44 August / September 2016

Chiral Amino Acid and Peptide Separations – the Next Generation

Denise Wallworth, Sigma-Aldrich, now part of Merck.

Chiral chromatography became well established in the 1980’s as a routine technique in pharmaceutical, food and environmental applications and now largely replaces earlier techniques that used derivatisation of the enantiomers with a chiral reagent and their separation on standard, achiral HPLC columns. For amino acids, the ability to separate as free amino acid enantiomers eliminates uncertainty in the determination of enantiomeric configuration compared to the use of a chiral derivatisation reagent. Several chiral stationary phases (CSPs) are available for this separation (Table 1), but whilst HPLC stationary phases have undergone a revolution with the introduction of sub-2µm and superficially porous particles (SPP), CSPs have generally remained on 3 and 5µm silica supports. This article reviews current methods, technology and new research that should bring UHPLC advantages to chiral HPLC.

Chiral Amino Acids and Peptides

Although all proteinogenic amino acids are present in nature as L-enantiomers (and so their biological interactions are stereochemical predictable), enzymatic posttranslational modifications can result in the incorporation of D-amino acids in some proteins, notably in small molluscs [1]. They are also abundant components of peptidoglycan cell walls of bacteria [2]. Further, D-Serine has been known for some time to act as a neurotransmitter, activating the N -methyl- D-aspartate (NMDA) receptor: it originates by synthesis in the brain from its L- enantiomer [3,4]. The ability to easily enantiomerically separate amino acids is therefore important to our understanding of nature and human biology.

Enantiomerically pure amino acids are frequently used as chiral building blocks in asymmetric synthesis such that monitoring both purity and reaction progress are important applications of chiral separations [5,6]. Additionally, because of the clinical monitoring of biomarkers [5] and peptides of therapeutic importance [7], interest in the effective separations of chiral amino acids has continued to grow over recent years.

For bioactive peptides, an analysis of their amino acid sequences is an essential key to their biological functions. Whilst amino acid analysis and tandem MS sequencing is available, the determination of amino acid stereochemistry in a peptide is critical to understanding possible post-translational

modifications and unexpected digressions from homochirality in which the natural L-isomer is replaced by the D-isomer. The action of peptidyl-aminoacyl-L/D- isomerases, for instance, can convert an L-enantiomer to its D-counterpart during biological peptide synthesis [8]. D-residues in a peptide linkage can also result from age-dependent racemization [9].

Chiral Separations

The separation of free amino acids has been made possible by several different CSPs that include Crown Ether, Ligand Exchange, while N-protected amino acids can be separated using brush type and cyclodextrin CSPs. Macrocyclic glycopeptide and chinchona alkaloid CSPs generally have the ability to separate both. Table 1 outlines the most commonly used CSPs for the direct separation of free and N-protected amino acids.

The mechanism of crown ether CSPs, available coated onto 5µm silica and (recently added) 3µm immobilised (commercially, CROWNPAK CR-I), relies on the multiple hydrogen bonding interactions between the primary amine of the amino acid and the ether moieties of the crown ether and so requires that the amine is cationic through the use of an acid (pH2- 3) in the mobile phase. In practice, amino acids with a secondary amine functionality (such as Proline) generally do not separate because of insufficient interactions [10]. This

CSP is compatible with LC-MS, as shown in a recent study [10], in which LC-TOFMS with an isocratic ACN/Water/TFA mobile phase enabled the separation of 18 of the proteinogenic amino acids without derivatisation.

Ligand Exchange separations use a more unusual mobile phase of CuSO4, useful if the amino acid does not have a UV chromophore. These CSPs utilise a bonded D- or L-amino acid (phenylalanine, in the case of Supelco CLC columns, for example) and so can be used for the reversal of elution order in trace analysis by simply changing columns [11,12]. Brush type and protein based CSPs have been also been utilised for the separation of some derivatised amino acids [13].

However, phases based on ionisable teicoplanin and the zwitterionic quinine/ quinidine selectors have become the broadest ranging and most useful today (see later sections on these). The main reason for this is the broad scope for separations as both separate free natural and synthetic α, β, γ-amino acids, whether primary or secondary, aliphatic or aromatic, cyclic or acyclic, in addition to small peptides (and of other amphoteric compounds). The prevalence of LC-MS is another reason; both are fully compatible. Interestingly, teicoplanin bonded CSPs have also been used in clinical applications to separate isobaric biomarker amino acids (example, glutamine and lysine[14].

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