Ristocetins

Table 2. Naturally Occurring Ristocetin-type Dalbaheptides...

Biosynthesis. Biochemical studies on dalbaheptides have been reviewed (92,97). Experiments with and H have shown that in vancomycin (39), D-tyrosine is the precursor of D-/> -hydroxyphenyiglycine and P-hydroxy-y -chlorotyrosine, and acetate the precursor of the two y jy -dihydroxyphenyiglycines (98). Similar results using either or radioactively labeled material have been reported for avoparcin (Table 5) (23), ristocetin (Table 2) (99,100), ardacin (Table 3) (101), and A47934 (102). 

Vancomycin, ristocetin A and teicoplanin are produced as fermentation products of Streptomyces orientalis, Nocardia lurida and Actinoplanes teichomyceticus, respectively. All three of these related compounds consist of an aglycone basket made up of fused macrocyclic rings and pendant carbohydrate moieties (Fig. 2-1). The macrocycles contain both ether and peptide linkages. The aglycones of vancomycin and teicoplanin contain two chloro-substituted aromatic rings, while the analogous portion of ristocetin A contains no chloro substituents. 

Fig. 2-4. The enantiomeric separation of a-hydroxy/halogen acids on ristocetin A CSP (250 x 4.6 mm) with the same mobile phase composition methanol with 0.02 % acetic acid and 0.01 % triethylamine (v/v). The flow rate was 1.0 mL min at ambient temperature (23 °C).

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

Fig. 2-9. Chromatograms of phensuximide in normal phase on vancomycin (A), teicoplanin (B), ristocetin A (C), vancomycin + teicoplanin (D), ristocetin A + vancomycin (E), ristocetin A + teicoplanin (F), and ristocetin A + vancomycin + teicoplanin (G). All columns were 100 x 4.6 mm. The numbers by the peaks refer to the retention time in minutes. The mobile phase was ethanol hexane (60/40 v/v) and the flow rate was 1.5 mL min at ambient temperature (23 °C).

Macrocyclic glycopeptides. The first of these CSPs - based on the cavity of the antibiotic vancomycin bound to silica - was introduced by Armstrong [25]. Two more polycyclic antibiotics teicoplanin and ristocetin A, were also demonstrated later. These selectors are quite rugged and operate adequately in both normal-phase and reversed-phase chromatographic modes. However, only a limited number of such selectors is available, and their cost is rather high. 

Figure 43 nOe buildup with respect to time, after irradiation of bound alanine methyl protons in ristocetin A-tripeptide complex for four different protons. (Reprinted from D. H. Williams et al., J. Am. Chem. Soc. 105, 1332, copyright (1983), with permission from The American Chemical Society, 1155 16th Street, N.W. Washington, D.C. 20036, U.S.A.)... 

Fig. 2-5. Examples showing the complementary separations on glycopeptide CSPs. (A) Separation of N-CBZ-norvaline on vancomycin (left) and teicoplanin (right). The mobile phase was methanol 1 % triethylammonium acetate (20/80 v/v) pH 4.1. (B) Separation of warfarin on teicoplanin (left) and vancomycin (right) CSPs. The mobile phase was acetonitrile 1 % triethylammonium acetate (10/90 v/v) pH 4.1. (C) Separation of naproxen on teicoplanin (left) and ristocetin A (right). The mobile phase was methanol 0.1 % triethylammonium acetate (30/70 v/v) pH 4.1. All columns were 250 x 4.6 mm i.d. The flow rate for all the separations was 1 mL min1 at ambient temperature (23 °C).

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