Diastereomeric HPLC separation

Although the separation of the diastereomeric alcohols 55 or 64 was not possible by flash chromatography we succeeded in a separation by preparative HPLC. The enantiomeric excess of the individual diastereoisomers was determined after saponification to the diol 54 or 63 by chiral GC. It turned out that the enantiomeric excess of both acetates was only 7% ee. The value was so low that we did not make an effort to continue with only marginally enantiomerically enriched material after HPLC separation. 

A general protocol for the HPLC separation of diastereomeric camphorsulfonamides derived from racemic ot-amino acids has been developed (eq 1). More complex amino acids, such as (8), were successfully analyzed by this procedure. ... 

Fig. 9.15 HPLC separation of diastereomeric esters formed from aliphatic...

The MaNP acid method has been successfully applied to various racemic alcohols listed in Table 9.3 for preparation of enantiopure secondary alcohols and the simultaneous determination of their absolute configurations. If the separation factor a is as large as in the case of l-octyn-3-ol 56 (entry 2 in Table 9.3, a. = 1.88), a large-scale HPLC separation of diastereomeric MaNP esters is feasible. For example, in the case of esters 64a and 64b derived from alcohol 56, ca. 0.85-1.0 g of the mixture was separable in one run by the HPLC (hexane/EtOAc = 20 1) using a silica gel glass column (22 x 300 mm) (Figs. 9.19 and 9.20). 

Table 9.3 HPLC > separation of diastereomeric esters formed from alcohols with MaNP acid (S)-(+)-3, determination of their absolute configurations by the H NMR anisotropy method, and absolute configurations of recovered chiral alcohols.

The HPLC separation data of diastereomeric esters prepared from other racemic alcohols 24, 36, 38, 39, and 57-63 with MaNP acid (.S)-(+)-3 are listed in Table 9.3. It should be emphasized that for most alcohols, their diastereomeric MaNP esters are clearly separated with a values of 1.10-1.88. Phenylacetylene alcohol 57 was separable as the MaNP esters 65a/65b (a = 1.30, entry 3). Substituted cyclohexanols 58 and 59 were also effectively separated as MaNP esters (entries 4 and 5). Especially, the a value of trans-2-isopropylcyclohexanol MaNP esters 66a/66b is as large as 1.88, which is comparable to that of fhe menthol case. On the ofher hand, in the case of trans-2-methylcyclohexanol MaNP esters 67a/67b, the a value is relatively small, a = 1.21. These results indicate that the combination of a longer and larger alkyl group on one side and a smaller alkyl group on fhe ofher side leads to better separation of two diastereomers, as seen in 2-hexadecanol esters 54a/54b (see Fig. 9.15) and tro s-2-isopropylcyclohexanol MaNP esters 66a/66b. 

As mentioned above, the different solubility of diastereomers offers a challenge for their separation by diastereomeric crystallization. The indirect HPLC separation of enantiomers relies on their different interaction with a (achiral) stationary phase. The interaction of the enantiomeric pair (R, S) with one (let us assume it to have S configuration) enantiomer of a chiral derivatizing reagent may be expressed as follows ... 

Chromatography HPLC separation of the diastereomeric derivatives of the phenols used in this paper was carried out on a Nucleosil 5C,s column (15 cm x 4.6 mm ID) eluted with water/acetonitrile (91 9) containing 0.005 M ammonia. 

In this method, chiral acid is covalently bonded to the racemic alcohol, and therefore, the obtained diastereo-meric esters can be separated by HPLC on silica gel. If the chromatogram shows a base-line separation and the chiral reagent used is enantiopure, the diastereomeric esters separated are enantiopure. So enantiopure alcohols can be recovered from the diastereomeric esters. Therefore, if the AC of one of diastereomeric esters can be... 

TABLE 55.2. Preparation of enantiopure alcohols by the CSDP acid method HPLC separation of diastereomeric CSDP esters and determination of their ACs by X-ray crystallography... 

Alcohol 43 (entry 31) is a starting material for the synthesis of (R)-(+)-[VCD( )984]-4-ethyl-4-methyloctane 51, a cryptochiral saturated hydrocarbon with a quaternary chirality center, as will be discussed in section 55.3.4 (see Figure 55.26). Enantiopure alcohol (l/ ,2/ )-(—)-43 was obtained by the CSDP acid method, and its AC was unambiguously determined by X-ray crystallography." However, the MaNP acid method is better than the CSDP acid method in this case because of more effective HPLC separation of diastereomeric esters (see the result in section 55.3.4). 

FIGURE 55.19. HPLC separation of diastereomeric esters formed from aliphatic alcohols and (5)-(+)-MaNP acid 3 (sihca gel, 22 [Pg.1645]

FIGURE 55.27. A large-scale HPLC separation of diastereomeric esters (5,.S)-(—)-118a and S,R)-(-)-118b. 

FIGURE 55.32. Preparation of diastereomeric amides from MaNP acid ( )-3 and phenylalaninol (5)-(—)-143, and their HPLC separation . 

TABLE 55.4. HPLC separation of diastereomeric amides and oxazolines... 

GC or HPLC, the diastereomeric derivatives may be separated by chromatographic means. 

One of the most powerful methods for determining enantiomer composition is gas or liquid chromatography, as it allows direct separation of the enantiomers of a chiral substance. Early chromatographic methods required the conversion of an enantiomeric mixture to a diastereomeric mixture, followed by analysis of the mixture by either GC or HPLC. A more convenient chromatographic approach for determining enantiomer compositions involves the application of a chiral environment without derivatization of the enantiomer mixture. Such a separation may be achieved using a chiral solvent as the mobile phase, but applications are limited because the method consumes large quantities of costly chiral solvents. The direct separation of enantiomers on a chiral stationary phase has been used extensively for the determination of enantiomer composition. Materials for the chiral stationary phase are commercially available for both GC and HPLC. 

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... 

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