Advances in chiral separations of small peptides by capillary electrophoresis and chromatography.

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Imran Ali1 Zeid A. Al-Othman2 Abdulrahman Al-Warthan2 Leonid Asnin3 Alexander Chudinov3 1 Department

of Chemistry, Jamia Millia Islamia (Central University), New Delhi, India 2 Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia 3 Perm National Research Polytechnic University, Perm, Russia Received May 28, 2014 Revised June 26, 2014 Accepted June 27, 2014

Review

Advances in chiral separations of small peptides by capillary electrophoresis and chromatography Many chemical and biological processes are controlled by the stereochemistry of small polypeptides (di-, tri-, tetra-, penta-, hexapeptides, etc). The biological importance of peptide stereoisomers is of great value. Therefore, the chiral resolution of peptides is an important issue in biological and medicinal sciences and drug industries. The chiral resolutions of peptide racemates have been discussed with the use of capillary electrophoresis and chromatographic techniques. The various chiral selectors used were polysaccharides, cyclodextrins, Pirkle types, macrocyclic antibiotics, crown ethers, imprinted polymers, etc. The stereochemistry of dipeptides is also discussed. Besides, efforts are made to explain the chiral recognition mechanisms, which will be helpful in understanding existing and developing new stereoselective analyses. Future perspectives of enantiomeric resolution are also predicted. Finally, the review concludes with the demand of enantiomeric resolution of all naturally occurring and synthetic peptides. Keywords: Capillary electrophoresis / Chiral recognition mechanisms / Chiral separation / Hybrid techniques / Peptides DOI 10.1002/jssc.201400587

1 Introduction Nowadays, the demand for chiral resolution is increasing continuously as one of the enantiomers/stereoisomers may be pharmaceutically active while the other may be toxic or ballast, creating various side effects and problems [1]. This is due to the fact that the metabolic and regulatory reactions in biological systems are stereoselective. This is because of the different stereoselective distribution rates, metabolism, excretion, and clearance of stereoisomers. In view of these facts, academicians, scientists, clinicians, industrialists, and government authorities are concerned for optically active drugs and other molecules of biological importance. The US-FDA, European Committee for Proprietary Medicinal Products, Health Canada, Pharmaceutical and Medical Devices Agencies of Japan, have banned the marketing of newly developed racemic drugs [2–4]. Small peptides (with the number of monomers n < 6) are of high importance due to their involvement in many metabolic processes. The biological activities of small peptides include mechanisms controlling protein syntheses, inflammation processes, fertilities, neurotransmissions, vital functions of pathogenic microorganisms, and many other functions relating to human health. This gives rise to the

Correspondence: Dr. Ali, Department of Chemistry, Jamia Millia Islamia, (Central University) JMI, New Delhi, India E-mail: [email protected], [email protected] Fax: +0091-11-26985507

Abbreviations: CD, cyclodextrin; TAG, teicoplanin aglycon

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

use of peptides and peptidomimetic structures in drug development [5–7] or biological markers in physiological research [8]. Another important area for small peptides is food and nutrition industries. Aspartame, carnosine, Val-Tyr are being produced at an industrial level [7]. The essential biological functions of peptides; especially those concerning enzymatic reactions; depend on peptide stereochemistry. Amino acids exist in D and L forms and constitute several peptide molecules. These form several stereoisomers, each with potentially different biological properties. The stereoisomers may experience mutual transformations (called racemization or epimerization depending on the position(s) of the stereogenic center(s) involved) during syntheses, storage or in the course of metabolic processes. All this may result in complex enantiomeric/epimeric compositions of respective industrial/commercial products or biological samples analyzed for research purposes. The above mentioned facts explain the importance of the enantiomeric resolution of small peptides in biological, medicinal, pharmaceutical, and industrial areas. Chiral separation of dipeptides can be achieved through various analytical techniques. The most important techniques are CE and different liquid chromatographic modalities. Czerwenka and Lindner [9] reviewed stereoisomeric analyses of dipeptides by HPLC, CE, and other techniques. However, this important publication is seven years old. A review on CE of peptides by Scriba [10] was published in 2003, which is also out of date. A recent review by Schmid [11] concerned only ligand-exchange CE. A brief account of chiral electrophoresis of peptides has been given by Kaˇsiˇcka [12]; without a representative bibliography on the issue. Therefore, there is a need to survey new literature on the enantiomeric resolution www.jss-journal.com

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of dipeptides by CE and chromatographic methods. The aim of the article is to describe the state-of-the-art of chiral separations of peptides using CE, chromatography, and hybrid techniques. Efforts have been made to discuss the mechanism of the chiral separations and future perspectives in this area.

2 Stereochemistry of Peptides Normally, peptides are polymers of amino acids bonded together by peptide (–CO–NH–) bonds. Out of 22 proteinogenic amino acids, only glycine is achiral. Isoleucine and threonine contain two chiral centers, while pyrrolysine has three asymmetric centers. The remaining amino acids contain only one chiral center at the ␣-carbon atom. Amino acids with one asymmetric center exist in two forms, i.e., L and D configurations. s-Chiral amino acids have s2 stereoisomers. The stereoisomers due to the ␣-C atom have the designation L and D regardless of configuration of the other stereogenic centers. All standard L amino acids, with the exception of cysteine, have an S configuration in the absolute Cahn–Ingold– Prelog nomenclature. Cystein is assigned an R configuration because of the sulfur atom in the side chain, which results in the change of priorities. Fisher nomenclature is used herein for amino acids. With two possible configurations of each stereogenic center, the number of stereoisomers for a polypeptide with n amino acids (m: glycines, o: pyrrolysines, and p: sum of threonines, and isoleucines) is 2(n−m+2o+p) [9]. The pairs LL/DD or LD/DL are complete mirror reflections of each other, i.e., enantiomers. Isomers whose configurations are not mirror reflections with respect to each other are called diastereomers. This division is important because diastereomers possess different physicochemical properties (and may be separated on this basis without applying stereoselective techniques [9]) whereas stereoisomers display the same properties unless they are in a chiral environment. Naturally, proteinogenic amino acids are found in L forms. Consequently, natural peptides have (L, . . . ,L) configuration. The synthetic peptides are equimolar mixtures of every stereoisomer, unless stereoselective synthesis fixing certain configuration was used. However, the occurrence of D amino acids is not as rare as believed earlier [13, 14]. Therefore, Dmonomer-containing peptides may occur in nature too. The biological properties of peptides depend on their stereochemical compositions. A classical example is the sweetener aspartame, which is a methyl ester of L-Asp-L-Phe (Fig. 1). The other three isomeric forms possess a bitter taste. Likewise, the interaction of optical isomers with a chiral selector is configuration dependent, which makes enantioselective separation possible.

3 Mechanisms of chiral separations This is relevant to preface a discussion of stereoisomers separation techniques with a brief introduction in the mechanisms of chiral separations. The key figure in enantiodiscrimination is the chiral selector—an entity that is able to interact C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Four diastereomers of N-(␣-aspartyl)-phenylalanine. The sweetener aspartame is the LL form.

differently with stereoisomers. This ability is explained by a special spatial configuration of entity referred to as a chiral surface that favorably traps one stereoisomer but is not adjusted to fit its optical antipode. Several phenomena may take part in effective binding, categorized by Lämmerhofer [15] are given below. www.jss-journal.com

Liquid Chromatography

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Steric fits, i.e., size and shape complementarily between stereoisomers and chiral selector. Electrostatic fits, i.e., a favorable geometric and spatial orientation of complementary functional groups allowing electrostatic type interactions such as ionic interactions, hydrogen bonding, coordination bonding, dipole–dipole interactions, ␲–␲ interactions, cation–␲ or anion–␲ interactions. Hydrophobic fits occur when hydrophobic regions of both binding partners can spatially match each other as to release in aqueous media. These are entropically unfavorable, structurally ordered water shell on the molecular surface, and eventually allow for close intermolecular contacts of the lipophilic moieties. Thus, these lead to the mutual saturation of their hydrophobic surfaces. Dynamic fits and induced fits meaning dynamic conformational adaptation in the course of complex formation directed to maximize binding interactions between analyte and selector.

