Amino selectors

Chiral stationary phases in tic have been primarily limited to phases based on normal or microcrystalline cellulose (44,45), triacetylceUulose sorbents or siHca-based sorbents that have been chemically modified (46) or physically coated to incorporate chiral selectors such as amino acids (47,48) or macrocyclic antibiotics (49) into the stationary phase. 

Diamide Chiral Separations. The first chiral stationary phase for gas chromatography was reported by GH-Av and co-workers in 1966 (113) and was based on A/-trifluoroacetyl (A/-TFA) L-isoleucine lauryl ester coated on an inert packing material. It was used to resolve the tritiuoroacetylated derivatives of amino acids. Related chiral selectors used by other workers included -dodecanoyl-L-valine-/-butylamide and... 

The first partial chiral resolution reported in CCC dates from 1982 [120]. The separation of the two enantiomers of norephedrine was partially achieved, in almost 4 days, using (/ ,/ )-di-5-nonyltartrate as a chiral selector in the organic stationary phase. In 1984, the complete resolution of d,l-isoleucine was described, with N-dodecyl-L-proline as a selector in a two-phase buffered n-butanol/water system containing a copper (II) salt, in approximately 2 days [121]. A few partial resolutions of amino acids and dmg enantiomers with proteic selectors were also published [122, 123]. 

However, it was not until the beginning of 1994 that a rapid (<1.5 h) total resolution of two pairs of racemic amino acid derivatives with a CPC device was published [124]. The chiral selector was A-dodecanoyl-L-proline-3,5-dimethylanilide (1) and the system of solvents used was constituted by a mixture of heptane/ethyl acetate/methanol/water (3 1 3 1). Although the amounts of sample resolved were small (2 ml of a 10 inM solution of the amino acid derivatives), this separation demonstrated the feasibility and the potential of the technique for chiral separations. Thus, a number of publications appeared subsequently. Firstly, the same chiral selector was utilized for the resolution of 1 g of ( )-A-(3,5-dinitrobenzoyl)leucine with a modified system of solvents, where the substitution of water by an acidified solution... 

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. 

Small chiral molecules. These CSPs were introduced by Pirkle about two decades ago [31, 32]. The original brush -phases included selectors that contained a chiral amino acid moiety carrying aromatic 7t-electron acceptor or tt-electron donor functionality attached to porous silica beads. In addition to the amino acids, a large variety of other chiral scaffolds such as 1,2-disubstituted cyclohexanes [33] and cinchona alkaloids [34] have also been used for the preparation of various brush CSPs. 

The majority of the original chiral selectors for brush-type CSPs were derived from natural chiral compounds. Selectors prepared from amino acids, such as phenyl... 

First, they compared CSPs 1 and 3 prepared by the two-step solid-phase methodology with their commercially available counterparts (CSPs 2 and 4) obtained by direct reaction of the preformed selector with a silica support. Although no exact data characterizing the surface coverage density for these phases were reported, all of the CSPs separated all four racemates tested equally. These results shown in Table 3-3 subsequently led to the preparation of a series of dipeptide and tripeptide CSPs 5-10 using a similar synthetic approach. Although the majority of these phases exhibited selectivities lower or similar to those of selectors built around a single amino acid (Table 3-3), this study demonstrated that the solid-phase synthesis was a... 

After this feasibility test, a library consisting of 50 types of beads each containing a different dipeptide selector attached through its C-terminal group was prepared and screened (Fig. 3-6) [84]. The first amino acid residue (aa 1) was chosen from a... 

The two best selectors resulting from Li s screening, DNB-L-ala and DNB-L-leu, were then prepared on a larger scale, attached to silica beads modified with 3-amino-propyl-triethoxysilane, and the CSPs were packed into columns. Respective separation factors of 4.7 and 12 were found for the separation of racemic naphthyl leucine ester 17 using these CSPs. 

In the next step, the best candidate from the series 2-oxo-4-(9-phenanthryl)-dihy-dropyrimidine 22 was prepared and isolated in enantiomerically pure form, then attached to a macroporous polymer support. To attach the isolated selector to the amino functionalized macroporous polymethacrylate support, a suitable reactive handle had to be introduced into the dihydropyrimidine. We chose to functionalize the methyl group at the C6 carbon atom by a simple bromination to afford (-)-22. Coupling of this compound to the amino functionalized support then gave the desired chiral stationary phase CSP 12 (Scheme 3-6) containing 0.20 mmol g of the selector. 

The mixture of deprotected amino acid derivatives in solution was then immobilized onto a polymeric solid support, typically activated 5-)xm macroporous poly(hydroxyethyl methacrylate-co-ethylene dimethacrylate) beads, to afford the chiral stationary phases with a multiplicity of selectors. Although the use of columns... 

As expected from the design of the experiment, the HPLC column packed with CSP 14 containing all 36 members of the library with tt-basic substituents separated 7t-acid substituted amino acid amides. Although encouraging since it suggested the presence of at least one useful selector, this result did not reveal which of the numerous selectors on CSP 14 was the most powerful one. Therefore, a deconvolution process involving the preparation of series of beads with smaller numbers of attached selectors was used. The approach is schematically outlined in Fig. 3-17. 

In contrast, there are fewer limitations from the chemical point of view. The preparation of large, well-defined, libraries that involve amino acid building blocks has been demonstrated many times. Carefully optimized reaction conditions for the preparation of other mixed libraries can also ensure that each desired compound is present in sufficient amount. However, the reaction rates of some individual selectors with the activated solid support may be lower than that of others. As a result, the more reactive selectors would occupy a majority of the sites within the beads. Since the most reactive selectors may not be the most selective, testing of a slightly larger number of specifically designed CSPs may be required to reduce the effect of falsenegative results. 

The enantioselectivity a is defined as the distribution ratio of one single enantiomer over the two chiral phases and has been determined experimentally for a variety of compounds (Table 5-1). It has been known from work by Prelog [66, 67] that tartaric acid derivatives show selectivities towards a-hydroxyamines and amino acids. However, from Table 5-1 it is obvious that tartaric acid derivatives show selectivity for many other compounds, including various amino bases (e.g. mirtazapine (10)) and acids (e.g. ibuprofen (11)). The use of other chiral selectors (e.g. PLA)... 

Free amino acids can be derivatized with isothiocyanates to phenyl- or methyl-thiohydantoin derivatives. The thiohydantoins can be separated on a CSP with poly-[Af-acryloyl-L-phenylalanine ethylester] (Chiraspher ) as a chiral selector [25]. This CSP offers a known selectivity for many five-membered heterocyclic rings. 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

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