Hydroxyproline resolution

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. 

For the separation of D,L-leucine, Ding et al. [62] used poly(vinyl alcohol) gel-coated microporous polypropylene hollow fibers (Fig. 5-11). An octanol phase containing the chiral selector (A-n-dodecyl-L-hydroxyproline) is flowing countercur-rently with an aqueous phase. The gel in the pores of the membrane permits diffusion of the leucine molecules, but prevents convection of the aqueous and octanol phase. At a proper selection of the flow ratios it is possible to achieve almost complete resolution of the D,L-leucine (Fig. 5-12). 

C Tao, TB Huang. Resolution of DL-a-amino acids on a L-hydroxyproline chiral phase by ligand-exchange chromatography. Chin Chem Lett 6 383-384, 1995. 

Schmid et al. [60] demonstrated the enantiomer separation of underivatized amino acids on a monolithic chiral ligand-exchange phase by rod-CEC. The chiral stationary phase was prepared in situ in the capillary by polymerization of methacrylic acid, piperazine diacrylamide, vinylsulfonic acid and /V-(2-hydroxy-3-alloxypropyl)-L-4-hydroxyproline. The monolithic separation bed was covalently linked to the internal capillary wall and thus no frits were required. Fig. 9.13 shows the enantiomer separation of phenylalanine by (A) pure CEC (30 kV), (B) nano-LC (12 bar) and (C) pressure supported CEC (30 kV, 12 bar at the inlet vial). The shortest elution time was clearly obtained by pressure supported CEC, while the highest resolution was found in the pure CEC mode (CEC Rs = 2.11 nano-LC Rs = 0.98 pressure supported CEC Rs= 1.60). 

By using a modification of a reaction discovered by Traube and Lehmann (61,62), Leuchs, Giua, and Brewster (63) synthesized 4-hydroxy-proline from a-carbethoxy-a, 5-dichloro-y-valerolactone from which Leuchs and Brewster (64) prepared Z-hydroxyproline by resolution with the aid of quinine. On methylation of a sample of Z-hydroxyproline thus prepared, Kiing (65) obtained a mixture of optically active betaines which he separated by fractional crystallization into a levorotatory betaine identical with betonicine and a dextrorotatory betaine identical with turicine. Betonicine is, therefore, the methyl betaine of 4-hydroxyhygrinic acid (XXIV). Betonicine and turicine possess different solubilities and can be separated... 

Treatment of asbestosis and idiopathic pulmonary fibrosis with colchicine (0.6 mg orally for 12 weeks) resulted in declines in dyspnoea index, selective improvement in several high-resolution computed tomography scans, but no statistically significant changes in bronchoalveolar lavage cells, cytokines, fibronectin, or hydroxyproline (Addriz-zo-Harris et al. 2002). However, there was a decline in hydroxyproline in the bronchoalveolar lavage fluid in 8 of 10 patients. 

Still higher resolution factors were found in systems in which a diastereomeric metal complex is responsible for chiral recognition. Thus, on Cu(L-proline)2 dissolved in the mobile phase (in HPLC), the enantiomers of valine were resolved with a = 4.8 at room temperature (AAG = —0.95 Kcal) [19] Davankov [28] has reported for proline on N-n-heptyl-L-hydroxyproline a = 10.9 (AAG = -1.4 Kcal). 

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