Enantioseparation amino acid enantiomers

Early examples of enantioselective extractions are the resolution of a-aminoalco-hol salts, such as norephedrine, with lipophilic anions (hexafluorophosphate ion) [184-186] by partition between aqueous and lipophilic phases containing esters of tartaric acid [184-188]. Alkyl derivatives of proline and hydroxyproline with cupric ions showed chiral discrimination abilities for the resolution of neutral amino acid enantiomers in n-butanol/water systems [121, 178, 189-192]. On the other hand, chiral crown ethers are classical selectors utilized for enantioseparations, due to their interesting recognition abilities [171, 178]. However, the large number of steps often required for their synthesis [182] and, consequently, their cost as well as their limited loadability makes them not very suitable for preparative purposes. Examples of ligand-exchange [193] or anion-exchange selectors [183] able to discriminate amino acid derivatives have also been described. 

The indirect method is an efficient technique for the enantioseparation of amino acids. However, it is essential that the chiral derivatization reaction proceeds completely in both enantiomers, and that the racemization reaction does not occur. Furthermore, if the optical purity of the derivatization reagent is not known, and/or is not taken into consideration, the optical purity of the amino acid will not be determined precisely. Thus, the indirect method is unsuitable for the analysis of amino acid enantiomers in a standard sample and pharmaceutical preparations, where a low amount of antipode, at a level of 0.1 or 0.05%, should be determined. 

Many chiral derivatization reagents have been developed for the enantioseparation of amino acids wherein ultraviolet-visible or fluorescence tags are introduced. The fluorescence derivatization is more effective for the determination of amino acid enantiomers in complex matrices in terms of sensitivity and/or selectivity. Table 1 shows the chiral derivatization reagents, whose structures are shown in Figure 2, used for the enantioseparation of amino acids. 

This report presents various methods developed primarily at our laboratory for chromatographic resolution of racemates of several pharmaceuticals (e.g., -blockers, NSAIDS, anta-acids, DL-amino acids, Bupropion, Baclofen, Etodolac, Carnitine, Mexiletine). Recently, we developed methods for establishing molecular dissymmetry and determining absolute configuration of diastereomers (and thus the enantiomers) of (/< .S )-Baclofcn, (/d.SJ-Bctaxolol with complimentary application of TLC, HPLC, H NMR, LCMS this ensured the success of diastereomeric synthesis and the reliability of enantioseparation. 

Later, a commercially available TAG CSP was tested in the enantioseparation of 10 secondary a-amino acids, by using RP mobile mode systems [154]. The chromatographic results, compared with those obtained on a native teicoplanin CSP, were given as the retention, separation, and resolution factors, together with the enanti-oselective free energy difference corresponding to the separation of the investigated enantiomers. 

In order to generally categorize the reaction schemes mentioned previously and the following ones in the course of indirect enantioseparation techniques, it has to be emphasized again, that the reciprocity principle should always be applicable. This means that if a chiral acid as the CDA can be used successfully to resolve the enantiomers of a chiral amine, then this optically pure amine as the CDA will equally well separate the enantiomers of the acid by the indirect method. The OPA reaction (see Figure 4) is therefore equally well suited for analyzing the optical purity of thiols, amines or amino acids. 

Lammerhofer and Lindner [90] explained the chiral resolution of N-derivatized amino acids by CEC. The authors explained the formation of the transient diastereomeric ion-pairs between negatively charged analyte enantiomers and a positively charged chiral selector by multiple intermolecular interactions which might be differentially adsorbed to the ODS stationary phase. Furthermore, they claimed that the enantioseparation was achieved because of different observed mobilities of the analyte enantiomers originating from different ion-pair formation rates of the enantiomers and/or differential adsorption of the diastereoisomeric ion-pairs to the ODS stationary phase [90]. 

The use of a convective macroporous polymer as an alternative support material instead of silica for the preparation of protein-based CSPs has successfully been demonstrated by Hofstetter et al. [221]. Enantioseparation was performed using a polymeric flow-through-type chromatographic support (POROS-EP, 20 pm polymer particles with epoxy functionalities) and covalently bound BSA as chiral SO. Using flow rates of up to 10 ml/min, rapid enantiomer separation of acidic compounds, including a variety of amino acid derivatives and drugs, could be achieved within a few minutes at medium efficiencies, typical for protein chiral stationary phases (Fig. 9.13). 

