Comparison of the Separation Performances of Cinchona Alkaloid-Based Zwitterionic Stationary Phases in the Enantioseparation of β2- and β3-Amino Acids

Ilisz, István; Grecsó, Nóra; Misicka, Aleksandra; Tymecka, Dagmara; Lázár, László; Lindner, Wolfgang; Péter, Antal

doi:10.3390/molecules20010070

Open AccessArticle

Comparison of the Separation Performances of Cinchona Alkaloid-Based Zwitterionic Stationary Phases in the Enantioseparation of β²- and β³-Amino Acids

by

István Ilisz

¹,

Nóra Grecsó

^1,2,

Aleksandra Misicka

³,

Dagmara Tymecka

³,

László Lázár

²,

Wolfgang Lindner

⁴ and

Antal Péter

^1,*

¹

Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, H-6720 Szeged, Hungary

²

Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös utca 6, H-6720 Szeged, Hungary

³

Department of Chemistry, University of Warsaw, Pasteura str. 1, 02-093 Warsaw, Poland

⁴

Department of Analytical Chemistry, University of Vienna, Währinger Strasse 38, 1090 Vienna, Austria

^*

Author to whom correspondence should be addressed.

Molecules 2015, 20(1), 70-87; https://doi.org/10.3390/molecules20010070

Submission received: 10 November 2014 / Accepted: 15 December 2014 / Published: 23 December 2014

(This article belongs to the Special Issue Dynamic Stereochemistry)

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Abstract

:

The enantiomers of twelve unusual β²- and β³-homoamino acids containing the same side-chains were separated on chiral stationary phases containing a quinine- or quinidine-based zwitterionic ion-exchanger as chiral selector. The effects of the mobile phase composition, the nature and concentration of the acid and base additives and temperature on the separations were investigated. The changes in standard enthalpy, ∆(∆H°), entropy, ∆(∆S°), and free energy, ∆(∆G°), were calculated from the linear van’t Hoff plots derived from the ln α vs. 1/T curves in the studied temperature range (10–50 °C). The values of the thermodynamic parameters depended on the nature of the selectors, the structures of the analytes, and the positions of the substituents on the analytes. A comparison of the zwitterionic stationary phases revealed that the quinidine-based ZWIX(−)™ column exhibited much better selectivity for both β²- and β³-amino acids than the quinine-based ZWIX(+)™ column, and the separation performances of both the ZWIX(+)™ and ZWIX(−)™ columns were better for β²-amino acids. The elution sequence was determined in some cases and was observed to be R < S and S < R on the ZWIX(+)™ and ZWIX(−)™ columns, respectively.

Keywords:

column liquid chromatography; β²-amino acids; β³-amino acids; Cinchona alkaloid-based chiral stationary phases

1. Introduction

In view of their diverse applications in various areas of organic and medicinal chemistry, β-amino acids have received increased attention in recent decades. They are present in numerous natural or synthetic bioactive molecules (e.g., taxane derivatives, sitagliptin, β-lactam antibiotics, etc.). β-Amino acids are applied in the synthesis of heterocyclic derivatives with wide structural diversity, and a rapidly growing class of oligomeric peptides with considerable biological and catalytic properties [1,2,3,4,5]. The newly developed enantioselective syntheses of β-amino acids [5,6,7,8] require analytical methods for a check on the enantiopurity of the final products.

The separation and identification of β-amino acid enantiomers have mainly been performed by indirect [9,10] and direct high-performance liquid chromatographic (HPLC) methods [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. Several types of chiral stationary phases (CSPs) have been applied for the enantioseparation of β-amino acids. Macrocyclic glycopeptide-based CSPs were utilized by D’Acquarica et al. [12] and Péter et al. [13,14]. Since the introduction of chiral crown ethers as CSPs by Cram et al. [15], a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based CSP has been successfully employed for the enantioseparation of β-amino acids by Hyun et al., [16,17,18,19,20,21] and Péter et al., [22,23]. A chiral ligand exchange column [24] and newly developed zwitterionic Cinchona alkaloid-based selectors [25,26,27] were recently used for the enantioseparation of β-amino acids.

Enantioselective retention mechanisms are well known to be influenced by temperature. Accordingly, as demonstrated in several papers [28,29,30,31,32,33], optimization of the column temperature is essential in enantioselective HPLC separations.

The dependence of the retention of an analyte on the temperature can be expressed by the van’t Hoff equation, which may be interpreted in terms of mechanistic aspects of chiral recognition:

l n k' = - \frac{Δ H^{o}}{R T} + \frac{Δ S^{o}}{R} + ln ϕ

(1)

where ∆H° is the standard enthalpy of transfer of the solute from the mobile phase to the CSP, ∆S° is the standard entropy of transfer of the solute from the mobile phase to the CSP, R is the gas constant, T is the temperature in Kelvin and ϕ is the phase ratio of the column (the volume of the stationary phase divided by the volume of the mobile phase). If ϕ is constant or independently measured, ∆H° can be determined by using the van’t Hoff plot (if ∆H° is invariant with temperature) [34,35]. Unfortunately, ϕ is not known in most cases, and even the definition of the stationary phase volume for bonded-phase columns is doubtful. However, if both enantiomers have access to the same stationary phase volume, the ∆(∆H°) and ∆(∆S°) values for the separated enantiomers can be determined from the modified equation:

l n α = - \frac{Δ (Δ H^{o})}{R T} + \frac{Δ (Δ S^{o})}{R}

(2)

where α is the selectivity factor (α = k₂/k₁), ∆(∆H°) is the difference in the standard enthalpy change, and ∆(∆S°) is the difference in the standard entropy change for the two enantiomers. With this simplified approach, a plot of R ln α vs. 1/T has slope −∆(∆H°) and intercept ∆(∆S°).

