Microbiological Preparation of 2H-Labeled L-Phenylalanine with Different Levels of Isotopic Enrichment for Medical Diagnostics of Hereditary Phenylketonuria

Abstract

It was carried out the microbiological preparation of deuterium labeled L-phenylalanine with using a Gram-positive aerobic facultative methylotrophic bacterium Brevibacterium methylicum, L-phenylalanine producer with ribulose-5-monophosphate (RuMP) cycle for carbon assimilation via conversion of low molecular weight substrates ([U-²H]MeOH and ²H₂O). For this purpose was the bacterium with improved growth characteristics on minimal salt media M9 supplemented with 2% (v/v) [U-²H]MeOH and increasing gradient of ²Н₂O concentration from 0; 24.5; 49.0; 73.5 up to 98% (v/v) ²Н₂O. L-phenylalanine was isolated from the growth medium after adding 5 M ²HCl (in ²Н₂О), pH = 2.0 by extraction with isopropanol and subsequent crystallization in ethanol (output 0.65 g/l). Alanine, valine, and leucine/isoleucine were produced and accumulated exogenously in amounts of 5–6 mmol in addition to the main product of biosynthesis. The method allows to obtain [²Н]phenylalanine with different levels of deuterium enrichment, depending on ²Н₂O concentration in growth media, from 17% ²Н (2 deuterium atoms) (on the growth medium with 24.5% (v/v) ²Н₂О) up to 75% ²Н (6 deuterium atoms) (on the growth medium with 98% (v/v) ²Н₂О) with introduction of deuterium to benzyl С₆Н₅СН₂-fragment of molecule that is confirmed with the data of electron impact (EI) mass spectrometry analysis of methyl ethers of N-5-dimethylamino(naphthalene)-1-sulfochloride [²H]amino acids after the separation by reverse-phase HPLC. ²H-labelled phenylalanine can be used in medical diagnostics of hereditary phenylketonuria.

Keywords: Brevibacterium methylicum, heavy water, biosynthesis, [²H]amino acids, hereditary phenylketonuria, EI mass spectrometry, HPLC.