthe rare rare case case the ofofrare positivepositive case of enthalpyenthalpy positive andand enthalpy entropyentropy and values,values, entropy i.e.i.e.,, the values,the gaingain i.e., inin entropyentropy the gain isis in aaccompaniedccompanied entropy is accompaniedbyby anan endothermicendothermic by an adsorption.adsorption.endothermic adsorption.

FigureFigureFigure 18.18.18. N N-(3,5-dinitrobenzoyl)-phenylglycineN-(3,5-dinitrobenzoyl)-phenylglycine-(3,5-dinitrobenzoyl)-phenylglycine (DNBPG) (DNB(DNBPG)PG) bonded bondedbonded to silica. toto Reprintedsilica.silica. ReprintedReprinted with permission withwith permissionpermissionfrom [81]. Copyright fromfrom [81].[81]. Copyright (1990)Copyright American ((1990)1990) Chemical AmericanAmerican Society. ChemicalChemical Society.Society.

TripodalTripodalTripodal 3,5-dinitrobenzoate3,5-dinitrobenzoate esterester ester andand and 2,4-dinitrophenyl2,4-dinitrophenyl etherether ether stationarystationary stationary phasesphases phases containingcontaining containing threethreethree πππ-acidic-acidic-acidic functionalitiesfunctionalities forfor for simultaneoussimultaneous multipoimultipoi multipointntnt supramolecularsupramolecular supramolecular interactioninteraction interaction withwith with fullerenesfullerenes fullerenes (‘Buckyclutcher’)(‘Buckyclutcher’)(‘Buckyclutcher’) werewere were developeddeveloped developed anan anddd theythey they providedprovided provided thethe the highesthighest highest retentretent retentionionion andand and thethe the greatestgreatest greatest separationseparation separation factor for the C60/C70 mixture [82]. A remarkable separation of 15 fullerene congeners in the range of factorfactor for for the the C C6060/C/C70 mixture70 mixture [82]. [82 A]. remarkable A remarkable separation separation of 15 of fullerene 15 fullerene congeners congeners in the in therange range of C60–C96, including enantiomerically enriched specimens, by HPLC on a syndiotactic poly(methyl Cof60–C C6096–C, including96, including enantiomerically enantiomerically enriched enriched specimen specimens,s, by byHPLC HPLC on on a asyndiotactic syndiotactic poly(methyl poly(methyl methacrylate)methacrylate)methacrylate) selectorselector selector inin in 4545 45 minmin min withwith with chlorochloro chlorobenzenebenzenebenzene asas eluenteluent hashas beenbeen describeddescribed [83].[83]. [83]. ((R(RR)-()-()-(−−−)-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic)-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic)-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic acid acidacid (TAPA) (TAPA)(TAPA) (Figures (Figure(Figure1b 1b,1b, and andand 19 ), FigureFigurea strong 19), 19),π a -electrona strong strong π π acceptor-electron-electron dueacceptoracceptor to the due due tetranitrofluorenylidene to to the the tetranitrofluorenylidene tetranitrofluorenylidene moiety, moiety, moiety, forms supramolecular forms forms supramolecular supramolecular charge charge transfer complexes with appropriate π-donor molecules. TAPA was used to separate C60 and C70 chargetransfer transfer complexes complexes with appropriate with appropriateπ-donor π-donor molecules. molecules. TAPA TAPA was used was toused separate to separate C60 and C60C and70 with C70 withwitha separation aa separationseparation factor factorfactorα up to αα 2.6 upup and toto 2.6 the2.6 and sameand thethe peculiar samesame increase peculiarpeculiar of incrincr retentioneaseease ofof with retentionretention increasing withwith temperature increasingincreasing temperaturetemperaturepreviously observed previouslypreviously with observedobservedπ-acidic withwithN-(3,5-dinitrobenzoyl)-phenylglycine ππ-acidic-acidic NN-(3,5-dinitrobenzoyl)-pheny-(3,5-dinitrobenzoyl)-pheny (DNBPG)lglycinelglycine was(DNBPG)(DNBPG) found waswas and foundfoundinterpreted andand interpretedinterpreted as arising fromasas arisingarising solute frfrom solvationom solutesolute at solvationsolvation low temperatures atat lowlow temperaturestemperatures [84]. The same [84].[84]. authorsTheThe samesame mentioned authorsauthors mentioned that C60 and C70 can behave as both π-electron donor and π-electron acceptor (the electron mentionedthat C60 and that C 70C60can and behave C70 can as behave both π -electronas both π donor-electron and donorπ-electron and π acceptor-electron (theacceptor electron (the affinity electron of affinity of C60 amounts to 2.6 eV). affinityC60 amounts of C60 amounts to 2.6 eV). to 2.6 eV). The enantioseparation of the tris-adduct C60[C(COOEt)2]3 and hexakis-adduct C60[C(COOEt)2]6 TheThe enantioseparation enantioseparation of of the tristris-adduct-adduct CC6060[C(COOEt)2]]33 andand hexakis-adduct CC60[C(COOEt)[C(COOEt)22]]66 entailedentailedentailed withwith with anan an inherentinherent inherent chiralchiral chiral substitutionsubstitution substitution pattepatte patternrnrn (Figure(Figure (Figure 20) 20)20 ) hashas has alsoalso also beenbeen been describeddescribed described onon on thethe the CSPCSP CSP ((R(RR)-()-()-(−−−)-2-(2,4,5,7-tet)-2-(2,4,5,7-tet)-2-(2,4,5,7-tetranitro-9-fluorenylideneaminooxy)propionicranitro-9-fluorenylideneaminooxy)propioranitro-9-fluorenylideneaminooxy)propionicnic acidacid acid (TAPA)(TAPA) (TAPA) bondedbonded bonded toto to silicasilica silica gelgel gel (Figure(Figure(Figure 19) 19)19) byby by micromicro micro HPLCHPLC HPLC [85].[85]. [85].

