Peptides diastereoisomers

COUPLING, 2-ALKYL-5(4H)-OXAZOLONE FORMATION AND GENERATION OF DIASTEREOISOMERS FROM ACTIVATED PEPTIDES... 

The enantiomeric purity of protected amino acids used in peptide synthesis can be determined by pre-column partial deprotection followed by derivatization with Marfey s reagent (116). The Marfey diastereoisomers can be easily resolved and determined by RP-HPLC using an ODS-Hypersil column288. Fifteen amino acids collected from mammalian tissues were derivatized with Marfey s reagent and subjected to two-dimensional TLC. Each individual spot (enantiomeric mixture of a diasteroisomer) was then resolved by RP-HPLC. Except for tyrosine (46) and histidine (117), subnanomole quantities of enantiomers could be analyzed289,290. 

Ermer, for example, utilized LCQ to monitor impurity profiles of various batches of ramorelix used in toxicological studies, clinical stndies and scale-up. Ramorelix is a synthetic glycosylated decapeptide with monoisotopic of 1530.7. The toxicological batch served as the benchmark against which all other batches were compared. Molecnlar weights of impurities were determined by ESI mass spectrometry, and nsed in conjunction with UV peak area % to gauge impurities in batches nsed in clinical trials. These impurity profiles were compared to those of batches used in the toxicologically qualified batch. Eour impurities were detected with the same value. They were believed to be diastereoisomers of ramorelix, i.e., a peptide sequence with one of the amino acids in the opposite enantiomeric form. 

A combined lactonization and deprotection to give 75 was achieved by refluxing 74 in 6N HCl. Peptide coupling with phenylalanine affords a mixture of diastereoisomers of the target compound 67. 

U-SNAr[15][CNlE] process was implemented. These para-cyclophanes 97 and 98 are interesting from the stereochemical point of view because they generate atropisomerism. Moreover, the stereogenic centres of the peptide backbone are configurationally unstable under the cyclization conditions, affording, under thermodynamic control, only two diastereoisomers (atropisomers) instead of the expected four. 

The antineoplastic, cyclic peptide dolastatin 3 (197) was isolated from the sea hare Dolabella auricularia in small quantities [187]. It bears much structural resemblance to the cyclic peptides of tunicates. Synthetic attempts indicated that the original published structure was incorrect [188]. Three reports of research directed towards synthesis of possible components of dolastatin 3 (197) failed to help with the correct structure [189-191]. Reisolation of 197 allowed the determination of the correct sequence of amino acids in this cyclic pentapeptide and the new structure was confirmed by synthesis [192]. Synthesis of dolastatin 3 (197) and the corresponding 12R diastereoisomer permitted study of the solution... 

One of the problems attaching to the HPLC separation of peptides is the analysis of stereoisomers (enantiomers and diastereoisomers), that is, of peptides that differ only in the configuration of their amino acid residues. 

The separation of enantiomers can be effected either by transforming them into diastereoisomers using a chiral reagent and separating them on conventional phases or by separating the enantiomers on chiral phases. The utilization of chiral phases has not yet become routine, but studies of enantiomeric dipeptides have been carried out (115,116). Pirkle et al. (117) and Hyun et al. (118) separated enantiomeric di- and tripeptides (methyl esters of /V-3-5-dinitrobenzoyl derivatives) on chiral stationary phases (CSPs) derived from (R)-a-arylalkylamines, (S)-N-(2-naphthyl) valine, or (S)-1 -(6,7-dimethyl-1 -naphthyl) isobutylamine. These workers were able to separate four peaks for each dipeptide derivative, corresponding to the two enantiomeric pairs (R,R)/(S,S) and (R,S)/(S,R). Cyclodextrin-bonded stationary phases and chiral stationary immobilized a-chymotrypsin phases were used to separate enantiomeric peptides (118a,b). 

In order to prevent S-alkylation in the repetitive acid-cleavage steps required in a multistep peptide synthesis, the protection of methionine residues as sulfoxides was proposed by Iselin.f This approach has since been widely used for peptide synthesis in solution and on solid supports.Generally, oxidation of L-methionine yields a mixture of S- and R-sulf-oxides and, depending on the conditions used, even a mixture of the related sulfone. Thereby, at least by oxidation with hydrogen peroxide the R-sulfoxide is formed in a preferred manner (80%), whereas oxidation of L-methionine by tetrachloroauric(III) acid has been reported to produce stereospecifically the 5-sulfoxide diastereoisomer. Isomeric pure Met(O) derivatives are obtained by isolation of the isomers from the 5,/ -sulfoxide mixture, taking advantage of the differences in solubility of the picrate salts.0 ... 

The 3EI NMR spectrum of (7) at low temperatures shows the presence of two diastereoisomers.35 Ab initio calculations have been made of 15N chemical shift differences induced by Ca2+ binding to EF-hand proteins.36 111 and 13 C NMR spectra were used to characterise calcium pyrrolates, [Ca (2-dimethylamino-methyl)pyrrolyl 2(D)n], where D = thf, py, n = 2, D = dmf, TMEDA, n = l.37 The 3H NMR spectra of (ri5-Gaz)M(thf)2, where M = Ca or Yb, and (r 5-Gaz)Yb(py)2, where Gaz = l,4-dimethyl-7-isopropylazulene, show exclusive formation of N2-ara, z-metallocenes.38 - N heteronuclear single quantum coherence spectra were used to study and compare the binding of Ca2+ and La3+ to calmodulin and a calmodulin-binding peptide.39... 

An interesting example taking the opposite approach was presented by Wieczorek et al. [24]. To separate a mixture of dipeptide diastereoisomers and their phosphonic analogues, achiral crown ethers were used as transport enhancers. In this case, the diastereomeric complex is formed between the chiral transported molecule and not the optically active carrier. The observed stereoselectivity depends on the peptide structure and was independent of the presence of carrier, but the application of carrier increased the transport rate of both diastereoisomers. 

The potential of reversed-phase HPLC in the chromatography of proteins and peptides has been amply demonstrated by results obtained with synthetic peptides (BIO, FI) and mixtures of proteins (Mil). Even diastereoisomers of short chain peptides can be separated by this method (K16, L7). 

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