3.1 Mechanisms in CE In CE, both chiral selector and racemate are added to the BGE. The chiral selector forms diastereomeric complexes with stereoisomers, which results in the resolution of stereoisomers. A simple mobility difference model explaining this phenomenon has been proposed by Wren and Rowe [16]. According to this model, the formation of transient diastereomeric complexes between a chiral selector S and the L and D enantiomers is an equilibrium process characterized by the complexation constants KL and KD : L + S ↔ LS K L =

[LS] [L] · [S]

D + S ↔ DS K D =

[DS] . [D] · [S]

(1)

(2)

The effective mobility (␮eff ) of either enantiomer can be written as J

␮eff =

␮0 + ␮JS K J [S] . 1 + K J [S]

(3)

Index J designates an enantiomer; J = L or D. ␮0 is the mobility of free enantiomer, which is the same for both enantiomers. ␮JS is the mobility of enantiomer–selector complex. An enantioseparation is observed when the effective mobilities of the enantiomers differ. According to Eq. (3), it is possible at different mobilities of the diastereomeric complexes, or the complexation constants differ, or both. Enantioseparation mechanisms in CE on molecular level have been discussed by Vespalec and Boˇcek [17] and most recently by Scriba [18]. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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3.2 Mechanisms in chromatography Chiral separation in chromatography is based on the interaction of stereoisomers with chiral selectors adsorbed or immobilized on the surface of the stationary phase. Equations (1) and (2) can be used to describe these interactions, keeping in mind that S is an immobilized or adsorbed chiral selector. The additional factors are nonselective adsorption sites, which also present on the surface of stationary phase [19, 20]. These sites do not differentiate stereoisomers. Adsorption equilibrium constants (Kns ) remain constant. Considering the simplest case of linear adsorption isotherm for illustrative purposes and omitting unnecessary mathematical manipulations (details can be found in Ref. [20]) the expressions for the chromatographic retention k of two enantiomers L and D are given below. ∗ kL = F q s∗ K L + q ns K ns

(4)

∗ kD = F q s∗ K D + q ns K ns .

(5)

∗ In these equations q s∗ and q ns are the concentrations of enantioselective and nonselective adsorption sites on the surface of stationary phase, respectively, with F as the phase ratio. Two enantiomers (or, in a general case, several stereoisomers) can be separated if their adsorption constants differ. A degree of enantioseparation not only depends on the difference between KL and KD but is also determined by the relative fractions of the enantioselective sites and the strength of the nonselective sites. Indeed, the selectivity ␣ of a chiral stationary phase (CSP) with respect to two enantiomers is given by

␣=

(q s /q ns ) K L + K ns (q s /q ns ) K D + K ns

(6)

In chiral chromatography the apparent enantioselectivity (␣) is opposed to the theoretical true enantioselectivity (␣true ), where ␣true = KL /KD [21]. Therefore, only the apparent enantioselectivity is used hereinafter. The above theory represents a macroscopic mechanism of enantioseparation, which is different from that for CE. Microscopic mechanisms of interaction of solute molecules with a chiral selector also differ in chromatography and CE. This is due to immobilization or adsorption of chiral selector in chromatography. As shown by Davankov [22], the surface of stationary phase can play an active role in enantioselective interactions between an optical isomer and a chiral selector. A detailed account of (molecular) enantioseparation mechanisms in chromatography is given in Ref. [23].

3.3 Features of the stereoselective binding of multichiral molecules One of the most important characteristic features of peptides is having more than one asymmetric center. Each center may www.jss-journal.com

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interact with a chiral selector in different ways. In other words, there may be competition between different chiral centers of one molecule for binding to the chiral selector. This scenario is more probable for inclusion type selectors such as cyclodextrins and crown ethers. An alternative mode is interaction of a molecule; containing more than one stereogenic center; with a selector as a single whole. It means that the points of interactions with a chiral selector are located in the different parts of a molecule. These are not associated with a neighborhood of a particular asymmetric center. Such a situation has been described by Czerwenka et al. [24, 25] for chiral separation of stereoisomers of derivatized oligoalanines on silica bound cinchona alkaloid derivatives. The discussed peculiarity of multichiral compounds has been recognized by researchers but not studied systematically. The only trial to review information on the enantioselective chromatography of multichiral compounds was attempted by Aboul-Enein [26] with limited accumulation of empirical data without addressing retention mechanisms. So far dominating concepts are (i) interactions of a molecule as a single whole as described above or (ii) binding via a certain chiral center. Therefore, the bindings of dipeptides to crown ethers are attributed entirely to the N terminus [27]. At the same time, empirical data [28, 29] suggested that the C terminus played a role in this interaction.

4.1 Capillary electrophoresis CE is a versatile technique of high speed, sensitivity, and efficiency. The high efficiency of CE is due to flat flow profile originated and a homogeneous partition of chiral selector in the BGE, which minimizes mass transfer. Other advantages of CE are simplicity, economic, selectivity, and high degree of matrix independence (due to high theoretical plate numbers normally ranging from 200 000 to 700 000). In chiral CE, a chiral compound called a chiral selector is added into BGE; intended for complexing the enantiomers imposing them through different electrophoretic mobilities. Commonly used chiral BGE additives are cyclodextrins, macrocyclic glycopeptide antibiotics, proteins, crown ethers, ligand exchangers, alkaloids etc. [36–45]. The best chiral selector should be water soluble, inexpensive, capable to form inclusion complexes through sufficient groups, atoms, grooves, cavities, etc. Besides, these should be chemoselective and not UV absorbing and EOF effecting in nature. Some old but still important reviews are available on chiral separations of small peptides by CE [10, 40]; more recent short contributions [46, 47] are also useful. Table 1 summarizes CE separations of small-peptide stereoisomers published since 2003 with the addition of some earlier studies not reflected in previous reviews. A detailed discussion of the topic is given in the following paragraphs. 4.1.1 Cyclodextrins

4 Chiral separation of small peptides Two broad groups of separation techniques are commonly used for the chiral separation of small peptides, i.e., LC and CE. Besides, hybrid techniques; combining features of electrophoresis and chromatography; such as CEC and MEKC are gaining more attention during recent years. These hybrid techniques are discussed in Section 4.2.2. Generally, the separation takes place on pseudostationary phase, which is a fundamental characteristic of chromatography. CE seems to be the most popular method for chiral separation of peptide stereoisomers. CE has achieved a good reputation in this area due to its inclusion into US and European Pharmacopeia. Moreover, a further expansion of this technique is expected into industrial analyses [10]. One of the differences between CE and chromatography, in enantiomeric separation, is location of chiral selector. It is located in the mobile and stationary phases in CE and chromatography, respectively. Almost all chiral selectors have been tested in both CE and chromatography. Some publications of interest are devoted to dipeptides, with few papers on tri-, tetra-, and larger peptides [24,30–35]. The lower number of publications on tri- and polypeptides is due to the low importance of such peptides. Other reasons are lack of commercially available standards and difficulty in the interpretation of the data obtained with multichiral compounds. It is worth mentioning that usually studied tripeptides contain one or two glycil residues. Such molecules contain two or one chiral centers, respectively. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Cyclodextrins (CDs) are cyclic and nonreducing oligosaccharides obtained from starch. Schardinger [48] identified three different forms of naturally occurring CDs, i.e., ␣-, ␤-, and ␥-CDs. The ability of CDs to form inclusion complexes with a wide variety of molecules is well documented. It is explained by the presence of a chiral hollow cavity in the structures of these molecules [49–54]. Hydrophobic interactions, hydrogen bonding, and van der Waals interactions play major roles in the host–guest binding of analytes. The enantioselectivity of CDs can be modulated by derivatization of peripheral hydroxyl groups. In CE, methyl, carboxymethyl, hydroxypropyl ethers as well as sulfated CDs have become especially popular. Water solubilities of hydrophobized CDs decrease. Therefore, addition of an organic solvent in BGE becomes necessary, if high concentration of chiral selector is desirable. Scriba et al. carried out remarkable work on the chiral separation of di- and tripeptide stereoisomers using CDs as chiral selectors [55–73]. All peptides resolved were unprotected at both termini. But Skanchy et al. [74] and Janini et al. [75] separated N-terminal-protected peptides with CDs as chiral selectors. Normally, CE chiral separation of small peptides with CDs was achieved under acidic conditions but some basic buffers were also tested [66]. Some authors reported a change in reversal order of elution by changing pH of BGE. It has also been observed that migration order depends on the charge states of peptides and their binding constants [56, 58, 66, 67, 76, 77]. Wan and Blomberg [78] used CD to separate some derivatized dipeptides. The highest plate number achieved was www.jss-journal.com


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Table 1. Enantiomeric resolution of dipeptides by CE

No. Peptidesa)

BGEs

Chiral Selectorsb)

References

1

Ala-Phe, Ala-Tyr, Asp-PheOMe

␤-CD

[182]

2

Gly-Asp-PheNH2 Phe-Asp-GlyNH2 Cyclo(Phe-Asp) Ala–Phe, Ala–Tyr

30–60 mM Na3 PO4 + 2 M urea + 0.01% w/v hexadimethrine bromide, pH 2.2–3.8 50 mM Na3 PO4 , pH 3 50 mM formic acid, pH 3 + 10% acetonitrile

CM-␤-CD S-␤-CD

[69]

␤-CD

[68]

DM-␤-CD

[70]

S-(␣/␤/␥)-CD SP-(␣/␤/␥)-CD CM-(␣/␤/␥)-CD ␤-CD

[183]

␤-CD

[178]

DM-␤-CD HP-␤-CD (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid (18C6 H4 ) (1)-(18-crown-6)-2,3,11,12tetracarboxylic acid (18C6 H4 ) L-histidine D-saccharic acid D-gluconic acid L-threonic acid

[184]

3

50 mM H3 PO4 (or citric acid, S,S-tartaric acid, R,R-tartaric acid, chloroacetic acid, glycine) + 2 M urea + 0.003% w/v hexadimethrine bromide, pH 2.2 and 3.8 60 mM Na3 PO4 , pH 2.2 40 mM Na3 PO4 , pH 3.0 30 mM sodium aspartate, pH 3.8 with and without 2 M urea 50 mM Na3 PO4 , pH 2.5 50 mM CH3 COONa, pH 5.3