Molecules 2016, 21, 1535 19 of 35 Molecules 2016, 21, 1535 19 of 35

FigureFigure 19. 19. StructureStructure of of (R (R)-()-(−−)-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic)-2-(2,4,5,7-tetranitro-9-fluorenylideneamino-oxy)propionic acid acid (TAPA) (TAPA) bondedbonded to silica gel [4,84]. [4,84].

60 TheThe enantioseparation enantioseparation of of bisbis-,-, tristris- -and and hexakishexakis-adducts-adducts of of C60 C (Figure60 (Figure 20, 20 top), top) has has been been achieved achieved by HPLCby HPLC on onthe theWhelk-O1-CSP Whelk-O1-CSP [86]. [86 As]. the As theWhelk-O1 Whelk-O1-CSP-CSP (Figure (Figure 3c) 3wasc) was originally originally optimized optimized for 60 racemicfor racemic naproxen naproxen [23], [it23 may], it maybe speculated be speculated that some that somebis-, trisbis- -,andtris hexakis- and hexakis-adducts-adducts of C60 may of C also60 may be enantioseparatedalso be enantioseparated by the silica-bonded by the silica-bonded naproxen naproxenselector (Figure selector 3a) (Figure via a twofold3a) via reciprocal a twofold recognition reciprocal scenario.recognition scenario.

Figure 20. Top: Structure of the tris-adduct C60[C(COOEt)2]3. Bottom: Enantioseparation of a hexakis- FigureFigure 20. 20. Top Top: Structure: Structure of the of thetris-adducttris-adduct C [C(COOEt) C60[C(COOEt)] . Bottom2]3. Bottom: Enantioseparation: Enantioseparation of a hexakis of a- adduct C60[C(COOEt)2]6 on the CSP TAPA bonded to aminopropyl silica gel by micro HPLC (α = 1.1). adducthexakis-adduct C60[C(COOEt) C60[C(COOEt)2]6 on the2 CSP]6 on TAPA the CSP bonded TAPA to bonded aminopropyl to aminopropyl silica gel silicaby micro gel byHPLC micro (α HPLC= 1.1). Packed(α = 1.1). fused-silica Packed fused-silica capillary capillary(20 cm × (20 0.25 cm mm× 0.25 I.D.), mm μ I.D.),L/minµ L/minacetonitrile/water acetonitrile/water (75/25, (75/25,v/v), roomv/v), temperature.room temperature. Reprinted Reprinted with permission with permission from [85]. from [85].

In order to optimize supramolecular recognition between fullerenes and polyaromatic In order to optimize supramolecular recognition between fullerenes and polyaromatic compounds compounds (PAHs), the reciprocal principle of changing the chromatographic roles of selector and (PAHs), the reciprocal principle of changing the chromatographic roles of selector and selectand selectand has been suggested by Saito et al. [87]. In liquid chromatography, fullerene selectors can be has been suggested by Saito et al. [87]. In liquid chromatography, fullerene selectors can be used used as supramolecular stationary phases either as native crushed specimen packed into capillaries or as supramolecular stationary phases either as native crushed specimen packed into capillaries covalently linked to a functionalized silica gel [78,88,89]. The chemically bonded C60 phase (Figure 21) or covalently linked to a functionalized silica gel [78,88,89]. The chemically bonded C60 phase [87] showed preferential interaction with triphenylene and perylene which possess partial structures (Figure 21)[87] showed preferential interaction with triphenylene and perylene which possess partial similar to that of C60. Interestingly, larger retention factors are observed for nonplanar vs. planar PAH structures similar to that of C60. Interestingly, larger retention factors are observed for nonplanar molecules of comparable molecular size. Thus effective molecular recognition occurs between the C60 vs. planar PAH molecules of comparable molecular size. Thus effective molecular recognition selector and nonplanar selectands with a similar molecular curvature of the fullerene [87]. Bonded C60 occurs between the C60 selector and nonplanar selectands with a similar molecular curvature of phases were also used for self-recognition, i.e., for the congener separation of C60 and C70 [87,88]. As the fullerene [87]. Bonded C60 phases were also used for self-recognition, i.e., for the congener compared to an octadecylsilica (ODS) reversed phase devoid of aromatic moieties, a C60 phase entailed separation of C60 and C70 [87,88]. As compared to an octadecylsilica (ODS) reversed phase devoid high retention factors for C60 and C70 and an enhanced separation factor (α = 2.9) [87]. of aromatic moieties, a C60 phase entailed high retention factors for C60 and C70 and an enhanced separation factor (α = 2.9) [87].

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FigureFigure 21.21. SyntheticSynthetic schemescheme forfor aa CC6060 bondedbonded silicasilica stationarystationary phase.phase. ReproducedReproduced withwith permissionpermission fromfrom [[87].87]. CopyrightCopyright Wiley-VCH,Wiley-VCH, Weinheim,Weinheim, Germany.Germany.

