N. Leo Benoiton

N-Methylamino Acids in Peptide Synthesis. IV. Racemization and Yields in Peptide-bond Formation

The racemization of an N-methylamino-acid residue during peptide-bond formation and mixed-anhydride activation has been investigated using Ala-MeLeu-Gly and Ala-MeLeu as model peptides. The results were compared with those for Ala-Leu-Gly, Ala-Leu, and Ala-Pro. The extents of racemization were determined by analysis of the diastereomeric products of the reactions after deprotection, using an amino-acid analyzer. Extensive racemization was detected after the hydrolysis of the mixed anhydrides of Bz-MeLeu, Z-Ala-MeLeu, and Z-Ala-Leu, but not of Boc-Ala-Pro and Z-MeIle. Significant racemization (2.8–39%) was observed when Z-Ala-MeLeu was coupled with Gly-OBzl by various methods in the presence of salts such as triethylamine hydrochloride or p-toluenesulfonate. Only coupling through the N-hydroxysuccinimide (HONSu) ester gave stereochemically pure product. In the absence of salt, less racemization was observed, but only couplings using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and N,N′-dicyclohexylcarbod...

N-Methylamino Acids in Peptide Synthesis. III. Racemization during Deprotection by Saponification and Acidolysis

The racemization of N-methylamino-acid derivatives in aqueous sodium hydroxide and hydrogen bromide in anhydrous acetic acid and other solvents has been investigated by determining the products of the reaction with an amino-acid analyzer after deprotection. Whereas MeIle-OMe, Z-MeIle, and the N-unmethylated derivatives were only slightly racemized (   N—H or carboxyl group whose ionization would suppress ionization of the neighboring α-C—H bond. Z-Melle and Z-Ala-MeLeu were substantially racemized (68% in 4 h and 34% in 1 h, respectively) by 5.6 N hydrogen bromide in acetic acid. The extent of racemization by acid varied with acid strength, polarity of solvent, and time. Incorporation of label into both isomers of Ala-MeLeu from a solution of the tritiated reagent established that ionization at the ...

Piperazinedione formation from esters of dipeptides containing glycine, alanine, and sarcosine: the kinetics in aqueous solution

Piperazinedione formation from the methyl esters of glycylglycine, glycyl-L-alanine, L-alanylglycine, glycyl-sarcosine, and sarcosylglycine and from the ethyl ester of glycylglycine in aqueous solution, pH 7·3–8·5, and 25·0° was studied. It was found to be a self-catalysed reaction and other amines also served as catalysts. Some concomitant ester hydrolysis occurred. The kinetics were analysed by regression analysis and the data fitted equation (i). Values of khyd′, {ks+kOH[OH+]}, kgb, and kgb′ were obtained. Glycylsarcosine methyl ester d[ester, total]/dt=[ester, free base]{ks+kgb‘[amine, free base]+kOH[OH–]}+[ester, free base]2kgb+[ester, total]khyd’(i) cyclised most rapidly (t½ca. 5 min), by more than an order of magnitude, and this was attributed to the much higher content of cis-isomer. The methyl esters of sarcosylglycine and glycylalanine cyclised considerably faster than the glycylglycine ester; sarcosylglycine methyl ester showed evidence of steric hindrance in its smaller self-catalytic (kgb) rate constant. Alanylglycine methyl ester cyclised the slowest and had a markedly low value of kgb. Saponification rates were similar to those of the esters of N-acylamino-acids. The mechanism of the cyclisation process, together with the effects of N- and C-methyl substituents on the reaction at the ester carbonyl carbon and on the ring opening of the piperazinediones are discussed.

2-ALKOXY-5 (4H) -OXAZOLONES AND THE ENANTIOMERIZATION OF N-ALKOXYCARBONYLAMINO ACIDS

N-Alkoxycarbonylamino acids are chirally stable under normal conditions of coupling. In the presence of tert.amine (tertiary amine), most activated N-alkoxycarbonylamino acids generate 2-alkoxy-5(4H)-oxazolone, which is not chirally stable in the presence of the base. Consequently, enantiomerization occurs in the presence of tert.amine if the activated residue is not immediately consumed by aminolysis. This paper reviews the situations in which 2-alkoxy-5(4H)-oxazolones are generated from N-alkoxycarbonylamino acids, the enantiomerization that occurs during and after the incorporation of N-alkoxycarbonylamino acids into peptides, the properties and methods of preparation of 2-alkoxy-5(4H)-oxazolones, and the practical use that has been made from our knowledge of the chemistry of 2-alkoxy-5(4H)-oxazolones.

Formation of meta-tyrosine from L-phenylalanine by beef adrenal medulla. A new biosynthetic route to catecholamines*

Summary Incubation of L-phenylalanine- 14 C, with beef adrenal medulla homogenate in the presence of a pteridine co-factor and a DOPA decarboxylase inhibitor gave rise to three radioactive products which were identified with an amino acid analyzer as tyrosine, m -tyrosine and DOPA. The formation of m -tyrosine was completely prevented by the tyrosine hydroxylase inhibitors α -methyltyrosine and 3-iodotyrosine. This demonstration of the conversion of phenylalanine to m -tyrosine, combined with our previously reported demonstration of the formation of L-DOPA from L- m -tyrosine, provides a new pathway for the biosynthesis of catecholamines in beef adrenals.

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.

Determination of n-methylamino acids and their optical purity with an amino acid analyzer

Abstract The ninhydrin color constants for N-methylamino acids obtained with a Model 120B Beckman amino acid analyzer were shown to increase 10–20 times to 18–93 % of those for the parent amino acid when the eluting buffer flow rate was decreased from 68 ml/h to 34 ml/h. The optical purity of N-methylamino acids can be established to within one part in 100 by determination with the analyzer of the diastereomeric dipeptides obtained by coupling l -alanine N-carboxyanhydride with the N-methylamino acid as devised for amino acids by Manning and Moore.

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 formation of 3,4-dihydroxy-L-phenylalanine from L-meta-tyrosine by rat liver and beef adrenal medulla.

Abstract Incubation of L- m -tyrosine with rat liver homogenate in the presence of a pteridine co-factor and a Dopa decarboxylase inhibitor gave rise to a new amino acid which was identified as 3,4-dihydroxyphenylalanine (DOPA) on the basis of its fluorescence spectrum, and chromatography. When L- m -tyrosine or L-tyrosine was incubated with a partially purified beef adrenal tyrosine hydroxylase preparation in the presence of the same additives, DOPA appeared in the medium in amounts greater than when no substrate was added. No DOPA was detected when the substrate was D- m -tyrosine. L- o -Tyrosine gave no new amino acid under the same conditions. The finding that partially purified rat liver phenylalanine hydroxylase and beef adrenal tyrosine hydroxylase preparations hydroxylate m -tyrosine is contrary to previous reports.

Racemisation of activated, urethane-protected amino-acids by p-dimethyl-aminopyridine. Significance in solid-phase peptide synthesis

Racemisation of Nα-t-butoxycarbonyl, fluoren-9-ylmethoxycarbonyl, and benzyloxycarbonyl amino-acid anhydrides by p-dimethylaminopyridine is shown to ba a significant side reaction during attachment of the first amino-acid to the resin in solid-phase peptide synthesis.