4

Ala-Tyr, Ala-Phe, Asp-PheOMe, Glu-PheNH2

5

15 dipeptides and 3 tripeptides

6

Ala–Phe, Ala–Tyr

7

Ala–Met, Ala-Leu, Ala-Val, Ala-Leu-Gly

8

Six dipeptides, Three tripeptides

9

Ala-Ala, Leu-Leu, Gly-Phe

3 M CH3 COOH, pH 2.0

10 11

Several tetra- to octapeptides Gly-Ala, Gly-Phe, Gly-Try

0.5 M acetic acid 25 mM CuSO4 , pH 12.0 50 mM CuSO4 , pH 10.0 30 mM CuSO4 , pH 5.0 (Ligand exchange conditions)

50 mM Na3 PO4 pH 2.5 and 3.5 50 mM Na3 PO4 pH 2.5 and 3.5 + 2 M urea 200 mM Na3 BO3 + 50 mM sodium dioxycholate + 15 % v/v 2-propanol 0.5 M acetic acid

[73]

[102]

[184] [109]

a) Including glycyl containing tripeptides; b) Abbreviations: M-␤-CD = methyl-␤-CD; DM-␤-CD = heptakis(2,6-di-O-methyl)-␤-CD; TM-␤-CD = heptakis(2,3,6-tri-O-methyl)-␤-CD; carboxymethyl-␤-CD = CM-␤-CD; CEt-␤-CD = carboxyethyl-␤-CD; hydroxypropyl-(␣/␤/␥)-CD = HP-(␣/␤/␥)-CD; succinyl-␤-CD = Succ-␤-CD; permethyl-␤-CD = PM-␤-CD; HDA-␤-CD = heptakis(2,3-di-O-acetyl)-␤-CD; HDAS-␤-CD = heptakis(2,3-di-O-acetyl-6-sulfo)-␤-CD; HDMS-␤-CD = heptakis(2,3-di-O-methyl-6-sulfo)-␤-CD; H6S-␤-CD = heptakis-6-sulfo-␤-cyclodextrin; S-␤-CD = sulfated ␤-CD; hS-␤-CD = highly sulfated ␤-CD; rS-␤-CD = randomly sulfated ␤-CD; HyTMAP-␤-CD = 2-hydroxy-3-trimethylammoniumpropyl-␤-CD; SB-␤-CD = sulfobutyl-␤-CD; SBE-␤-CD = sulfobutylether-␤-CD4; SP-(␣/␤/␥)-CD = sulfopropyl-(␣/␤/␥)-CD c) Deriviatized at the N-terminal end with the 8-guanidinooctanoyl group. d)cHxAla = cyclohexyl-Ala. d) Buffers containing ␤-CD were prepared with 2 M urea. f)Substitution degree 4.

106 plates/m. The BEG used was borate buffer (40 mM, pH, 9.2) with 15.0 mM SDS. van de Griend [79] exploited uncharged CDs for the enantiomeric resolution of 13 glycyl dipeptides. The BEG was 0.1 M phosphoric acid, 0.088 M triethanolamine with 10 mM CD. Interestingly, some nonaromatic dipeptides were resolved. The authors developed mobility difference plots for Gly-DL-Leu and Gly-DL-Phe with heptakis(2,6-di-O-methyl)-␤-CD, which showed relatively flat profiles in a large concentration range. The authors also tried malonic acid and triethanolamine to achieve the best separation at pH 3.0. Furthermore, van de Griend [80] utilized


eight neutral CDs for enantiomeric separation of alanyl and leucyl dipeptides at pH 3.0. Seven out of eight CDs provided a good separation of one or more of the dipeptide enantiomer pairs. The best results were obtained with heptakis(2,6-di-Omethyl)-␤-cyclodextrin. The separated dipeptides were aromatic and lipophilic aliphatic ones. Aromatic dipeptides had higher affinities for CD than nonpolar and aliphatic dipeptides; as determined by mobility difference plots at pH 3.0 with malonic acid/triethanolamine as BGE. Li and Waldron [77] resolved Ala-Phe and Leu-Phe using a ␤-CD chiral selector and urea as solubility enhancer, respectively. Weak

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inclusion complex formation was proposed between the dipeptides and chiral selector. The elution order was independent of pH. The binding constants for four Ala-Phe stereoisomers were K = 42–66 and 4–41 M−1 for the cationic and zwitterionic forms, respectively. Xu et al. [81] employed carboxymethyl-␤-cyclodextrin polymer (CM-P-CD polymer) as a chiral selector for resolving several underivatized dipeptides with benzene moieties. The chiral separation of a mixture of DL-Ala-DL-Phe and DL-Leu-DL-Phe was obtained using 27 mg/mL CM-␤-CD polymer in the running buffer at pH 5.12. The authors evaluated the effects of different cyclodextrin types, selector concentration, buffer pH, and organic additive. The stereoseparation was dependent on selector concentration, buffer pH, and modifier. Poor resolution was observed at high concentration of organic modifier. The authors observed better resolution of dipeptides with short chains in the vicinity of the second chiral carbon atom using CM-␤-CD polymer than either carboxyethyl-␤-CD or succinylated-P-CD. Süß et al. [60] used sulfated and sulfonated cyclodextrins for separating dipeptides. The sulfated ␤-CD and single isomer derivatives heptakis-6-sulfato-␤-CD and heptakis(2,3-diacetyl-6-sulfato)-␤-CD were better chiral selectors than sulfobutylether-␤-CD and heptakis-(2,3-dimethyl-6-sulfato)␤-CD. As per the authors, order of elution was DD enantiomers followed by LL antipodes using sulfobutylether-␤-CD or heptakis-(2,3-diacetyl-6-sulfato)-␤-CD as chiral selector. On the other hand, elution order got reverse using heptakis-6sulfato-␤-CD. In another study, Süß [65] used neutral single isomer of cyclodextrin derivative [heptakis-( [2,3]-di-O-acetyl)␤-CD (DIAC-␤-CD)] for resolving dipeptides. The authors reported that the DD enantiomer migrated faster than the LL stereoisomer; with only one exception. As per the authors, the order of elution was same using ␤-CD and sulfated single isomer derivatives heptakis-(2,3-di-O-acetyl-6-sulfo)-␤-CD (HDAS-␤-CD) and heptakis-6-sulfo-␤-CD (HS-␤-CD) in presence of 2,3-disubstituted derivatives DIAC-␤-CD and HDAS␤-CD. On the other hand, reversed elution order was observed for ␤-CD and HS-␤-CD as compared to DIAC-␤-CD and HDAS-␤-CD. These experiments suggested the significance of substitution pattern on wider side of CD cavity toward chiral recognition of dipeptide stereoisomers. This has been confirmed by NMR spectroscopy. Furthermore, Süß [67] used sulfated ␤-cyclodextrin, heptakis-6-sulfato-␤CD and heptakis-(2,3-diacetyl-6-sulfato)-␤-CD as chiral selectors for enantiomeric resolution of dipeptides at pH 5.3. As per the authors, the elution order was reversed upon the reversal of the applied voltage and increasing buffer pH from 2.5 to 5.3, separately and respectively. The authors explained migration behavior on the basis of complexation constants and the mobilities of the peptide-CD complexes. Hammitzsch-Wiedemann and Scriba [68] studied the effect of urea on chiral separations of dipeptides Ala-Phe and Ala-Tyr using ␤-CD in the pH range of 2.5–3.8. According to the authors, a little effect on resolution factor was observed due to buffer concentration. However, improved selectivity at pH 2.5 was observed but decreased at pH 3.8 by adding C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 2. Enanatiomeric resolution of peptides. BGE: 25 mM 18C6H4 (pH 2.0), voltage: 20 kV [100].