FullerenesFullerenes havehave alsoalso beenbeen usedused asas stationary phasesphases in gas chromatography (GC).(GC). A glass capillary columncolumn (15(15 mm ×× 0.290.29 mm mm I.D.) I.D.) was was coated coated with with pure C60 (film (film thickness 0.1 μµm) using a high-pressure staticstatic methodmethod [90[90].]. The The thermostable thermostable solid solid C60 C60 phase phase behaved behaved like like the the liquid liquid phase phase squalane squalane in its in van its dervan Waalsder Waals interaction interaction with withn-alkanes. n-alkanes. C60 linked to poly(dimethylsiloxane) (Figure 22, top) was coated on a 10 m × 0.25 mm I.D. fused C60 linked to poly(dimethylsiloxane) (Figure 22, top) was coated on a 10 m × 0.25 mm I.D. fusedsilica column silica column and used and for used the for selective the selective separation separation of isomeric of isomeric hexachlorobiphenyls hexachlorobiphenyls differing differing in ortho in- orthochlorine-chlorine substitution substitution pattern. pattern. Thermodynamic Thermodynamic parameters parameters were were obtained obtained by by the the retention-increment retention-increment methodmethod RR0′ [[91,92].91,92]. It separates supramolecular interactions from non-specific non-specific interactions of of the the linker. linker. ItIt waswas shownshown thatthat thethe retentionretention behaviourbehaviour ofof PCBsPCBs dependsdepends stronglystrongly onon thethe degreedegree ofof orthoortho-chlorine-chlorine substitutionsubstitution whichwhich inin turnturn isis linkedlinked withwith non-planaritynon-planarity (Figure(Figure 2222,, bottom).bottom). The deviationdeviation fromfrom planarity of PCBs strongly decreases the supramolecular interaction with C60 [93]. Since planar PCBs planarity of PCBs strongly decreases the supramolecular interaction with C60 [93]. Since planar PCBs are more toxic than non-planar PCBs, a tentative link of retention behaviour on C60 and toxicity has are more toxic than non-planar PCBs, a tentative link of retention behaviour on C60 and toxicity has beenbeen advancedadvanced [[93].93]. In Table 4 the selective interaction of aromatic compounds with C60 linked to poly(dimethylsiloxane) In Table4 the selective interaction of aromatic compounds with C 60 linked to poly(dimethylsiloxane) (Figure(Figure 22,22, top) top) is is expressed expressed by bythe theretention-increment retention-increment R’, i.e.,R0 ,a i.e.,value a of value two indicates of two indicates that the retention that the of the PCB congener on C60 is twice as compared to a reference column devoid of the fullerene retention of the PCB congener on C60 is twice as compared to a reference column devoid of the fullereneselector [94]. selector [94].

Table 4. Retention-increments R’0 for the selective interaction of aromatic compounds with C60. Table 4. Retention-increments R for the selective interaction of aromatic compounds with C60. Complexation column (k): 10 m x 0.25 mm I.D. fused silica column coated with the C60 phase (0.25 μm). Complexation column (k): 10 m x 0.25 mm I.D. fused silica column coated with the C60 phase Reference column (k0, without C60): 10 m × 0.25 mm I.D. fused silica column coated with dimethyl- (0.25 µm). Reference column (k0, without C60): 10 m × 0.25 mm I.D. fused silica column coated withacetylaminopropyl(methyl)polysiloxane dimethyl-acetylaminopropyl(methyl)polysiloxane (0.25 μm) [94]. (0.25 µm) [94].

Analyte T (°C) R’ = (k − k0)/k0 Analyte T (◦C) R0 = (k − k )/k 1.2-Dichlorobenzene 80 0.61 0 0 1.2-Dichlorobenzene2.6-Dichlorotoluene 80 80 0.69 0.61 2.6-Dichlorotoluene 80 0.69 NitrobenzeneNitrobenzene 80 80 0.95 0.95 2-Nitrotoluene2-Nitrotoluene 80 80 0.91 0.91 AnilineAniline 80 80 1.58 1.58 PhenolPhenol 90 90 0.50 0.50 Naphthalene 90 0.73 1-MethylnaphthaleneNaphthalene 90 120 0.73 0.83 2-Methylnaphthalene1-Methylnaphthalene 120 120 0.83 0.70 2-MethylnaphthaleneIndole 120 120 0.70 1.28 QuinolineIndole 120 120 1.28 1.87 Isoquinoline 120 2.94 4-ChlorophenolQuinoline 120 120 1.87 0.34 2.4-DichlorophenolIsoquinoline 120 120 2.94 0.77 2.4.6-Trichlorophenol4-Chlorophenol 120 120 0.34 1.29 2.4-Dichlorophenol 120 0.77 2.4.6-Trichlorophenol 120 1.29

Molecules 2016, 21, 1535 21 of 35 Molecules 2016, 21, 1535 21 of 35

60 FigureFigure 22. Top Top:: Synthesis Synthesis of of CC60 linkedlinked toto aminopropylaminopropyl poly(dimethylsiloxane).poly(dimethylsiloxane). BottomBottom:: Gas Gas chromatographicchromatographic elution elution order order of of hexachlorobiphenyl hexachlorobiphenyl congeners congeners with with different different degree degree of of orthoortho-chloro-chloro substitution.substitution. The The increased increased retention retention time time of of the the seco secondnd eluted eluted enantiomer enantiomer is is marked marked with with an an arrow. arrow. ◦ 1010 m m ×× 0.250.25 mm mm I.D. I.D. fusedfused silica silica column column coated coated with with the the C C6060 phasephase (0.25 (0.25 μµm),m), 190 190 °C,C, carrier: carrier: 0.6 0.6 bar bar (at(at gauge) gauge) helium, helium, electron electron capture capture detection. detection. Reprinted Reprinted with with permission permission from from [93]. [93].

5.5. The The ReciprocalReciprocal Selectand/SelectorSelectand/Selector System System of of Fullerenes/Cyclodextrins Fullerenes/Cyclodextrins