Also, it is expected that the micellar size is controlled easier than with a conventional low-molecular-mass surfactant (EMMS). The first report on enantiomer separation by MEKC using a chiral HMMS appeared in 1994, where poly(sodium A-undecylenyl-L-valinate) [poly(L-SUV)] was used as a chiral micelle and binaphthol and laudanosine were enantioseparated. The optical resolution of 3,5-dinitrobenzoylated amino acid isopropyl esters by MEKC with poly(sodium (10-undecenoyl)-L-valinate) as well as with SDVal, SDAla, and SDThr was also reported. 

Successful enantioseparation of individual N -protected amino acids stimulated the development of a rapid method of their simultaneous enantioseparation and quantification in a mixture. A feasibility study on this topic has been recently published by Welsch et al. [69]. The two-dimensional HPLC method involves online coupling of a narrow-bore C18 reverse phase (RP) column in the first dimension (separation of racemic amino acids) to a short enantioselective column based on nonporous 1.5 pm particles modified with t-BuCQD in the second dimension (determination of enantiomer composition). Using narrow-bore column resulted in fast analysis time for example, the mixture of nine racemic N-DNB-protected amino acids was completely analyzed within 16 min. 

D-Enantiomers of amino acids in living organisms are attributed to many important bioprocesses or can be markers of certain disorders. Therefore, accurate quantification of the low levels of the D-form in the presence of a large amount of L-form is of considerable interest. Branched D-amino acids in mammalian tissues and body fluids were quantified recently using a sophisticated two-dimensional HPLC system containing a narrow-bore reversed phase and t-BuCQN column for the enantioseparation of l- and D-enantiomers. Target analytes were determined as their fluorescent derivatives, precolumn labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole. D-Val, alio-lie, lie, and Leu were determined at nmol/ml level, making this a valuable method for quantification of D-enantiomers of amino acids [80]. 

The theory and experiment of direct crystallization of enantiomers is quite well understood at present [10]. There are a number of variables which affect the resolution by direct crystallization in practice. Several technological schemes based on this principle are realized on the commercial scale. These are, for example, the Merck process used for the production of antihypertensive drug methyldopa [11], a process developed by Harman and Reimer for (-)-menthol, which is separated as an ester [12], the process patented by Industria Chimica Profarmaco for the resolution of naproxen enantiomers as the ethylamine salt [13], the production of L-glutamic acid by the Japanese company Ajinomoto on a scale in excess of 10000 tons annually as early as the 1960s [14], etc. In general, it seems that spontaneous crystallization is a very useful technique for the enantioseparation of the naturally occurring a-amino acids. All of them may be resolved either directly or as derivatives [10]. 

The model chiral phases, iV-(tert-butylaminocarbonyl)-(5 )-valylaminobutane (Phase 1) and (J )-l-(a-naphthyl)ethylaminocarbonyl-glycylaminobutane (Phase 2) are shown in Figure 8.1. Phase 1 was used for the enantioseparation of N-acetylamino acid methylesters and [R]- and (5)-4-nitrobenzoyl amino acids, but Phase 2 could not separate these enantiomers. The enantiomer selectivities of N-(5 )-l-(a-naphthyl)ethylaminocarbonyl-(5)-valylaminobutane (Phase 3), N-(5 )-l-(a-naphthyl)ethylaminocarbonyl-(P)-valylaminobutane (Phase 4), N-[R]-1-(oc-naphthyl)ethylaminocarbonyl-(R)-valylaminobutane (Phase 5), and N-[R)-l- a-naphthyl)ethylaminocarbonyl-(5 )-valylaminobutane (Phase 6), which all have two chiral centers, were examined by computational chemical analysis. The structures of model Phases 3-6 are also shown in Figure 8.1. 

Crown ethers are synthetic macrocyclic polyethers that can form selective complexes with various cations. Chiral crown ethers such as (diphenyl-substituted l,l -binaphthyl) crown ether or (-Fill 8-crown-6)-2,3,11,12-tetracarboxylic acid, which are bound to silica gels or coated on reversed-phase materials, were utilized for the enantioseparation of underivatized primary amino acids and their esters. On the other hand, the enantiomers of underivatized and derivatized amino acids enter into... 

