Diastereoisomerism 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-... 

Numerous HPLC analyses have been carried out on biologically active diastereoisomeric peptides (encephalins, endorphins, hormones) in order to isolate them so that their activity can be studied however, the separation of stereoisomers is not yet common in the field of food science. 

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). 

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-... 

A very straightforward approach in route C (Fig. 2) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [18]) by enantioselective aminolysis of diester 9 with (S)-tert-leucine methylamide 13 or even racemic 13. This would combine three synthetic objectives the resolution of (rac)-9, the resolution of (roc)-13 and the coupling step. In orientating experiments monoester 10 was tested as a model substrate. It was contacted with an equal amount of (S)-amine 13 in the presence and absence of an organic solvent. Solid or liquid subtilisin Carlsberg preparations (Alcalase 2.0 T or Alcalase 2.5 L, respectively) were used as the catalyst. Only with the liquid enzyme preparation was the formation of minor amounts of one of two possible diastereoisomeric peptides observed [19], whereas most of the ester was hydrolyzed to the acid. Likewise, a few selected lipases also provided negative results. 

Scheme 20 yielded the target 3 -phosphono-L-tyrosine as its trimethyl ester benzyl ether (194). The stereoisomeric composition of the product was determined by the generation of the diastereoisomeric peptides (195), the h.p.l.c. of which indicated the S,S)/ R S) ratio as 87.4 12.6. ... 

An elegant example of the latter reaction was its application to peptide chemistry for inducing structural changes within the peptide chain. Peptide 23 readily cyclized to the 4-methylproline-containing derivative 24 in 64% yield (Sch. 15). The diastereoisomeric ratio was found as 2 1 in favor of the cis-isomer for this example. The Us-selectivity reflects the approach of the... 

Since all naturally occurring phencyclopeptines have the same stereochemistry at C-9, a synthetic route involving peptide cyclization of the p-nitrophenyl ester (22) to give a single diastereoisomeric cyclic peptine (23) suggests a feasible route to the total synthesis of these natural products.18 Pubescine A (24), obtained from the... 

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. 

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). 

Peptide formation with an A-protected, optically pure aminocarboxylic acid is an alternative means of obtaining diastereoisomeric derivatives of chiral aminoalkylphos-phonic acids, the diastereoisomeric products being distinguishable on a quantitative basis by P NMR spectroscopy for example, A-boc-L-alanine was used to distinguish the enantiomers of diethyl (a-aminobenzyl)phosphonate. The reaction between an enan-tiomerically enriched sample of 125 (R = H) and L-leucine methyl ester hydrochloride in the presence of DCC was followed by HPLC separation of the l,l- and D,L-peptides 126 (R = H ratio 95.3 4.7), and essentially the same procedure was applied to an analysis of 125 (R = OCH2Ph), as prepared using the bislactim ether procedure (see Section IV. C.2.b) when the final product consisted of a mixture of R,S and S,S diastereoisomers in the ratio 87.4 12.6 ... 

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