A supramolecular selectand/selector system par excellence is represented by fullerenes (Cn, n = A supramolecular selectand/selector system par excellence is represented by fullerenes (Cn, numbern = number of carbon of carbon atoms) atoms and) andcyclodextrins cyclodextrins (CDn (CD, n =n number, n = number of D-glucose of D-glucose building building blocks). blocks).It also lendsIt also itself lends to itself the principle to the principle of reciprocal of reciprocal recognition.recognition. According According to Figure to Figure 23, the 23 role, the of role selector of selector and selectandand selectand can be can reversed. be reversed. TheThe non-chromatographic non-chromatographic supramolecular supramolecular reco recognitiongnition phenomenon phenomenon between between fullerenes fullerenes and and cyclodextrinscyclodextrins is is well well established. established. γγ-Cyclodextrin-Cyclodextrin (CD8) (CD8) forms forms water- water-solublesoluble bicapped bicapped 2:1 2:1 association association complexes with [60]fullerene (C60) [95–101]. Also β-cyclodextrin (CD7) may undergo complexation complexes with [60]fullerene (C60)[95–101]. Also β-cyclodextrin (CD7) may undergo complexation with C60 [102,103]. with C60 [102,103]. Shape selectivity governs the HPLC separation of C60 and C70 on native γ-cyclodextrin chemically Shape selectivity governs the HPLC separation of C60 and C70 on native γ-cyclodextrin chemically bonded to silica [104]. C70 was much more strongly retained than C60 on this stationary phase whereas bonded to silica [104]. C70 was much more strongly retained than C60 on this stationary phase whereas nono separation separation of the twotwo fullerenesfullerenes occurred occurred on on the the corresponding corresponding unmodified unmodified silica silica indicating indicating that that the theseparation separation is due is due to theto the selective selective supramolecular supramolecular interaction interaction with with the the cyclodextrin cyclodextrin selector selector [104 [104].]. In In a a reciprocal strategy, Bianco et al. used a C60-bonded phase (Figure 24) in an aqueous/organic medium reciprocal strategy, Bianco et al. used a C60-bonded phase (Figure 24) in an aqueous/organic medium forfor the the high-efficiency high-efficiency HPLC HPLC separation separation of of αα-,-, ββ-- and and γγ-cyclodextrins-cyclodextrins (CD6-CD8) (CD6-CD8) [105]. [105]. As As expected, expected, thethe trace trace shows shows increased increased retention retention with with increased increased molecular molecular weight of CD n (Figure 25).25). WithWith the the advent advent of of the the enzymatic enzymatic access access to to large- large-ringring cyclodextrins cyclodextrins [106,107], [106,107], separation separation of of the the congenerscongeners CD CDnn representedrepresented a a considerable considerable analytical analytical chal challenge.lenge. Koizumi Koizumi et et al. al. separated separated CD6-CD85, CD6-CD85, obtained by the action of cyclodextrin glycosyltransferase from Bacillus macerans, on synthetic amylose by high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric

Molecules 2016, 21, 1535 22 of 35 obtained by the action of cyclodextrin glycosyltransferase from Bacillus macerans, on synthetic amylose Molecules 2016, 21, 1535 22 of 35 byMolecules high-performance 2016, 21, 1535 anion-exchange chromatography (HPAEC) with pulsed amperometric detection22 of 35 using a 25 cm × 4 mm I.D. Dionex CarboPac PA-100 column (Figure 26)[108], whereas Bogdanski et detection using a 25 cm × 4 mm I.D. Dionex CarboPac PA-100 column (Figure 26) [108], whereas al.Bogdanski separated et CD6-CD21 al. separated by liquid-chromatographyCD6-CD21 by liquid-chromatography with electrospray with ionizationelectrospray mass ionization spectrometric mass detection (LC/ESI-MS) using a 25 cm × 4 mm I.D. LiChrospher NH column [109]. With one exception spectrometric detection (LC/ESI-MS) using a 25 cm × 4 mm I.D. LiChrospher2 NH2 column [109]. With (CD9one on exception HPAEC) (CD9 the congenerson HPAEC) CD then congenerswere eluted CD fromn were the eluted stationary from the phases stationary according phases to according the degree of polymerizationto the degree of polymerization (Figure 26). (Figure 26).

FigureFigure 23. 23.Schematic Schematic representation representationof ofthe the reciprocalreciprocal principle between between fulleren fullereneses and and cyclodextrins. cyclodextrins. ReproducedReproduced from from A. A. Bogdanski, Bogdanski, Doctoral Doctoral Thesis, Thesis, UniversityUniversity of Tübingen, Tübingen, Tübingen, Germany, Germany, 2007. 2007.

FigureFigure 24. 24.Synthetic Synthetic pathway pathway to to a a 6,6-[60]fulleropyrrolidine 6,6-[60]fulleropyrrolidine derivative derivative by by aziridine aziridine ring ring opening opening via via 1,3-dipolar addition to C60 and grafting the fullerene-containing triethoxysilane to silica microparticles 1,3-dipolar1,3-dipolar addition addition to to C 60C60and and grafting graftingthe the fullerene-containingfullerene-containing tr triethoxysilaneiethoxysilane to to silica silica microparticles microparticles leading to a chemically homogeneous material with defined structure and high chromatographic leadingleading to to a chemicallya chemically homogeneous homogeneous materialmaterial withwith defineddefined structure structure and and high high chromatographic chromatographic efficiency. Reprinted with permission from [105]. Copyright (1997) American Chemical Society. efficiency.efficiency. Reprinted Reprinted with with permission permission from from [ 105[105].]. CopyrightCopyright (1997) American American Chemical Chemical Society. Society.

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Molecules 2016, 21, 1535 23 of 35

Figure 25.25. SeparationSeparation ofof αα-,-, ββ-- andand γγ-cyclodextrins-cyclodextrins (CD6,(CD6, CD7,CD7, CD8)CD8) onon immobilizedimmobilized CC6060 by HPLC. Figure 25. Separation of α-, β- and γ-cyclodextrins (CD6, CD7, CD8) on immobilized C60 by HPLC. 250250 × 1.81.8 mm mm I.D. I.D. stainless stainless steel steel column column packed packed with with [60]fulleropyrrolidine-based [60]fulleropyrrolidine-based silica (Hypersil, silica (Hypersil, 5 μm), 250 × 1.8 mm I.D. stainless steel column packed with [60]fulleropyrrolidine-based silica (Hypersil, 5 μm), 5eluent:µm), eluent:0.25 mL/min 0.25 mL/min water/methanol/tetrahydrofuran water/methanol/tetrahydrofuran (80/10/10 for (80/10/10 3 min and for then 3 min linear and thengradient linear to eluent: 0.25 mL/min water/methanol/tetrahydrofuran (80/10/10 for 3 min and then linear gradient to ◦ gradient40/30/3040/30/30 toin 40/30/30 25in min,25 min, at in at25 25 25 °C. min, °C. Reprinted Reprinted at 25 C. withwith Reprinted permissionrmission with from permissionfrom [105]. [105]. Copyright from Copyright [105 (1997)]. Copyright(1997) American American (1997) AmericanChemicalChemical Society. Chemical Society. Society.