2.0 M urea. The authors suggested that addition of urea resulted in a decrease of the apparent complexation constants between ␤-CD and BOC-Phe enantiomers. De Boni et al. [69] resolved Gly-Asp-PheNH2 , Phe-Asp-GlyNH2 , and Cyclo(PheAsp) peptides using CM-␤-CD and S-␤-CD. The BGEs used were 50 mM Na3 PO4 (pH 3), 50 mM formic acid, and pH 3.0 + 10% acetonitrile. The LODs reported were 0.005– 0.1 mM. An example illustrating possibilities of CE in the stereoseparation of dipeptides is shown in Fig. 2. 4.1.2 Chiral crown ethers Crown ethers are synthetic macrocyclic polyethers and appear as crown. Pedersen [82] synthesized these polyethers in 1967. Ether oxygen atoms that are electron donors remain inside the crown cavity and surrounded by methylene groups in a collar fashion. IUPAC nomenclature of these ethers is complex and, hence, trivial names are commonly used. For example, 2,3,11,12-dibenzo-1,4,7,10,13,16 hexaoxacyclooctadeca-2,11-diene is called dibenzo-18-crown6 ether, where dibenzo, 18 and 6 indicate the substituent groups, total number of atoms in the ring and the number of the oxygen atoms, respectively. The chirality in the crown ether is developed by introducing the chiral moieties and, hence, the developed crown ether is called as the chiral crown ether. The most important chiral groups, used for this purpose, are binaphthyl [83–86] biphenanthryl [87–89], hericene derivatives [90], tartaric acid derivatives [91], carbohydrate moiety [92], chiral carbon atom with a bulky group directly incorporated in crown ring [92, 93] aromatic bicyclo derivatives [3.3.1] nonane derivatives [94, 95], hexahydrochrysese or tetrahydroindenoinden [96], and 9,9 -spirobifluorene groups [97]. The capabilities of crowning selectivities of these chiral crown ethers and their stereospecific configurations made them suitable chiral selectors in CE. In the 1990s, 18C6 H4 was used as the popular chiral selector for enantiomeric resolution of dipeptides [55, 98, 99]. www.jss-journal.com


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Kuhn et al. [99] reported chiral resolution of dipeptides using chiral crown ether as chiral selector in the BGE. The baseline resolution of dipeptides with amine group located as far as four bonds from stereogenic center was obtained. The optimized variables were crown ether concentrations, buffer pH, and temperatures. Schmid et al. [100] used (+)-18crown-6-tetracarboxylic acid (18C6 H4 ) as a chiral selector and resolved racemic glycyldipeptides and diastereomeric dipeptides. The authors observed the influence of buffer compositions, crown ether concentrations and organic modifiers. Salami et al. [101] used (+)-18-crown-6-tetracarbonic acid for resolving dipeptides. The addition of acetonitrile and tetra-nbutylammonium bromide resulted in improved chiral separation. Xia et al. [102] used a bare fused-silica capillary with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C6 H4 ) for resolving dipeptides using CE–ESI-MS. 4.1.3 Macrocyclic glycopeptide antibiotics Macrocyclic antibiotics are newest and probably most varied class of chiral selectors [103] introduced by Armstrong [104]. The antibiotics have been found to have a good potential for chiral resolution. It may be due to their specific structures and the capabilities to work under varied BGEs. Besides, their relatively small sizes and well known structures are key points to carry out chiral recognition easily and exactly. These are often complimentary in type of compounds these can separate. For example, rifamycin B (an ansamycin) is enantioselective for many positively charged analytes, whereas vancomycin (a glycopetide) can resolve a variety of chiral compounds containing free carboxylic acid functional groups. Additionally, the resolution of enantiomers by antibiotics is not very sensitive and, hence, highly robust. The antibiotics used for chiral resolution are vancomycin, vancomycin aglycon, Teicoplanin, Teicoplanin aglycon, Ristocetin A, thiostrepton, rifamycin, fradiomycin, streptomycin, kanamycin, and avoparcin. However, the most commonly used antibiotics are vancomycin, Teicoplanin, Teicoplanin, aglycon, and Ristocetin A. Wan and Blomberg [78, 105–107] used macrocyclic glycopeptides antibiotics for enantiomeric resolution of dipeptides. The authors used a BGE of pH above the pIs of macrocyclic antibiotics to avoid their adsorption onto the capillary wall. By adopting this approach, the authors used vancomycin for successful chiral separation of N-terminal-protected dipeptides. Wan and Blomberg [106] used vancomycin for the enantiomeric separation of dipeptides. The concentration effects of vancomycin and 2-propanol (organic modifier) and pH in BGE were studied. The best resolution was at high vancomycin concentrations, while optimum efficiency was at low vancomycin concentrations. According to the authors, due to the high enantioselectivity of vancomycin, low concentration (below the amount necessary for maximal resolution) could be used. Low separation efficiency could be attributed to adsorption of selector at the capillary wall. It was observed that adsorption was decreased at pH above the zero mobility pH value, low vancomycin concentrations, and the presence of 2-propanol. Wan and Blomberg [78] used vancomycin C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

7

and Teicoplanin to separate some derivatized dipeptides. The highest plate numbers were 106 plates/m. The BEG used was borate buffer (40 mM; pH, 9.2; with 15 mM SDS). The plate number was lower, 0.5 × 106 plates/m, but the resolution was high. All the tested peptides were fully resolved with the antibiotics as chiral selectors except Leu-Ala, Leu-Leu, and Arg-Gly. 4.1.4 Ligand exchange Ligand exchange involves the breaking and formation of coordinate bonds among the metal ion of complex, the ligands, and the enantiomers. Therefore, ligand-exchange CE is useful for the chiral resolution of molecules containing electrondonating atoms such as oxygen, nitrogen, and sulfur. Dipeptides have these types of groups and, hence, ligand exchange is quite a good chiral selector for their enantiomeric resolution. Copper(II) is the most commonly used metal in this sort of chiral selector. Schmid et al. [108] used N-(2-hydroxyoctyl)L-4-hydroxyproline and Cu(II) as the chiral selector in the BGE for the enantiomeric resolution of dipeptides. Hödl [109] used Cu(II), Co(II), Ni(II), and Zn(II) complexes of D-gluconic acid, D-saccharic acid, and L-threonic acid as chiral selectors for the chiral resolution of glycyl dipeptides. According to the authors, the Cu(II)-D-gluconic complex acid gave the best results.

4.2 Chromatography Chromatography is a dynamic chiral separation technique involving CSPs surfaces, while analyte molecules are being transported in the mobile phase. In the classic chromatography, flow of mobile phase is driven by pressure drop between the column(s) inlet and outlet. The mobile phase may be gas, liquid or supercritical fluid. In CEC, the mobile phase is driven by the EOF. The significant advantages of liquid and supercritical fluid chromatographies, as compared to other chromatographic modalities and electrophoresis, are capabilities of large-scale separations and reproducibilities. Besides, chiral selector is not wasted in chromatographic separation (including CEC) and resolved stereoisomers are obtained in pure form, unlike in CE, which yields a complex of an analyte and a chiral selector. These features make LC and SFC the methods of choice for preparation of large amounts of pure stereoisomers for further clinical trials in drug discovery [110]. 4.2.1 Gas chromatography GC is a suitable technique for volatile compounds at the working temperature of a gas chromatograph. Small peptides are not volatile compounds, which need derivatization prior to GC analysis. Trifluoroacetyl or pentafluoropropionyl serve as a substitution group at the N terminus and a small alkyl radical is used at the C terminus [111, 112]. Alternatively, dipeptides may be trimethylsilylated [113]. Czerwenka www.jss-journal.com

8

I. Ali et al.

and Lindner [9] reviewed the GC separation of dipeptide stereoisomers on a Chirasil-Val stationary phase (valine derivative) and on a trivaline based Shumichiral OA-300 stationary phase. Oi et al. [114] have reported a modification of a later CSP with higher melting point, called Shumichiral OA-310. The separations of peptide diastereomers can be achieved on an achiral stationary phase as diastereomers differ in physicochemical properties [10]. The importance of the method has declined since the 1980s. Only few reports dealing with the stereoseparation of dipeptides on ChirasilVal [115–117] and cyclodextrin based Lipodex-E were published in the 2000s [115].

J. Sep. Sci. 2014, 00, 1–20

Fluorenylmethyloxycarbonyl chloride (Fmoc) functionalized dipeptides were more strongly retained on smaller ␣-CD than on ␤- and ␥-CDs whereas stereoseparation was better on ␥-CD, followed by ␣-CD and ␤-CD [118]. Interestingly, that elution order was dependent on the type of CD [118, 119]. The effect of RP composition on the retention of small peptides on a ␥-CD-based column was carried out by Chang et al. [120]. The authors considered effect of water/methanol ratio, pH, ionic strength and addition of Cu(II). The mobile phase concentration dependence of k had a cup-like shape with the minimum at around 80% of methanol. The stereoselectivity absent in water rich solvents appeared after that point and increased to the maximum in neat methanol.

4.2.2 Liquid chromatography The HPLC method is more popular in the stereoseparation of small peptides. Reversed-phase conditions are used in the separation of both protected and unprotected small-peptide diastereomers on achiral (C8 -, C18 -, Phenyl-type) columns [10, 34]. CSPs necessary for the resolution of racemates are typically polar surface adsorbents. These are used in combination with both aqueous and nonaqueous (both polar and nonpolar) solvents (Table 2). Nonderivatized small peptides are ionogenic compounds with pKa in an approximate range of 3 to 5 and pKa (ionization of the amino group) in a range of 8 to 10. Therefore, buffer compositions affect the analyte charge and consequently their adsorption properties. When the chiral selector is an ionogenic group, the mobile phase pH affects its state too. As a result, the choice of mobile phase pH will be trade-off between activity of selector and favorability to resolve the ionic state of an analyte. A survey of the literature (Table 2) shows that different diapasons of pH were used with different chiral selectors. CSPs available in HPLC are based on CDs, crown ethers, macrocyclic glycopeptides antibiotics, polysaccharides, Pirkle-type selectors, ligand exchangers, and molecularly imprinted polymers (MIPs). With the exception of MIPs, CSPs are obtained by the immobilization either by covalent binding or by coating of a chiral selector onto the surface of a solid support, usually silica. Table 2 summarizes the literature data on the use of HPLC for the enantioseparations of small peptides, with detailed information given in the following sections.