For the purposes of this study, the classical van’t Hoff approach assuming only one site interaction was used. For a more realistic approach to the thermodynamic calculations, the contributions of enantioselective and non-selective sites should be distinguished. This can be achieved through the application of non-linear characterization methods [36,37].

The aim of the present work was to investigate the effectiveness of Cinchona alkaloid-based CSPs for the separation of a series of isobaric β²- and β³-homoamino acids. For comparison purposes, most of the separations were carried out at constant mobile phase compositions at different temperatures. The influence of specific structural features of the analytes and selectors and the effects of temperature on the retention will be discussed on the basis of the experimental data. The elution sequence was in some cases determined by spiking the racemate with an enantiomer with known absolute configuration.

2. Results and Discussion

The two classes of analytes, β²- and β³-homoamino acids, differ in the positions of their substituents (Figure 1). In both classes, analogues 1, 2 and 3 bear alkyl groups, which may have different steric effects. This influences the hydrophobicity, bulkiness and rigidity of the molecules. Compounds 4, 5 and 6 possess aromatic rings, potentially able to undergo π-π, steric/rigid or other interactions with the aromatic structure elements of the chiral selector and CSP (Figure 2).

Figure 1. Structures of investigated β² and β³ amino acids: 3-Amino-2-methylpropanoic acid (β²-1), 2-aminomethylbutanoic acid (β²-2), 2-aminomethyl-3-methylbutanoic acid (β²-3), 3-amino-2-(naphthalen-2-ylmethyl)propionic acid (β²-4), 3-amino-2-(4-methyl- benzyl)propionic acid (β²-5) and 3-amino-2-(4-chlorobenzyl)propionic acid (β²-6), 3-aminobutanoic acid (β³-1), 3-aminopentanoic acid (β³-2), 3-amino-4-methylpentanoic acid (β³-3), 3-amino-4-(naphthalen-2-yl)butanonic acid (β³-4), 3-amino-4-(4-methylphenyl)butanoic acid (β³-5), 3-amino-4-(4-chlorophenyl)butanoic acid (β³-6).

Figure 2. Structures of quininine- [ZWIX(+)™] and quinidine-based [ZWIX(−)™] CSPs.

2.1. Effect of the Mobile Phase Composition

The Cinchona-derived CSPs have often been used in non-aqueous polar ionic mode (PIM), with MeOH and MeCN as bulk solvent, together with acid and base additives [38]. The polar protic character of MeOH weakens the H-bonding interactions, while MeCN as an aprotic solvent weakens the aromatic π-π interactions and strengthens the ionic interactions. To ensure primary ionic interactions with the selector (SO), an acid and base are generally added to the non-aqueous polar organic solvents, acting as ion pair and charge-giving components of the ionizable ampholytic SO and the selectand (SA).

The influence of the composition of bulk solvents on the chromatographic parameters was investigated for analytes containing an alkyl (1) or aryl (4) side-chain, on ZWIX(+)™ and ZWIX(−)™ in the presence of MeOH as protic and MeCN as aprotic solvent. The mobile phase system consisted of MeOH/MeCN, with increasing amounts of MeCN in MeOH (25%, 50% and 75%), and contained 50 mM AcOH and 25 mM propylamine (PRA) or 50 mM AcOH and 25 mM tripropylamine (TPRA), the acid-to-base ratio being kept constant at 2:1 (Figure 3 and Supplementary Material Tables S1 and S2). As in earlier studies [39,40] on both columns in a MeOH/MeCN bulk solvent system containing 50 mM AcOH and 25 mM PRA, the retention of zwitterionic amino acids increased substantially with increasing MeCN content (Figure 3). Similar results were obtained on both columns in the MeOH/MeCN eluent system containing 50 mM AcOH and 25 mM TPRA (Supplementary Material, Tables S1 and S2). This trend was observed for all the investigated analytes on both stationary phases.

Figure 3. Effects of the compositions of the bulk solvents on the chromatographic parameters for compounds β²-1, β³-1, β²-4 and β³-4 on ZWIX(+)™ or ZWIX(−)™ CSPs. Chromatographic conditions: column, Chiralpak ZWIX(+)™ and ZWIX(−)™; mobile phase, a, MeOH/MeCN (75/25 v/v) containing 25 mM PRA and 50 mM AcOH, b, MeOH/MeCN (50/50 v/v) containing 25 mM PRA and 50 mM AcOH, c, MeOH/MeCN (25/75 v/v) containing 25 mM PRA and 50 mM AcOH d, MeOH/MeCN (75/25 v/v) containing 25 mM TPRA and 50 mM AcOH, e, MeOH/MeCN (50/50 v/v) containing 25 mM TPRA and 50 mM AcOH, f, MeOH/MeCN (25/75 v/v) containing 25 mM TPRA and 50 mM AcOH; flow rate, 0.6 mL·min⁻¹; detection, 215 and 230 nm or corona detector; temperature 25 °C; symbols, k₁: Molecules 20 00070 i001