Dipeptides, enantiomeric separation

Sanger-van de Griend, C. E. (1999). Enantiomeric separation of glycyl dipeptides by capillary... 

Finally, we would like to describe the enantiomeric separation of racemic methyl phenyl sulfoxide by the dipeptide (1). Optically active sulfoxides are utilized as a... 

A suspension of the dipeptide (1) (1 mmol) in water (2 ml) was stirred together with racemic methyl phenyl sulfoxide (2 mmol) at room temperature for one day. The formed inclusion compound was collected by filtration and washed with water (20 ml) and dichloromethane (20 ml). From the inclusion compound, we recoverd the included methyl phenyl sulfoxide by extraction with dichloromethane to give (/ )-methyl phenyl sulfoxide. The dipeptide (1) remained as a solid. When the recovered dipeptide (1) was again subjected to the formation of inclusion compound with racemic methyl phenyl sulfoxide, (A1 (-methyl phenyl sulfoxide was obtained as summarized in Table 5. Thus, it was shown that the dipeptide (1) can be used repeatedly for enantiomeric separation of methyl phenyl sulfoxide. 

Xia S, Zhang L, Lu M, Qiu B, Chi Y, Chen G. Enantiomeric separation of chiral dipeptides hy CE-ESI-MS employing a partial filling technique with chiral crown ether. Electrophoresis 2009 30 2837-2844. 

Amino acid derivatives can be examined for enantiomeric purity by the same procedures after removal of the protecting groups. Another approach is to couple them directly with another derivative to give protected dipeptides whose diastereomeric forms are usually easy to separate by HPLC (see Section 4.11). An A-protected amino acid is coupled with an amino acid ester, and vice versa. Use of soluble carbodiimide as reagent (see Section 1.16), followed by aqueous washes, gives clean HPLC profiles. It is understood that the derivative that serves as reagent must have been demonstrated to be enantiomerically pure.43 84-89... 

The classical methods for detection and quantitation of racemization require analysis of the chiral purity of the product of a peptide-bond-forming reaction. For example, the Anderson test is used to explore a variety of reaction conditions for the coupling of Z-Gly-Phe-OH to H-Gly-OEt (Scheme 6). 9 The two possible enantiomeric tripeptides are separable by fractional crystallization, so that gravimetric analysis furnishes the racemization data. This procedure has a detection limit of 1-2% of the epimerized tripeptide. A modification by Kemp,1"1 utilizing 14C-labeled carboxy components, extends the detection limit by two to three orders of magnitude by an isotopic dilution procedure. The Young test 11 addresses the coupling of Bz-Leu-OH to H-Gly-OEt, and the extent of epimerization is determined by measurement of the specific rotation of the dipeptide product. 

The separation of enantiomers can be effected either by transforming them into diastereoisomers using a chiral reagent and separating them on conventional phases or by separating the enantiomers on chiral phases. The utilization of chiral phases has not yet become routine, but studies of enantiomeric dipeptides have been carried out (115,116). Pirkle et al. (117) and Hyun et al. (118) separated enantiomeric di- and tripeptides (methyl esters of /V-3-5-dinitrobenzoyl derivatives) on chiral stationary phases (CSPs) derived from (R)-a-arylalkylamines, (S)-N-(2-naphthyl) valine, or (S)-1 -(6,7-dimethyl-1 -naphthyl) isobutylamine. These workers were able to separate four peaks for each dipeptide derivative, corresponding to the two enantiomeric pairs (R,R)/(S,S) and (R,S)/(S,R). Cyclodextrin-bonded stationary phases and chiral stationary immobilized a-chymotrypsin phases were used to separate enantiomeric peptides (118a,b). 

The use of homochiral complexation agents to separate the fluorine-19 chemical shifts of enantiomers containing fluorine has also been examined. Addition of the supported dipeptide 9 to a solution of the racemic iV-acylamino acid ester 10 in carbon tetrachloride leads to the appearance of two CF3 peaks corresponding to the two enantiomers. The separation is obviously concentration-dependent, but peak separation was sufficient for mixtures of enantiomers to give enantiomeric excesses comparable with those determined by gc analysis55. 

A resolution method involving the aminolysis of a racemic oxazolone intermediate 158 with L-Pro-NHMe hydrochloride allowed the incorporation of enantiomerically pure ( R,2S)-and (lS,2f )-l-amino-2-hydroxycyclohexane-l -carboxylic acids (c6Ser) into Xaa-Pro dipeptide, a ffording d iastereomers 159 and 160 separated by column chromatography the... 