4.2.2.1 Cyclodextrin CSPs CDs are not as popular in HPLC as in CE. Czerwenka and Lindner [9] reported six studies on the direct stereoseparation of di-, tri-, and tetrapeptides using RP and polar-organic conditions. Retention mechanisms for these two types of mobile phases were different. While in aqueous solvents, the hydrophobic moiety of an analyte molecule formed an inclusion complex with a CD ligand, in nonaqueous systems solvent molecules occupied the selector cavity. Then the solute molecules interacted with peripheral groups of a CD ligand, hydroxyl groups in native CDs [118, 119]. The size of CD ring plays an important role in this interaction. Therefore, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

4.2.2.2 Crown ether CSPs The capabilities of crown ethers for the host–guest complexation of protonated primary amines make this type of selectors suitable for the stereoselective separation of peptides with unprotected N termini [27]. The requirement of a protonated amino group stipulates the use of crown ether based CSP with strongly acidified mobile phases, typical pH 1.5–2.5. Such aggressive media may be dangerous to a CSP; especially with covalently bonded ligands. The first commercial crown ether column applied to the stereoselective separation of small peptides was Crownpak; based on (3,3 -diphenyl-1,19-binaphthyl)-20-crown-6 coated onto C18 silica [10]. It was observed that distance between primary amine group and stereogenic center is the main factor governing chiral resolution [121, 122]. The Crownpak column was not compatible with organic-rich eluents as chiral layer and was subject to leaching when the content of organic fraction exceeded 15% [27]. The covalently bonded silica (18crown-6)-2,3,11,12-tetracarboxylic acid, commercialized under the trade name Chirosil, is free of this drawback. Conrad et al. [28] used a Chirosil SCA with [(–)-crown ether] for the stereoseparation of 12 dipeptides and four tripeptides. Asnin et al. [29, 123] used Chirosil RCA [with (+)-crown ether] for the separations of Ala-Ala and Gly-Leu stereoisomers. Both groups used water/methanol solvents acidified with millimolar sulfuric acid. The retention time increased with methanol concentration, reflecting the negative effect of methanol on solvation of protonated amino group by mobile phase. It was found that the major contribution to stereoseparation was from the interactions of the N-terminal residue with the oxygen atoms of the crown ether cavity. The interactions of the C-terminal residue with the side groups of chiral ligand affected the stereoselectivity to a degree too [29]. 4.2.2.3 Macrocyclic glycopeptide antibiotic CSPs This class of chiral adsorbents appears to be the most popular and the best studied in the separation of peptide stereoisomers [10, 35, 124–126]. Antibiotic CSPs may be used in the reversed phase, polar organic as well as in the normal phase modes. Stereorecognition mechanisms may vary in different mobile phase modes, which increase versatility of these CSPs. However, only reversed phase mode has been used in chiral chromatography of peptides. Generally, www.jss-journal.com


46 oligopeptides, n = 5–13

22 dipeptides, three tripeptides

16

17

Silica bonded Pirkle-type selectors 18 N-DNB-derivatized methyl esters, eight dipeptides, six tripeptides, Phe-Gly-Phe-Ala 19 N-DNB-derivatized methyl esters, 12 dipeptides, two tripeptides

16 dipeptides, Ala-Leu-Gly, Leu-Gly-Phe Seven enkephaline peptides

14 15

20 mM NH4 Ac/MeOH (40:60), pH 7 1% TEAA/MeOH (80:20) H2 O/MeOH (50:50)

(S)-1-(6,7-dimethyl-l-naphthyl) isobutylamine (home- made column) (R)-(-)-N-(2-naphthyl)valine (R)-1-(6,7-dimethyl-1-naphthyl)-ethylamine (home-made colums)

Ristocetin A (home-made column)

Teicoplanin aglycone (Chirobiotic TAG) Teicoplanin (Chirobiotic T) Ristocetin A (Chirobiotic R)

Teicoplanin (home-made column) -doTeicoplanin aglycone, Teicoplanin (home-made column) Teicoplanin (Chirobiotic T) Teicoplanin (Chirobiotic T)* Ristocetin A (Chirobiotic R)**

[29]

-do-

Silica bonded antibiotics 11 Ala-Ala 12 Leu-Val, Leu-Leu, Leu-Phe, Leu-Asn 13 12 Gly-dipeptides

[136] [135]

2-propanol/hexane (20:80)

[129]

[35]

[128] [34]

[126] [127] [124]

[188]

[187]


(22:78) → 0.1% HCOOH/MeCN (30:70) **H2 O/MeCN (20:80) + 0.1% NH4 CF3 COO H2 O/MeCN (85:15–65:35) 5–40 mM HCOONH4 , pH 3/MeCN (90:10–70:30) 0.06–0.1 % HCOOH/MeCN (40:60–25:75) 0.75–1% TEA, pH 2.8/MeCN (80:20–60:40) H2 O/MeOH (50:50) H2 O/MeCN (50:50)

H2 O/EtOH (40:60) * Stepwise gradient 3 mM NH Ac, pH 3.8/MeCN 4

AcOH/TA/MeOH/MeCN (1:2:5:495) TA = TEA, tripropylamine MeCN/MeOH/AcOH/TEA (480:20:1:2) MeCN/MeOH/AcOH/TEA (450:50:1:4)

[185]

H2 O/MeOH (10:90) TEAA/MeOH (10:90), pH 4 AcOH/TEA/MeCN (3:12:1000)

[186]

[118] [120] [140]

MeCN/MeOH/TEA/AcOH (850:150:14:1) 10 mM (NH4 Ac +AcOH)/MeOH (20:80–100:0) 50 mM TEAA/MeOH (10:90–50:50), pH 4

[123]

References

H2 O/MeOH (40:60–10:90) + 5 mM H2 SO4

Mobile phasesc)

[28]

Chiral selector (Column trade name)

H2 O/MeOH (0:100 – 100:0) + (0–10mM) H2 SO4

Dipeptidesa),b)

Silica bonded inclusion type selectors: Cyclodextrins and crown ethers 1 FMOC derivatives, 14 Gly dipeptides ␣-CD, ␤-CD, ␥-CD (home-made columns) 2 11 dipeptides, seven tripeptides ␤-CD, ␥-CD (home-made columns) 3 11 cyclic dipeptides, Gly-Phe, Leu-Tyr, Leu-Phe, ␤-CD (Cyclobond I) Leu-Leu 4 Four cyclyc Gly-dipeptides, Phe-Gly, ␤-CD (Cyclobond I) homoPhe-Gly, PhenylGly-Gly ␥-CD (Cyclobond II) 5d) FMOC derivatives, 5 dipeptides, 8 tripeptides, -doAla-Leu-Gly-Gly 6e) Three Ala-dipeptides, 4 Leu-dipeptides, ␤-CD (home-made column) Leu-Gly-Phe 7 Six dipeptides, Leu-Gly-Phe ␤-CD (Cyclobond I) R- or S-naphthylethyl carbamated-␤-CD (Cyclobond I 2000 RN or SN) 8 14 dipeptides, 4 tripeptides (–)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (Chirosil SCA(–)) 9 Ala-Ala (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (Chirosil RCA(+)) 10 Ala-Ala, Ala-Pro, Gly-Ala, Gly-Leu -do-

No.

Table 2. Enantiomeric resolution of small peptides by LC

J. Sep. Sci. 2014, 00, 1–20

Liquid Chromatography 9

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11 cyclic dipeptides, Gly-Phe, Leu-Tyr, Leu-Phe, Leu-Leu DNB-Alan, n = 2–10

Rh) -Alan, n = 2—6

DNB-Alan, n = 3, 4

Ala-Ala, Ala-Gly, Gly-Gly, 4 tripeptides Alan , Valn , Phen, n = 2–4

20

21

22

23

24 25

9 Gly-dipetides, 3 Ala-dipeptides, 4 Leu dipeptides, 2 tripeptides

32

Phe-GlyAn

Leu5 -enkephalin MIP Leu5 -enkephalinAn MIP Boc-Leu5 -enkephalin MIP Boc-Met5 -enkephalin MIP (home-made column) L-Phe-GlyAn-MIP, L-PheAn-MIPL-PheβNAc) ,L-LeuβNAc) (home-made columns)

Proline (Chiral-Pak WH) L-Proline L-4-hydroxyproline N-(2-hydroxycyclohexyl)-L-proline N-(2-hydroxycyclohexyl)-L-4-hydroxyproline (home-made columns) N-decyl-L-4-hydroxyproline(home-made column)

Chiralcel OD-H Chiralpak AD-H Chiralcel OD

-doZWIX CSPs, Cinchona alkaloid-based zwitterion N-butyl-carbamoylated quinidine

3,5-dinitrobenzoyl phenylglycine (D-Phenylglycine) Quinine, quinidine, and quinine and cinchonidine derivatives (home-made columns) tert-butylcarbamoylquinine, 6’(1-adamantylmethoxy)-9-)-tertbutylcarbamoylcinconidine, 1,4-bis(9-O-quinidinyl)phthalazine (home-made columns) tert-butylcarbamoylquinine (home-made column)

