Francis M.F. Chen

Determination of epimeric peptides for assessing enantiomeric purity of starting materials and studying racemization in peptide synthesis using high-performance liquid chromatography☆

Abstract Chromatography of the epimeric peptide pairs for the series Xxx-Lys, Lys-Yyy, Gly-Xxx-Lys, Gly-Lys-Yyy (Xxx is the activated residue during coupling, Yyy is the residue aminolysing the activated residue forming the peptide bond) and some neutral, acidic and N-methylated peptides on a μBondapak C18 column is described. Good resolution was achieved in most cases using isocratic elution with aqueous ammonium acetate, or buffers of lower pH. Observations on factors affecting retention of peptides are made, and the potential use of these model compounds for determining enantiomeric content in amino acid derivatives and studying racemization are discussed.

The dependence of asymmetric induction on solvent polarity and temperature in peptide synthesis

Abstract When a racemic 2,4-dialkyl-5(4 H )-oxazolone reacts with an L-amino acid ester, the DL epimer is formed in excess in apolar solvents and the LL epimer is formed in excess in polar solvents, the proportion of LL isomer increasing with decreasing temperature.

The relative susceptibility to racemization of L- and D-residues in peptide synthesis

Abstract An activated D-residue of a protected dipeptide when coupled racemizes twice as much as an activated L-residue, which is contrary to the case for couplings of acylamino acids.

Not the alkoxycarbonylamino-acid O-acylisourea

The reaction of equivalent amounts of N-benzyl-oxycarbonyl-L-valine and N,N′-di-isopropylcarbodi-imide in deuteriochloroform gives an equimolar mixture of the symmetrical anhydride of the N-protected amino-acid, N,N″-di-isopropylurea, and unchanged carbodi-imide and not the O-acylisourea adduct as reported in the literature.

Peptide synthesis in partially aqueous solution: Preparation of a Leu—Ala—Gly—Val using symmetrical anhydrides of N-alkoxycarbonylamino acids in aqueous N,N-dimethylformamide

Peptide synthesis by incremental addition is normally carried out in organic solvents in which the activated residue is soluble and coupling reactions proceed smoothly. Addition of a single residue to amino groups in proteins, however, is less straightforward because the acylating agents are not soluble in, and the usual coupling procedures are not compatible with, the aqueous solvents required to dissolve proteins, The practise has been to use mixed solvents, the most popular activated form of the protected amino acid being an activated ester (review [ 1 I). Large excesses of reagent have to be used; water-soluble activated esters have been introduced to try to overcome their inefficiency in coupling in aqueous solvents [2]. Mixed anhydrides will acylate amino groups in partially aqueous solution, however secondary acylation products are formed [3,4];N-carboxyanhydrides can lead to the addition of two residues [S]. A coupling procedure which is efficient in partially aqueous solution and which leads to only one acyiation product would be valuable since it would allow the synthesis of products requiring a minimum of purification. We describe here a method of acylating amino groups in partially aqueous solution which satisfies these criteria. It involves the use of symmetrical anhydrides of N-alkoxycarbonylamino acids in 80% aqueous NJ-dimethylformamide,

Studies on the use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for the synthesis of acylamino acid anilides and p-nitroanilides.

The use of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) for coupling acyl-L-amino acids with aniline and p-nitroaniline has been investigated. Optically pure anlides are obtained in good yield but partial racemization occurred in one case. p-Nitroanlides cannot be obtained under similar conditions but if the reaction solvent is removed and the residue left for several days, coupling occurs giving a racemic product in moderate yields. Reaction mechanisms are proposed on the basis of isolated and characterized intermediates. It has been found that N-benzoylamino acid anilides and p-nitroanilides are cleaved within a few minutes by hydrogen bromide in acetic acid to give the N-benzoylamino acid and the amine.

Reaction mechanisms in peptide synthesis. Part 2. Tautomerism of the peptide bond

SummaryWe had concluded in previous work that ring opening of a 2-alkyl-5(4H)-oxazolone by water or ammonia leads to transient high-energy imidol intermediates which instantly tautomerize to the native amides. Using the MOPAC molecular orbital program, detailed geometric and energetic characteristics of the tautomerism of a peptide bond have been determined on the AM1 level. The results demonstrate that tautomerism of a peptide bond comprises a three-stage process involving three successive transition states and a bimolecular mechanism: (i) E→Z peptide bond isomerization followed by dimerization, (ii) concerted double-hydrogen exchange leading to an α-hydroxyimine (imidic acid) followed by splitting of the dimer, and (iii) Z→E N-methylimine inversion. While pathway (iii→ii→i) is predicted as a feasible route terminating in the formation of a peptide bond, the inverse route (iii←ii←i) is excluded as a possible initial step in the generation of a 5(4H)-oxazolone intermediate.

Reaction of N-t-butoxycarbonylamino acid anhydrides with tertiary amines and carbodi-imides. New precursors for 2-t-butoxyoxazol-5(4H)-one and N-acylureas

N-t-Butoxycarbonylamino acid anhydrides react with tertiary amines to give 2-t-butoxyoxazol-5(4H)-ones, and with N,N′-dialkylcarbodi-imides to give the oxazol-5(4H)-ones and the N-acyl-N,N′-dialkylureas.

Reaction mechanisms in peptide synthesis. Part 1. Semiquantitative characteristics of the reactivity of 2-methyl-5(4H)-oxazolone with water and ammonia in the gas phase and weakly polar media

Summary2,4-Dialkyl-5(4H)-oxazolones are well-recognized intermediates in some aminolysis reactions in peptide synthesis. Using the MOPAC molecular orbital programs, detailed geometric and energetic characteristics of the elementary reaction pathways for the additions of water and ammonia to 2-methyl-5(4H)-oxazolone have been determined at the AM1 level. The results demonstrate that the additions must be parsed into a two-step mechanism involving formation of the α-hydroxyimine followed by tautomerization to the parent N-acetylamino acid or amide.