Preparation and Characterization of [5‐¹³C]‐(2S,4R)‐Leucine and [4‐¹³C]‐(2S,3S)‐Valine − Establishing Synthetic Schemes to Prepare Any Site‐Directed Isotopomer of L‐Leucine, L‐Isoleucine and L‐Valine

Abstract

In this paper a chemo‐enzymatic method has been developed that gives access to any isotopomer of the essential amino acids isoleucine and valine. The method gives the correct introduction of the second chiral center in (2S,3S)‐isoleucine and allows for discrimination between the two prochiral methyl groups in valine as shown by the preparation of (2S,3S)‐[4‐13C] valine. For the preparation of (2S)‐leucine in any isotopomeric form, the O’Donnell method to prepare optically active amino acids has been used. The protected glycine scaffold used in this method has been prepared by a strategy that allows access to any isotopomeric form. The preparation of [5‐13C]‐(2S,4R)‐leucine shows that the O’Donnell method in combination with the Evans method to obtain chiral 2‐methylpropyl iodide leads to a good discrimination between the two prochiral methyl groups. The O’Donnell strategy for the preparation of α‐amino acids is preferred over other methods since the reaction conditions are mild, the chiral auxiliary can be easily recovered and the optically active product can be easily separated. For the preparation of isotopically enriched valine and isoleucine the O’Donnell method is not suitable, because the alkyl substituents involved have a secondary halide substituent which is sterically too hindered to give an effective reaction with the protected glycine. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Synthesis of Isotopically Labeled α‐Amino Acids

“…The use of deuteriated ammonia and D 2 O gave (2S,3R)-½3-2 H 1 aspartic acid 5 in 67% yield (Scheme 11.4). In a similar manner, b-methylaspartase has been utilized for the synthesis of (2S,3S)-½4- 13 Cvaline and (2S,4R)-½5- 13 Cleucine using mesaconic acid and 2-ethylfumaric acid as substrates, respectively [15].…”

“…a-Amino acids specifically labeled with stable ( 2 H, 13 C, 15 N, 17 O, and 18 O) and radioactive ( 3 H, 11 C, 14 C, 13 N, 15 O, and 35 S) isotopes are valuable tools for a range of bioorganic chemical studies. These include the elucidation of biosynthetic pathways, the investigation of peptide and protein conformations using nuclear magnetic resonance (NMR) spectroscopy as well as a wide range of clinical applications.…”

Access to Any Site Directed Stable Isotope (2H, 13C, 15N, 17O and 18O) in Genetically Encoded Amino Acids

“…The amino and carboxyl groups of glycine ( 6 ) are protected using benzyl chloroformate and tert -butyl bromide, respectively, to afford N -protected glycine tert -butyl ester 7 [47]. Hydrogenation of N -benzyloxycarbonyl glycine tert -butyl ester ( 7 ) with palladium on charcoal yielded the tert -butyl ester of glycine with a free amino group.…”

“…Hydrogenation of N -benzyloxycarbonyl glycine tert -butyl ester ( 7 ) with palladium on charcoal yielded the tert -butyl ester of glycine with a free amino group. Reaction with commercially available 1,1-diphenylmethyl-eneimine afforded N -(diphenylmethylene)glycine tert -butyl ester XIV [47]. …”

Selective Isotope Labelling of Leucine Residues by Using α‐Ketoacid Precursor Compounds

“…[10] Subsequent saponification and decarboxylation yielded labelled isobutyric acid (8), which was further reduced to the corresponding alcohol with LiAlH 4 . [11] Oxidation to isobutyraldehyde under PfitznerMoffat conditions proved to be superior to conventional Swern oxidation in terms of reaction reproducibility. The product was directly frozen into a cooling trap in THF at 90 mbar and subsequently converted into 5-isobutylidenehydantoin (9).…”