Chiral selector (Column trade name)

[140] [153]

0.25 M CuSO4 10−5 M CuSO4

AcOH/MeCN (7.5:92.5–12:88) gradient (0→30 %) AcOH:MeCN

AcOH/MeCN (5:95)

[143]

[149]

[154]

[132]


0.1 mM CuSO4

[133]

[25] [164]

[33]

[32]


0.5 M NH4 Ac/MeOH (20:80) + AcOH, pH 6 MeCN/MeOH (80:20) + 400 mM AcOH + 4 mM TEA 0.1 M NH4 Ac/MeOH (20:80) + 0.3% AcOH MeOH, 50 mM HCOOH, 25 mM diethylamine

0.5 M NH4 Ac/MeOH (20:80), pH 6

[140]

2-propanolg) /hexane 0.2% Na2 CO3 , pH 7/MeOH (50:50) 0.5 M NH4 Ac, pH 6/MeOH (20:80)

[24, 30]

References

Mobile phasesc)

I. Ali et al.

Molecular imprinted polymers 31 Leu-enkephalin, Leu-enkephalinAn, Boc-Leu-enkephalin, Tyr-D-Ala-Gly-Phe-Leu, Tyr-D-Ala-Gly-Phe-D-Leu, Phe-GlyAn,

30

Polysaccharide CSPs 26 3-Rf-2-iodine-propionyl-Phe-Et Rf = CF3 , n-C3 F7 , i-C3 F7 , n-C6 F13 27 Z-Asp(␤-Bzl)-PheOCH3 Ligand exchange CSPs 28 11 cyclic dipeptides, Gly-Phe, Leu-Tyr, Leu-Phe, Leu-Leu 29 11 Gly-dipeptides, 5 Leu-dipeptides, Ala-Ser, Ala-Val

Dipeptidesa),b)

No.

Table 2. Continued

10 J. Sep. Sci. 2014, 00, 1–20

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N-AcPhe-TrpOMe N-AcPhe-TrpOMe Z-Ala-AlaOMe

Z-Ala-AlaOMe, Z-Ala-Gly-PheOMe

Z-Ala-AlaOMe (*)Z-Ala-Gly-PheOMe (**)Boc-Phe-GlyOEt (***)

Z-Asp-PheOMe, Z-␤-Asp-PheOMe Phe-Phe

33 34 35

36

37

38 39

LL-Analyte-MIP (home-made column) LL-Analyte-MIP DD-Analyte-MIP MIPs are based on a CD-containing matrix (home-made column)

LL-Analyte-MIP (home-made column)

LL-Analyte-MIP (home-made column)

LL-Analite-MIP (home-made column) LL-Analite-MIP (home-made column) LL-Analyte-MIP (home-made column)

Chiral selector (Column trade name) AcOH/CHCl3 (1:99) H2 O/MeCN (0:100–8:92) + AcOHg) AcOH/CHCl3 (0.25:99.75) CHCl3 gradient (0.25→1 %) AcOH:CHCl3 Different mobile phase compositions are specified in the Experimental part and in the Results part (*)AcOH/CHCl3 (0.25:99.75) (**)AcOH/CHCl3 (0.5:99.5) (***) CHCl3 Gradient elution: (*)(0.25→1 %) AcOH/CHCl3 (**)(1→10 %) AcOH/CHCl3 (***) (0.1→1 %) AcOH/CHCl3 AcOH/MeCN (1:99) 50 mM HCOONH4 , pH 6.9

Mobile phasesc)

[147] [150, 151]

[148]

[146]

[144] [145] [142]

References

a) Including glycyl containing tri and tetrapeptides. b) Abbreviations: An = anylide, BOC = tert-butyloxycarbonyl, β NA = ␤-naphthylamid, DNB = 3,5-dinitrobenzoyl, FMOC = 9-fluorenylmethyl chloroformate, Phth = Phthaloyl, TEA = triethylamine, TEAA = triethylamine acetate, Z = benzyloxycarbonyl. c) Unless otherwise specified, elution conditions orrespond to the isocratic mode. For gradient elution, the initial and final concentration is specified, separated by the symbol "arrow". d) The analytes were derivatized with benzoyl chloride or similar type reagents at the N-terminal end. e) Fmoc derivatives. f)cHxAla = cyclohexyl-Ala. f) Concentration of a polar modifier was not specified. g) Protecting groups of different nature. h) Capillary electrochromatography.

Dipeptidesa),b)

No.

Table 2. Continued

J. Sep. Sci. 2014, 00, 1–20

Liquid Chromatography 11

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12

J. Sep. Sci. 2014, 00, 1–20

I. Ali et al.

Table 3. Enantioselectivity of different CSPs with respect to several dipeptides

S. No.

CSP/Chiral selector

Mobile phases

Ala-Ala

Leu-Leu

Gly-Leu

1 2 3 4 5 6 7 8 9 10

␤-CD Chirosil RCA(+) Chirosil SCA(−) Ristocetin A Ristocetin A Teicoplanin Teicoplanin Teicoplanin aglycon L-4-Hydroxyproline Chiralpak WH

H2 O/MeOH (90:10) 5 mM H2 SO4 in 70% MeOH 10 mM H2 SO4 in 70% MeOH H2 O/MeOH (50:50) H2 O/MeCN (50:50) H2 O/EtOH (40:60) H2 O/MeOH (50:50) H2 O/MeOH (50:50) 10−5 M CuSO4 0.25 M CuSO4

1.33 1.17 1.57 2.54 -

2.00 2.31 1.53 1.34 1.68

1.05 1.84 1.17 2.90 3.90 4.87 1.67 -

stereoselectivity of antibiotic based CSPs is comparable to that of the CSPs based on the host–guest complexation selectors, i.e., CDs and crown ethers (Table 3). Among various macrocyclic glycopeptide antibiotics, teicoplanin, teicoplanin aglycon (TAG) and Ristocetin A are used for small peptides. The teicoplanin selector demonstrated better stereoseparation ability than ristocetin A. TAG as a selector is characterized by a higher stereoseparation than teicoplanin [124]. However, it is achieved at the expense of peak broadening and increased retention time. A large volume of empirical data on separation of peptide stereoisomers on silica bonded antibiotics have been acquired by Armstrong and co-workers [35, 127–129]. A useful review has been presented by Illisz et al. [125]. Schmid et al. [124] reported successful applications of these adsorbents in a microbore column. Zhang et al. [35] and Soukup-Hein et al. [34] described the applications of teicoplanin, TAG, and ristocetin A columns for the separation of diastereomers of several peptides with n ࣙ 5, including enkephalin pentapeptides, dynorphins etc. Cavazzini et al. [126] used method of nonlinear chromatography to study the adsorption of Ala-Ala on a Teicoplanin CSP. The authors revealed an essential difference in the adsorption models of weakly retained L-Ala-L-Ala and strongly retained D-Ala-D-Ala. The former enantiomer obeyed a homogeneous Langmuir model while the latter followed Toth’s model. The authors explained a significant, roughly hundredfold, difference in the retention factors of the stereoisomers by the fact that antibiotic macrocyclic Teicoplanin behaved as a molecular filter (at receptorial level) toward the stereoisomers of the dipeptide tested. In other words, antibiotic macrocycle was designed by nature in such a way to recognize a D-Ala-D-Ala fragment while not responding to its optical antipode [125]. The binding capabilities of vancomycin family antibiotics, teicoplanin, and ristocitin A with dipeptide amino acids resemble D-Ala at the C terminus. The molecular aspects of this property are discussed by Ciogli et al. [130]. 4.2.2.4 Polysaccharide-based CSPs Polysaccharide-based CSPs have also been tried for stereoseparation of dipeptides. Wu et al. [131] used Chiralcel OD C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

140 29 28 129 129 128 124 124 153 140

column for stereoseparation of Z-Phe-Ala-OBzl dipeptides. The mobile phase used was n-hexane-isopropanol (5:1, v/v). Lin et al. [132] reported chiral separations of dipeptides on cellulose carbamate derivative CSP (Chiralcel OD). The resolved dipeptides were LL-, LD-, DL-, and DD-(Z)-Asp(␤-Bzl)Phe-OCH3 ; an aspartame precursor. Tonoi et al. [133] have applied two CSPs of this group, Chiralcel OD-H and Chiralpak AD-H, for the separation of fluoro-dipeptide stereoisomers under normal phase conditions.