FigureFigure 26. Separation 26. Separation of of cyclodextrin cyclodextrin congeners congeners (CD6–C (CD6–CD85)D85) enzymatically enzymatically obtained obtained by cyclodextrin by cyclodextrin glycosyltransferaseglycosyltransferase from fromBacillus Bacillus macerans maceranson on syntheticsynthetic amylose amylose by by high high-performance-performance anion-exchange anion-exchange Figurechromatography 26. Separation (HPAEC) of cyclodextrin with pulsed congeners amperometric (CD6–C detectionD85) enzymatically using a 25 cm obtained × 4 mm I.D.by cyclodextrinDionex chromatography (HPAEC) with pulsed amperometric detection using a 25 cm × 4 mm I.D. Dionex glycosyltransferaseCarboPac PA-100 from column. Bacillus Reprinted macerans with on permission synthetic from amylose [108]. by high-performance anion-exchange CarboPac PA-100 column. Reprinted with permission from [108]. chromatography (HPAEC) with pulsed amperometric detection using a 25 cm × 4 mm I.D. Dionex CarboPacIn striking PA-100 contrast, column. an Reprinted unusual withsupramolecular permission fromselectivity [108]. pattern has been observed for the Inreciprocal striking separation contrast, of anCD6–CD15 unusual congeners supramolecular on a fullerene selectivity C60 selector pattern anchored has been to silica observed particles for the (Figure 27), which strongly deviates from the usually observed correlation of retention and molecular reciprocalIn strikingseparation contrast, of CD6–CD15 an unusual congeners supramolecular on a fullerene selectivity C60 selector pattern anchoredhas been toobserved silica particles for the weight [110]. Thus, whereas CD6 and CD7 exhibit a low retention and are eluted in ~5 min, CD8–CD10 (Figurereciprocal 27 separation), which strongly of CD6–CD15 deviates congeners from the usually on a fullerene observed C correlation60 selector anchored of retention to andsilica molecular particles are eluted only after a long retention gap of about 15 min with the elution order CD8 < CD10 < CD9 in weight(Figure [11027),]. which Thus, strongly whereas deviates CD6 and from CD7 the exhibit usually a low observed retention correlation and are eluted of retention in ~5 min, and CD8–CD10 molecular ~20 min. The higher CD11-CD15 congeners are eluted together with CD6 in ~5 min (Figure 27). The are eluted only after a long retention gap of about 15 min with the elution order CD8 < CD10 < CD9 weightdrop [110]. of retention Thus, whereas between CD6CD10 and and CD7 CD11 exhibit of 15 mi an low is indeed retention remarkable. and are The eluted unusual in ~5 elution min, CD8–CD10 order inare ~20 elutedhas min. been only The detected after higher a longwith CD11-CD15 retentionLC-electrospray gap congeners of aboutionizati are 15on-mass elutedmin with togetherspectrometry the elution with (ESI-MS) order CD6 inCD8 ~5of

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Figure 27. OverlaidOverlaid LC-ESI-MS LC-ESI-MS traces traces of ofthe the selective selective separation separation of CD6-CD15 of CD6-CD15 sodium sodium ion adducts ion adducts on C60 60 on Ca 60250on × a4 250mm× I.D.4 mmstainless I.D. stainlesssteel column steel packed column with packed silica-bonded with silica-bonded C stationary C60 phase,stationary 0.5 mL/min phase, 0.5water mL/min to acetonitrile/tetrahydrofuran water to acetonitrile/tetrahydrofuran gradient elut gradiention, 25 °C elution, [110]. Reproduced 25 ◦C[110]. Reproducedfrom A. Bogdanski, from A. Bogdanski,Doctoral Thesis, Doctoral University Thesis, of University Tübingen, of Tübingen Tübingen,, Germany, Tübingen, 2007. Germany, 2007.

The retention-increment method [91,92] has been employed to quantify the unusual selectivity. The retention-increment method [91,92] has been employed to quantify the unusual It separates supramolecular interactions from non-specific interactions of the linker. Thus two selectivity. It separates supramolecular interactions from non-specific interactions of the linker. stationary phases, i.e., one containing the spacer alone and one containing the C60 selector bonded via Thus two stationary phases, i.e., one containing the spacer alone and one containing the C60 selector the spacer were prepared (Figure 28). Nucleosil (5 μm, 100 Å) was reacted in toluene with bonded via the spacer were prepared (Figure 28). Nucleosil (5 µm, 100 Å) was reacted in toluene with 3-glycidoxypropyltrimethoxysilane to yield 3-glycidoxypropyl-silica and (i) the epoxide was reacted 3-glycidoxypropyltrimethoxysilane to yield 3-glycidoxypropyl-silica and (i) the epoxide was reacted with bis(1-aminohexyl)malonate to the silica-bonded spacer without selector and (ii) the same epoxide with bis(1-aminohexyl)malonate to the silica-bonded spacer without selector and (ii) the same epoxide was reacted with bis(1-aminohexyloxycarbonyl)malonate-dihydro[60]fullerene [111]. The striking was reacted with bis(1-aminohexyloxycarbonyl)malonate-dihydro[60]fullerene [111]. The striking selectivity change of CDn occurred only on the C60-selector but not on the silica-bonded spacer devoid selectivity change of CDn occurred only on the C60-selector but not on the silica-bonded spacer of the C60 selector. Due to the necessary use of an LC elution gradient only apparent relative devoid of the C60 selector. Due to the necessary use of an LC elution gradient only apparent relative complexation constants Krel (related to CD6) were obtained (Table 5). They nevertheless highlight the complexation constants K (related to CD6) were obtained (Table5). They nevertheless highlight remarkable stability differencesrel for the CD6-CD12 congeners in their supramolecular interaction with the remarkable stability differences for the CD6-CD12 congeners in their supramolecular interaction C60 [110]. The striking selectivity pattern (Table 5) may be subject to molecular modelling studies in with C60 [110]. The striking selectivity pattern (Table5) may be subject to molecular modelling studies the future. The well-known deviation from the ideal ‘doughnut’ structure of small CDn (n < 10) due in the future. The well-known deviation from the ideal ‘doughnut’ structure of small CDn (n < 10) to conformationally strain-induced band flips and kinks in CDn (n > 10) [112] may reveal clues of due to conformationally strain-induced band flips and kinks in CDn (n > 10) [112] may reveal clues of molecular recognition pattern. molecular recognition pattern.