Bile salts are natural and chiral anionic surfactants which form helical micelles of reversed micelle conformation. The first report on enantiomer separation by MEKC using bile salts was the enantioseparation of dansylated DL-amino acids (Dns-o,L-AAs) and, since then, numerous papers have been available. Nonconjugated bile salts, such as sodium cholate (SC) and sodium deoxycholate (SDC), can be used at pH > 5, whereas taurine-conjugated forms, such as sodium taurocholate (STC) and sodium taurodeox-ycholate (STDC), can be used under more acidic conditions (i.e., pH > 3). Several enantiomers, such as diltiazem hydrochloride and related compounds, carboline derivatives, trimetoquinol and related compounds, binaphthyl derivatives, Dhs-dl-AAs, mephenytoin and its metabolites, and 3-hydroxy-l,4-benzodiazepins have been successfully separated by MEKC with bile salts. In general, STDC is considered as the the most effective chiral selector among the bile salts used in MEKC. 

Ding et al. described the chiral separation of enantiomers of several dansyl-amino acids by HPLC in the reversed-phase mode. The natural logarithms of selectivity factors (In a) of all the investigated compounds depended linearly upon the reciprocal of temperature (1/T). For most processes of enantioseparation, enantioselectivity, a, decreased with increasing of temperature, and the processes of chiral recognition were enthalpy-controlled. It is very interesting that enantioselectivity, a, increased with increasing temperature for dansyl-threonine (Dns-Thr) at a... 

As a result, the baseline enantioseparation was obtained, as shown in Figure 19.1a. Monomeric Pro is one of these rare amino acids, which develop yellow (and not bluish) color when visualized with ninhydrin. l-Pto used as an external standard (Figure 19.1b) confirmed the identity of the lower yellow spot number 2 as enantiomer L and the upper yellow spot number 3 as enantiomer d, as shown in Figure 19.1a. The respective Rp values were 0.57 0.02 and 0.74 0.02. Brownish-purple spot number 1 (Rp = 0.32 0.02 Figure 19.1) apparently originates from the Pro-derived peptides and it is also fully separated from the monomeric l-Pto spot number 2. The presence of peptides in the two freshly prepared Pro samples wimesses to rapid peptidization of this amino acid (although contamination of the commercial monomeric DL-Pro and L-Pro samples with peptides cannot be excluded). 

The first cellulose enantioseparation was of a racemic amino acid by paper chromatography [8]. Early cellulose TLC smdies were aimed at repeating paper chromatography enantiomer separations more quickly and with better resolution. Cellulose TLC has mostly been used for amino acids, their derivatives, and peptides and the following are examples of successful enantioseparations. Aromatic amino acids were separated on polyester-backed 20 x 20 cm plates by elution with methanolic aqueous 0.1 M HCl (A) or ethanol/pyridine/water (1 1 1) (B). Typical respective R values were 0.75/0.81 for l- and o-tyrosine in mobile phase A and 0.37/0.46 for L- and D-5-methyl tryptophan in B [9,10]. 

Macrocyclic antibiotics, for example, erythromycin and vancomycin, have also been used as impregnating agents for TLC resolution of enantiomers of dansyl amino acids by Bhushan and Parshad [6] and Bhushan and Thiong o [7]. Separation in this case is due to chirality of macrocyclic antibiotic molecule, which is ionizable and contains hydrophobic and hydrophilic moieties as well providing enantioseparation via n-n complexation, H-bonding, inclusion in a hydrophobic pocket, dipole stacking, steric interactions, or combination thereof. In view of the scope of the chapter and nonavailability of reports related to NSAIDs, these are not being discussed here. 

For aromatic amino and hydrazino acids and several other structurally related compounds, the influence of MeOH content in both RP and POM was investigated on a teicoplanin CSP [90]. Using a hydroorganic mobile phase, complete enantiosep-arations of a-methylamino acids were not attained. However, this type of separation was suitable for the enantiomers of dopa. Further experiments performed by the same authors in POM allowed the complete enantioseparation of a-methyl-ooPA enantiomers [91]. 

In another approach, reactive monodisperse porous poly(chloromethylstyrene-co-styrene-co-divinylbenzene) beads have been employed for the preparation of chiral HPLC packings. Thus, reactive chloromethyl groups were derivatized to yield amino functionalized beads onto which both rt-basic and rt-acidic type chiral. selectors, (/ )- -(l-naphthyl)ethylamine and (/ )-A -(3.5-dinitrobenzoyl)phenylglycine, respectively, were attached. The resulting chiral particles were chromatographically tested for the enantioseparation of model SAs. Despite the presence of strongly competitive it-TT-binding sites of the styrenic support these chirally modified beads afforded baseline separations for 2,2,2-trifluoro-l-(9-anthryl) ethanol and Af-(3.5-dinitro-benzoyl) leucine enantiomers, respectively [369. ... 

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