4.2.2.5 Pirkle-type CSPs In a narrow sense, Pirkle-type stationary phases are chiral adsorbents that contain low-molecular-weight donor–acceptor selectors grafted to a solid support. A key feature of such selectors is the ability to retain adsorbate molecules via several interaction modes simultaneously due to the presence of several functionalities. The interaction modes involved might be hydrogen bonding, ␲–␲ stacking, steric repulsion, etc. ˆ with colleagues [134] seem to be the first who emOi ployed grafted small synthetic selectors, which later would become known as Pirkle-type phases, in the stereoseparation of peptidomimetic compounds. Later, Pirkle et al. [135, 136] described the successful chiral resolution of N3,5-DNB dipeptide esters on different naphthylalkylamineor naphthylvaline-derived CSPs under normal conditions. Dobashi and Hara [137–139] used N-acyl valines bound to aminopropyl silica gel under normal mobile phase for stereoseparation of fully protected dipeptides. All these studies were performed with homemade columns. Florance et al. [140] tested a commercial column with grafted 3,5-DNB phenylglycine with little success. Only racemate of cyclo (TrpTyr) was resolved out of 15 dipeptides studied. The authors explained the lack of resolution of analytes bearing charges impeding stereoselective interactions with the chiral selector. The group of Lindner have developed semisynthetic chiral selectors on the basis of cinchona alkaloids. These phases were able to separate enantiomers (all L versus all D) of N-protected oligoalanine peptides with the number of monomers of 2–10 [30]. The mechanisms of enantiorecognition on these CSPs were elucidated using a combination of chromatographic, NMR spectroscopy, and molecular www.jss-journal.com

J. Sep. Sci. 2014, 00, 1–20


13

Figure 3. Envisaged interactions between the various parts of the selector and a peptide analyte, shown for DNB-Ala-Ala-Ala as an example [25].

modeling techniques [24]. Of special elegance is the study [25], in which the authors investigated the chromatographic behavior of all the possible all L and all D di- and tripeptides containing Ala and Gly monomers. As glycine is an achiral amino acid, the position of Ala monomer is responsible for stereorecognition. The authors developed a model of selector– selectand interactions schematically (Fig. 3).

4.2.2.6 Molecularly imprinted polymers MIP CSPs are bulk polymer solids with chiral cavities left after washing out chiral templates. The concept of molecular imprinted polymerization implies the existence of single-type chiral cavities with well established geometries distributed in an achiral matrix. In practice, the imperfectness of polymerization methods results in manufactured solids with a number of distinct enantioselective and nonselective sites [141]. The first application of the MIP technique in the stereoseparation of peptides came from the group of Mosbach, who developed a series of MIP CSPs by imprinting PheGly anilide and fully protected Phe-Trp, Ala-Ala, and Ala-Gly dipeptides [142–147]. The separations of stereoisomer pairs LL/DD were studied. The separations of all four stereoisomers of N-Ac-Phe-TrpOMe were demonstrated in this work [144]. Kempe [148] described a MIP selective to stereoisomers of Z-protected Ala-Gly-Phe methyl ester. Andersson et al. [149] synthesized a few MIP CSPs capable of recognizing diastereomers of an enkephalin-mimicking pentapetide. One of these CSPs was able to resolve the stereoisomers of Phe-Gly anilide. Most of the studies were carried out with acidified organic eluents. The presence of an acid was necessary to maintain polymer carboxylic acid residues in the protonated form. Nicholls et al. [145] showed that addition of water essentially reduced stereoselectivity. Akiyama et al. [150] and Hishiya et al. [151] developed silica-coated MIP adsorbents [150,151] compatible with aqueous solvents. These types of solids had a thin layer of imprinted CD-methylenebisacrylamide copolymer over silica gel beads. The authors prepared materials using LL or DD Phe-Phe as a template. The resulting CSPs demonstrated low but measurable stereoselectivity. Although MIPs can demonstrate remarkable enantioselectivity [144], frequently enantioselectivity coefficients for these CSPs are rather moderate, ranging between 1 and 2. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 4. Chromatogram of the chiral resolution of a racemic Ac-Phe-Trp-OMe (i.e., 1 and 2), using chloroform (l.0% AcOH) as eluent. The attenuation was increased by ten times after 13 min to provide a clearer view of the peak of 1 [144].

An essential disadvantage of this sort of chromatographic phases is an undesirable strong retention of a template analyte; frequently requiring harsh conditions for complete removal. Besides, the peaks of the retained compounds are usually diffused. A typical chromatogram of a stereoisomers pair in Fig. 4 illustrates the above mentioned drawbacks. 4.2.2.7 Ligand-exchange CSPs Ligand-exchange chromatography of small peptides has been reviewed [10, 152]. Nevertheless, there is still room for some relevant comments and generalizations. In general, the enantioselectivity of ligand-exchange systems involving small peptides is low to moderate. Gübitz et al. [153] have achieved stereoselectivity greater than 2.0 only for two Gly-X dipeptides of 12 studied. The authors used a covalently bonded L-4-hydroxyproline as the CSP. The similar results were reported by Florance et al. [140] for the stereoisomers of Leu-Leu and Leu-Tyr (␣ ࣈ 1.7 for either dipeptide). Schmid et al. [154] also reported stereoseparations a series of dipeptides on dynamically coated ligand-exchange columns. Such level of stereoselectivities allowed a partial resolution of four stereoisomers of dipeptides [153]. On the other hand, in some cases a baseline resolution of all four stereoisomers was possible [155, 156]. Many ligands have been suggested for this mode of chromatography [152]. Proline and its derivatives remain the main workhorse of ligand-exchange separation (Table 2). One of the drawbacks of ligand exchange separation is a long analysis time (20–60 min). Schmid et al. [154] turned this difficulty by applying C18 monolithic columns coated with ligand exchange selectors. The monolithic columns allowed high flow rate resulting in faster elution of samples. Figure 5 compares the separation of Gly-L-Leu and Gly-D-Leu www.jss-journal.com

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Figure 5. Chromatograms of enantiomeric resolution of Gly-Leu on N-decyl-L-4-hydroxyproline monolithic column at a flow rate of 8.0 mL/min (A) and 0.5 mL/min (B) and on a particulate ligand exchange column at a low flow rate of 0.5 mL/min (C). (A, B) Stationary phase: RP-18 monolithic column coated with N-decyl-L-4-hydroxyproline. Mobile phase: 0.1 mM Cu(II) sulfate. (C) Stationary phase: 3 ␮m RP-18 silica coated with N-decyl-L-4-hydroxyproline. Mobile phase: 0.1 mM Cu(II) sulfate [154].

on such a coated monolithic column and on a particulate ligand-exchange column.

demonstrated the usefulness of these CSPs for the stereoseparation of several dipeptides and Phen -oligopeptides (Fig. 6).

4.2.2.8 Miscellaneous CSPs Besides traditional CSPs, some less popular or new materials have been tried in the chiral chromatography of peptides. Hirayama et al. [157] used synthetic amino acid polymer CSPs for the separation of stereoisomers of Z-Leu-Phe methyl ester. Jadaud and Wainer [158, 159] reported chiral separation of dipeptides on ␣-chymotrypsin based CSP. A new type of CSP was introduced about a decade ago, which is based on oligonucleotides sequences [160]. Aptamers (possessing an antibody like affinity) are suited for enantioseparation, considering strong dependence of nucleic acid–protein interactions on stereochemical factors [161]. It was proven by Michaud et al. [161] who separated all L and all D stereoisomers of nonapeptide arginine-vasopressin. Recently, Huang et al. [162] demonstrated the capabilities of aptamer-based materials in separating amino acid enantiomers. They suggested that stereoisomers of larger molecules such as dipeptides and tripeptides could also be resolved. An extension to family of cinchona alkaloid CSPs (zwitterionic cinchona-based CSPs) was manufactured in the laboratory of Lindner [163]. This type of CSP was prepared by fusing a cinchona alkaloid entity bearing a positive charge (anion exchanger) and a sulfonic acid residue; bearing a negative charge (cation exchanger). Such selectors facilitate the retention of nonderivatized amphoteric molecules under weakly acidic and polar organic conditions. It is based on simultaneous double ion pairing between the acidic and basic sites of the selector and solute [164]. Wernisch and Lindner [164]

4.3 Thin-layer chromatography


TLC is an inexpensive alternative to LC and useful in qualitative or semiquantitative separation. The method is routinely used in organic synthesis for an immediate control of reaction products or in phytochemistry for the screening of plant extracts. Enantioselective TLC can be performed by either using chiral stationary phases or chiral selectors in the mobile phases. The former are prepared either from chiral materials (e.g. microcrystalline cellulose triacetate [165]) or by impregnating an achiral silica or RP plate with a chiral reagent [166]. Ligand-exchange plates employing copper complexes of proline derivatives were commercialized under the trade names of Chiralplate (Macherey and Nagel) and CHIR (EMD Merck) [167]. The chiral mobile phases are frequently prepared with CD additives [166]. Chiral TLC has occasionally been used in the stereoisomer separation of dipeptides in the past [10]. Wall [167] gave an example of the resolutions of D-Leu-L-Leu and L-Leu-DLeu stereoisomers on a CHIR high-performance thin-layer chromatography plate using methanol/1-propanol/water as the mobile phase. Rare publications in this area in recent years are attributed due to disadvantages of TLC. These include mostly manual operation and that it is a nonquantitative technique [10]. At the same time, chiral TLC finds many applications in the area of amino acid analysis at present [166]. Thus, one may expect that expansion of research in small peptide chemistry may create interest in TLC. www.jss-journal.com

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Figure 6. Chromatograms of enantioseparation of Phe-4 peptides on a quinine-based zwitterionic CSP, Mobile Phase: MeOH, 50 mM FA, 25 mM DEA, 1.0 mL/min [164].