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Figure 28. Structure of the silica-bonded spacer stationary phase (top) and the silica-bonded C60 stationary Figure 28 28.. StructureStructure of the of thesilica-bonded silica-bonded spacer spacer stationary stationary phase ( phasetop) and (top the) andsilica-bonded the silica-bonded C60 stationary C phase (bottom) [110]. Reproduced from A. Bogdanski, Doctoral Thesis, University of Tübingen,60 phasestationary (bottom phase) [110]. (bottom Reproduced)[110]. Reproduced from A. Bogdanski, from A. Bogdanski,Doctoral Thesis, Doctoral University Thesis, Universityof Tübingen, of Tübingen, Germany, 2007. Tübingen, Tübingen,Germany, 2007. Germany, 2007.

Table 5. Apparent complexation constants Krel of cyclodextrins and silica-bonded [60]fullerene (C60) Table 5.5. ApparentApparent complexationcomplexation constantsconstants KKrel of cyclodextrins and silica-bondedsilica-bonded [60]fullerene (C60)) related to CD6 (α-cyclodextrin) [110]. rel 60 related to CD6 (α-cyclodextrin) [[110].110]. CD6 CD7 CD8 CD9 CD10 CD11 CD12 CD6 CD7 CD8 CD9 CD10 CD11 CD12 Krel 1 CD6 4.5 CD739 CD8 CD946 CD10 CD1142 CD12 1 2 Krel 1 4.5 39 46 42 1 2 Krel 1 4.5 39 46 42 1 2 It has been speculated [110] that by using a single [78]fullerene selector (C78), the most strongly It has been speculated [110] that by using a single [78]fullerene selector (C78), the most strongly interacting CD-congener can be identified and, according to the reciprocal principle, could then interactingIt has beenCD-congener speculated can [110 be] thatidentified by using and, a single according [78]fullerene to the selectorreciprocal (C principle,78), the most could strongly then applied as a silica-bonded resolving agent for the separation of all five isomeric [78]fullerenes appliedinteracting as CD-congenera silica-bonded can beresolving identified agent and, for according the separation to the reciprocal of all principle,five isomeric could [78]fullerenes then applied including the direct enantioseparation of chiral D3-[78]-fullerene [113]. includingas a silica-bonded the direct resolving enantioseparation agent for theof chiral separation D3-[78]-fullerene of all five isomeric [113]. [78]fullerenes including the direct enantioseparation of chiral D3-[78]-fullerene [113]. 6. Chromatography of Calixarenes and the Reciprocal Principle 6. Chromatography of Calixarenes and the Reciprocal Principle 6. ChromatographyGutsche reviewed of the Calixarenes application and of thecalixarenesReciprocal (FigurePrinciple 29) as reciprocal selectors and selectands Gutsche reviewed the application of calixarenes (Figure 29) as reciprocal selectors and selectands [114].Gutsche reviewed the application of calixarenes (Figure 29) as reciprocal selectors and selectands [114]. [114].

Figure 29. The structure of calix[4]arene. Reproduced with permission of the Royal Society of Figure 29.29.The The structure structure of calix[4]arene.of calix[4]arene. Reproduced Reproduced with permissionwith permission of the Royalof the Society Royal of Society Chemistry of Chemistry from [114]. Chemistryfrom [114]. from [114].

Following the purification of C60- and C70-fullerenes with calix[8]arene [115,116], a reciprocal Following the purificationpurification of CC6060-- and C70-fullerenes-fullerenes with with calix[8]arene calix[8]arene [115,116], [115,116], a a reciprocal separation of calix[4]arene, calix[6]arene and calix[8]arene (Figure 30, top) by microcolumn liquid separation of calix[4]arene, calix[6]arene and calix[8]arene (Figure 30,30, top) by microcolumn liquid chromatography (μ-LC) on a chemically-bonded C60 silica stationary phase (Figure 21) has been chromatography (μ-LC) on a chemically-bonded C60 silica stationary phase (Figure 21) has been reported by Saito et al. [117]. An improved sseparation of the same calixarene congeners by μ-LC on reported by Saito et al. [117]. An improved sseparation of the same calixarene congeners by μ-LC on

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µ Moleculeschromatography 2016, 21, 1535 ( -LC) on a chemically-bonded C60 silica stationary phase (Figure 21) has26 been of 35 reported by Saito et al. [117]. An improved sseparation of the same calixarene congeners by µ-LC aon 6,6-[60]fulleropyrrolidine a 6,6-[60]fulleropyrrolidine silica silica stationary stationary phase phase (Figure (Figure 24) 24 was) was later later repo reportedrted by by Bianco Bianco et et al. al. (Figure 3030,, bottom) [[105].105].

