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

Oleg Mosin Ignat Ignatov


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.


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ISSN 2222-4807 (online)  ISSN 2222-5668 (Paper)

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