4.4 Hybrid techniques 4.4.1 Capillary electrochromatography CEC combines the high selectivity of stationary phases for LC and the high performance efficiency (resulting in narrow peaks) of CE. CEC separation can be performed in packedbed capillaries (particle size is typically 3–3.5 ␮m), in monolithic capillary columns prepared by in situ polymerization or in open-tube capillaries with chiral selectors grafted onto the capillary walls [168]. Schmid et al. [169] compared CEC with micro-HPLC in the stereoseparation of glycyl dipeptides using TAG immobilized on 3.5 ␮m silica particles. These authors observed that the former method is more efficient as characterized by greater selectivities and shorter analysis time than micro-HPLC. The authors used slightly acidic hydro-organic mobile phases. The organic modifier appeared to play a significant role in separation. Ethanol resulted in superior enantioselectivity but long analysis time. Acetonitrile decreased analysis time with reduced enantioselectivity. The same authors developed a particle-loaded monolithic capillary column with the same selector [170,171], but no improvement was observed compared to the packed-bed capillary. Pittler et al. [172] described a ligand-exchange CSP for chiral separation of dipeptides in CEC. The CSP had L-4hydroxyproline covalently bonded to 3.0 ␮m silica particles. The enantioselectivities reported were rather low. The values for seven glycyl dipeptides of eight studied were lower than 1.2 (except 1.53 for Gly-Nvl). The diastereomeric dipeptides and tripeptides were partly separated. The migration time of the last eluted diastereomer ranged from 13 to 28 min. The authors also tested a dynamically coated L-4-hydroxyprolinebased system, which demonstrated even worse results. One may conclude that ligand-exchange CEC is inferior to ligandexchange HPLC in terms of resolution power but allows roughly two times faster analysis with sufficient resolution. Ludewig et al. [173] prepared a hydrophobic monolithic capillary column, which was capable to separate diastereomers C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

of several dipeptides and tripeptides, both unfunctionalized and peptide esters. The elution order with rare exceptions could be explained based on pKa values of the peptide diastereomers. Less acidic L stereoisomers eluted faster than DL or LD diastereomers. More information on CEC separations is given in Table 4. 4.4.2 Micellar electrokinetic chromatography MEKC is an electrophoretic method based on the distribution of analytes between micelles and the free solution. The micelles formed by a surfactant above its critical micellar concentration play a role of pseudostationary phase only, unlike a real stationary phase in LC. The micelles travel through capillary driven by the superposition of EOF and electrophoretic mobility [174, 175]. The BGE is usually an alkaline solution with pH 9–10 to maintain a strong EOF. MEKC enantioseparations can be carried out using two basic approaches. The first mode imparts enantioselective properties to micelles formed by a chiral surfactant. The alternative approach consists of achiral micelles and chiral selector in the running buffer [10, 17]. This second method has been preferentially used in direct separation of peptide stereoisomers. SDS is a typical surfactant employed in a combination with a CD (␤and ␥-CD [76] and ␥-CD [176]) as a chiral selector. Skanchy et al. [74] described the effect of some anionic and neutral surfactants on separation efficiency of acylated dipeptide stereoisomers using sulfobutyl ether-␤-CD as chiral selector. Wan and Blomberg [105] used vancomycin as a chiral selector in SDS-MEKC of Fmoc-derivatized dipeptides. Besides SDS, sodium deoxycholate was used by Chen et al. [177] in combination with 2-hydroxypropyl-␤-CD for the stereoisomeric separation of Tyr-Phe, Tyr-Leu, and Ala-Gln derivatized with naphthalene-2,3-dicarboxyaldehyde. Shen et al. [178] resolved Ala-Met, Ala-Leu, Ala-Val, and Ala-Leu-Gly using a combination of ␤-CD and sodium dioxycholate. The indirect peptide chiral separation in combination with MEKC has been popular since 2000. It consists of www.jss-journal.com

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Table 4. Enantiomeric resolution of small peptides by capillary electrochromatography and micellar electrokinetic chromatography

No.

Dipeptides

Chiral Selectors

BGEs

1

Capillary 12 Gly-dipeptides

ElectroTeicoplanin aglycone

2

Ala-Ala, Ala-Gly, Ala-Gly-Gly, Ala-Phe, Ala-Met

Teicoplanin aglycone

3

12 Gly-dipeptides

Teicoplanin aglycone

4

O-9-(tert-butylcarbamoyl) quinidine (home column)

5

homo-Phe␺(PO2 HCH2 )Phe, DNP-homo-Phe␺(PO2 HCH2 )Phe, Z-homo-Phe␺(PO2 HCH2 )Phe, pseudodipeptides Leu-Phe, Leu-Trp

Chromatography (0.2 % TEA + AcOH pH 4.1)/MeCN (50:50) 0.2% TEAA, pH 4.1/MeOH (90:10) 0.2% TEAA, pH 4.1/MeCN (70:30) 0.2% TEAA, pH4.1/MeCN/EtOH (50:30:20) 0.2% TEAA, pH 4.1, 5.8, 7.35 0.2% TEAA, pH 4.1/MeOH (80:20) 0.2% TEAA, pH4.1/MeCN/EtOH (50:30:20) MeCN/MeOH (50:50 to 100:0) + 400 mM (acetic acid + formic acid) + 4 mM TEA

6

8 Gly-dipeptides

L-4-Hydroxyproline

7

7 dipeptides (Separation of diastereomeres)

Hydrophobic monolithic column

Micellar Tyr-Phe, Tyr-Leu, Ala-Gln

Electrokinetic 100 mM Tris-H3PO4 + 40–50 mM sodium deoxycholate, pH 8.5, 9.0, 9.2 Vancomycin 2-Hydroxypropyl-␤-CD

Chromatography 2HP-␤-CD

[177]

SDS SDS, sodium deoxycholate

[105] [177]

␤-CD

Sodium dioxycholate

[178]

8

9. 10.

11.

FMOC-derivatized dipeptides Tyr-Phe, Tyr-Leu and Ala-Gln derivatized with naphthalene-2,3dicarboxyaldehyde Ala-Met, Ala-Leu, Ala-Val and Ala-Leu-Gly

L-Hydroxyproline (monolithic column)

derivatization of an analyte with a chiral reagent to produce a diastereomeric mixture, which can be separated using an achiral MEKC. The different approaches to the indirect technique were reviewed [10]. The method seems to have lost its importance as no further application of this technique in MEKC analyses of peptide stereoisomers had been reported in recent years. Some applications of MEKC are given in Table 4.

5 Concluding remarks and future prospects Chiral separation methods for small peptides have gained interest since the early 1990s with the realization of the importance of enantiopure drugs and food additives. The importance of D amino acid containing peptides for cell metabolism has been achieved [13, 179], which stimulated interest to stereoisomeric composition of biological samples. The first decade of active research in peptide stereoisomer analyses (1990–2000) was devoted to search for appropriate separation systems. The results of this work were switched


Refs.

MeCN/0.50 mM Cu(Ac)2 + 50 mM NH4 Ac (7:3), pH 6.5 25 mM NH4 Ac + 0.5 mM Cu(Ac)2 , pH 6 30 mM phosphate buffer, pH 3.0/MeCN (60:40)

[171] [189]

[169]

[190]

[191] [172] [173]

from indirect methods to direct chiral separations. CDs and macrocyclic antibiotics were the most important findings of this “Sturm and Drang” period. These still play a pivotal role in the stereoisomer separation of small peptides. In the last decade, research activities shifted to open new possibilities through developing new separating materials (aptamers, zwitterionic CSPs). These studies brought new techniques into this area such as combinations of CE and HPLC with MS detectors, 2D-LC [180] and the development of miniaturized analytical devices. Two other important tendencies are to be noted, i.e., reducing analysis time and complication of analyzed samples. The former one has obvious economical justification. The first successful attempt to achieve this goal has been demonstrated [156]. A fast UHPLC instrument has not been tested in the chiral chromatography of peptides. However, it had been used to separate enantiomers of derivatized amino acids [181]. Therefore, the use of UHPLC is expected in near future for chiral separations of peptides. The second mentioned tendency of analyzing mixtures (containing diastereomers of several small peptides) arose in physiological research. In metabolic processes small stereoisomeric oligopeptides with the number of monomers

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of four to ten are involved. Besides, multistereoisomer mixtures of dipeptides are found in the food and pharmaceutical industries. The separation of such mixtures in one run is economically beneficial and a challenge to develop analytical techniques further. To the best of our knowledge, only one report described a preparative chiral separation of dipeptides with poor results [140]. At the same time, preparative chiral chromatography is extensively used for the purification of amino acid enantiomers. One can assume the needs of large-scale separation of peptide stereoisomers. Briefly, it is expected that more attention is required in this area due to the growing demand of oligopeptide stereoisomers. The authors are thankful to the Department of Science and Technology (DST), New Delhi, India (project No. DST/INT/RFBR/P-147), the Russian Foundation of Basic Research (RFBR), Russia (Grant No. 13-03-92692) and King Saud University, Riyadh, Saudi Arabia (Visiting Professor Program) for funding.


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