With racemic a-alkylated amino acids an enzymatic racemate resolution is possible. There are several methods to access racemic a-alkylated amino acids in high yields [38]. Different microorganisms have been applied, and the products are obtained in very high enantiomeric purities [39]. Because a-alkylated amino acids are used as building blocks for different active substances, methods for the synthesis of large quantities have been developed, especially in industry [40]. Other effective racemic resolution techniques have been described recently. Disubstituted azlactones of type 28 can react with the phenylalanine derivative 29 [41]. The diastereo-mers of the protected dipeptide 30 are then separated. The easy access of compounds of type 28, together with the optimized reagent 29, ensures... 

Formyl-5-hydroxy[2.2]paracyclophane (153) was used as a chiral auxiliary in the synthesis of a-amino acids [98]. The reported enantiomeric excess was in the range of 90-98%. Racemic 153 was first prepared by Hopf and Barrett [99]. To separate the enantiomers, their Schiff bases with the dipeptide (S)valyl-(S)valine was prepared. The diastereomeric copper(II) complexes of this compound show different solubility in 2-propanol. Alternatively they can be separ-... 

Separation performance can be expressed by the separation factor a, which is only dependent on the retention times of the two enantiomers, or by the resolution factor Rs which also takes into account the breadth of the chromatographic peaks. The higher these factors are, the better separation is. Remarkable selectivities have been obtained, for instance with an enantiomeric mixture of the dipeptide JV-acetyl-Trp-Phe-O-Me (non-covalent system, Rs= 17.8) [141] and... 

Acid catalyzed hydrolysis followed by the identification of D-amino acids in the hydrolysate is equally useful. To make this possible the amino acids in the mixture are acylated with an enantiomerically pure amino acid, for instance with the N-carboxyanhydride of L-leucine. In the resulting mixture of dipeptides any racemized residue is revealed by the formation of two dipeptides that are diastereoisomers of each other, for instance L-leucyl-L-phenylalanine and L-leucyl-D-phenylalanine. Since these are compounds with different physical properties they are separable and appear as a doublet on recordings of an amino acid analzyer. In recent years the conversion to diastereoisomers became unnecessary because the availability of chiral supports now permits separation of enantiomers by high pressure liquid chromatography (HPLC) and also by thin layer chromatography on plates covered with a chiral layer. 

Enantiomeric ions can be separated in normal phase liquid-solid systems with one antipode of a chiral counter-ion added to the non-polar mobile phase. The chiral selector should have properties such that several interaction points with the enantiomeric solutes are obtained. Electrostatic interaction, hydrogen bonding and a steric influence from a bulky structure in the vicinity of the asymmetric centre seem to be needed. Pettersson has developed systems for the separation of enantiomers of amino alcohols used as /8-adrenoceptor blocking drugs, with (-l-)-lO-camphorsulfonic acid [54] and a dipeptide derivative, N-benzoxycarbonylglycyl-L-proline [25], as selectors, the latter giving higher chiral selectivity. 

Gunther et al. (19) reported for the first time the separation of enantiomeric dipeptides on Chiralplate (20). Typical examples of these separations are given in Table 5. It was observed that the... 

The research listed in Table 2 (81-98) deals mainly with separation problems concerning amino acids, amino acid derivatives, and dipeptides, focusing on the influence of the structure of the chiral support and the eluent temperature on the separation behavior of the racemates. Separation of the aromatic amino acids phenylalanine, P-2-thienylalanine, 4-fluorophenylalanine, and tyrosine could not be achieved on microcrystalline or amorphous cellulose tryptophan isomers, however, could be reproducibly resolved on microcrystalline cellulose layers (83). Lowering the eluent temperature from 30 C to O C enhances enantiomeric resolution. However, developing times of 10 1 h (O C), 7.5 0.5 h (10°C), 5 0.5 h (20°C), and 3.5 h (30°C) have to be tolerated hydrophobic eluent combinations further enhance separation, because they improve formation of the helical cellulose conformation (87). Separation of racemic 3,4-dihydroxyphenylalanine, tryptophan, and 5-hydroxy-tryptophan can be achieved in only 2 h, on a cellulose HPTLC plate (89) these experiments will be described in detail in Section FV.C. 

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