Figure 30. Top: Structures of tert-butyl-calix[n]arenes. Bottom: HPLC trace for the separation of Figure 30. Top: Structures of tert-butyl-calix[n]arenes. Bottom: HPLC trace for the separation of tert-butylcalix[n]arenes on a 6,6-[60]fulleropyrrolidine stationary phase (Figure 24). 250 × 1.8 mm I.D. tert-butylcalix[n]arenes on a 6,6-[60]fulleropyrrolidine stationary phase (Figure 24). 250 × 1.8 mm I.D. stainless steel column. Eluent: CH2Cl2/2-propanol (99.5/0.5), flow rate 0.3 mL/min; T: 25 °C; UV detection◦ stainless steel column. Eluent: CH2Cl2/2-propanol (99.5/0.5), flow rate 0.3 mL/min; T: 25 C; at 280 nm. Reprinted with permission from [105]. Copyright (1997) American Chemical Society. UV detection at 280 nm. Reprinted with permission from [105]. Copyright (1997) American Chemical Society. Achiral chromatographic selectivity has been achieved for various amino acid esters on silica-bonded calix[4]arene tetraester [118,119]. The enantioseparation of binaphthyl atropisomers on Achiral chromatographic selectivity has been achieved for various amino acid esters on the chiral acylcalix[4]arene amino acid derivatives (N-l-alaninoacyl)calix[4]arene and (N-l-valinoacyl) silica-bonded calix[4]arene tetraester [118,119]. The enantioseparation of binaphthyl atropisomers on calix[4]arene) by electrokinetic chromatography (EKC) has been reported [120]. By enantioselective the chiral acylcalix[4]arene amino acid derivatives (N-l-alaninoacyl)calix[4]arene and (N-l-valinoacyl) liquid chromatography a chiral calixarene functionalized with (−)-ephedrine at the lower rim and calix[4]arene) by electrokinetic chromatography (EKC) has been reported [120]. By enantioselective chemically bonded to silica has been used to separate racemic 1-phenyl-2,2,2-trifluoroethanol [121]. liquid chromatography a chiral calixarene functionalized with (−)-ephedrine at the lower rim and A chiral resorc[4]arene selector [122] of the basket-type containing four ω-unsaturated alkyl chains chemically bonded to silica has been used to separate racemic 1-phenyl-2,2,2-trifluoroethanol [121]. was synthesized from resorcinol and 1-undecenal [123]. Afterwards chiral L-valine-tert-butylamide A chiral resorc[4]arene selector [122] of the basket-type containing four ω-unsaturated alkyl chains moieties were attached to the eight hydroxyl groups. The chiral resorc[4]arene was then linked via was synthesized from resorcinol and 1-undecenal [123]. Afterwards chiral L-valine-tert-butylamide the ω-unsaturated alkyl chains to poly(dimethyl-methylhydro)siloxane via platinum-catalyzed moieties were attached to the eight hydroxyl groups. The chiral resorc[4]arene was then linked hydrosilylation to yield chemically bonded Chirasil-Calix-Val (Figure 31) [123]. On a 20 m × 0.25 mm via the ω-unsaturated alkyl chains to poly(dimethyl-methylhydro)siloxane via platinum-catalyzed I.D. fused silica capillary column coated with Chirasil-Calix (0.25 μm) the N(O,S)-trifluoroacetyl-D,L- hydrosilylation to yield chemically bonded Chirasil-Calix-Val (Figure 31)[123]. On a 20 m × 0.25 mm α-amino acids Ala, Abu, Val, Ile, Leu, Pro, Phe, Glu, Tyr, Orn and Lys were enantioseparated by GC I.D. fused silica capillary column coated with Chirasil-Calix (0.25 µm) the N(O,S)-trifluoroacetyl-D,L- with Rs > 2 [122]. α-amino acids Ala, Abu, Val, Ile, Leu, Pro, Phe, Glu, Tyr, Orn and Lys were enantioseparated by GC with Rs > 2 [122].

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Figure 31. SynthesisSynthesis of the CSP Chir Chirasil-Calix.asil-Calix. Reprinted Reprinted with permission from [123]. [123].

In the dual chiral recognitionrecognition systemsystem Chirasil-Calix-Val-DexChirasil-Calix-Val-Dex, a resorc[4]arene containing L-valine-valine diamide diamide (Calix-Val) (Calix-Val) and permethylated β-cyclodextrin-cyclodextrin (CD) (CD) were were chemically linked to poly(dimethylsiloxane) [124]. [124]. The The select selectoror system system belongs belongs to to the category of mixed chiral stationary phases [125]. [125]. On On th thisis binary binary CSP CSP both both N-TFA-valineN-TFA-valine ethyl ethyl ester ester (due (due the presence the presence of Calix-Val) of Calix-Val) and chiraland chiral unfunctionalized unfunctionalized 1,2-trans 1,2-trans-dialkylcyclohexanes-dialkylcyclohexanes (due (due to tothe the presence presence of of the the CD) CD) were simultaneously enantioseparated enantioseparated (Figur (Figuree 32)32)[ [124].124]. In In order order to account for matched and mismatched enantioselectivities imparted byby thethe twotwo selectors,selectors, the the chirality chirality of of Calix-Val Calix-Val could could be be changed changed from from to toL to L Dto. WhenD. When racemic racemic Calix-LD-Val Calix-LD-Val were we used,re used, any any observed observed enantioselectivity enantioselectivity could could only ariseonly arise from fromthe presence the presence of CD of [124 CD]. [124]. In the example above, the chiral resorc[4]arene is usedused asas anan enantioselectiveenantioselective selector.selector. In In a reversed fashion the enantiomers enantiomers of of a a chiral chiral resorc[4 resorc[4]arene]arene compound compound can can also also be be studied studied as as selectands. selectands. Thus, tetrabenzoxacine resorc[4]arene resorc[4]arene (Figure (Figure 33,33 ,top) top) was was enantioseparated enantioseparated by by HPLC HPLC on on the CSP Chiralpak AD developed by Okamoto et al. [[126].126]. The chromatograms show temperature-dependent interconversion profiles profiles due due to to enantiomerization duringduring enantioseparation enantioseparation (Figure (Figure 33,33, bottom) bottom) [127]. [127]. By peak-form analysis featuring plateau formation [128], [128], the inversion barrier was determined to Δ∆G = 92 92 ±± 22 kJ/mol kJ/mol (298K) (298K) [127]. [127 ].

