Amino acid diastereoisomeric peptides

Separation of diastereoisomeric peptides by HPLC is more common. Since each diastereo-isomer has different physicochemical and biological properties, this is of great interest. Separations of diastereoiosomeric di- and tripeptides have usually been performed on reversed-phase columns. Cahill et al. (119) separated diastereoisomeric amino acids and derivatized dipeptides using esters of the /V-hydroxysuccinamide of f-butyl carbonyl-L-amino acid on Cl8 and C8 columns. Linder et al. (120) separated amino acid and peptide derivatives on an RP-C8 column, adding a metal chelate. Mixtures of DL and LD-dipeptides can be separated by RP-HPLC into two peaks, one containing LL- and DD-isomers, the other containing LD and DL-isomers. Sep-... 

The enhanced reactivity of chelated amino acid esters towards attack by other nucleophiles has been used to advantage in the sequential synthesis of small peptides equation (4l).225 Formation of the amide bond takes only seconds to minutes at room temperature in DMSO as solvent, and the peptide can be easily recovered by reducing the metal to the Co" state. Recent studies have shown that the A and A diastereoisomeric reactants are selective in their couplings to (2 ) and (S) amino acid esters and that mutarotation at the asymmetric centre of the chelated ester reactant varies from 0-6%.226 Isied and coworkers have described the use of the Co(NH3)3+ as a C-terminal protecting group for the sequential synthesis of peptides (equation 42).227 This procedure has advantages over other methods in some cases. 

In order to be able to predict the retention behavior of peptides of different composition, of peptides of the same composition but different sequence (positional isomers), and of diastereoisomeric peptides, a knowledge of the incremental contribution of each amino acid to the overall contact area term is required not only for each well-defined stationary phase but also for each mobile-phase condition. Group retention coefficient summation approaches based on the assumption that selectivity differences can be ascribed predominantly to amino acid sequence differences, have been developed by Meek (46a, 52b) and Su et al. (45a). These treatments have subsequently been applied to a number of different elution systems (52c-52e). A comparative analysis of the different amino acid group contribution coefficients derived for phosphate, perchlorate, pyridine/acetate, trifluoroacetate, and bicarbonate buffer systems has been reported (52f). 

Racemization of chiral centers during the isolation or synthesis of a particular peptide will lead to diastereoisomeric mixtures. In addition, in peptide synthesis it is often more efficient to use DL-amino acids, particularly if they are specifically labeled with C, H, S, etc. Rapid... 

Similar stereochemical arguments can be extended to larger peptides. For example, the elution order of diastereoisomeric nonpolar peptides of the type l-X-l-Y-l-Z, l-X-l-Y-d-Z, l-X-d-Y-l-Z can be predicted on conformational grounds to be lll < lld < ldl and this has been observed experimentally, e.g., enkephalin analogs 112). Furthermore, the introduction of the glycinyl residue into an endo position between two chiral amino acids decreases both the k and a values for the dias-tereoisomers 46). 

Similarly, reversed-phase HPLC can be used as an Eilternative to the racemization test for amino acids as developed by Manning and Moore (115). Rivier and Burgus (109) have suggested the use of L-phenylalanine, coupled via the N-carboxyanhydride method to a hydrolysate, to monitor racemization during synthesis, although other hydrophobic L-amino acids should also prove equally effective. The use of /eri-butyloxycarbonyl-L-amino acid-Af-hydroxysuccinimide esters in the separation of enantiomeric amino acids and diastereoisomeric peptides has been described (110). Ultimately, these methods may not prove as versatile as the use of chiral stationary phases made by stereoselective control of the bonding process or, alternatively, with surface-active reagents similar to the D-... 

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