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FigureFigure 32.32. The mixedmixed CSPCSP Chirasil-Calix-Val-Dex.Chirasil-Calix-Val-Dex. Figure 32. The mixed CSP Chirasil-Calix-Val-Dex.

Figure 33. Top: Interconverting enantiomers of tetrabenzoxacine resorc[4]arene by HPLC on the CSP FigureChiralpakFigure 33. 33. TopAD. Top: InterconvertingBottom: Interconverting: Distorted enantiomers enantiomers peak profiles of of tetrabenzoxacine caused tetrabenzoxa by enantiomerizationcine resorc[4]arene resorc[4]arene (dotted by by HPLC HPLC line: on onexpected the the CSP CSP ChiralpakpeakChiralpak profiles AD. AD. devoidBottom Bottom of: interconversion) Distorted: Distorted peak peak profiles. Reprinted profiles caused caused with by permission by enantiomerization enantiomerization from [127]. (dotted (dotted line: line: expected expected peakpeak profiles profiles devoid devoid of interconversion).of interconversion) Reprinted. Reprinted with with permission permission from from [127 [127].]. This finding is recalled here, because one may speculate to employ interconverting calixarene This finding is recalled here, because one may speculate to employ interconverting calixarene enantiomersThis finding in a reciprocal is recalled fashion here, as because a dynamic one CSP may in speculate the spirit toof a employ scenario interconverting of column deracemization calixarene enantiomers in a reciprocal fashion as a dynamic CSP in the spirit of a scenario of column deracemization enantiomersdescribed by in Welch a reciprocal [129].fashion Thus when as a dynamic an enantiomeric CSP in theally spirit pure of selectand, a scenario ofwhich column interacts deracemization with high described by Welch [129]. Thus when an enantiomerically pure selectand, which interacts with high describedaffinity and by enantioselectivity Welch [129]. Thus with when an interconverting an enantiomerically selector pure present selectand, in the which stationary interacts phase, with is added high affinity and enantioselectivity with an interconverting selector present in the stationary phase, is added affinityto the mobile and enantioselectivity phase, it may cause with deracemization an interconverting of the selector selector, present thereby in thecreating stationary a nonracemic phase, is CSP added for to the mobile phase, it may cause deracemization of the selector, thereby creating a nonracemic CSP for tosubsequent the mobile enantiomeric phase, it may separations cause deracemization of the selectand ofin theits racemic selector, form. thereby This creatingintriguing a scenario nonracemic was subsequent enantiomeric separations of the selectand in its racemic form. This intriguing scenario was CSPdemonstrated for subsequent by HPLC enantiomeric with a conformationally separations of la thebile selectand naphthamide in its racemicatropisomer form. [129]. This First intriguing it was demonstrated by HPLC with a conformationally labile naphthamide atropisomer [129]. First it was scenarioshown that was the demonstrated naphthamide by HPLCcan be with enantiosepar a conformationallyated on the labile Whelk-O1 naphthamide CSP (Figure atropisomer 3c). Under [129]. stopped-flowshown that conditionsthe naphthamide [130], deracemizationcan be enantiosepar took atedplace on in the Whelk-O1mobile phase. CSP With (Figure the 3c).reciprocal Under conceptstopped-flow the racemic conditions naphthamide [130], deracemization was then linked took toplace silica in whereasthe mobile the phase. soluble With analog the reciprocalof the concept the racemic naphthamide was then linked to silica whereas the soluble analog of the

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First it was shown that the naphthamide can be enantioseparated on the Whelk-O1 CSP (Figure3c). Under stopped-flow conditions [130], deracemization took place in the mobile phase. With the reciprocal concept the racemic naphthamide was then linked to silica whereas the soluble analog of the enantiomerically pure Whelk-O1 selector was added to the mobile phase. After deracemization of the CSP, the racemic analog of the Whelk-O1 selector was enantioseparated, however, with the expected subsequent decrease of the separation factor α due to gradual racemization of the CSP [129]. The reciprocal concept of interconverting molecules in separation methods could also be extended to the liquid chromatographic investigation of conformational cis/trans-diastereomers of L-peptidyl-L-proline. Studies of proline-containing peptides separated on calixarene-bonded silica gels have been investigated by Gebauer et al. [131] and interconversion barriers of L-proline containing dipeptides by dynamic CE were determined [132].

7. Conclusions Since the first reports by Mikeš and Pirkle on the use of a reciprocal approach in chromatography through changing the role of selectands and selectors, many examples have been reported in different areas of selective supramolecular interactions including combinatorial advances. Advantages and limitations of the reciprocal concept are outlined. The reciprocal strategy clearly aids the development of optimized selectors for difficult separations of selectands including enantiomers. Various scenarios of the interchanging role of selectors and selectands in separation science are discussed in the hope that this will stir further endeavors devoted to the principle of reciprocity in supramolecular recognition. The present account should be seen as part of the emerging field of supramolecular separation methods [1,2] with further emphasis also to molecular sensor approaches and surface related recognition phenomena. The optimization of selector/selectand systems is the precondition for meaningful mechanistic studies by various molecular modelling approaches.

Acknowledgments: This work was partially funded by the German Research Council (DFG Schu 395/18-2) in 2005 on ‘Combinatorial Chiral Selectors for the Enantiomeric Separation by Chromatography and Electromigration’. Conflicts of Interest: The author declares no conflict of interest.

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